Human Secondary Lymphoid Tissue Research Articles

Robust memory humoral immune response to SARS-CoV-2 in the tonsils of adults and children.

Adaptive humoral immunity against SARS-CoV-2 has mainly been evaluated in peripheral blood. Human secondary lymphoid tissues (such as tonsils) contain large numbers of plasma cells that secrete immunoglobulins at mucosal sites. Yet, the role of mucosal memory immunity induced by vaccines or natural infection against SARS-CoV-2 and its variants is not fully understood. Tonsillar mononuclear cells (TMNCs) from adults (n=10) and children (n=11) were isolated and stimulated using positive SARS-CoV-2 nasal swabs. We used endpoint enzyme-linked immunosorbent assays (ELISAs) for the measurement of anti-S1, -RBD, and -N IgG antibody levels and a pseudovirus microneutralization assay to assess neutralizing antibodies (nAbs) in paired serum and supernatants from stimulated TMNCs. Strong systemic humoral response in previously SARS-CoV-2 infected and vaccinated adults and children was observed in accordance with the reported history of the participants. Interestingly, we found a significant increase in anti-RBD IgG (305 and 834 folds) and anti-S1 IgG (475 and 443 folds) in the stimulated TMNCs from adults and children, respectively, compared to unstimulated cells. Consistently, the stimulated TMNCs secreted higher levels of nAbs against the ancestral Wuhan strain and the Omicron BA.1 variant compared to unstimulated cells by several folds. This increase was seen in all participants including children with no known history of infection, suggesting that these participants might have been previously exposed to SARS-CoV-2 and that not all asymptomatic cases necessarily could be detected by serum antibodies. Furthermore, nAb levels against both strains were significantly correlated in adults (r=0.8788; p = 0.0008) and children (r = 0.7521; p = 0.0076), and they strongly correlated with S1 and RBD-specific IgG antibodies. Our results provide evidence for persistent mucosal humoral memory in tonsils from previously infected and/or vaccinated adults and children against recent and old variants upon re-exposure. They also highlight the importance of targeting mucosal sites with vaccines to help control infection at the primary sites and prevent potential breakthrough infections.

The role of lymphoid tissue SPARC in the pathogenesis and response to treatment of multiple myeloma.

Despite the significant progress in the treatment of multiple myeloma (MM), the disease remains untreatable and its cure is still an unmet clinical need. Neoplastic transformation in MM is initiated in the germinal centers (GCs) of secondary lymphoid tissue (SLT) where B cells experience extensive somatic hypermutation induced by follicular dendritic cells (FDCs) and T-cell signals. We reason that secreted protein acidic and rich in cysteine (SPARC), a common stromal motif expressed by FDCs at the origin (SLTs) and the destination (BM) of MM, plays a role in the pathogenesis of MM, and, here, we sought to investigate this role. There were 107 BM biopsies from 57 MM patients (taken at different time points) together with 13 control specimens assessed for SPARC gene and protein expression and compared with tonsillar tissues. In addition, regulation of myeloma-promoting genes by SPARC-secreting FDCs was assessed in in vitro GC reactions (GCRs). SPARC gene expression was confirmed in both human primary (BM) and secondary (tonsils) lymphoid tissues, and the expression was significantly higher in the BM. Sparc was detectable in the BM and tonsillar lysates, co-localized with the FDC markers in both tissues, and stimulation of FDCs in vitro induced significantly higher levels of SPARC expression than unstimulated controls. In addition, SPARC inversely correlated with BM PC infiltration, ISS staging, and ECOG performance of the MM patients, and in vitro addition of FDCs to lymphocytes inhibited the expression of several oncogenes associated with malignant transformation of PCs. FDC-SPARC inhibits several myelomagenic gene expression and inversely correlates with PC infiltration and MM progression. Therapeutic induction of SPARC expression through combinations of the current MM drugs, repositioning of non-MM drugs, or novel drug discovery could pave the way to better control MM in clinically severe and drug-resistant patients.

IL‐10 induces IgG4 production in NOD‐scid Il2rγnull mice humanized by engraftment of peripheral blood mononuclear cells

IL‐10 induces IgG4 production in NOD‐<i>scid</i> Il2rγ^null mice humanized by engraftment of peripheral blood mononuclear cells

Abstract A074: Spatially resolved deep antigen profiling of single cells in FFPE tissue samples through CODEXTM

Abstract Spatially resolved, deep antigen profiling of tissue specimens is crucial for investigating the architecture and cell diversity in complex matrices, such as the tumor microenvironment (TME). The correlation of these two parameters with the progression of a variety of pathologies, ranging from cancer to autoimmune diseases, is still vastly unknown due to the lack of technologies that enable the detection of both high content and spatial resolution. Formalin-fixed, paraffin-embedded (FFPE) tissues constitute the ideal sample for these investigations as they retain tissue morphology at room temperature for long periods of time, which is convenient and cost-effective, and are available in vast specimen archives. Akoya Biosciences, Inc., is commercializing CODEXTM (CO-Detection by indEXing), a multiparameteric imaging platform that performs the concurrent detection and quantification of dozens of antigens with single cell resolution within a tissue specimen. The CODEX platform uses a DNA-based barcode library to label antibodies and iterative cycles of adding and removing cognate dye-labeled oligonucleotides to reveal the staining pattern for a subset of the target markers per cycle. The entire data acquisition process is fully automated using the CODEX instrument, which integrates with existing microscopes. To support high-multiplexing capabilities, more than 45 different antibody clones have been screened and validated for the detection of target epitopes in human FFPE secondary lymphoid tissues for both healthy and cancerous specimens using the CODEX platform. Proof-of-concept data have also been obtained for different cancer types, including melanoma and head and neck cancer in both normal and TMA formats. Analysis software has been developed to extract meaning from CODEX datasets and is included as part of the CODEX platform. The analysis pipeline includes image drift compensation, deconvolution and segmentation to measure integrated fluorescence intensity for each cell across tens of parameters. In this manner, infiltrating lymphocytes and other immune cells have been identified within the TME. With the high multiplexing capabilities, a variety of T-cells can be identified to detect the ratios of both immunosuppressive and immune-activating subtypes. Analysis of FFPE tissues using the CODEX platform demonstrates the unparalleled opportunities for simultaneously detecting tens of antigens with single-cell resolution within a tissue specimen. Citation Format: Maria Elena Gallina, Atri Choksi, Nadya Nikulina, Jaskirat Singh, Gajalakshmi Dakshinamoorthy, Joseph Kim, Sejal Mistry, Julia Kennedy-Darling. Spatially resolved deep antigen profiling of single cells in FFPE tissue samples through CODEXTM [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A074.

Identification of tonsillar CD4+CD25−LAG3+ T cells as naturally occurring IL-10-producing regulatory T cells in human lymphoid tissue

IL-10-producing regulatory T cells (IL-10-producing Tregs) are one of the regulatory T cell subsets characterized by the production of high amounts of IL-10, the lack of FOXP3 expression and the strong immunosuppressive capabilities. IL-10-producing Tregs have been primarily reported as induced populations thus far, in part because identifying naturally occurring IL-10-producing Tregs was difficult due to the lack of definitive surface markers. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue that we have identified as being expressed on IL-10 producing Tregs. In human PBMC, LAG3 combined with CD49b efficiently identifies IL-10-producing Tregs. However, naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue have not been described.In this report, we identified CD4+CD25−LAG3+ T cells in human tonsil. This T cell subset produced high amounts of IL-10 and expressed low levels of FOXP3. Surface markers and microarray analysis revealed that this is a distinct tonsillar CD4+ T cell subset. CD4+CD25−LAG3+ T cells expressed interleukin 10 (IL10), PR/SET domain 1 (PRDM1), and CD274 at high levels and chemokine receptor 5 (CXCR5) at low levels. CD4+CD25−LAG3+ T cells suppressed antibody production more efficiently than CD4+CD25+ T cells, and CD4+CD25−LAG3+ T cells induced B cell apoptosis. Moreover, analysis of humanized mice revealed that this cell subset suppressed a graft-versus-host disease (GVHD) reaction in vivo. Our study reveals the existence of naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue and their function in immune regulation.

A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets

The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.

A Novel CD94+ Immature NK Cell Population Based on NKp80 Expression with ILC3-like Features in Human Secondary Lymphoid Tissue

Natural killer (NK) cells are important for their innate ability to identify and kill cancer cells without previous stimulation. However, many of the mechanisms concerning their differentiation from immature precursor cells are still unknown. Elucidation of these pathways are critical to improving targeted therapies that can both activate as well as overcome inhibitory pathways that prevent an effective response. NK cells are thought to develop in secondary lymphoid tissue (SLT) through five stages distinguished by the expression of CD34, CD117, CD94, and CD16. While virtually all NK cells in blood express the pan-NK cell activating receptor, NKp80, we have discovered a distinct Lin CD94+NKp80-CD16- population that is selectively enriched in SLT. The NKp80- population appears to comprise those cells “in transit” between stage 3 (CD117+CD94-) and stage 4 (CD117loCD94+) when evaluating CD94 and CD117 expression among Lin-CD34- cells by flow cytometry. These ex vivo data suggest that this NKp80- population may represent a novel NK cell developmental intermediate (NKDI). SLT-derived NKp80- cells lack cytolytic granules, show minimal cytotoxicity, and produce negligible amounts of interferon-gamma when compared to NKp80+ NK cells in SLT and blood. In addition, NKp80- cells have lower mRNA and protein expression of TBET and EOMES compared to NKp80+ NK, while expressing these factors at higher levels compared to stage 3 cells. Unexpectedly, the NKp80- population also demonstrates Group 3 innate lymphoid cell (ILC3)-like features ex vivo with expression of surface interleukin-1 (IL-1) receptor 1 and CD127 and mRNA and protein expression of the ILC3-associated transcription factors, AHR and RORC. Moreover, following overnight stimulation with IL-1-beta and IL-23, NKp80- cells produce IL-22, albeit at lower concentrations compared to stage 3 cells. Collectively, these data identify NKp80 as a surrogate marker of functional competence during in vivo human NK cell development and provide evidence for the existence of at least two distinct maturation steps within the previously described stage 4 NK cell population in SLT. Since CD94 expression is currently considered to be NK-restricted, these findings raise new questions regarding the developmental relationship between NK cells and ILC3 in humans. In addition, as ILC3s are primarily used to stimulate an anti-microbial defense while NK cells are functional anti-cancer effector cells, defining the differentiation patterns of these cells can have impacts in both of these areas of research. Studies are ongoing to investigate the in vitro differentiation potential(s) of this novel SLT-derived Lin-CD94+NKp80-CD16- population. DisclosuresNo relevant conflicts of interest to declare.

CD117+CD3−CD56−OX40Lhigh cells express IL‐22 and display an LTi phenotype in human secondary lymphoid tissues

Here, we identify cells within human adult secondary lymphoid tissues that are comparable in phenotype and location to the lymphoid tissue inducer (LTi) cells that persist in the adult mouse. Identified as CD117(+) CD3(-) CD56(-) cells, like murine LTi cells, they lack expression of many common lineage markers and express CD127, OX40L and TRANCE. These cells were detected at the interface between the B- and T- zones, as well as at the subcapsular sinus in LNs, the location where LTi cells reside in murine spleen and LNs. Furthermore, like murine LTi cells, these cells expressed high levels of IL-22 and upregulated IL-22 expression upon IL-23 stimulation. Importantly, these cells were not an NK cell subset since they showed no expression of IFN-γ and perforin. Interestingly, a subset of the CD117(+) CD3(-) CD56(-) OX40L(+) population expressed NKp46, again similar to recent findings in mice. Finally, these cells supported memory CD4(+) T-cell survival in an OX40L-dependent manner. Combined, these data indicate that the CD117(+) CD3(-) CD56(-) OX40L(+) cells in human secondary lymphoid tissues are comparable in phenotype, location and function to the LTi cells that persist within adult murine secondary lymphoid tissues.

Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-γ

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56brightCD16− NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.

T cell–dependent survival of CD20+ and CD20− plasma cells in human secondary lymphoid tissue

AbstractThe signals mediating human plasma cell survival in vivo, particularly within secondary lymphoid tissue, are unclear. Human tonsils grafted into immunodeficient mice were therefore used to delineate the mechanisms promoting the survival of plasma cells. Tonsillar plasma cells were maintained within the grafts and the majority were nonproliferating, indicating a long-lived phenotype. A significant depletion of graft plasma cells was observed after anti-CD20 treatment, consistent with the expression of CD20 by most of the cells. Moreover, anti-CD52 treatment caused the complete loss of all graft lymphocytes, including plasma cells. Unexpectedly, anti-CD3, but not anti-CD154, treatment caused the complete loss of plasma cells, indicating an essential role for T cells, but not CD40-CD154 interactions in plasma cell survival. The in vitro coculture of purified tonsillar plasma cells and T cells revealed a T-cell survival signal requiring cell contact. Furthermore, immunofluorescence studies detected a close association between human plasma cells and T cells in vivo. These data reveal that human tonsil contains long-lived plasma cells, the majority of which express CD20 and can be deleted with anti-CD20 therapy. In addition, an important role for contact-dependent interactions with T cells in human plasma cell survival within secondary lymphoid tissue was identified.

Potent Systemic Antitumor Immunity Induced by Vaccination with Chemotactic-Prostate Tumor Associated Antigen Gene-Modified Tumor Cell and Blockade of B7-H1

We previously reported that several DNA fragments from human prostate-specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate-specific antigen (hPSA) genes were selected and fused to create a novel hPSM-mPAP-hPSA fusion gene (named 3P gene), and human secondary lymphoid tissue chemokine (SLC), 3P, and human IgG Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC-3P-Fc. In this report, to establish a more efficient treatment for immunotherapy against prostate cancer, the construct was transfected into B16F10 to generate gene-modified tumor cell vaccine (named B16F10-SLC-3P-Fc). In poorly immunogenic B16F10 mouse melanoma model, the immunization with B16F10-SLC-3P-Fc resulted in a strong antitumor response and 50% of tumor-bearing mice achieved long-term survival (>120 days). In vivo depletion of lymphocytes indicated that CD8(+) T cells were involved in the direct tumor killing, whereas CD4(+) T lymphocytes were required for the induction of CD8(+) CTL response in B16F10-SLC-3P-Fc-immunized mice. Splenocytes from B16F10-SLC-3P-Fc-immunized mice specifically recognized and lysed PSM, PAP, PSA, and 3P expressing tumor cells. The combined therapy of B16F10-SLC-3P-Fc plus anti-B7-H1 MAbs further enhanced the immune response. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy. These observations suggest pSLC-3P-Fc-modified tumor cells could serve as a vaccine against prostate cancer, and the therapy combined with anti-B7-H1 MAbs further enhanced the antitumor immune response.

NK cells of human secondary lymphoid tissues enhance T cell polarization via IFN‐γ secretion

Human secondary lymphoid tissues harbor NK cells that predominantly secrete cytokines in response to activation. Here, we demonstrate that these immunoregulatory NK cells assist in the Th1 polarization of primary immune responses, induced by dendritic cells. Tonsilar, but not peripheral blood NK cells enhanced the expansion of IFN-gamma-producing CD4+ T cells via their superior ability to produce IFN-gamma. Addition of IFN-gamma increased Th1 polarization while antibody blocking of this cytokine abolished NK cell-dependent Th1 polarization. Our data suggest that NK cells in secondary lymphoid organs assist priming of Th1 cells via cytokine secretion and this effect should be harnessed during vaccination against viruses and tumors.

Differential expression of oestrogen receptors in human secondary lymphoid tissues

Many autoimmune diseases including rheumatoid arthritis (RA), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) occur much more frequently in women than in men. There is much evidence that oestrogen is the major cause of this gender difference. Interestingly, oestrogen relieves the symptoms of RA and SS but it exacerbates SLE. This contradictory effect of oestrogen on autoimmune diseases is not well understood. Most of the effects of oestrogen are mediated by two receptors: oestrogen receptor alpha and beta (ERalpha and ERbeta). To determine whether these contradictory effects of oestrogen relate to the involvement of distinct effects of the two ERs, we investigated expression of ERalpha and ERbeta in human secondary lymphoid tissues. We observed that, in tonsils, ERbeta is expressed in lymphocytes of germinal centres (GC) and the follicular mantle zone as well as in granulocytes, while ERalpha is expressed only in activated germinal centres but not in the follicular zone. ERbeta is the predominant ER in human leucocytes from peripheral blood, spleen and in leucocytes infiltrating cancers in both males and females. In addition, in different human lymphoma cell lines including Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma, ERbeta is abundant while ERalpha is not detectable. Our results indicate that ERbeta is the predominant type of ER in mature lymphocytes. We suggest that ERalpha and ERbeta have distinct roles in secondary lymphoid tissues and that further studies with ERbeta-specific agonists will help to elucidate the role of ERbeta in these tissues.

The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic.

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.

IL-12 secreting dendritic cells are required for optimum activation of human secondary lymphoid tissue T cells.

Successful immunization requires that mature dendritic cells (mDCs) prime T cells in secondary lymphoid tissue (LT). Previously, the authors have shown that LT T cell activation has an increased costimulatory threshold for a proliferative response as compared with peripheral blood (PB) T cells. Therefore, to optimize mDC immunogenicity, DC maturation was studied using LT T cells as responders. While mDCs obtained with soluble CD40Ligand (sCD40L) or a sCD40L/IFNgamma combination similarly expressed the CD83 and CCR7 molecules on their membrane, only the latter secreted IL-12. sCD40L/IFNgamma mDCs, as compared with sCD40L mDCs, enhanced allogeneic LT T cell proliferation, LT CD4+ cell IFNgamma production and LT CD8+ cell cytotoxicity. Enhancement could be predominantly ascribed to IL-12 secreted by sCD40L/IFNgamma mDCs and to additional costimulatory signals as shown remarkably in the IFNgamma response when IL-12 was neutralized. Therefore, in addition to their membrane phenotype, mDCs to be used in immunization protocols should be assessed for IL-12 secretion as a surrogate marker for an optimum costimulatory potential.

Morphological Interactions of Interdigitating Dendritic Cells with B and T Cells in Human Mesenteric Lymph Nodes

Interdigitating dendritic cells (IDC) of the human mesenteric lymph nodes (LN) were examined by two-color immunofluorescent microscopy and flow cytometry to clarify their exact localization, immunophenotype, and relationships with T and B cells. IDC were identified as HLA-DR(bright) large dendriform cells of the T cell areas co-expressing CD40, CD54 (ICAM-1), CD80 (B7/B7-1), CD83, and CD86 (B70/B7-2). The majority of IDC directly attached to a few IgD+ naive B cells as well as to numerous CD4+ T cells. When LN cells were singly suspended and briefly incubated in vitro, IDC formed clusters with IgD+ IgM+ naive B cells, but not with IgA+ or IgG+ B cells. When suspended LN cells were cultured, clustered B cells disappeared within 7 days, and on prolonged culture, some IDC developed into extensively dendriform cells forming stable complexes with several or sometimes numerous CD4+ IL-2R+ CD40L+ activated T cells. These findings indicate that resting naive B cells actually interact with IDC directly in T cell areas of human secondary lymphoid tissues. IDC have a non-antigen (Ag)-specific, strong affinity for resting naive B cells, but this affinity is transient and IDC cannot form stable complexes with B cells, although they can form stable complexes with activated T cells. It is suggested that the stable IDC/Ag-activated T cell complexes make it possible to capture and to stimulate rare Ag-specific resting naive B cells with high efficiency.

Distribution of laminin variants and their integrin receptors in human secondary lymphoid tissue. Colocalization suggests that the alpha 6 beta 4-integrin is a receptor for laminin-5 in lymphoid follicles.

Laminins are a family of multifunctional basement membrane glycoproteins. Over the last years, many laminin isoforms have been characterized, which were shown to be composed of distinct combinations of variant alpha, beta and gamma chains. Some of these isoforms show remarkable tissue specificity, which suggests functional involvement in local processes. In this study the previously described mAb 4C7, which recognize epithelial basement membranes as well as endothelial basement membranes in lymphoid follicles, was identified as an anti-laminin-5 antibody. Using a set of mAbs against various variant laminin chains we established that specifically the gamma 2 chain of laminin-5 was confined to the endothelial basement membranes of vessels in lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid tissue. Additionally, the expression of the known integrin receptors of laminin-5 was also examined. The alpha 6 beta 4 integrin-receptor for laminin was found to be colocalized with the laminin-5 gamma 2 chain on the abluminal surface of endothelial cells, whereas the alpha 3 integrin chain could not be detected in lymphoid follicles. This finding suggests that the alpha 6 beta 4 integrin (and not the alpha 3 beta 1 integrin) serves as a laminin-5 receptor on endothelial cells in the follicular compartment of lymphoid tissue. Furthermore, alpha 6 beta 4 was also found in the same punctuated pattern on FDCs as laminin-5. The function of the laminin-alpha 6 beta 4 complex in this particular localisation is still obscure, but a role in the maintainance of the follicular compartment via hemidesmosome-like attachment sites is postulated.

Regulation of tissue-selective T-lymphocyte homing receptors during the virgin to memory/effector cell transition in human secondary lymphoid tissues.

Conventional virgin T cells efficiently and homogeneously recirculate through all secondary lymphoid tissues, but not "extralymphoid" effector sites. In contrast, memory/effector populations are composed of distinct subsets with differential, often tissue-selective, migratory capability to both secondary lymphoid tissues and effector sites. In keeping with these observations, CD45RA(high)/RO(low) virgin T cells in human peripheral blood uniformly express the peripheral lymph node (PLN) homing receptor (HR) L-selectin, and lack the skin-selective HR CLA, whereas among the CD45RA(low)/RO(high) "memory/effector" population, differential expression of these HR yields three predominant subsets: L-selectin+/CLA+, L-selectin+/CLA-, L-selectin-/CLA-. Although these subsets are of approximately equal size in the peripheral blood, the vast majority of T cells obtained from cutaneous chronic inflammatory sites display the L-selectin+/CLA+ phenotype. To investigate the mechanisms responsible for the generation of these memory/effector T-cell subsets, we developed a multiparameter flow cytometric technique that defines a common pathway of postthymic T-cell differentiation in secondary lymphoid tissues: the virgin to memory/effector transition. Our analyses indicate that these HR are differentially regulated during the virgin to memory/effector transition in a tissue-specific fashion. The great majority of memory/effector T cells produced in PLN retain high levels of L-selectin expression, and 50 to 60% upregulate CLA. In contrast, memory/effector T cells produced in appendix and tonsil are generally L-selectin(low), and CLA is upregulated on less than 10% of newly formed memory/effector T cells in appendix and on about 30 to 35% of such cells in tonsil.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-specific suppression of anti-influenza antibody production in man. Possible role of a membrane-antigen complex.

E rosette-forming (E+) cells from human secondary lymphoid tissue were incubated with high dose influenza A virus (Mem-Bel) in an attempt to generate suppressor T cells. Suppression was assayed by transferring the antigen-pulsed E+ cells into effector cultures consisting of E+ and E- cells stimulated with immunogenic amounts of either the inducing virus Mem-Bel) or the non-cross-reacting influenza B virus (B/HK). The transfer resulted in marked inhibition of IgG, IgA and IgM antibody production to Mem-Bel but not to the control antigen, B/HK virus. The suppressive effect was specific at the level of induction as well as expression since E+ cells exposed to high dose Mem-Bel could provide help to an effector culture containing E- cells and optimal dose of B/HK virus. However, metabolically active cells did not appear to be required for suppression. Thus, it could be elicited (a) after only 15 min incubation of E+ cells with high-dose virus and (b) by E+ cells exposed to irradiation, incubated in the presence of metabolic inhibitors, or disrupted by repeated freeze thawing. In contrast, treatment of E+ cells with pronase reversed the suppressive effect. Interestingly, virus heated to 70 degree C failed to induced suppression, while retailing the ability to elicit a normal helper response. Suppression induced by exposure to standard amounts of high-dose antigen was mediated by T cells of both helper/inducer (Leu-3a+) and suppressor/cytotoxic subsets (Leu-2a+), but not by B cells. Two groups of observations pointed to the B cell as the target of suppression. First, suppression could still be transferred to effector cultures in which helper T cells had been replaced by T cell-replacing factor or suppressor T cells removed by irradiation. Second, significant inhibition of antibody production was obtained when the transfer of antigen-pulsed E+ cells was delayed for up to 120 h after initiation of the effector culture. Taken together the results suggest that suppression in this system is due to the formation of an antigen bridge between specific receptor sites on the T cell membrane and the target. Although not dependent on triggering of metabolically active suppressor T cells the phenomenon highlights the need for care in interpreting the mechanism of suppression by high-dose antigen and could, in addition, represent a biologically important control mechanism capable of rapid inhibition of effector T cells and B cells in sites of high antigen concentration.

Human antigen-specific IgA responses in blood and secondary lymphoid tissue: an analysis of help and suppression.

Antigen-specific IgA responses to influenza virus were obtained from lymphocytes derived from human secondary lymphoid tissue but not from the circulating pool (blood). T helper cells for IgA were present in both the circulating pool and secondary lymphoid tissue, were of the Leu-3A+ subset, and were relatively radioresistant. In addition, T alpha cells were shown to be important, but not crucial, for IgA helper responses. T cell-dependent suppression of IgA responses was demonstrated with T cells from both mucosal and systemic lymphoid tissue and was mediated by a radiosensitive Leu-2a+ cell. In contrast to T cells, functional B cell precursors for antigen-specific IgA responses were found only in secondary lymphoid tissue and not in the circulating pool.