CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

Efficacious control of cytomegalovirus infection after long-term depletion of CD8+ T lymphocytes.

Although the relative contribution of different immune effector functions to clearing tissues of cytomegalovirus is controversial, the contribution of CD8+ T lymphocytes has generally been accepted as essential. In this report, we show that under certain conditions the CD8+ T-lymphocyte subset can be dispensable for clearance of cytomegalovirus. Mice depleted of the CD8+ T-lymphocyte subset eliminated infectious virus with a clearance kinetics similar to that of normal mice. Adoptive transfer studies revealed that the limitation of virus spread required the cooperation between the CD4+ subset and other cells. Comparison between protective functions generated in fully immunocompetent and in CD8- mice demonstrated that elimination of the CD8+ subset before infection altered the quality of the antiviral immune response. The compensatory protective activity gained by CD4+ cells in CD8- mice was absent in normal mice recovering from virus infection.     

Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine.

BALB/c mice vaccinated with a temperature-sensitive mutant (TS-4) of Toxoplasma gondii develop complete resistance to lethal challenge with a highly virulent toxoplasma strain (RH). This immunity is known to be dependent on IFN-gamma synthesis. In vitro and in vivo T cell depletions were performed in order to identify the subsets responsible for both protective immunity and IFN-gamma production. When stimulated with crude tachyzoite Ag in vitro, CD4+ cells from vaccinated mice produced high levels of TH1 cytokines (IL-2 and IFN-gamma) but not TH2 cytokines (IL-4 and IL-5). CD8+ cells, in contrast, produced less IFN-gamma and no detectable IL-2. Nevertheless, they could be induced to synthesize IFN-gamma when exposed in culture to exogenous IL-2. In vivo treatment with anti-CD4 plus anti-CD8 or anti-IFN-gamma antibodies during challenge infection completely abrogated resistance to T. gondii. In contrast, treatment with anti-CD4 alone failed to reduce immunity, whereas anti-CD8 treatment partially decreased vaccine-induced resistance. These results suggest that although IFN-gamma and IL-2-producing CD4+ lymphocytes are induced by vaccination, IFN-gamma-producing CD8+ T cells are the major effectors of immunity in vivo. Nevertheless, CD4+ lymphocytes appear to play a synergistic role in vaccine-induced immunity, probably through the augmentation of IFN-gamma synthesis by the CD8+ effector cells. This hypothesis is supported by the observation that when giving during vaccination, as opposed to after challenge, anti-CD4 antibodies are capable of blocking protective immunity.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

Class I MHC genes and CD8+ T cells determine cyst number in Toxoplasma gondii infection

To determine roles of MHC class I and II genes in protection against Toxoplasma gondii, H-2 congenic and mutant mice were infected perorally with bradyzoites of T. gondii and brain cysts were enumerated 30 days later. As B10 mice (H-2b) are cyst susceptible and B10.A mice (H-2a) are cyst resistant, B10 congenic mice having the same alleles but different H-2 haplotypes were used to locate the controlling gene. Genes located at H-2L (i.e., class I genes) were found to regulate the number of brain cysts which form following peroral infection with T. gondii (p less than 0.001) with Ld being resistant and Lb being susceptible. The regulatory function of the H-2L gene product was confirmed through the study of D mutant (dm) mice. B10.D2-H-2dm1 (dm1) mice have a gain-loss mutation in Dd and Ld (i.e., recombination of Ld and Dd) and BALB/c-H-2dm2 (dm2) mice have a deletion of the Ld gene. Both these dm strains were cyst susceptible (p less than 0.001). These results provide the first direct evidence that class I genes regulate numbers of T. gondii cysts that form. In vivo ablation of CD8+ T cells with mAb YTS 169.4 converted cyst resistant B10.BAR12 mice to cyst susceptible. This result is consistent with a role for MHC restricted CD8+ cytotoxic (or suppressor) T cell regulation of cyst formation. A mutation in Ia in B6.C-H-2bm12 (bm12) mice amplified cyst numbers in susceptible mice, which is consistent with the importance of helper/inducer T cells in the induction of cytotoxic T cells. These findings are relevant to understanding the complex immunologic mechanisms that protect against T. gondii infection, development of protective preparations, and provide a conceptual basis for determining whether similar immunogenetic regulation of susceptibility is also operative in humans.     

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.     

T cell-derived interferon-γ is required for host defense to Toxoplasma gondii

Interferon-γ (IFN-γ) is important for host defense against various intracellular organisms including a protozoan pathogen Toxoplasma gondii. Various immune cells are recently shown to produce IFN-γ in T. gondii infection, however, it remains elusive which cell types are important for anti-T. gondii host defense so far. Here we generate a new IFN-γ reporter "GREVEN" mouse line in which a fusion protein of Venus and NanoLuc to analyze IFN-γ producing cells during T. gondii infection and find that CD4⁺, CD8⁺, γδ T cells and natural killer cells express Venus in a time dependent manner. Furthermore, Lck-Cre/Ifng^fl/fl mice are highly susceptible to T. gondii infection. Taken together, our results demonstrate that T cell-derived IFN-γ plays an important role in anti-T. gondii host defense.     

CD8+ T lymphocytes are not required for murine resistance to human filarial parasites.

Mice are resistant to the establishment of infection with the nematode parasite Brugia malayi, an etiologic agent of human lymphatic filariasis. We have recently shown that T and B lymphocyte-deficient C.B.-17 scid/scid mice are permissive for infection with this parasite, whereas coisogenic C.B.-17+/+ mice are resistant. This observation suggests that T and B lymphocytes that comprise the antigen-specific immune system orchestrate murine resistance to B. malayi. In order to define the component of the antigen-specific immune response that is responsible for this resistance, we have tested the susceptibility of beta 2M-/- mice to infection with B. malayi L3 larvae. These mice are homozygous for insertional disruption of their B2m genes, which encode beta 2-microglobulin, the small subunit of the major histocompatibility (MHC) antigens. They do not express beta 2-microglobulin and, as a consequence, fail to express the class I major histocompatibility antigens, and they do not develop the CD8+ class I MHC-restricted cytotoxic T cell subset. We find that these mice are completely resistant to B. malayi, indicating that the CD8+ T lymphocyte subset is not an obligate requirement for murine resistance to human filarial parasites.     

Syphilitic infection impairs immunity by inducing both apoptosis and pyroptosis of CD4+ and CD8+ T lymphocytes

Syphilis is an important health problem worldwide; however, few studies have probed the impact of syphilitic infection on T cell turnover. The mechanisms behind the frequency of T cell subset changes and the associations between these subsets during syphilitic infection remain unclear. Herein, we used a cell-staining method and flow cytometry to explore changes in T cell subpopulations and potential contribution of apoptosis and pyroptosis that triggered therein. We investigated caspase-1-mediated pyroptosis and caspase-3-mediated apoptosis of CD4⁺ and CD8⁺ T cells, the major effector lymphocytes with pivotal roles in the pathogenesis of infectious diseases. We found that the levels of caspase-1 and caspase-3 increased in both the circulation and intracellularly in CD4⁺ and CD8⁺ T cells. Caspase-1 showed a continual increase from early latent stage infection through to phase 2 disease, whereas caspase-3 increased through to phase 1 disease but declined during phase 2. In addition, serum levels and intracellular expression of caspase-1 and caspase-3 were positively correlated. Overall, this study increases our understanding of how syphilitic infection influences CD4⁺ and CD8⁺ T-cell turnover, which may help with designing novel and effective strategies to control syphilis infection and prevent its transmission.     

Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Augmentation of the CD8+ T cell response by IFN-gamma in IL-12-deficient mice during Toxoplasma gondii infection

The importance of IFN-gamma in regulating the host CD8+ T cell response during microbial infection has not been delineated. Mice deficient for the p40 chain of the IL-12 heterodimer have impaired IFN-gamma production and are susceptible to infection with the intracellular parasite Toxoplasma gondii. The administration of exogenous IFN-gamma to parasite-infected p40-/- mice increases survival and up-regulates the depressed CD8+ T cell response following infection. CD8+ T cells isolated from cytokine-treated p40-/- mice exhibit an increase in both precursor CTL frequency and IFN-gamma production compared with untreated controls. The enhancement of the CD8+ T cell response is independent of CD4+ T cell help. These CD8+ T cells induce protective immunity against a lethal challenge when adoptively transferred into naive p40-/- and IFN-gamma-/- mice. These observations indicate that IFN-gamma can regulate the CD8+ T cell response during T. gondii infection.     

Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes

The relationship of T cell activation to HIV entry and generation of viral DNA intermediates was studied in freshly isolated CD4+ T lymphocytes. Unstimulated cells exposed to infectious virus for up to 48 h did not synthesize any detectable unintegrated HIV DNA duplex forms or integrated genomic provirus. However, activation of these cells with either PHA or OKT3 (anti-CD3) mAb before viral exposure resulted in the generation of unintegrated HIV DNA after 6 h and integrated copies after 24 h. Cell-to-cell fusion studies showed significantly attenuated fusion between freshly isolated resting T cells and T cells constitutively expressing high levels of HIV envelope glycoprotein (HXB/gpt) compared with T cells first stimulated with either PHA or OKT3 mAb. The baseline fusion observed with resting T cells is believed to be a consequence of allogeneic stimulation by the HXB/gpt cell line. These results provide evidence that HIV entry and HIV envelope-dependent cell-to-cell fusion require T cell activation.     

Functional cure of SIVagm infection in rhesus macaques results in complete recovery of CD4+ T cells and is reverted by CD8+ cell depletion

Understanding the mechanism of infection control in elite controllers (EC) may shed light on the correlates of control of disease progression in HIV infection. However, limitations have prevented a clear understanding of the mechanisms of elite controlled infection, as these studies can only be performed at randomly selected late time points in infection, after control is achieved, and the access to tissues is limited. We report that SIVagm infection is elite-controlled in rhesus macaques (RMs) and therefore can be used as an animal model for EC HIV infection. A robust acute infection, with high levels of viral replication and dramatic mucosal CD4(+) T cell depletion, similar to pathogenic HIV-1/SIV infections of humans and RMs, was followed by complete and durable control of SIVagm replication, defined as: undetectable VLs in blood and tissues beginning 72 to 90 days postinoculation (pi) and continuing at least 4 years; seroreversion; progressive recovery of mucosal CD4(+) T cells, with complete recovery by 4 years pi; normal levels of T cell immune activation, proliferation, and apoptosis; and no disease progression. This functional cure of SIVagm infection in RMs could be reverted after 4 years of control of infection by depleting CD8 cells, which resulted in transient rebounds of VLs, thus suggesting that control may be at least in part immune mediated. Viral control was independent of MHC, partial APOBEC restriction was not involved in SIVagm control in RMs and Trim5 genotypes did not impact viral replication. This new animal model of EC lentiviral infection, in which complete control can be predicted in all cases, permits research on the early events of infection in blood and tissues, before the defining characteristics of EC are evident and when host factors are actively driving the infection towards the EC status.     

Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcalpha2-6Gal sequence, and a corresponding increase in glycans carrying the Galalpha1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, alpha1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.     

Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.