Characterization of germinal center dendritic cells in follicular lymphoma

A subset of dendritic cells called germinal center dendritic cells (GCDC) has recently been described inside germinal center from reactive lymphoid organs. We investigated this newly recognized population in follicular lymphoma (FL), which is considered to be the pathologic counterpart of germinal center B cells. Immunohistochemistry analysis with a panel of antibodies demonstrated the presence of a cell population with the peculiar GCDC phenotype in FL biopsies and a similar localization of these cells inside tumoral and reactive follicles. Therefore, we analyzed the relationships between GCDC and the other cell subsets of the tumor follicles. Some of CD4+ and CD8+ T lymphocytes present inside the follicle were found to be in close association with GCDC, suggesting a potential implication of GCDC in their activation. In addition, the distribution of GCDC inside FL and reactive follicles did not appear disrupted, in contrast to follicular dendritic cells, the other follicle dendritic cell type. Finally, we demonstrated that GCDC could be detected from FL lymph node cell suspension by flow cytometry. Taken together, these results indicate that FL development is not associated with a disappearance of GCDC or with a lack of physical interactions between GCDC and T cells inside the follicles. In addition, the fact that GCDC can be observed in FL samples by flow cytometry should allow their purification to further study their putative role in FL development and maintenance.     

The relative resistance of HIV type 1-infected chimpanzees to AIDS correlates with the maintenance of follicular architecture and the absence of infiltration by CD8+ cytotoxic T lymphocytes

Lymphoid tissues are the focus of critical events in HIV pathogenesis. Persistent and high levels of virus production, extensive trapping of virus particles in germinal centers, and progressive degenerative changes in lymph node architecture are characteristics of progressive HIV-1 infection. Infiltrates of granzyme B- and TIA-expressing CD8+ "cytotoxic" T lymphocytes (CTLs) precede involution of germinal centers in humans who develop AIDS. Similar to humans, HIV-1 infection in chimpanzees is active and persistent. However, in contrast to humans, they remain relatively resistant to AIDS. Lymph node biopsies from chimpanzees infected with HIV-1 or a related chimpanzee lentivirus were studied for the level and pattern of virus expression, changes in lymphoid architecture, CD8+ T cell infiltrates and the presence or absence of CTL markers. In stark contrast to HIV-1-infected humans, lymph nodes from infected chimpanzees had little virus deposition in germinal centers and a paucity of virus-expressing cells. Although some of the lymph nodes examined from infected animals had moderate follicular hyperplasia with infiltrating CD8+ T cells, none had evidence of follicular fragmentation. Most importantly, in marked contrast to infected humans, CD8+ T cells infiltrating the germinal center were negative for the CTL marker granzyme B. This evidence suggests that the infiltration of CD8+ CTLs into the germinal centers of lymph nodes may be a key determinant in AIDS pathogenesis.     

In situ quantitation of lymph node helper, suppressor, and cytotoxic T cell subsets in AIDS

We have used the novel monoclonal antibodies 9.3 and anti-Leu-8 in conjunction with other T cell markers to quantify T cell subpopulations in the paracortex, mantle, and germinal center compartments of frozen sections of lymph nodes from seven homosexual men with acquired immunodeficiency syndrome (AIDS) and five heterosexual controls. Antibody 9.3 allows dissection of the Leu-2+ cytotoxic/suppressor subset (Tcs) into 9.3+ cytotoxic cells (Tc) and 9.3- suppressor cells (Ts). Anti-Leu-8 allows dissection of the Leu-3+ helper subset (TH) into functionally distinct subpopulations. The data indicate that the T cells in patients with AIDS exhibit normal antigen expression but altered subset ratios. In this series, the data suggested that the reversal of the paracortical TH-Tcs ratio was due to an increase in Ts with a concomitant decrease in TH and Tc. These changes were also reflected in a reversal of the normal paracortical Tc-Ts ratio (3.0) to less than 1.0. Furthermore, the data suggested a marked decrease in paracortical Leu-3+8+TH, which are known to have inducer function in cellular immune reactions and exert feedback inhibition of immunoglobulin production through a suppressor T cell intermediary. In contrast, there was preservation of the Leu-3+8-TH population within the germinal center. This T cell subset is known to help B cell differentiation. This microenvironmentally specific constellation of T cell subset alterations within lymph nodes may in part explain several of the immunologic findings associated with AIDS.     

Simian immunodeficiency virus (SIVsm) infection of cynomolgus monkeys: effects on follicular dendritic cells in lymphoid tissue.

We studied follicles in sections of lymph nodes and spleen from cynomolgus monkeys (Macaca fascicularis) after infection with simian immunodeficiency virus (SIVsm), by (immuno)histology and (immunogold) electron microscopy. Also isolated follicular dendritic cells (FDC) were investigated. Histology showed ranged from follicular hyperplasia to follicle fragmentation. FDC showed desmin and vimentin, characteristic of mesenchymal cells. Except for two animals who got experimental chemotherapy in the first postinfection period, the cells expressed SIV gag p28 protein. Electron microscopy showed SIVsm-like particles in the germinal centers. A number of cell types in the germinal center, including FDC, showed tubuloreticular structures, indicative of alpha-interferon synthesis during an antiviral response. In immunogold electron microscopy, SIV p28 label was observed on the surface of FDC, on SIVsm-like particles, and in the cytoplasm of macrophages. A relatively high density of CD8-positive cells (T cytotoxic-suppressor phenotype) was observed around and in germinal centers, especially areas depleted of FDC. Cells immunoreactive for serine esterase granzyme-B, a protein occurring in granules of cytotoxic cells, occurred around germinal centers, but not in germinal centers at areas where FDC and SIV p28 label localized. This argues against a role of cytotoxic T cells in mediating follicle destruction.     

Normal T cell subsets in homosexual men living in a community without endemic AIDS

The cause of the abnormal T lymphocyte subsets reported in healthy homosexual men is not known. Frequent sexually transmitted infections including human T cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) are possible causes. To determine if the T lymphocyte subsets were abnormal in this population in an area without endemic acquired immune deficiency syndrome (AIDS), T lymphocyte subsets of 52 homosexual men in Rochester, New York, were enumerated, and evidence of infections known to cause these abnormalities was sought. Unlike the findings in previous reports, relative numbers of T helper and T suppressor cells and helper/suppressor T cell ratios were normal. Prevalence of cytomegalovirus infection (86 percent) was similar to that found in analogous populations, but only 9 percent had seropositive results for HTLV-III/LAV. Men with serologic evidence of nonprimary cytomegalovirus disease had lower helper/suppressor T cell ratios (1.5 +/- 0.2 versus 2.2 +/- 0.2; p less than 0.01). Hence, despite frequent infections with cytomegalovirus and other sexually transmitted pathogens, T cell subsets are normal in homosexual men in an area without endemic AIDS. Therefore, HTLV-III/LAV is primarily responsible for the T cell abnormalities observed elsewhere.     

Association of HTLV-III with Epstein-Barr virus infection and abnormalities of T lymphocytes in homosexual men

Homosexual men were studied for associations among human T-lymphotropic virus type III (HTLV-III) infection, Epstein-Barr virus (EBV) infection, and T cell abnormalities. The presence of IgG antibody to EBV capsid antigen and antibody to EBV early antigen was significantly associated with augmented counts of suppressor T cells in healthy HTLV-III-seronegative men. HTLV-III-seropositive asymptomatic subjects had significantly enhanced titers of antibody to EBV and lower ratios of helper to suppressor T cells compared with HTLV-III-seronegative homosexual men. Of three men who seroconverted to HTLV-III, two had a greater than fourfold increase in titer of IgG antibody to EBV capsid antigen after seroconversion. These results suggest that the interaction of HTLV-III and EBV and their immunologic perturbations are significant in the natural history of this retrovirus infection in homosexual men.     

Persistent infection with human T-lymphotropic virus type III/lymphadenopathy-associated virus in apparently healthy homosexual men

A group of 14 apparently health homosexual men with serologic evidence of human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) infection were studied to determine the duration of their seropositivity, their immunologic status, and the frequency of isolation of HTLV-III/LAV from their peripheral blood. The men were selected from a larger sample of patients who attended a clinic for treatment of sexually transmitted diseases in San Francisco because they did not have acquired immunodeficiency syndrome (AIDS), signs or symptoms suggestive of the prodrome of AIDS, or laboratory evidence of anemia or leukopenia. 4 or more serum samples were available from previous clinic visits. The men ranged in age from 26-41 years, and had a median number of sexual partners in the last year of 23. The estimated duration of seropositivity ranged from 4-69 months (median, 33 months). 11 of the 14 had T-helper: T-suppressor cell ratios below 1 (the lower limit of normal), and low ratios were significantly correlated with duration of seropositivity. HTLV-III/LAV was isolated in peripheral blood samples from 8 of 12 men tested. Culture-positive and culture-negative men did not differ significantly in terms of age, presence of a palpable lymph node, T helper:T-suppressor cell ratio, or duration of seropositivity. These findings suggest that some seropositive men may remain asymptomatic for at least 5 years. However, the isolation of HTLV-III/LAV from the peripheral blood of most of these men indicates persistent infection may be common among asymptomatic seropositive men at risk for AIDS. It should be assumed that these men have the potential to transmit HTLV-III/LAV infection.     

The Germinal centre-derived lymphomas seen through their cellular microenvironment

The human lymph node is a complex tissue resulting from the microenvironmental organisation of different cell populations linked by topographical and/or functional relationships. Germinal centres (GCs) of lymphoid follicles contain a meshwork of follicular dendritic cells in addition to B-cells and some CD4⁺ T cells. Moreover, there is a sharp demarcation around the whole follicle centre, which is highlighted by fibroblastic reticulum cells. On the whole, GC exerts a role in B cell physiology and malignancy. In GC-derived lymphomas, gene expression profiling studies have raised the possibility that survival of the affected patients may be associated with signatures preferentially expressed in non-malignant T cells and macrophages and/or dendritic cells. Immunohistological analyses in lymphoma biopsy samples have confirmed that the biological behaviour and tumour progression may be influenced by the tumour microenvironment. This review will examine GC-derived lymphomas, including follicular lymphomas, Hodgkin lymphomas and angioimmunoblastic T-cell lymphoma, through their integrated cellular microenvironment, highlighting those findings which may serve as a useful surrogate marker for tumour diagnosis or tumour progression, together with key molecules involved in tumour development.     

Gastrointestinal tissue cultures for HIV in HIV-infected/AIDS patients. The University of Calgary Gastrointestinal/HIV Study Group.

OBJECTIVE: To determine the prevalence of HIV in endoscopic biopsies from the esophagus, stomach, duodenum, and rectum of homosexual and bisexual men at various stages of HIV infection as part of a comprehensive study of gastrointestinal dysfunction in HIV infection. METHODS: After repeated washings and mechanical disruption, biopsies obtained from 58 volunteers were individually cocultured with pooled peripheral blood lymphocytes from healthy HIV-seronegative blood donors. RESULTS: HIV was isolated from at least one site in 40 out of 49 patients. Esophageal biopsies were most frequently found positive (46%), followed by duodenal biopsies (44%), rectal biopsies (43%), and gastric biopsies (27%). Recovery of HIV was not related to any gastrointestinal signs or symptoms. HIV was recovered in the biopsies both from asymptomatic patients with CD4 lymphocyte counts greater than 500 x 10(6)/l and also from patients with more advanced disease receiving zidovudine therapy. CONCLUSIONS: The entire gastrointestinal tract appears to be a target site throughout the course of HIV infection in homosexual and bisexual men.     

Immunohistochemical, electron microscopic and in situ hybridization evidence for the involvement of lymphatics in the spread of HIV-1

To investigate the role of the lymphatic vessels and the sinus systems of the lymph node in the spread of HIV-1, we evaluated 15 lymph nodes from patients with persistent generalized lymphadenopathy (PGL). Fifteen lymph nodes taken from patients with follicular hyperplasia not related to HIV-1 infection served as controls. Immunohistochemical and in situ hybridization techniques revealed infected cells within the sinuses and the efferent lymphatics of the PGL lymph nodes. In contrast, infected cells could not be detected within the walls of the high endothelial venules nor in the areas immediately adjacent. The parenchymal side of the marginal sinus was lined by a discontinuous endothelium. Macrophages and lymphocytes were located within the gaps of this endothelium. More importantly, when the enlarged follicle extended as far as the wall of the marginal sinus, the processes of follicular dendritic cells could be seen extending through the gaps into the lumen of the sinus. This suggests that these cells could transport antigens (including HIV-1) from the sinuses directly to the germinal centers. In addition, HIV-1 particles within cytoplasmic vacuoles were seen in infected macrophages located in the submarginal zone. Positive cells were also found in the extrafollicular lymphoid parenchyma, especially in the area between the marginal sinus and the follicles. The observed distribution of the virus-positive cells within the PGL lymph nodes strongly implicates the lymphatic vessels in the spread of HIV-1 infection.     

Growth-factor receptor-bound protein-2 (Grb2) signaling in B cells controls lymphoid follicle organization and germinal center reaction

Grb2 (growth-factor receptor-bound protein-2) is a signaling adaptor that interacts with numerous receptors and intracellular signaling molecules. However, its role in B-cell development and function remains unknown. Here we show that ablation of Grb2 in B cells results in enhanced B-cell receptor signaling; however, mutant B cells do not form germinal centers in the spleen after antigen stimulation. Furthermore, mutant mice exhibit defects in splenic architecture resembling that observed in B-cell-specific lymphotoxin-β-deficient mice, including disruption of marginal zone and follicular dendritic cell networks. We find that grb2(-/-) B cells are defective in lymphotoxin-β expression. Although lymphotoxin can be up-regulated by chemokine CXCL13 and CD40 ligand stimulation in wild-type B cells, elevation of lymphotoxin expression in grb2(-/-) B cells is only induced by anti-CD40 but not by CXCL13. Our results thus define Grb2 as a nonredundant regulator that controls lymphoid follicle organization and germinal center reaction. Loss of Grb2 has no effect on B-cell chemotaxis to CXCL13, indicating that Grb2 executes this function by connecting the CXCR5 signaling pathway to lymphotoxin expression but not to chemotaxis.     

The evolution of lymphadenopathy and hypergammaglobulinemia are evidence for early and sustained polyclonal B lymphocyte activation during human immunodeficiency virus infection.

To examine whether polyclonal activation of B lymphocytes as measured by hypergammaglobulinemia contributes to lymphadenopathy in human immunodeficiency virus (HIV) infection, correlates of adenopathy were examined in 240 homosexual men. Lymph node size was measured in 12 sites semiannually over 4 years. Both adenopathy and hyperglobulinemia developed within 1 year after seroconversion and persisted at high levels. Adenopathy declined near diagnosis of AIDS whereas serum IgG decreased 8-16 months after diagnosis. Adenopathy attributable to HIV occurred in all palpable node groups. By logistic regression, HIV-positive men were best discriminated from HIV-negative men by size of posterior cervical nodes and the number of sites with enlarged nodes. In a repeated measures model of covariance, adenopathy in HIV-positive men was associated with more CD4+ cells (P less than .002), elevated serum globulins (P less than .01), and lower platelet counts (P less than .05). Adenopathy declined over time (P less than .001) and with diagnosis of AIDS or AIDS-related complex (P less than .03). Thus, adenopathy and hypergammaglobulinemia are correlated and follow a similar course through various stages of HIV infection, suggesting that both are caused by polyclonal B cell activation.     

Association of CXCL13 and CCL21 expression with the progressive organization of lymphoid-like structures in Sjögren's syndrome

OBJECTIVE: Ectopic lymphoneogenesis can occur in the salivary glands of Sj?gren's syndrome (SS) patients and is associated with local antigen-driven B cell responses, autoantibody formation, and potential lymphomatous transformation. CXCL13 and CCL21 have been identified in salivary glands, but their role in ectopic lymphoneogenesis in SS remains unclear. This study aimed to evaluate the microanatomic association between CXCL13 and CCL21 expression and the acquisition of lymphoid features in periductal foci. METHODS: Salivary glands from 37 SS patients and 9 chronic sialadenitis patients were analyzed by immunohistochemistry for T cell/B cell segregation, CD21+ follicular dendritic cell networks, and peripheral lymph node addressin (PNAd)-positive high endothelial venules (HEVs) in relationship to the size of the aggregates and the expression of CXCL13 and CCL21 within infiltrating cells, epithelium, and endothelium. RESULTS: Grade 1 aggregates (10-50 lymphocytes) demonstrated predominance of nonorganized CD3+ cells, while grade 2 (>50 lymphocytes) and grade 3 (>50 with germinal centers) showed a progressive increase in CD20+ B cells and T cell/B cell segregation. This higher degree of lymphoid organization was significantly related to an increased expression of CXCL13 within infiltrating cells and PNAd+ HEV-associated CCL21-producing cells. Conversely, no association between lymphoid organization and lymphoid chemokine expression by epithelial cells was observed. CONCLUSION: The acquisition of lymphoid features by inflammatory foci in SS is critically associated with the enlargement of the inflammatory foci and with the expression of CXCL13 and CCL21 within the infiltrate, but is not associated with their expression by epithelial cells. These data strongly support an active participation of CXCL13 and CCL21 in regulating the progressive organization and maintenance of periductal foci.     

Abnormalities in intestinal mucosal T cells in homosexual populations including those with the lymphadenopathy syndrome and acquired immunodeficiency syndrome

Enteric infections are common in homosexual men. We have characterized the phenotypic distribution of small intestinal mononuclear cells among healthy homosexual men, homosexual men with lymphadenopathy syndrome, homosexual men with acquired immunodeficiency syndrome (AIDS), and a group of healthy heterosexual men. Total numbers of T lymphocytes in the small intestinal mucosa were significantly decreased in homosexual men with lymphadenopathy syndrome and AIDS. This decrease was most striking among the Leu-3a T-cell subset usually associated with helper/inducer function. The proportion of mucosal T cells reacting with Leu-2a (cytotoxic/suppressor phenotype) and lymphoid cells having the T305 antigen was significantly increased only in AIDS subjects. Both lymphadenopathy syndrome and AIDS subjects had a significant reversal of the normal mucosal helper/suppressor T-cell ratio. Mucosal helper/suppressor T-cell ratios and the distribution of mucosal mononuclear cells were normal in healthy homosexual men, although the same individuals had reversed helper/suppressor ratios among circulating T cells. Enteric infections in healthy homosexual men likely reflect sexual practices, and not a primary abnormality in intestinal mucosal immunity. In contrast, specific abnormalities in intestinal mucosal immunity may contribute to the persistent and opportunistic enteric infections that occur in AIDS.     

Disrupted homeostasis of Langerhans cells and interdigitating dendritic cells in monkeys with AIDS

Langerhans cells (LCs) are immature dendritic cells (DCs) that capture antigen in peripheral tissues and migrate to draining lymph nodes, where they reside in the paracortex as interdigitating dendritic cells (IDCs). We studied the effects of simian immunodeficiency virus (SIV) on LCs and IDCs during different stages of infection in monkeys. LCs isolated from monkeys with acute SIV infection or acquired immunodeficiency syndrome (AIDS) underwent normal maturation in vitro, including a switch in chemokine receptor expression from CCR5 to CXCR4 and CCR7. LCs migrated normally from skin in response to contact sensitization in monkeys with acute SIV infection. In contrast, LC migration from skin was markedly impaired during AIDS, associated with a reduction in antigen-bearing DCs in draining lymph nodes. Lymph node IDCs were increased in proportion during acute SIV infection and had an activated phenotype, whereas during AIDS IDCs had significantly lower expression of CD40 and the activation marker CD83. IDCs from monkeys with AIDS were refractory to stimulation with CD40L, demonstrating a functional consequence of decreased CD40 expression. SIV-infected DCs were not identified in lymph nodes or skin of monkeys with AIDS, suggesting an indirect effect of infection on DC populations in vivo. These data indicate that DCs are mobilized to lymph nodes during acute SIV infection, but that during AIDS this process is suppressed, with LC migration and IDC activation being impaired. We conclude that disruption of DC homeostasis may play a role in immunopathology induced by human immunodeficiency virus and suggest that therapeutic strategies targeting DCs may have limited efficacy during AIDS.     

Vascular cell adhesion molecule 1 and alpha 4 and beta 1 integrins in lymphocyte aggregates in Sj?gren's syndrome and rheumatoid arthritis.

OBJECTIVES--Interactions between vascular cell adhesion molecule 1 (VCAM-1) and its ligand, the alpha 4/beta 1 integrin, have been shown to be important in a number of cellular events in vitro. To assess the importance of such interactions in the development of lymphocytic infiltration in diseased tissue the distribution of the two ligands has been studied immunohistochemically. METHODS--Cryostat sections of labial tissue from patients with Sjögren's syndrome, normal labial tissues, rheumatoid synovia, and normal tonsils were stained using antibodies to VCAM-1, alpha 4 and beta 1 integrin chains, and markers for T cells, B cells, macrophages, and follicular dendritic reticulum cells (FDRCs), visualised using alkaline phosphatase and fast red. RESULTS--Staining patterns for VCAM-1 and integrin chains in lymphocyte aggregates in synovial and labial tissues were similar. VCAM-1 staining was found on both vascular and ramifying dendritic cells at the centre of large T cell aggregates and in all aggregates where there was a central clustering of B cells. VCAM-1 colocalised with, but also extended beyond, staining for the FDRC marker R4/23. Staining for the alpha 4 and beta 1 integrin chains was more widespread than staining for VCAM-1, with no significant increase in staining at sites of maximum VCAM-1 staining. In tonsils VCAM-1 and R4/23 codistributed in germinal centres, but staining for the alpha 4 and beta 1 integrin chains was chiefly seen in T lymphocyte areas. CONCLUSIONS--VCAM-1 may be more important in determining the distribution of B than T lymphocytes in lymphocytic infiltration of non-lymphoid tissue. Unlike the follicles of lymphoid tissue, ectopic follicle-like structures in non-lymphoid tissues may form by immigration of B cells via VCAM-1+ vessels at the centre of T cell aggregates.     

Human herpesvirus 8 cellular immune responses in homosexual men.

Little is known about cellular immunity to human herpesvirus 8 (HHV-8), the virus associated with Kaposi's sarcoma (KS). T cell proliferative responses to purified HHV-8 were measured in homosexual men, a group with elevated HHV-8 seroprevalence and high risk of KS. None of 20 blood donor controls had T cell responses to HHV-8. Among human immunodeficiency virus (HIV)-negative homosexual men, 8 (42%) of 19 HHV-8 seropositive men responded as did 4 (16%) of 25 HHV-8 seronegative men. Among HIV-positive homosexual men, however, none of 21 HHV-8 seropositives had T cell responses to HHV-8, even though most responded to common recall antigens, and 10 had >/=400 CD4 cells/mm3. The results suggest that HHV-8 T cell proliferative responses are common in HIV-negative homosexual men and that HIV infection may be associated with diminished HHV-8 cellular immunity, possibly before there is substantial depletion of CD4 cells. If correct, this could explain why KS occurs relatively early in HIV infection/AIDS.     

Peripheral T cell lymphoma with cytotoxic phenotype: an emerging disease in HIV-infected patients?

Recently, a 15-fold increased risk of T cell lymphomas has been estimated in HIV-infected populations. This increase has been observed for all T cell lymphoma subtypes. In the present report we describe clinical and pathological features of three consecutive cases of peripheral T cell lymphoma (PTCL) with cytotoxic phenotype in HIV-positive patients that came to our attention in May-September 2002. The diagnosis of PTCL was made in lymph node (two cases) and in needle biopsies from liver and bone marrow of the same patient. The patients were two females (31 and 45 years old) and one male (49 years old). The risk factor for each patient was heterosexual, injecting drug user, and homosexual, respectively. CD4 cell counts were low (79-81 cells/mm3). Two patients were naive for antiretroviral therapy. At histological examination, all the involved tissues were effaced by a neoplastic proliferation of CD3+/CD8+ medium to large pleomorphic cells containing TIA-1+ cytotoxic granules and a few granzyme B+ granules. Neoplastic cells were not infected by EBV or by HHV-8. They were negative for the B cell antigens CD20 and CD79a, for CD30 and for CD56. Clonal T cell receptor-g (TCR-g) rearrangements were demonstrated in the three cases.     

Lymph node B lymphocyte trafficking is constrained by anatomy and highly dependent upon chemoattractant desensitization

B lymphocyte recirculation through lymph nodes (LNs) requires crossing endothelial barriers and chemoattractant-triggered cell migration. Here we show how LN anatomy and chemoattractant receptor signaling organize B lymphocyte LN trafficking. Blood-borne B cells predominately used CCR7 signaling to adhere to high endothelial venules (HEVs). New B cell emigrants slowly transited the HEV perivenule space, and thereafter localized nearby, avoiding the follicle. Eventually, the newly arrived B cells entered the basal portion of the follicle gradually populating it. In contrast, newly arriving activated B cells rapidly crossed HEVs and migrated toward the lymph node follicle. During their LN residency, recirculating B cells reacquired their sphingosine-1 phospate receptor 1 (S1P1) receptors and markedly attenuated their sensitivity to chemokines. Eventually, the B cells exited the LN follicle by entering the cortical lymphatics or returning to the paracortical cords. Upon entering the lymph, the B cells lost their polarity, down-regulated their S1P1 receptors, and subsequently strongly up-regulated their sensitivity to chemokines. These results are summarized in a model of homeostatic trafficking of B cells through LNs.