The Lymph Node RevisitedDevelopment Morphology Functioning and Role in Triggering Primary Immune Responses.код для вставкиСкачать
THE ANATOMICAL RECORD 293:320–337 (2010) The Lymph Node Revisited: Development, Morphology, Functioning, and Role in Triggering Primary Immune Responses GUY SAINTE-MARIE* Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec, Canada ABSTRACT Scenarios have been proposed to explain how lymphoid components of a lymph node favor the encounter of a drained antigen with a circulating competent naı̈ve lymphocyte to trigger a primary immune response. However, these scenarios rest on incorrect concepts about the organ. This situation resulted from a loss of interest for studies on in vivo lymphoid organs due to a widespread switch, decades ago, to work on suspended lymphoid cells. However, an in vivo holistic study of the organ continued in our laboratory. The present review synthesizes resulting knowledge on lymph node morphology and global functioning. We show that the opening of an afferent lymphatic vessel into the subcapsular sinus is the focal point from which the related portion of a lymph node—a node compartment—is developed. As to the formation of a compartment’s lymphoid components, it is neonatally orchestrated by the dichotomic nature and distribution of antigens in this subcapsular sinus, which determines a dichotomic recruitment of circulating cells and the compartment’s architectural complexity. The transport process of an antigen from a given tissue territory into restricted sites of the draining compartment further deﬁnes its local morphological features and activities, while providing the possibility to reduce the wandering of a short-lived naı̈ve cell through innumerable target-devoid sites. We also explain that the nodal lymphoid components are not implicated in the triggering of primary responses, but are rather products of such responses. Scenarios for the triggering of primary responses, consistent with real node morphology and functioning, C 2010 Wiley-Liss, Inc. are proposed. Anat Rec, 293:320–337, 2010. V Key words: lymph node development; lymph node morphology; lymphnode functioning; lymphocyte quest; triggering of primary immune responses; rat In 1984, Nossal wrote ‘‘A readership consisting of primarily anatomists has every right to question the favorite sport of research workers in cell immunology. This is to take a lymphoid tissue and totally destroy its beautiful and elaborately designed architecture to obtain simple cell suspension of lymphocytes, which are then asked to do more or less all the jobs of the original anatomic masterpiece.’’ Nonetheless, in vitro work prevailed largely because of the great difﬁculty of in vivo experimentation on lymphoid tissues. More recently, Gretz et al. (1996) recalled that while in vitro studies yielded notions on the activation of T and B cells at the subcelluC 2010 WILEY-LISS, INC. V *Correspondence to: Guy Sainte-Marie, Faculté de médecine, Département de pathologie et biologie cellulaire, Université de Montréal, C.P. 6128 succursale Centre-ville, Montréal, QC, Canada H3C 3J7. Tel: 450-449-1319. Fax: 418-775-0740. E-mail: email@example.com Received 23 December 2008; Accepted 18 September 2009 DOI 10.1002/ar.21051 Published online in Wiley InterScience (www.interscience.wiley. com). THE LYMPH NODE REVISITED lar level, the efﬁciency of the immune system in vivo depends on a higher level of orchestration that occurs in lymph nodes, where most primary immune responses are presumed to be triggered. They added that activation requiring the encounter of an antigen (or an antigen-presenting cell: APC) and a rare competent circulating naı̈ve lymphocyte—and how that encounter occurs—depend on morphological features of the lymph node’s parenchymal components, so that knowledge of lymph node morphology is essential for understanding its immune function. This opinion was shared by Cyster (1999), Crivaletto et al. (2004), and Sixt et al. (2005) who along with Gretz et al. (1996) proposed scenarios for the triggering of a primary response in the lymph node. However, these scenarios may directly transpose in vitro ﬁndings to the in vivo condition, while ignoring the organ’s complexity. Moreover, as will be demonstrated below, the lymph node’s assumed morphology rests on some inadequate morphological concepts and it is inconsistent across the various scenarios, reﬂecting confusion in the ﬁeld. This review describes the basic morphology of a normal lymph node. It explains how some of the more popular misconceptions about lymph node morphology and functioning arose, and why they are inadequate as a basis for elaborating scenarios on the triggering of a primary response. Alternative views are presented that are derived mainly from a synthesis of our long-term, mostly in vivo work on the rat lymph node undergoing various spontaneous immunological states that reveal natural reactions. We explain how drained antigens and the inﬂuenced subcapsular sinus play a primordial role in the neonatal emergence of nodal lymphoid components and in orchestrating their activity. We show how antigen dichotomy—the partitioning of antigens into those causing either a cellular or humoral response—can account for the organ’s complexity and dichotomic recruitment of circulating lymphocytes. These features facilitate the encounter of lymph-carried and blood-borne related immunogenic elements, and therefore, the triggering of a response. We also consider how the recruitment of circulating cells may have become more efﬁcient through the elimination of time-consuming aspects of a lymphocyte quest for its target. Finally, we elaborate new and morphologically more realistic proposals on the triggering of primary cellular and humoral responses in the lymph node. MORPHOLOGY OF THE LYMPH NODE1 Since the morphological features of the lymph node are essential for understanding its functions, they are summarized here. Though our work focused on the rat, nodal morphology is basically similar among eight examined mammal species including the human and the mouse (Bélisle and Sainte-Marie, 1981a). However, interpretation of single lymph node sections can be complicated by: (1) irregular occurrence, but more often in 1 Note that this review uses the terminology we have adopted over the years to designate lymph node components; other terms may be used when necessary but are put into quotation marks. Lymph node terminology has been quite labile over the years, and changes in terminology often have not been well justiﬁed, contributing to confusion in the ﬁeld. 321 some species and in large than in small lymph nodes, of variably developed connective tissue trabeculae or septae arising from the capsule, which may partition a lymph node to various extents (Yoffey and Courtice, 1970); (2) changes in the appearance of structural components with cutting angle because lymph node and compartment architectures are non-uniform; (3) difﬁculty of clearly outlining the diverse nodal lymphoid components in standard tissue sections; and (4) intrinsic and extensive variability in the number, size, shape, and content of these components within and among compartments, lymph nodes, and individuals (see for example, the extreme variability in lymphocyte content of the subcapsular sinus described in Sainte-Marie et al., 1982; Sainte-Marie and Peng, 1990b; Sainte-Marie, 2001). These complications, combined with often limited organ samples, contribute to explain why the lymph node has remained poorly understood and why contradictory reports, conclusions, and functional hypotheses abound in the literature. Although random sections of lymph nodes may be variable and puzzling in appearance, their basic architectural organization is simple and consists of a structure called a compartment. The compartment is the portion of a lymph node associated with the opening of each afferent lymphatic vessel, or each of its terminal branches, into the subcapsular sinus (Bélisle and SainteMarie, 1981b; Aijima et al., 1986; Hoshi et al., 1997). The smallest lymph nodes have a single afferent lymphatic vessel and a single compartment (Bélisle and Sainte-Marie, 1981b). In larger organs, the number of compartments equals the number of afferent lymphatics in small mammals or the number of terminal branches of afferent lymphatics in large mammals (Bélisle and Sainte-Marie, 1981b; Aijima et al., 1986). Here and there at the margin of each compartment, cortical gaps connect the subcapsular sinus to medullary sinuses (Fig. 1A, B). A compartment also has a subsinus layer, a peripheral and a deep cortex, as well as medullary cords and sinuses (Fig. 1C, D). Compartmentation was demonstrated by the subcutaneous injection of a small dose of tracer (Sainte-Marie et al., 1982). The tracer entered the subcapsular sinus of the single compartment associated with the lymphatic vessel draining the injected tissue territory. The tracer then ﬂowed from the lymphatic opening towards and into the closest cortical gaps (Fig. 1C). Thus, the drained material from a given tissue territory usually inﬂuences only those components of the connected compartment (Sainte-Marie et al., 1982). This is contrary to the common view of the lymph node being one functional unit, such that the content of any of its afferent lymphatics is assumed to spread throughout and inﬂuence the whole organ. This view arose from observations made after locally injecting a large dose of tracer that ﬁlled all sinuses of the draining lymph node (Drinker et al., 1934). The dose was administered to demonstrate the lymph node’s great ﬁltration capacity, but it was physiologically unrealistic. Recently, Gretz et al. (1997) recognized the existence of the compartment, but renamed it a ‘‘lobule.’’ Their description of a ‘‘lobule’’ in the rat lymph node is compatible with ours; however, their statement that the mouse lymph node has a single ‘‘lobule’’ disagrees with observations in Bélisle and Sainte-Marie (1981a). Gretz 322 SAINTE-MARIE et al. (1997) reported that in larger mammals, the ‘‘lobules’’ are outlined by radial ﬁbrous trabeculae (more correctly, septae). Although septae may indeed be seen in some species (Yoffey and Courtice, 1970), they are not a basic feature of lymph nodes with multiple compartments (Sainte-Marie et al., 1982). For example, there are few septae in the rat’s multi-compartmented lymph nodes. Peripheral Cortex and Subsinus Layer Fig. 1. Lymph node compartment. A: Minute lymph node with a single compartment. (c) Indicates the capsule, (s) subcapsular sinus, (o) opening of the afferent lymphatic into the sinus, (p) peripheral cortex, (d) deep cortex, and (e) efferent lymphatic. The drained lymph, carried into the subcapsular sinus via the opening, enters medullary sinuses at the margin of the peripheral cortex (arrows). B: A lymph node with three compartments. The drained lymph further reaches medullary sinuses via cortical gaps (g) between individual compartments. An often wider expansion of peripheral cortex beyond the deep cortex center and details of the medulla are not shown for the sake of simplicity. C: Compartment details. The peripheral cortex has folliculo-nodules (fn) and an extrafollicular zone (e). The deep cortex has a center (DCC) and a periphery (DCP). The arrowhead points to a deep cortex sinus arising as a culde-sac in the periphery. A compartment also has anastomosed medullary cords (mc) surrounded by medullary sinuses (ms) that fuse to form the efferent lymphatic at the lymph node’s hilus. The horizontal arrows above the ﬁgure delimit the two domains of the subcapsular sinus and peripheral cortex: one above the deep cortex center (aDCC) and one beyond it (bDCC). The thick arrows trace lymph ﬂow whereas the others show the migration pathways of recruited circulating lymphocytes. From the subcapsular sinus and HEVs (ovals) of the extrafollicular zone–aDCC, cells committed to cellular responses (thin, solid arrows) migrate to the DCC. Cells committed to humoral responses (thick, stippled arrows) migrate to medullary cords by way of the DCP for those recruited aDCC. Unconcerned drained cells (broken arrow) leave the subcapsular sinus via cortical gaps. D: Left: HEVs (dark) run in the extrafollicular zone and deep cortex periphery. At the corticomedullary junction, they become regular medullary venules (shaded). Right: Capsule-borne coarse reticular ﬁbers extend in parallel across the subcapsular sinus and extrafollicular zone; those above the deep cortex center bend and extend into the deep cortex periphery. Reproduced with permission from Anat Rec;209:95–104 and Anat Rec;245:593–620. Until about 1962, the lymphoid population present between the subcapsular sinus and the medulla of a lymph node was termed cortex, which was further resolved into a peripheral and a vaguely demarcated deep cortex. It was then found that lymphocytes comprise B and T cells, and that T cells, responsible for cellular responses, are located in the deep cortex (Oort and Turk, 1965). Subsequently, components that had been so far termed peripheral and deep cortex became known as the ‘‘cortex’’ and ‘‘paracortex,’’ respectively. For reasons that will become apparent below, we continued to use the original terms of cortex including a peripheral and a deep cortex. The peripheral cortex underlies the subcapsular sinus entirely. It tends to stretch symmetrically around the lymphatic opening and to thin with increasing distance (Fig. 1A). It contains a superﬁcial row of folliculo-nodules, each formed of a B-cell follicle covering to various extents a nodule (or germinal center) (Sainte-Marie and Sin, 1970). Folliculo-nodules are separated by an extrafollicular zone (Fig. 1C, D); they are not discussed further since they are not implicated in primary responses. An elusive subsinus layer is sandwiched between the subcapsular sinus and the peripheral cortex. Its ﬁber network supports reticular-like cells interspaced by lymphocytes and APCs (Lind, 1968). The subsinus layer is about 25 lm thick over the extrafollicular zone but thins over outwardly bulging folliculo-nodules. This layer complements the screening activity of the sinus wall (Sainte-Marie and Peng, 1985a). Deep Cortex The ‘‘paracortex’’ was initially deﬁned as a uniform layer of T cells underlying the whole peripheral cortex of a lymph node (Gutman and Weisman, 1972). Actually, the deep cortex of a compartment is hemispherical or semi-ovoid, centered on the lymphatic opening (Fig. 1A), and it usually does not extend horizontally as much as the peripheral cortex above (Bélisle and Sainte-Marie, 1981c; Aijima et al., 1986; Okada et al., 2002; WillardMack, 2006). The deep cortex has a center, where T cells of cellular responses are engendered, and a periphery underlying the center (Fig. 1C) (Bélisle and SainteMarie, 1981d). The periphery is actually an extension of the extrafollicular zone of the overlying peripheral cortex, with the additional feature of lymphatic culs-de-sac, termed deep cortex sinuses, which are continued by medullary sinuses. Hence, the deep cortex is not totally unlike the peripheral cortex and it is, therefore, inappropriate to call it ‘‘paracortex,’’ which etymologically implies a different component situated beside (para) the cortex. Note that because the deep cortex does not form a continuous layer in a lymph node, we previously THE LYMPH NODE REVISITED referred to its discontinuous portions as deep cortex ‘‘units’’ (Bélisle and Sainte-Marie, 1990). Considering that each lymph node compartment has a single portion of deep cortex, the word unit is superﬂuous and is now discarded except when quoting. Representation of the deep cortex and of its relationship to the peripheral cortex in single tissue sections may be puzzling, due to variable development of individual compartments and the cutting plane (Fig. 2A–D). In some lymph nodes, the peripheral cortex of neighboring compartments does not extend beyond their respective deep cortex, and therefore, these deep cortices may appear to form a continuous layer (Bélisle and SainteMarie, 1981a). In some compartments, to the contrary, the peripheral cortex extends far beyond the limit of the deep cortex, and then, depending on the cutting plane, tissue sections may exhibit only peripheral cortex so that a compartment appears to be devoid of deep cortex (Fig. 2C). Various other conﬁgurations may occur (Sainte-Marie et al., 1990). The fact that the deep cortex usually underlies only part of the peripheral cortex of a compartment makes the cortex as a whole non-uniform, contrary to the state of uniformity implicit in the popular concept of ‘‘paracortex.’’ This difference is of paramount importance, for either state implies different patterns for recruitment of circulating cells. A uniform cortex would result in uniform recruitment. Instead, the peripheral cortex and subcapsular sinus are actually divided into domains, one above the deep cortex center (aDCC) and one extending beyond the deep cortex center (bDCC), that recruit differently (Fig. 1C). The aDCC recruits elements involved in both cellular and humoral responses, whereas the bDCC recruits only elements committed to humoral responses (see below). A third domain located above the deep cortex periphery was also distinguished (Sainte-Marie and Peng, 1996), but it is not essential to the present discussion. High Endothelial Venules These peculiar vessels recruit blood cells and were called ‘‘postcapillary venules’’ (Pirro, 1954; Burwell, 1962) before the term HEV was largely adopted (Anderson and Anderson, 1975). HEVs were thought to be restricted to, and scattered throughout, the ‘‘paracortex’’ (Anderson and Anderson, 1975; Ford and Smith, 1982). It was and continues to be claimed that lymphocytes enter lymph nodes via HEVs only and at random (Gowans and Knight, 1964; Rouse et al., 1984). How these misconceptions arose will be examined in a later subsection. Actually, HEVs occur in the extrafollicular zone of the peripheral cortex and the continuing deep cortex periphery; they transform into regular venules upon entering the medullary cords (Fig. 1C) (Sainte-Marie and Sin, 1970). Exceptionally, an HEV may cross the deep cortex center and directly enter a cord; nonetheless, lymphocytes traveling in the HEV perivascular channels do not mix with lymphocytes in the center as demonstrated in athymic animals (Sainte-Marie et al., 1984). In addition, HEVs are commonly described as being uniform, but they have generally been studied under conditions that do not reveal the great plasticity in the features of their individual endothelial (or HEV) cells. Striking differences may be apparent even in adjacent cells (for a general review on HEVs, see Sainte-Marie and Peng, 1996). 323 Fig. 2. Normal rat lymph nodes stained using the Dominici technique. A: Cervical lymph node cut in a plane showing a single compartment whose peripheral cortex extends greatly beyond the deep cortex (d) (about 60). B: Parathymic lymph node. The cutting plane shows the deep cortex of two compartments but no cortical gap in the upper part of the organ; gaps are at other levels (about 20). C: Cervical lymph node cut in a plane showing no deep cortex, the peripheral cortex appears underlaid by medulla only; a small deep cortical element is at another level (about 15). D: Cervical lymph node whose cutting plane shows the deep cortex (d) of three compartments overlaid by a seemingly undisrupted peripheral cortex. The differences in the density of their dark lymphocyte population betray variations in the intensity of their individual activity (about 60). Reproduced with permission from Am J Anat;164:275–309 and Anat Rec; 199:45–59. Hence, HEV cells can vary markedly in shape and size: the lowest are squamous, the highest are narrowed and may exhibit a blastoid aspect (Dabelow, 1938; Burwell, 1962; Sainte-Marie, 1966). Lymphocytes are present in variable numbers in individual interendothelial spaces (Norberg and Rydgren, 1978) and in the expandable subendothelial space present between each HEV cell and the basement membrane (Fig. 3A) (Sainte-Marie, 1966; Mikata and Niki, 1971). Note that in comparison to the rat, the architecture of the lymph node of the commonly investigated mouse is more difﬁcult to interpret because the peripheral cortex and associated HEVs are less developed, and this in turn may explain why the opinion that HEVs are absent from the peripheral cortex is so persistent. Reticular Fiber Network and Pathways of Transcortical Cell Migration Coarse ﬁbers 1–4 lm wide, lined by pleomorphic ﬁbroblast- or reticular-like cells, arise perpendicularly from 324 SAINTE-MARIE Fig. 3. A: HEV wall. Between each distorted HEV cell and its very dark basement membrane, an expandable subendothelial space shelters subendothelial lymphocytes in varying number per space; one space has an unusual abundance of them. Perivascular channels, outlined by reticular ﬁber material, cuff the vessel. The ﬁrst channel, next to the endothelium, is narrower and exhibits ﬂattened lymphocytes. B: Prevailing view of the process of random blood lymphocyte recruitment. A luminal lymphocyte (0) is attracted to an HEV cell and rolls on it (1–2), crossing the basement membrane at the interendothelial junction (3). C: Author’s proposal: The lymphocyte (0) is already rolling on the endothelium. At step 3, rather than crossing the basement membrane, it may enter a subendothelial space (4). After interaction there, it crosses the basement membrane (5) and enters the ﬁrst perivascular channel (6) where also it might interact and/or enter the second channel (7). Reproduced with permission from Anat Rec; 245:593–620. the capsule but almost only over the extrafollicular zone (Fig. 1D). They run parallel through the subcapsular sinus, subsinus layer, and extrafollicular zone. Between the coarse ﬁbers in the extrafollicular zone, ﬁne ﬁbers knit small communicating alveoles, each hosting a few lymphocytes (Sainte-Marie and Sin, 1970). Fiber material outlines one, but more often two or three, roughly concentric perivascular channels that surround each HEV (Sainte-Marie, 1966). The ﬁrst channel, immediately cufﬁng the HEV endothelium, is narrow and hosts distorted lymphocytes. The second and third channels are dilated to varying degrees and contain more rounded lymphocytes. At the ﬂattened face of the deep cortex center, the parallel ﬁbers and HEVs bend and course into the deep cortex periphery to join medullary cords below. The extrafollicular zone and continuing deep cortex periphery connect both gateways—the subcapsular sinus and HEV network—for entry of lymphocytes into the compartment. Together, they form a continuing pathway of parallel ﬁbers and perivascular channels that orient the migration of incoming lymph-carried or blood-borne cells. This pathway guides a competent cell committed to either a cellular or a humoral response towards the deep cortex center or a medullary cord, respectively, where it yields effector-cells of a response (Fig. 1C). In other respects, it is noteworthy that the pattern of the ﬁber network differs in the different components of the compartment (Fig. 1D), allowing them to be better delimited with silver impregnation (Sin, 1967; Sainte-Marie and Sin, 1970). The Concept of ‘‘Paracortical Cords’’ Kelly (1975) suggested that ‘‘paracortical cords,’’ favoring T cell activation, are the basic elements of a ‘‘paracortical area.’’ A ‘‘cord’’ was said to comprise a central HEV with a perivascular cuff of lymphocytes enclosed in a reticulin framework surrounded by an alleged ‘‘paracortical sinus.’’ The area shown in Kelly’s supporting Fig. 2 is in fact the deep cortex of a compartment, whose center is the site of formation of effector-cells of cellular responses. However, no ‘‘cord’’ is apparent in the illustrated deep cortex center and this is consistent with the demonstrated absence of HEVs in the center (refer previous section). More recently, Gretz et al. (1997) reviewed lymph node organization and contemplated how a primary cellular response might be triggered in the ‘‘paracortex.’’ THE LYMPH NODE REVISITED Their review was offered as a functionally-oriented summary of their understanding of ‘‘paracortex’’ architecture and presented a hypothesis regarding its microanatomy and the rules governing an orchestrated movement of cells and soluble factors within it. Actually, they adopted Kelly’s general concept of ‘‘paracortical cords,’’ while stating as a caveat that they had not been able to identify deep cortex ‘‘units,’’ and therefore, could not integrate them in their hypothesis. However, Fig. 7B in Gretz et al. (1997) shows an accumulation of lymphocytes under peripheral cortex, which is probably a deep cortex ‘‘unit’’ that appears round due to the cutting plane. Intriguingly, in an article published only a year earlier, Gretz et al. (1996) had acknowledged the existence of deep cortex ‘‘units.’’ Moreover, as pointed out above, the existence of deep cortex ‘‘units’’ was conﬁrmed by others through histology (Ajima et al., 1986) and more recently by plastic casting of lymph nodes (Okada et al., 2002). Gretz et al. (1997) modiﬁed Kelly’s (1975) concept of ‘‘paracortical cords.’’ They correctly recognized the narrow ﬁrst perivascular channel cufﬁng an HEV endothelium as described earlier (Sainte-Marie, 1966). However, they renamed it ‘‘perivenular’’ channel, renamed the second and third channels ‘‘corridors,’’ and renamed Kelly’s ‘‘paracortical’’ sinus a ‘‘cortical’’ sinus and added that such sinuses ﬁll the extrafollicular zone. Figure 4 of Gretz et al. (1997) was presented to support the existence of ‘‘cortical’’ sinuses, but none can be seen. Recently, Sixt et al. (2005) reported that drained dextrans accumulate in ‘‘paracortical’’ sinuses, but none were illustrated and to our knowledge they do not exist. Gretz et al. (1997) said ‘‘that the elegant form’’ of the paracortical cords ‘‘dictates, in large part, the marvelous efﬁciency of secondary lymphoid tissues in facilitating and regulating immune responses.’’ Ultimately, they asked ‘‘If the foregoing description of the paracortical cord is substantially correct, why has its existence been such a well kept secret?’’ The answer was that its complex geometry probably ‘‘precludes simple assessment of precisely how much of the paracortex consists of paracortical cords.’’ They stated that Kelly’s (1975) serendipitous study had revealed that cords are the essential constituent of ‘‘paracortex.’’ It is not clear how this could be concluded since Kelly had not carried out a tridimensional analysis of the presumed cords, a necessary task given their reportedly complex geometry and the acknowledged difﬁculty of distinguishing them in normal lymph nodes (Kelly, 1975; Gretz et al., 1997). Moreover, it is disconcerting that the ‘‘paracortical cords’’ were not mentioned 1 year earlier in a proposal on how ‘‘paracortex’’ morphology facilitates the triggering of a cellular response (Gretz et al., 1996) or a few years later in an article examining APC-lymphocyte interaction (Gretz et al., 2000). In our opinion, the existence of these cords is unsubstantiated. FUNCTIONAL ASPECTS OF THE LYMPH NODE COMPARTMENT Transport of Soluble Immunogenic Factors by the Reticular Fiber Network How do antigens penetrate the compartment’s parenchyma from the subcapsular sinus? An antigen causing 325 Fig. 4. Development of a compartment’s cortex; drawings based on silver-impregnated sections, which blackens reticular ﬁbers and lymphocytic nuclei present in the ﬁber-poor deep cortex center and folliculo-nodules. A: Rat, about 1 min old. The cortex to be has simply a framework of ﬁne ﬁbers. B: Rat, about 1 day old. A tiny deep cortex center has emerged, aligned with the overlying lymphatic opening. It has a decreased density of ﬁbers and a high level of large blastrelated lymphocytic cells. C: Rat, about 1 week old. The deep cortex exhibits some mature features. Compared to day one, the density of ﬁbers and of blast-related cells have decreased. HEV-precursors (oval-like) appear in the nascent peripheral cortex and deep cortex periphery, both of which have a high density of ﬁbers. D: Rat, about 2 week old. The now typical deep cortex has a further decreased density of ﬁbers and blast-related cells in its center but a greater level of lymphocytes. Lymphatic sinuses, loaded with emigrating lymphocytes, form as culs-de-sac in the deep cortex periphery. Follicles, still without a nodule (or germinal center), have emerged in the peripheral cortex. Modiﬁed with permission from Am J Anat;164:275–309 and Histol Histopathol;16:771–783. a cellular response is carried by an APC, one causing a humoral response is transported by ﬁbers. In 1963, Moe suggested that the lymph node’s reticular ﬁbers transport various soluble substances. Anderson and Anderson (1975) later reported that drained horseradish peroxidase is dispersed in the reticulum and sheaths around HEVs before permeating their interendothelial spaces and entering their lumen. However, we could not detect the tracer in these spaces and lumen in their supporting Fig. 19. Besides, permeation obtained with horseradish 326 SAINTE-MARIE peroxidase was suspicious because this tracer alters vascular permeability (Contran and Karnovsky, 1967). Injecting instead a foreign albumin, Sainte-Marie and Peng (1986) observed its transport by ﬁbers running through the thickness of the subcapsular sinus, subsinus layer, extrafollicular zone, deep cortex periphery, and adjoining HEV basement membranes; no endothelial permeation was detected. Recently, Gretz et al. (2000) stated that drained ﬂuorophore-labeled low MW dextrans and proteins diffused from peri-HEV ﬁbers in between HEV cells and into an HEV’s lumen. However, in the absence of counterstaining with a standard technique, it is questionable whether the small pale structures in their supporting Fig. 3A, B are HEVs. This uncertainty also applies to the only magniﬁed structure presented in their Fig. 3D, which shows no HEV cells— an essential feature for identifying an HEV. In addition, no interendothelial ﬂuorescence is detectable to support permeation as the delivery mode for the astonishingly abundant ﬂuorescent material seen in the presumed HEV lumen. Hence, in our opinion, HEV permeation has not been demonstrated. Still, a ﬁber-carried soluble factor can reach HEV cells, as conﬁrmed by the presence in ﬁbers and HEV basement membranes of typical metachromatic products spontaneously released by mast cells in the extrafollicular zone (Sainte-Marie and Peng, 1990a). Incidentally, on the basis of a conductive activity, the reticular ﬁber network was renamed ‘‘reticular conduit’’ by Gretz et al. (1996), which is a reductionist term unlike the accepted one. Transport of soluble immunogenic factors is unidirectional. A barrier, present in each capsule-borne parallel ﬁber at the outer wall of the subcapsular sinus, prevents transported antigens from disseminating in the capsule and perinodal tissue (Sainte-Marie and Peng, 1986). Conversely, antigens deposited on the capsule spread in its ﬁbers but not in their sinusal extensions. By contrast, antigens deposited on the thymic capsule are transported by capsule-borne ﬁbers into the thymic parenchyma (Sainte-Marie et al., 1986a). The quest is further facilitated by restricting the delivery of those antigens causing cellular responses only to the domain of the subcapsular sinus and peripheral cortex above the deep cortex center (see below). This prevents the wasteful wandering of concerned T cells into the peripheral cortex beyond the center, where only humoral responses are dealt with. Moreover, if T cells committed to cellular responses entered the peripheral cortex extending beyond the deep cortex center, obstacles would prevent them from reaching the center. First, T cells would have to migrate around the large folliculonodules and HEVs in this domain of peripheral cortex and cross its numerous reticulin walls set perpendicularly to the path of their migration towards the center. Second, such a migration of T cells would be against the ﬂow of numerous cells committed to humoral responses moving towards the medulla, thus hindering both migrations. Third, cell congestion would increase with proximity to the deep cortex center as all incoming T cells moved towards an increasingly smaller volume of peripheral cortex, rendering opposing migrations even more difﬁcult. Clearly, the entry of concerned T cells in the peripheral cortex above the deep cortex center, demonstrated below, provides the most expedient and effective pathway for these cells to reach the deep cortex center. The above constraints on T cell quest/migration have not been perceived so far because of the widely held concepts that cells enter a lymph node only at HEVs and that HEVs occur only in the deep cortex. Consequently, a randomly incoming T cell would directly enter the deep cortex. These concepts have endured, even though a few studies have demonstrated them to be incorrect, because it is difﬁcult to perceive how unrealistic they are, especially when one is unaware of their genesis as is common nowadays. Therefore, it is appropriate to review the origin of these concepts and examine alternative views. Restricted Lymphocyte Wandering for a Successful Quest Around 1960, the role of thymic lymphocytes was revealed by neonatal thymectomy (Miller, 1961); this breakthrough eventually allowed great advances in knowledge of immune reactions at the cellular and subcellular levels. But it also initially generated a rush of in vivo experiments, implicating lymph nodes whose morphology and functioning were still poorly understood. This resulted in many premature observations that could not be put into proper morphological context, and therefore, the interpretations or functional concepts that followed were questionable. A striking example is an initial report that lymph nodes of thymectomized mice ‘‘showed no proper structures and no plasma cells, often being reduced to a small piece of adipose tissue’’ (Miller, 1962). Reports followed stating instead that the deep cortex of the lymph nodes of thymectomized mice is devoid of lymphocytes, hence, it was called a ‘‘thymusdependent zone’’ (Parrott et al., 1966) but also a ‘‘T cell area’’ or a ‘‘paracortex’’ (Goldschneider and McGregor, 1973), however, these terms still conveyed partly unrealistic concepts. The term ‘‘thymus-dependent zone’’ is a misnomer (Fossum, 1990) because the deep cortex does form in athymic animals, except that its center is devoid of T cells while its periphery is still populated by B cells To trigger a response, a naı̈ve lymphocyte must contact its target, which may or may not be present in a body, and when present usually occurs in only a small territory. In addition, at best one out of 100,000 circulating lymphocytes may be competent to react with any one of a large mosaic of antigens (Ager, 1994). This context, at face value, makes it highly unlikely that a naı̈ve cell will encounter its target. Gretz et al. (1997) realized that this apparent hurdle had been overcome by an evolved process of antigen transport from tissues into secondary lymphoid organs. Instead of a lymphocyte circulating throughout the entire body, they argued, it needs only to circulate through these organs and mainly through lymph nodes, where virtually all primary responses would be triggered. This is especially important for rare circulating naı̈ve cells that survive only a few days if inactivated (Stites et al., 1994). Sainte-Marie and Peng (1996) further concluded that a circulating cell enters only a compartment hosting its target, not excluding the possibility that a cell might enter for an activity other than triggering a response. Origin of Popular Concepts THE LYMPH NODE REVISITED (Sainte-Marie et al., 1984). As for the term ‘‘T cell area,’’ it implies that the deep cortex contains only T cells, which is true only of its center. The inadequacy of the term ‘‘paracortex’’ was explained above. Incidentally, in two of our earlier works (Sainte-Marie et al., 1981, 1984), we mistakenly labeled the area of peripheral cortex over the deep cortex center (aDCC) as being thymusdependent, because of the occurrence of numerous T cells there. We should have stated simply that the extrafollicular zone aDCC is the site of entrance and migration of T cells towards the deep cortex center. In the absence of a thymus, the extrafollicular zone aDCC is still entered by B cells as is the continuing deep cortex periphery. Some of the most enduring misconceptions arose from the results of an intravenous transfer of labeled thoracic duct cells to determine whether lymphocytes re-circulate from blood to lymph (Gowans and Knight, 1964). At the organismal level, it was reported that labeled cells appeared to occur in equal concentrations in the various lymph nodes of the host, which was taken to indicate random entry. This interpretation was not supported by quantitative data. Regardless, even if the mix of T and B cells of various antigen-speciﬁcities had been more or less equally distributed among the various lymph nodes, this would not prove or disprove either a random or an antigen-speciﬁc entry. At the level of individual lymph nodes, the earliest observation at 24 hr after transfer, revealed that labeled cells present in a host’s lymph node were mostly located in the deep cortex, where some were associated with HEVs, and that only ‘‘trivial’’ numbers occurred in the subcapsular sinus and peripheral cortex. These observations yielded the concepts that circulating lymphocytes enter lymph nodes only at HEVs and that these venules are restricted to the deep cortex. The concept that cells do not enter a lymph node at its subcapsular sinus was immediately questionable because in the course of a study of the thymus (Sainte-Marie and Leblond, 1958) lymphocytes were observed in the subcapsular sinus of parathymic lymph nodes. This discrepancy prompted an analysis of the experimental basis for this and other concepts summarized above, which showed them to be unfounded, largely due to a lack of observations made at short time-intervals within the ﬁrst 24 hr after the transfer (Sainte-Marie, 1975). To resolve the mode of lymphocyte entry into lymph nodes, we performed an intravenous transfer of labeled thoracic duct cells and followed them through high-frequency observations starting soon after injection (Sainte-Marie et al., 1975). Labeled cells were ﬁrst seen in the subcapsular sinus and peripheral cortex, and later they accumulated in the deep cortex. However, concerns about the mode of lymphocyte entry were cast into shadow by a blinding interest for the process of lymphocyte recirculation. The early questionable concepts continued to be uncritically accepted and became entrenched in the literature despite later divergent ﬁndings (Sainte-Marie et al., 1975; Elves, 1977), which were overlooked but never rebutted. Moreover, the great difﬁculty of in vivo work on lymph nodes soon fostered a switch of research efforts to in vitro work using suspended lymphoid cells. As a result, understanding of the lymph node stagnated. Hence, Gowans wrote in 1996, that it is still not possible to explain the evolution of any immune response in terms of the highly dynamic struc- 327 ture of lymphoid tissue in vivo and of the migratory pathways of co-operating lymphocytes within it, qualifying this situation a ‘‘disgraceful gap in medical knowledge.’’ His review unfortunately did not mention some studies discussing the route of lymphocyte entry into lymph nodes and intranodal migration pathways (Sainte-Marie and Peng, 1980, 1983, 1990), which did contribute to partly ﬁll this gap (Fig. 2). The entrenchment of the concept that cells enter the lymph node only via HEVs diverted interest away from the subcapsular sinus and subsinus layer. Similarly, reported variability of individual HEV cells and the existence of subendothelial spaces and lymphocytes were overlooked because the concept of random cell entry was accepted. These oversights formed a serious obstacle to understanding lymph node functioning since these elements and features are of primordial importance, as will become apparent in the next three subsections. Entry of Circulating Lymphocytes at the Subcapsular Sinus Recent ﬁndings revealed that for several days after birth, lymphocytic cells colonizing the lymphocyte-free lymph node compartment enter it virtually only at the subcapsular sinus (Sainte-Marie, 2001). Thereafter, to a variable extent, lymphocytes continue to enter the compartment via this route as evidenced by many facts (Sainte-Marie and Peng, 1996), some of which are recalled here. First, is the frequent occurrence of lymphocytes in the afferent lymphatic vessels (as seen with a favorable cutting plane of tissue in Fig. 19 in SainteMarie and Sin, 1970) and the subcapsular sinus of older animals. Entry by this route is also supported by the presence in this sinus of newly formed marrow B cells (Brahim and Osmond, 1973) and of lymphocytes soon after transfusion (Sainte-Marie et al., 1975). At times, moreover, lymphocytes are retained on the wall of the subcapsular sinus while their number in the corresponding area of subsinus layer and outer stratum of the extrafollicular zone is reduced (Sainte-Marie and Peng, 1990b). Additionally, in some lymph node compartments of Xid mice almost devoid of B and T cells, occasional B cells occur in areas of the subcapsular sinus and accumulate in corresponding areas of the subsinus layer while the almost HEV-devoid underlying peripheral cortex remains quite lymphocyte-free (Sainte-Marie and Peng, 1985b). A contribution of drained cells to the lymphocytic population of the adult lymph node is supported as well by unusual events in some compartments under an athymic state. One is the replacement of normal cells of this population by drained altered lymphocytes (Sainte-Marie and Peng, 1990c). A second event is the atrophy of the extrafollicular zone in a compartment whose subcapsular sinus is deprived of drained lymphocytes (Sainte-Marie and Peng, 1990d). A third event is the formation of a compartment replica on the outer wall of the subcapsular sinus, which represents a compensatory reaction to the degradation of the original compartment (SainteMarie and Peng, 1987). Brieﬂy, a replica can arise next to the lymphatic opening as an intracapsular lymphocytic islet with a high level of blast-related cells, which recalls the neonatal situation. In the absence of HEVs in 328 SAINTE-MARIE the capsule, the colonizing cells of the islet should come from the sinus, as they do in the neonatal state. Dichotomic Recruitment of Circulating Lymphocytes due to Antigen Dichotomy One factor that facilitates the encounter of related elements for triggering an immune response is the topographically dichotomic recruitment of circulating cells arising from antigen dichotomy, that is, the partitioning of antigens into groups causing either a cellular or a humoral response. After birth, the earliest drained antigens induce the lymphocytic colonization and development of a compartment. As the compartment develops, antigen dichotomy causes the functional division of both the subcapsular sinus and nascent peripheral cortex into two domains, with each domain having a different recruitment pattern (Fig. 1C). The result is a non-uniform cortex. The formation of the two cortex domains is induced by sequential delivery and differential spreading in the subcapsular sinus of antigens provoking cellular and humoral responses, as follows. Although popular concepts imply that colonization happens by way of blood lymphocytes entering randomly at HEVs (Harris and Ford, 1963), in fact only T cells are initially implicated (Eikelenboom et al., 1979). Moreover, they enter at the subcapsular sinus, mostly as blast-related elements, settling on a small surface of the sinus wall opposite the lymphatic opening (Sainte-Marie, 2001). From there, they gather at a short distance beneath the wall and form a tiny spheric deep cortex center within a day (Fig. 4A–D) (Bélisle and Sainte-Marie, 1981e). Days after, the sinus domain above the center begins to also handle late-arriving drained antigens causing humoral responses that induce the emergence of peripheral cortex above the center, which is initially constituted of extrafollicular zone alone. As the latter thickens, it ﬂattens the outer half of the center and extends under it, preﬁguring the deep cortex periphery. Hence, the tardy arrival of B cells prevents mixing of T and B cells in the emerging center, whereas the subsequent formation of the deep cortex periphery continues to preserve the integrity of the center’s milieu: B cells do not cross the center to reach the underlying medullary cords, traveling instead via the periphery as conﬁrmed by immunoﬂuorescence (van Roojen, 1987). Meanwhile, HEV-precursors of the nascent peripheral cortex and deep cortex periphery develop HEV traits and a cell-recruiting activity, thus enhancing colonization. Progressively, the sinus domain above the center becomes somehow saturated with antigens causing humoral responses. As such antigens continue to enter the sinus, they spread beyond the center into a nascent second sinus domain, which will thus deals only with antigens causing humoral responses. Therefore, beneath this second domain one ﬁnds only peripheral cortex underlaid by plasmocytic medullary cords. Indeed, antigens retained by a sinus site enter the portion of peripheral cortex directly beneath, stimulating the development of HEV-precursors and inducing them to recruit relevant circulating cells. Thus, antigen dichotomy establishes a topographically dichotomic pattern of antigen presence and inﬂuence in the sinus as well as in the nascent peripheral cortex, which results in a correspond- ing dichotomic pattern of recruitment of circulating cells. A few factors account for this scenario. At birth, antigens causing humoral responses are controlled for some time by maternal antibodies, probably then halting their drainage towards lymph nodes. This explains why APCcarried antigens causing cellular responses and relevant T cells are the ﬁrst to be drained from tissues and to enter a lymph node compartment. There, APCs contact ﬁbers crossing the subcapsular sinus and settle, with attracted T cells, on the sinus wall opposite to the lymphatic opening. This leads to the emergence beneath of the deep cortex center with a peculiar milieu propitious to the development of effector-cells of cellular responses. By contrast, antigens causing humoral responses are minute, soluble, and possibly more abundant, so they eventually ﬂow farther than APCs, that is, beyond the center, where they promote the expansion of only peripheral cortex beneath the sinus. In axenic animals there is very little such expansion (Bélisle et al., 1982) because the sinus above the deep cortex can cope with an unusually small amount of antigens of both categories. Paradoxically at ﬁrst glance, such expansion is also very limited in some neighboring compartments of normal animals, and particularly, in their mesenteric lymph nodes. The reason for this peculiarity is a marked development of humoral responses directly in the gut, as revealed by abundant plasmocytes there, which reduces the arrival of antigens causing humoral responses to draining mesenteric compartments. Consequently, the peripheral cortex does not expand much beyond the deep cortices, which are therefore close to one another (Sainte-Marie et al., 1982). This situation is a vestige of the phylogenesis of the immune system: the formation of gut plasmocytes precedes the development of lymph nodes (Good and Finstad, 1967). By contrast, the peripheral cortex expands considerably in some compartments at other sites and this is matched underneath by formation of plasmocytic medullary cords (Fig. 2A, C). It thus appears that the extent to which peripheral cortex and plasmocytic cords are developed beyond the deep cortex is proportional to the supply of antigens causing humoral responses to individual compartments. The lymph node is to be viewed as a complement, not a substitute, to ancestral immune tools. A multitude of potential encounter sites of related elements improves the chance of triggering a response. For greatest efﬁciency of the system, a naı̈ve cell should be activated wherever it encounters its target and essential mediators within a basic territory of the immune system. Such a territory comprises: (1) a portion of body tissue that is drained by (2) a collecting lymphatic vessel, termed an afferent lymphatic at its junction with (3) a compartment of a local lymph node, over which it spreads its content. Within a territory, a continuum of events unfolds that is initiated by the intrusion of an antigen in the drained tissue, leading to the triggering of a response in the tissue itself and/or in the compartment where the lymphatic carries drained immunogenic elements. That activation can also occur in the drained tissue is evidenced postnatally by a late entry of blastrelated cells in the subcapsular sinus and their presence in the sinus beyond the deep cortex, with these cells yielding plasmocytes in underlying medullary cords (Sainte-Marie, 2001). In the adult, moreover, humoral THE LYMPH NODE REVISITED responses appear to unfold to a greater degree in some tissues rather than in draining compartments, as is the case of the gut where plasmocytes are known to be abundant. Antigen-Speciﬁc Recruitment of Circulating Lymphocytes A process that would further favor the encounter of a naı̈ve cell and its target is the antigen-speciﬁc recruitment of circulating cells, which provides a rational explanation for a suite of intriguing observations on HEVs in particular (reviewed in Sainte-Marie and Peng, 1996). As explained above, antigen-speciﬁc recruitment is a corollary of the beneﬁcial directed transport of antigens from a tissue territory into a single lymph node compartment or even limited sites of that compartment. Moreover, speciﬁcity is strongly supported, for instance, by the lymphocytic colonization of neonatal compartments (Sainte-Marie, 2001). In a random process, colonization by B and T cells would be expected to occur concomitantly, as both cells would occur irrespective of the presence or absence of proper targets in a compartment. Instead, T cells account for nearly all colonization activity during the ﬁrst postnatal week (Eikelenboom et al., 1979). Moreover, blast-related forms represent 84% of colonizing cells at 16-20 hr after birth, declining to 21% at day 21, mostly due to an accumulation of their maturing progeny-lymphocytes prior to emigration from the lymph node. Such percentages are not compatible with a random entry of circulating cells of which 0.001% at most are competent to respond to any given antigen (Goodman, 1994). In other respects, a speciﬁc recruitment of lymph-carried cells is consistent with the observation that a drained soluble antigen adheres to the wall of the subcapsular sinus (Sainte-Marie and Peng, 1985c). An APC does so as well, and may also become associated with the subsinus layer (Hendricks et al., 1980). There, an APC could release exosomes by long cytoplasmic processes (Murk et al., 2002; Théry et al., 2002) to expose its antigens at the surface of the sinus wall. A relevant lymphocyte, probing this wall, may thus contact its target and be selectively recruited there. As to the speciﬁc recruitment of blood-carried cells, it is supported by the non-uniform (i.e., focalized and selective) adherence pattern of suspended live nodal lymphocytes to HEVs in sections of frozen lymph nodes (Sainte-Marie and Peng, 1995) as well as by other observations on HEVs and lymph nodes under various states, which can be explained only by speciﬁc recruitment (Sainte-Marie and Peng, 1996). In addition, the marked variations in the features of individual and even adjacent HEV cells (Fig. 3A) as well as in the numbers of lymphocytes hosted in their subendothelial spaces reﬂect the individualism of their activity (Fig. 3C). By itself, the existence of subendothelial lymphocytes betrays a speciﬁc recruitment: the presence of lymphocytes in a subendothelial space makes little sense unless they are related cells, recruited by a concerned HEV cell to participate in a speciﬁc interaction. Actually, these lymphocytes are in contact with abluminal endothelial processes and, moreover, they interact in groups (Soderström, 1967; Bailey and Weiss, 1975), which implies a common competence. In turn, the interaction of related cells in a given space requires that 329 they group there, that is, that they be recruited speciﬁcally. Indeed, the number of circulating cells that can at a given time enter one of a multitude of exiguous subendothelial spaces is quite limited, so that selection is the only process by which rare cells competent to carry out a particular interaction could become associated in a same space. That HEV cells are stimulated individually and react speciﬁcally is also indicated by an unusual excess of lymphocytes in the subendothelial space of an isolated HEV cell(s) of a restricted HEV area(s). Such excessive loading betrays recruitment of related lymphocytes by HEV cells inﬂuenced by the same material, the recruited cells then unusually being retained in their subendothelial spaces due to an anomaly that prevents them from entering the compartment’s parenchyma. This explanation is consistent with the fact that lymphocyte retention is by far most often observed under the state of immunodeﬁciency (Sainte-Marie et al., 1986b); moreover, the phenomenon mimics excessive lymphocyte retention in the subsinus layer of immunodeﬁcient Xid mice (SainteMarie and Peng, 1985b). Finally, it is noteworthy that subendothelial lymphocytes have never been included in models of random cell recruitment (see Ulrich and Thorsten, 2003; and Fig. 3B), with which they are incompatible. It was suggested that drained cytokines permeating into an HEV lumen can ‘‘either in solution or immobilized on the luminal endothelial surface act directly on lymphocytes’’ of the blood (Gretz et al., 1996), but the authors omitted to consider a comparable role for drained soluble antigenic materials despite having reported that they too permeate the HEV endothelium. Although one may doubt that antigens usually do so, they nonetheless can reach an HEV cell via its basement membrane as seen above. There, the antigen could be immobilized on the luminal face of the membrane and/or the abluminal face of the HEV cell, becoming accessible to blood cells probing its subendothelial space (Fig. 3C). An antigenic material causing a cellular response could similarly inﬁltrate a subendothelial space through the release of exosomes by cytoplasmic processes of an APC migrating in a perivascular channel. A contact between a probing blood cell and its target would be the decisive signal in the cascade of steps leading to cell recruitment (Sainte-Marie and Peng, 1996). DEVELOPMENT OF ENTRANCES FOR CIRCULATING LYMPHOCYTES Additional aspects of the subcapsular sinus and HEVs will be examined, especially their interrelated and comparable mode of development or expansion. The almost concomitant formation of a new sinus area and of ﬁberlinked HEV areas beneath, both inﬂuenced by the same antigenic material, enhances the recruitment of competent cells and the chances of triggering of responses. Moreover, consideration of the mode of expansion of the subcapsular sinus and HEVs can provide clues on their functioning. Subcapsular Sinus Despite rather uniform appearance, the subcapsular sinus is functionally complex. Because it is shallower above the bulging folliculo-nodules, lymph ﬂows more 330 SAINTE-MARIE freely over the extrafollicular zone where the cell-lined ﬁbers crossing the sinus are concentrated. This favors the screening of the lymph content there, so that selected drained elements by far mostly enter the extrafollicular zone (Sainte-Marie and Peng, 1985c, 1986a). Besides, antigen distribution and recruitment of drained cells differ in the two sinus domains, as discussed earlier. Furthermore, recruitment of particular cells can involve only part of a domain: in some compartments, for instance, the sinus contains numerous mast cells entering the extrafollicular zone selectively next to cortical gaps (Sainte-Marie and Peng, 1990a). How does the sinus develop or expand after antigen dichotomy has neonatally delimited it into two functional domains? After the existing areas of the sinus domain above the deep cortex become almost or completely saturated with antigens, the entry in the sinus of additional antigenic material causing humoral responses promotes its expansion. Sinus expansion results from the addition of new sinus area at the margin of a compartment to handle2 excesses of such material and is accompanied by the emergence of peripheral cortex and plasmocytic medullary cords beneath. This mode of expansion strongly suggests that the added area selectively recruits drained cells related to the antigens that initiated its emergence, an interpretation supported by a neonatal event. Days after birth, there is a high level of blast-related cells in the subcapsular sinus and the nascent peripheral cortex beneath. Thereafter, the lymphocyte level increases, greatly predominating at about day 13. Nonetheless, blastic forms in the sinus of some compartments are still abundant beyond the deep cortex, where they can yield a trail stretching from a limited sinus area to the plasmocytic medullary cords directly beneath (Sainte-Marie, 2001). This betrays a focalized and speciﬁc recruitment of these cells, which are probably activated in the drained tissue by the same antigens that contributed to induce the formation of the sinus area where they are selectively recruited. Expansion of the subcapsular sinus further indicates that the capacity of a sinus area to handle lymph-carried elements is limited. This capacity is at least partly constrained by the correlated diameter and length of local ﬁbers crossing the sinus. In the neonate, the foremost antigens causing humoral responses are transported beneath the subcapsular sinus by short extensions of the ﬁne sinus ﬁbers. These extensions are connected to the basement membranes of underlying HEV-precursors. The antigens carried by these extensions stimulate the development of HEV features in the linked areas of the HEV-precursors and the recruitment of relevant blood cells which, in conjunction with drained cells recruited from the sinus, contribute to the emergence of a thin nascent peripheral cortex. With further antigenic stimuli, the peripheral cortex thickens while ﬁbers and HEVs elongate, matching the growing thickness of the peripheral cortex, as these elements evolve in unison (Bélisle and Sainte-Marie, 1981e). Fiber diameter simultaneously increases, probably due to the increasing volume of transported drained factors. Fiber growth and 2 Handling refers to all the events implicated in the screening of lymph-carried immunogenic elements for entry into the parenchyma. peripheral cortex thickening continue until antigenic stimuli peak or some maximal handling capacity of a sinus area is reached. These events apply as well to the sinus ﬁbers of the peripheral cortex above the deep cortex center. There, moreover, expansion of the sinus wall favors more crossings of APCs at spaces between the parallel ﬁbers, thus allowing a matching expansion of the deep cortex center. Clearly, the neglected subcapsular sinus plays a primordial role in the organization and functioning of the compartment, by screening the content of its afferent lymph and offering passage to focally selected elements into the compartment’s parenchyma. Moreover, the rarely observed and almost ignored lymphatic opening is the central point from which this architecture is spatially established; this knowledge is essential for understanding the lymph node compartment. High Endothelial Venules An HEV arises from the transformation of squamous endothelial cells of a regular venule under the inﬂuence of an antigenic stimulation. Observations made under various conditions indicate that development of HEV cells results from focal and individual cell stimulation, which is manifest both neonatally and after wherever a new HEV area emerges (Sainte-Marie and Peng, 1996). Macrophages were proposed to be the main stimulators of HEV formation (Hendricks et al., 1980). However, HEV formation is hindered in Xid animals with abundant macrophages but few lymphocytes (Sainte-Marie and Peng, 1985b) and more recent observations point to an essential stimulatory role for drained lymphocytes and, in particular, antigen-presenting T cells (Pichler and Wisscoray, 1994). Hence, it appears that development of HEV cells necessitates stimulation involving antigenic material, drained lymphocytes, and mediators: where they meet in a vessel, a squamous cell becomes an HEV cell (Sainte-Marie and Peng, 1996). Neonatally, the foremost antigens causing humoral responses induce initial HEV cell development in a small area of regular venules slightly below the subcapsular sinus (Sainte-Marie and Sin, 1970), and possibly on one side only of a vessel (Bailey and Weiss, 1975). Moreover, the extent of individual cell transformation differs, reﬂecting uneven individual stimulation. Subsequent antigens can further transform the earliest HEV cells to the extent that they can still be inﬂuenced; they also transform squamous cells in a contiguous area of the same vessels. Thus, HEV elongation mimics subcapsular sinus expansion. Similarly, the HEV scenario also implies limits to the number of HEV cells that can be stimulated at a given time by a given material and to the extent to which a given cell can be stimulated, with these limits partly depending on the amount of material handled by the sinus area above. The scenario of HEV elongation is repeated until the associated sinus area has attained a maximal handling capacity, thereby ending elongation of underlying HEVs. However, the HEV does not remain in a steady state after. Since the inﬂuence of a stimulation is transient (Hendricks et al., 1980), an HEV cell regresses when stimulation fades and redevelops if re-stimulated. In fact, such dynamics concern all cell populations of a compartment that are involved in ongoing responses. Because these THE LYMPH NODE REVISITED populations have a defensive role, they would atrophy if stimuli waned and were not followed by new ones. Later in life, new antigenic stimuli accentuate the transformation of lesser developed HEV cells (Henry and Beverly, 1976), which are either variably understimulated or regressed cells. The stimulus can also form new HEV cells in areas of transition from an HEV to a regular vessel, mainly at the corticomedullary junction (Burwell, 1962), where once again the neonatal features betraying individual stimulation of squamous cells can be observed. The fact that a new stimulation adds areas of HEV cells at this junction should increase the recruitment of competent blood cells. This would likely be the case if a new HEV area selectively recruits cells related to the antigens having caused its emergence, as proposed for a sinus area added by the same stimulus. Indeed, the HEV network starts as ﬁne branches fusing into progressively larger ones that form an HEV trunk upon nearing the junction (Sainte-Marie and Sin, 1970). Thus, if a competent blood cell has not been recruited prior to reaching the trunk, it may be recruited there in an area of new HEV cells; otherwise it would exit the compartment through the continuing medullary venule in an inactivated state—a waste of time and energy. Hence, the HEV layout matches that of a new sinus area under the inﬂuence of the same stimulation. Their combined features and advantages should ensure the triggering of a response. Functional Link Between a Subcapsular Sinus Area and Associate HEV Areas As we have seen above, the handling of antigens causing cellular responses by the subcapsular sinus domain around the lymphatic opening determines the emergence of the underlying rounded deep cortex center where cells of these responses form. Hence, the nature of the antigens handled in a sinus area determines the nature of the responding effector cells formed beneath it. By analogy, since a newly formed sinus area and underlying HEV area(s) are stimulated by the same antigens, it is reasonable to propose that they recruit the same competent cells to yield the same responses. This would represent a further step in the optimization of the immune process, consisting in the transport of a given tissue antigen into a restricted site(s) of the same sinus and peripheral cortex of the draining compartment. Such a layout would increase the likelihood of the encounter of related elements by eliminating their wandering in target-devoid sites of the peripheral cortex. A common stimulation of a sinus and HEV area(s) occurs because of topographical and functional links between them, whose modality will now be examined. When a humoral response is in cause, ﬁbers transport the antigen and ensure a functional link between a new sinus area and the new underlying area(s) of HEV(s). When a cellular response involving an APC-carried antigen is in cause, two functional links are plausible. One is the release of an antigen from exosomes extruded by an APC associated with an area of sinus wall or subsinus layer. The antigen could then be transported by local ﬁbers as above. A second possibility is that the APC migrates in the perivascular channel of an underlying HEV or HEV-precursor. Upon reaching one or more endothelial cells open to stimulation, the APC inﬂuences 331 it or them by way of antigenic material released from exosomes excreted by cytoplasmic processes extending through the HEV’s basement membrane. Since the inﬂuence of an antigen is transient, a functional link produced by a given antigen evolves following scenarios orchestrated by the importance and duration of its presence in the afferent lymph. For instance, when an antigen handled by a given sinus area fades, that area eventually can handle another material proportionally to its regained capability, with the cells of functionally linked HEV areas behaving likewise (Sainte-Marie and Peng, 1996). Such a dynamic state causes variability in the antigen-speciﬁcity of recruitment at a given sinus and HEV sites, entailing a variably important distribution of sites recruiting the same cells. This makes it difﬁcult to experimentally prove cell recruitment speciﬁcity, as does the varying degree of stimulation of a given HEV cell by diverse antigenic materials. Although not strictly comparable, the transient neonatal recruitment of neutrophils by HEVs illustrates a similar variability in recruitment (Sainte-Marie and Guay, 1995). After birth, neutrophils are recruited in the initial HEV area(s) of a regular venule. Recruitment gradually declines there and is shifted towards the venule’s next developing HEV area(s); this is repeated for about a month until neutrophil recruitment ceases altogether. This betrays a temporal change in the nature of cells recruited at a given HEV site. To summarize, the proposed scenario represents a more efﬁcient mode for lymphocyte searching through the transport of antigens from a tissue territory into only one compartment of the draining lymph node and, in the case of antigens causing cellular responses, into only one domain of its subcapsular sinus and peripheral cortex. One can reasonably infer that evolution also led to a further improvement consisting in the handling of a given antigenic material by a restricted site(s) of this sinus and peripheral cortex, thus limiting the recruitment of relevant cells to these linked sites. Experimental proof will be difﬁcult. Meanwhile, considering that complex and efﬁcient means to ensure the encounter of related elements have evolved in the brain, one may wonder why comparable reﬁnements would not have occurred in the lymph node compartment as well, especially considering that its reticular ﬁber network seems well suited for that task and that prompt immune responses can be critical to individual survival. ANTECHAMBERS TO THE LYMPH NODE PARENCHYMA At both entrances by which circulating cells can access the lymph node compartment, evolution may have further favored the formation of a conﬁning place for the rapid encounter of an antigen and recruited competent cells, thus optimizing encounter. Such antechambers to the parenchyma of a compartment exist: the subsinus layer and subendothelial spaces. Subsinus Layer Because of the common belief that lymphocytes do not enter the lymph node via the subcapsular sinus, this elusive component receives very little attention. Overall, observations indicate that it complements the screening 332 SAINTE-MARIE of drained cells by the sinus, which can explain why some may be retained in this layer, delaying or preventing their passage into the extrafollicular zone. Thus, in some lymph node compartments of Xid mice, B cells accumulate in the subsinus layer while the cortex remains lymphocyte-free (Sainte-Marie and Peng, 1985b). This means that an element that would normally allow their entry into the cortex is lacking or deﬁcient. In fact, these B cells fail to undergo blastogenesis and do not switch to IgG production (Mond et al., 1980). In other respects, since drained immunogenic elements can penetrate the subsinus layer, activation can occur there. required for an essential interaction is lacking or inefﬁcient, so that recruited cells are withheld. The B nature of the retained cells is indicated by the predominance of the phenomenon next to medullary cords, where plasmocytes form. The missing element is probably thymic helper-cells and/or factors, especially since the phenomenon occurs mainly in athymic animals (Sainte-Marie and Peng, 1996). In other respects, as factors essential to activation can reach a concerned subendothelial space and the narrow adjacent area of the ﬁrst perivascular channel where lymphocytes also interact (Schoeﬂ, 1972), activation can occur in both places. Subendothelial Spaces ROLE OF THE LYMPH NODE IN TRIGGERING PRIMARY IMMUNE RESPONSES A ‘‘Paracortical’’ Hypothesis The subendothelial spaces also are largely ignored, as it is assumed that the interaction of circulating lymphocytes with an HEV cell happens exclusively at its luminal face. However, this interaction also proceeds at its abluminal face that is exposed to subendothelial lymphocytes, to factors transported by ﬁbers and conceivably to antigens presented by APCs. In a subendothelial space, endothelial processes contact probing blood lymphocytes. There, these cells form groups that may include drained lymphocytes having migrated into a ﬁrst perivascular channel (Sainte-Marie and Peng, 1996). The groups reﬂect cell interaction (Soderström, 1967; Bailey and Weiss, 1975), which is likely a prerequisite, allowing passage into the parenchyma of blood cells entering an endothelial space rather than directly entering the ﬁrst perivascular channel. Consistent with these observations, suspended live nodal lymphocytes bind selectively to individual HEV cells or restricted HEV sites in sections of frozen lymph nodes (Sainte-Marie and Peng, 1995). They bind to the plasma membrane at both faces of HEV cells as well as to subendothelial spaces, basement membranes and ﬁrst perivascular channels, where they likely become attached to lymphocytes or antigenic materials. Moreover, lymphocytes that bind to a same site are interconnected by cytoplasmic bridges. Note that an endothelial space can also serve as a waiting room for asynchronously recruited partner-cells (see below). Subendothelial lymphocytes predominate in HEV trunks, supporting the idea that a subendothelial space is a site of essential cell interaction. HEV cells in trunk areas have often been stimulated by new antigenic material, which suggests that blood cells entering their subendothelial spaces are often naı̈ve and in need of an essential interaction. This idea is also supported by the fact that unusually large accumulations of subendothelial lymphocytes occur primarily in HEV trunks where they exhibit a peculiar distribution (Sainte-Marie and Peng, 1996). Indeed, such accumulations can occur under an isolated HEV cell, a few to several cells in a small HEV area, and/or many cells in a larger area and possibly on one side only of an HEV. This distribution pattern is similar to that of the foremost HEV cells arising in a neonatal regular vessel under the inﬂuence of the earliest antigenic stimuli (see above). By analogy, therefore, the similar distribution pattern of accumulated subendothelial cells betrays a stimulation of the implicated HEV cells by the same material and their recruitment of the same lymphocytes. Like lymphocyte retention in the subsinus layer of Xid mice, a subendothelial accumulation must imply that an element A proposal by Gretz et al. (1996) dealt with features of the ‘‘paracortex’’ that could facilitate the triggering of a primary cellular response within it. The proposal is summarized using their terminology and conception of lymph node organization as follows: APCs, which have processed antigens in a tissue and are lymph-carried into the subcapsular sinus of a local lymph node, migrate from the sinus through the ‘‘intrafollicular’’ zone to the ‘‘paracortex’’ where they cling to the ﬁber network. Circulating lymphocytes come from the direction opposite to arriving APCs, by transmigrating into the ‘‘paracortex’’ from HEVs; then T cells committed to cellular responses progress along the ﬁber network studded with APCs. Activation would occur in this meshwork of intertwining ﬁbers of the ‘‘paracortex’’ when a proper APC and naı̈ve T cell meet. However, the ﬁber network spreading between the subcapsular sinus and the HEVs in Gretz et al.’s supporting Fig. 1 lies next to a folliculo-nodule (‘‘follicle’’ in their terminology), and therefore, represents a portion of the extrafollicular zone of a compartment’s peripheral cortex, not its deep cortex (‘‘paracortex’’ in their terminology). Hence, if both APCs and transmigrating naı̈ve T cells did indeed move into the deep cortex, which in fact lies below the extrafollicular zone, then both would move in the same direction, not in opposite directions. In a later article, Gretz et al. (1997) repositioned HEVs inside presumptive ‘‘paracortical cords’’ forming the ‘‘paracortex’’ (see above). In this case, a cell leaving an HEV would settle within the ‘‘paracortex,’’ thus not needing to move towards it from any particular direction. Gretz et al. (1996) remarked that the dense ﬁber network of the structure presumed to be ‘‘paracortex’’ in their Fig. 1 could facilitate contact between a clinging APC and a relevant wandering naı̈ve T cell. We argued earlier that this structure is in fact a portion of the ﬁber-rich extrafollicular zone. The deep cortex center itself is actually poor in ﬁbers (Bélisle and Sainte-Marie, 1981d; Okada et al., 2002). It is therefore unlikely that these scarce ﬁbers play an important role in mediating contact between rare wandering competent T cells and numerous APCs. Moreover, a quest in the deep cortex center would be hindered by congestion arising from the many proliferating or maturing lymphocytes and by abundant randomly-incoming circulating cells. This is particularly true if one considers the minuteness of a naı̈ve cell with respect to the large volume of a deep THE LYMPH NODE REVISITED cortex center to be probed. Moreover, one wonders what mechanism could guide a minute rare cell through this large volume and permit it to encounter a relevant APC that may be present, but most likely is not. Almost as a closing remark, Gretz et al. (1996) recognized that the ﬁber network is looser in areas of cell proliferation such as the deep cortex ‘‘units,’’ adding that activation in these areas has already progressed enough that rapid access to information transported by ﬁbers is no longer required. Since the ﬁber-poor center represents a substantial portion of the deep cortex, the question of where the site of activation is localized in Gretz et al.’s (1996) scheme remains wide open. In another respect, Gretz et al. (1996) initially considered that T cells enter a lymph node only at HEVs, in agreement with a popular view, but soon after Gretz et al. (1997) stated that the afferent lymph carries lymphocytes from tissues and upstream lymph nodes. Why then did they not examine the possibility of a triggering of responses by lymph-carried T cells? Probably because one of the authors had postulated that virtually only memory cells exercise tissue surveillance (Adams and Shaw, 1994). If so, naı̈ve cells would be absent from the afferent lymph so that drained cells need not be considered in a proposal dealing with primary responses. However, the concept of tissue surveillance based on memory cells alone is questionable, especially since it is assumed to ensure an optimal immune function. Quite to the contrary, such a surveillance mode would compromise the survival of neonates in particular, for memory cells do not exist when an antigen intrudes for the ﬁrst time. Besides, lymphocytes occur in the gut lamina propria and in the subcapsular sinus of draining lymph nodes soon after birth, indicating that naı̈ve cells assume surveillance during this crucial period (Sainte-Marie, 2001). In addition, Gretz et al. (1996) recognized that the afferent lymph of central lymph nodes can contain some efferent lymph from peripheral lymph nodes. On the basis of random entry of cells into lymph nodes, a concept accepted by Gretz et al. (1996), this afferent lymph would contain naı̈ve T cells having already entered and left a peripheral lymph node because it was devoid of their target. Hence, the participation of lymph-carried T cells should have been considered in the elaboration of a proposal on T cell activation. However, such participation implies lymphocyte intranodal migration in the same direction as lymph-carried APCs, not the opposite direction as proposed by Gretz et al. (1996). A Chemokine Hypothesis Cyster (1999) ascribed a central role to chemokines in the intranodal encounter of circulating cells with their target. It was proposed that T and B cells leaving HEVs at random would be oriented towards a T- or a B-area, respectively, by corresponding attracting chemokines produced there. It is hardly possible to conceive how such an orientation process could work in vivo. For instance, to attract into the deep cortex center T cells committed to cellular responses—which are hypothetically recruited by HEVs situated anywhere in the peripheral cortex—chemokines produced in the center would need to diffuse throughout the peripheral cortex. Since the center underlies only a portion of the peripheral cortex, chemokines would have to ﬁrst diffuse out- 333 wardly into this portion and then laterally while forming a gradient potent enough to attract all incoming T cells from as far as a possibly distant compartment margin. These numerous cells would then all have to migrate towards the center, something also inconceivable as explained above. Thereafter, each cell would have to wander throughout the center in search of a relevant APC, a quest hindered by cellular congestion. In addition, an encounter could happen only if a naı̈ve cell had randomly entered not only a rare relevant lymph node hosting its target, but also the node’s relevant compartment. This seems unlikely, for the blood vascular network of a compartment represents a small fraction of the network of a whole lymph node which is usually multi-compartmented, and an even quite smaller fraction of the body’s whole vascular network. Hence, such a scenario would be grossly inefﬁcient for rare competent short-lived naı̈ve T cells, which with increasing age become even rarer due to thymic involution. In other respects, it must be considered how a naı̈ve T cell, having randomly entered a center devoid of its target, could succeed in exiting it rather than endlessly and vainly wandering in it due to the local elevated concentration and steep gradient of attracting chemokines. If somehow the cell succeeded in exiting, it would have to repeat, over and over again, the same hazardous scenario in any of a great number of compartments, few (if any) of which contain its target. The cell would likely die before such an encounter. Cyster (1999) stressed that a lymph node contains an immensely complex chemokine landscape, its multiple gradients attracting a cell in opposite directions. If so, a resulting erratic migration would jeopardize the beneﬁt provided by a compartment architecture well organized for an efﬁcient directed migration. He added that it will be exciting to see how many ‘‘turns’’ a cell might make as it travels through such multiple gradients. He did not indicate where a cell would turn. In the case of a T cell committed to a cellular response, turns would not be useful outside of a deep cortex center where there is at least a minute probability that its target might be present. In addition, the randomness of such turns would decrease a cell’s chance of encountering its target. Turns would furthermore represent an additional waste of time, which would be a detrimental behavior, especially for short-lived cells. All of this does not ﬁt with a condition identiﬁed by Cyster (1999), that, for a mature dendritic cell to function in an immunogenic manner, it must interact rapidly with competent T cells. In fact, Cyster’s proposal could work only if the compartment morphology was as simple as presented in his Fig. 3, where a ‘‘T area’’ is reduced to a cell islet close to the subcapsular sinus with HEVs conveniently situated next to it. The reality, as we have seen, is far different, yet this does not preclude a role for chemokines. However, this role must be consistent with the orderly design of the compartment, which imposes deﬁnite patterns for factor transport and cell migration. This context suggests that a chemokine has a focal or local inﬂuence, as illustrated by the recruitment of T cells committed to cellular responses occurring only above the deep cortex center. The proliferation of unrealistic proposals based on a misunderstanding of lymph node morphology and oversight of relevant literature is self-fostering and will 334 SAINTE-MARIE continue. This trend is illustrated by a recent proposal by Crivaletto et al. (2004). These authors also emphasized that a proper understanding of lymph node morphology is paramount for proposing relevant hypotheses and theoretical models for immune responses based on in vitro experiments in cell and molecular immunology. However, their conception of lymph node morphology is inadequate in many ways. For instance, in their Fig. 1, the afferent lymphatics are incorrectly shown to be as numerous as folliculo-nodules whereas the extrafollicular zone between these structures is delimited by an ‘‘intermediate sinus’’—a component that simply does not exist. A Morphologically Realistic Proposal Previous proposals on the triggering of primary immune responses attempted to explain how morphological features of components of a developed lymph node, mainly its HEVs and ‘‘paracortex,’’ allow triggering of a primary response, concluding that ‘‘forms dictate function’’ (Gretz et al., 1997). In reality, antigens (i.e., function) dictate compartment morphology as we have seen above. From the outset, previous proposals are fundamentally unrealistic because the components (i.e., forms) invoked do not exist prior to the occurrence of the ﬁrst postnatal antigenic stimuli and immune responses: these components are products of responses, not prerequisites. Therefore, to be plausible, a proposal on the triggering of primary responses must above all be consistent with the level of compartment organization at birth, a requirement not considered so far. At birth, only lymphatic sinuses exist (Fig. 4A) and among them the subcapsular sinus is the ﬁrst to be exposed to the foremost antigens of the afferent lymph. Sinusal cells handling these incoming antigens contribute to the foremost immunological reactions of the lymph node compartment. This neonatal potential for triggering primary responses in the absence of developed nodal components (i.e., independently of form) persists throughout life. Since each primary response is speciﬁc, its triggering at all ages requires particular lymphocytes to be recruited by sinusal or HEV cells inﬂuenced by a new antigen. These recruiting cells are newly formed and/or existing cells having regained the ability to be inﬂuenced by new antigenic material. Hence, existing sinusal and HEV cells of a developed compartment can be implicated in the triggering of a primary response because of a newly acquired recruiting property that is elicited by a new antigen. To formulate a proposal on events associated with the triggering of a response in a developed lymph node compartment, one must consider that the latter is basically constituted of (1) a receptacle for drained immunogenic elements—the subcapsular sinus, (2) distinct entrances for lymph-borne and blood-carried lymphocytes—the subcapsular sinus and HEVs, respectively, (3) an antechamber to the parenchyma for each type of entrance— the subsinus layer and subendothelial spaces, (4) a migration pathway—the extrafollicular zone and the continuing deep cortex periphery—which is connected to the antechambers and guides a recruited competent lymphocyte to the structure where it will yield its progeny, and (5) distinct structures propitious to the production of effector-cells of either cellular or humoral responses— the deep cortex center and medullary cords, respectively. Where does activation occur? Observations indicate that a recruited T or B cell is activated before it enters the structure where it yields its progeny. Neonatally, a large fraction of the cells entering the nascent deep cortex center or medullary cords are blast-related, that is, already activated cells. Thereafter, plasmablasts still congregate in the initial portion of cords, followed deeper by progressively less immature plasmocytes (SainteMarie, 1964). These facts point to an activation occurring somewhere upstream of the center and cords. The immediate upstream component is the migration pathway. For sake of efﬁciency, this pathway for cell trafﬁc should not be a place where a rare short-lived naı̈ve cell searches for a related, yet by far most often absent, element; it should instead serve only to guide an already activated cell directly to either the center or a cord. Therefore, activation should ideally happen even more upstream, in components providing a conﬁned milieu favoring the rapid encounter of related elements and where a naı̈ve cell can be exposed to all factors essential to activation. Such components are the subsinus layer and the subendothelial spaces of relevant HEV cells or the adjacent ﬁrst perivascular channel. This allows the subsequent direct migration of an activated cell towards either the center or a cord, the cell being oriented by ﬁbers and/or perivascular channels of the migration pathway studded with guiding determinants. On its way, the activated cell undergoes blastogenesis, thus explaining the presence of possibly numerous blastrelated cells in the extrafollicular zone and deep cortex periphery (Sainte-Marie, 1966). If unﬁnished, blastogenesis is completed in the center or a cord. Cellular Response After having processed an antigen in a tissue and having been carried by the lymph into the subcapsular sinus of the draining compartment, an APC probes the sinus over the extrafollicular zone present above the deep cortex center until it reaches a receptive site where it attaches to or enters the subsinus layer. The APC’s attracting chemokines induce drained T cells committed to cellular responses to probe the sinus there. Chemokines are also transported by local ﬁbers towards the center. Somewhere along their course, these ﬁbers join the basement membrane of HEVs overlying the center, so that the transported chemokines attract similar blood T cells to probe the endothelium of these same HEVs, as can do chemokines released by APCs migrating in their perivascular channels. Thus, the recruitment of T cells committed to cellular responses is restricted to the domain of subcapsular sinus and peripheral cortex present above the center, so as to reach the center with minimal delay (Fig. 1C). A lymph-carried naı̈ve T cell encountering a relevant APC associated with the sinus wall or the subsinus layer can be activated in either place, where they are exposed to all essential factors (Fig. 5A, left). This ensures that a mature APC and a competent T cell will interact quickly. The activated T cell then migrates to the center where it engenders its progeny. The APC can likewise travel to the center (Fig. 5A, left), so that, from a given site of the subsinus layer, both cells are guided by the same ﬁbers 335 THE LYMPH NODE REVISITED Fig. 5. A–E: Schematics of proposed modes of intranodal encounter of cooperating elements and their migration pathways for the triggering of primary immune responses. Explanations are in the last two subsections of this review. to the same place in the center where their interaction may continue. An observation suggests that an APC may cling to an element of the loose ﬁber network of a deep cortex center, from where it could inﬂuence related T cells close by. Indeed, in a large center with a reduced population of T cells, the latter concentrate into broadly spaced ribbons (Bélisle and Sainte-Marie, 1981d). Conceivably, factors from APCs clinging to distant ﬁbers attract T cells in their proximity explaining the fewer T cells between ribbons. As for competent naı̈ve blood T lymphocytes, they are recruited by relevant HEV cells, that is, cells that are presenting the implicated antigenic material (Fig. 5A, right). Activation can occur in the subendothelial spaces or in the adjacent ﬁrst perivascular channel since both places may contain all essential factors. The activated T cell (and perhaps a related APC) then migrates to the center. This leads to their aggregation at the same place of the center where related drained cells also gather, all being guided by the same ﬁbers or channels. Finally, it is conceivable that in the center an APC might activate newly formed memory cells, rapidly reinforcing a response by sparing them the quest for their target. Humoral Response In the case of a thymo-independent response, a drained naı̈ve B cell can be recruited at a site of the sub- capsular sinus exposing its target. Activation may occur there or next to it in the subsinus layer (Fig. 5B, left). The activated cell is then guided by local ﬁbers to an underlying medullary cord, either indirectly via the deep cortex periphery for a cell recruited above the deep cortex center or directly for a cell recruited beyond it. The activation of a B cell recruited by a relevant HEV cell can occur in the subendothelial space or in the adjacent perivascular channel; the activated B cell then migrates to the underlying medullary cord (Fig. 5B, right). Reaching a cord, the activated cell completes blastogenesis if it is unﬁnished. Thus, resulting plasmoblasts congregate at the entrances of cords. In the case of a thymo-dependent response, triggering implicates a T helper-cell and a naı̈ve B cell. In the subcapsular sinus, both cells can be recruited over the extrafollicular zone at a site presenting their target and there, they can enter the subsinus layer. When all essential factors are encountered in the sinus or the layer, activation unfolds. The activated B cell then travels to an underlying medullary cord, guided by local parallel ﬁbers (Fig. 5C). If instead either only the B or the T helper-cell is recruited on the sinus wall where the target is encountered, the recruited cell may wait there or in the subsinus layer for the arrival of a partner. Alternatively, it may leave this site in search of a partner, which could have been recruited by an underlying relevant HEV cell (Fig. 5D, E). To do so, the recruited cell moves along local ﬁbers connecting to a perivascular channel until it reaches a relevant HEV cell. If, instead, a partner is present in the subendothelial space or the ﬁrst perivascular channel, activation can happen when all essential factors are present. The activated B cell then proceeds towards an underlying medullary cord. If a partner is absent where a relevant HEV cell is ﬁrst encountered, the recruited cell may still adhere to its target, bind to the abluminal face of the relevant HEV cell or a facing site of its basement membrane, and wait for a partner to arrive. Alternatively, in the absence of a partner there, the recruited B cell may eventually move to a relevant HEV cell located deeper, thus possibly reaching the HEV trunk where recruitment of cells concerned in the triggering of a primary response is most intense. In a simpler scenario, a naı̈ve blood B cell and T helper-cell are both recruited by a same relevant HEV cell (Fig. 5F). To conclude, although knowledge of the lymph node compartment is still incomplete, it is possible to elaborate proposals on its global functioning and on basic tissue and cellular events related to the triggering of primary responses there. Nevertheless, these proposals would beneﬁt from improved knowledge, in particular, on the neonatal emergence of the compartment parenchyma, which could provide further clues on the unfolding of primary responses. 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