CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30(+) T cells elicited with allogeneic APC are a major source of IFN-gamma and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30(+) T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30(+) T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30(+) cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.     

Continued antigen stimulation is not required during CD4(+) T cell clonal expansion

Peptide Ag initiates CD4(+) T cell proliferation, but the subsequent effects of Ag on clonal expansion are not fully known. In this study, murine CD4(+) T cells were labeled with the fluorescent dye CFSE and were stimulated with specific peptide Ag. Activation occurred, as CFSE-associated fluorescence was reduced 2-fold with each cell division. Separation of proliferating cells based upon CFSE fluorescence intensity showed that daughter cells from each cell division proliferate even after removal of Ag. A limited exposure (2 h) to peptide programmed the cells to proliferate independently of Ag. Although not required for cell division, Ag increased the survival of proliferating cells and increased the total number of cell divisions in the expansion process. These results indicate that Ag exposure begins a program of cell division that does not require but is modified by further TCR stimulation.     

Homeostatic proliferation as an isolated variable reverses CD8+ T cell anergy and promotes tumor rejection

Although recent work has suggested that lymphopenia-induced homeostatic proliferation may improve T cell-mediated tumor rejection, there is little direct evidence isolating homeostatic proliferation as an experimental variable, and the mechanism by which improved antitumor immunity occurs via homeostatic proliferation is poorly understood. An adoptive transfer model was developed in which tumor-specific 2C/RAG2(-/-) TCR transgenic CD8+ T cells were introduced either into the lymphopenic environment of RAG2(-/-) mice or into P14/RAG2(-/-) mice containing an irrelevant CD8+ TCR transgenic population. RAG2(-/-), but not P14/RAG2(-/-) recipients supported homeostatic proliferation of transferred T cells as well as tumor rejection. Despite absence of tumor rejection in P14/RAG2(-/-) recipients, 2C cells did become activated, as reflected by CFSE dilution and CD44 up-regulation. However, these cells showed poor IFN-gamma and IL-2 production upon restimulation, consistent with T cell anergy and similar to the hyporesponsiveness induced by administration of soluble peptide Ag. To determine whether homeostatic proliferation could uncouple T cell anergy, anergic 2C cells were transferred into RAG(-/-) recipients, which resulted in vigorous homeostatic proliferation, recovery of IL-2 production, and acquisition of the ability to reject tumors. Taken together, our data suggest that a major mechanism by which homeostatic proliferation supports tumor rejection is by maintaining and/or re-establishing T cell responsiveness.     

Anti-CD3/28 mediated expansion of macaque CD4+ T cells is polyclonal and provides extended survival after adoptive transfer

BACKGROUND: Our lab has previously shown that adoptive transfer of in vitro expanded autologous purified polyclonal CD4(+) T cells using anti-CD3/CD28 coated beads induced antiviral responses capable of controlling simian immunodeficiency virus (SIV) replication in vivo. RESULTS: Expansion on anti-CD3/28 coated beads was found to induce a true polyclonal expansion as CFSE labeled cells uniformly showed dilution of the dye over several days of culture, in contrast to aliquots of the same cells subjected to mitogen stimulation. Of interest was the finding that CD4(+) T cells collected before and during early chronic SIV infection or AIDS stage did not show any or only modest differences in proliferative response or expansion kinetics. The reason for such excellent expansion properties was analyzed by the quantitation of telomerase activity in aliquots of expanding CD4(+) T cells from sample collected at various times post-infection. First, anti-CD3/28 expanded CD4(+) T cells exhibited telomerase levels 2- to 20-fold higher than the starting population of CD4(+) T cells. Moreover, while telomerase activity in ex vivo tested CD4(+) T cells was found to decrease following SIV infection and disease progression, anti-CD3/28 expansion appeared to restore significant levels of telomerase activity as no difference was noted in telomerase expression between CD4(+) T cells expanded from samples collected before or during the chronic SIV infection. When such expanded and CFSE labeled T cells were autologously transferred to monkeys, evidence for extended survival in vivo was provided as CFSE labeled cells were detected to relatively high levels in blood and spleen at 1 week post-infection. CONCLUSION: In summary, the data suggest that anti-CD3/28 mediated expansion of CD4(+) T cells retains its immunotherapeutic potential not only during the early stages of lentiviral infection but also at more advanced stages of disease.     

Characterisation of the Immunomodulatory Effects of Meningococcal Opa Proteins on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells

Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs) and purified CD4⁺ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4⁺ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins.     

A previously unrecognized large fraction of cytotoxic lymphocyte precursors is present in CD4+ human peripheral blood T cells

We describe a limiting dilution (LD) culture system in which cell sorter-purified CD4+ (and CD8+) peripheral blood T cells are cocultured with irradiated, anti-CD3 mab-producing OKT3 hybridoma cells. Under these conditions, one out of 2-3 CD4+ (and CD8+) T cells is induced to clonal proliferation. In striking contrast to previously described LD culture systems, every growing CD4+ cell clone displayed cytotoxic activity when tested in a lectin-facilitated 51Cr release assay against P815 target cells. This contrasts with the development of cytotoxic CD4+ T cells in alloantigen-stimulated LD cultures, where only one out of 15-20 proliferating CD4+ cells killed P815 in the presence of PHA, and one out of 300-500 proliferating CD4+ cells displayed alloantigen-specific cytotoxic activity. Furthermore, we have established antigen-specific proliferating CD4+ T cell clones which do not exert antigen-specific cytotoxicity but can be cytotoxic when crosslinked to target cells via lectin or monoclonal antibody (anti-CD3, anti-TCR). Our results show that a previously unrecognized large fraction (at least 30-50%) of all peripheral blood CD4+ T cells can give rise to cytotoxic effector cells. The mode of CD4+ T cell activation (OKT3 hybridoma versus alloantigen) thus determines whether the intrinsic cytotoxic capacity of CD4+ T cells is functionally activated or not.     

A closer look at homeostatic proliferation of CD4+ T cells: costimulatory requirements and role in memory formation

Ag-specific proliferation of CD4+ T cells is regulated, in part, by costimulatory signals through CD28. The proliferative response during primary activation is an important determinant of the ability of the T cell to respond to Ag re-encounter. Proliferation of mature CD4+ T cells during lymphopenia (homeostatic proliferation) requires interaction with endogenous peptide MHC. However, the role of costimulation during homeostatic proliferation is unclear, as is the ability of homeostatic proliferation to regulate secondary T cell responses. Using a TCR transgenic system and serial adoptive transfers we find that homeostatic proliferation of CD4+ T cells occurs for at least 5 wk after adoptive transfer into recombination-activating gene (RAG)-/- recipients. Two discrete populations of proliferating T cells can be resolved, one that is highly proliferative and dependent on CD28 signaling, and the other that contains cells undergoing low levels of CD28-independent proliferation. Importantly, naive CD4+ T cells that have undergone homeostatic proliferation acquire both phenotypic and functional characteristics of true memory cells. These studies indicate that functional memory T cells can be generated by encounters with endogenous Ags only. This mechanism of T cell regeneration is possibly active during lymphopenia due to viral infections, such as HIV, transplantation, or cancer therapy, and may explain selected autoimmune diseases.     

Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells: development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8(+) T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8(+) T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8(+) T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8(+) T cells by either BLC or HUVEC derived from the same donor. CD8(+) T cells expanded by allostimulation were identified as CD8(+), CFSE(low) cells and were categorized as CTL by the expression of intracellular perforin and IFN-gamma. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8(+) T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8(+) T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.     

Memory T cells are enriched in lymph nodes of selectin-ligand-deficient mice

Fucosyltransferase-IV and -VII double knockout (FtDKO) mice reveal profound impairment in T cell trafficking to lymph nodes (LNs) due to an inability to synthesize selectin ligands. We observed an increase in the proportion of memory/effector (CD44(high)) T cells in LNs of FtDKO mice. We infected FtDKO mice with lymphocytic choriomeningitis virus to generate and track Ag-specific CD44(high)CD8 T cells in secondary lymphoid organs. Although frequencies were similar, total Ag-specific effector CD44(high)CD8 T cells were significantly reduced in LNs, but not blood, of FtDKO mice at day 8. In contrast, frequencies of Ag-specific memory CD44(high)CD8 T cells were up to 8-fold higher in LNs of FtDKO mice at day 60. Because wild-type mice treated with anti-CD62L treatment also showed increased frequencies of CD44(high) T cells in LNs, we hypothesized that memory T cells were preferentially retained in, or preferentially migrated to, FtDKO LNs. We analyzed T cell entry and egress in LNs using adoptive transfer of bone fide naive or memory T cells. Memory T cells were not retained longer in LNs compared with naive T cells; however, T cell exit slowed significantly as T cell numbers declined. Memory T cells were profoundly impaired in entering LNs of FtDKO mice; however, memory T cells exhibited greater homeostatic proliferation in FtDKO mice. These results suggest that memory T cells are enriched in LNs with T cell deficits by several mechanisms, including longer T cell retention and increased homeostatic proliferation.     

Cutting edge: B7/CD28 interactions regulate cell cycle progression independent of the strength of TCR signaling

The role of B7/CD28 signals in Ag-induced cell cycle progression of CD4(+) T cells was examined using the technique of CFSE dye dilution and flow cytometry. In wild-type T cells, proliferation was directly related to the concentration of Ag available to the APC. Consistent with this, the rate of G(0)-->G(1) cell cycle progression varied with the concentration of Ag. However, cell division by T cell blasts occurred at a constant rate, independent of Ag concentration. G(0)-->G(1) phase progression by CD28-deficient CD4(+) T cells or wild-type T cells cultured in the presence of neutralizing anti-B7 mAbs was slowed, confirming that a synergy does exist between TCR and CD28 signaling in the initial activation of the T cells. However, unlike the TCR, the strength of CD28 stimulation was also shown to play a unique role in controlling the rate of cell division by T cell blasts.     

IL-7-regulated homeostatic maintenance of recent thymic emigrants in association with caspase-mediated cell proliferation and apoptotic cell death

Homeostasis of T cells is essential to the maintenance of the T cell pool and TCR diversity. In this study, mechanisms involved in the regulation of cytokine-mediated expansion of naive T cells in the absence of Ag, in particular the role of caspase activation and susceptibility to apoptosis of recent thymic emigrants (RTEs), were examined. Low level caspase-8 and caspase-3 activation was detected in proliferating IL-7-treated cells in the absence of cell death during the first days of culture. Caspase inhibitors suppressed IL-7-induced expansion of RTEs. Low level expression of CD95 and blocking Ab experiments indicated that this early caspase activation was CD95 independent. However, CD95 levels subsequently became dramatically up-regulated on proliferating naive T cells, and these cells became susceptible to CD95 ligation, resulting in high level caspase activation and apoptotic cell death. These results show a dual role for caspases in proliferation and in CD95-induced cell death during Ag-independent expansion of RTEs. This method of cell death in IL-7-expanded RTEs is a previously unrecognized mechanism for the homeostatic control of expanded T cells.     

Human NK cells proliferate and die in vivo more rapidly than T cells in healthy young and elderly adults

NK cells are essential for health, yet little is known about human NK turnover in vivo. In both young and elderly women, all NK subsets proliferated and died more rapidly than T cells. CD56(bright) NK cells proliferated rapidly but died relatively slowly, suggesting that proliferating CD56(bright) cells differentiate into CD56(dim) NK cells in vivo. The relationship between CD56(dim) and CD56(bright) proliferating cells indicates that proliferating CD56(dim) cells both self-renew and are derived from proliferating CD56(bright) NK cells. Our data suggest that some dying CD56(dim) cells become CD16(+)CD56(-) NK cells and that CD16(-)CD56(low) NK cells respond rapidly to cellular and cytokine stimulation. We propose a model in which all NK cell subsets are in dynamic flux. About half of CD56(dim) NK cells expressed CD57, which was weakly associated with low proliferation. Surprisingly, CD57 expression was associated with higher proliferation rates in both CD8(+) and CD8(-) T cells. Therefore, CD57 is not a reliable marker of senescent, nonproliferative T cells in vivo. NKG2A expression declined with age on both NK cells and T cells. Killer cell Ig-like receptor expression increased with age on T cells but not on NK cells. Although the percentage of CD56(bright) NK cells declined with age and the percentage of CD56(dim) NK cells increased with age, there were no significant age-related proliferation or apoptosis differences for these two populations or for total NK cells. In vivo human NK cell turnover is rapid in both young and elderly adults.     

Pro-IL-16 regulation in activated murine CD4+ lymphocytes

Prior DNA microarray studies suggested that IL-16 mRNA levels decrease following T cell activation, a property unique among cytokines. We examined pro-IL-16 mRNA and protein expression in resting and anti-CD3 mAb-activated primary murine CD4(+) T cells. Consistent with the microarray reports, pro-IL-16 mRNA levels fell within 4 h of activation, and this response is inhibited by cyclosporin A. Total cellular pro-IL-16 protein also fell, reaching a nadir at 48 h. Pro-IL-16 comprises a C-terminal cytokine domain and an N-terminal prodomain that are cleaved by caspase-3. Pro-IL-16 expressed in transfected tumor cells was previously shown to translocate to the nucleus and to promote G(0)/G(1) arrest by stabilizing the cyclin-dependent kinase inhibitor p27(Kip1). In the present study, we observed increased S-phase kinase-associated protein 2 mRNA expression in IL-16 null mice, but basal expression and activation-dependent regulation of p27(Kip1) were no different from wild-type mice. Stimulation with anti-CD3 mAb induced transiently greater thymidine incorporation in IL-16-deficient CD4(+) T cells than wild-type controls, but there was no difference in cell survival or in the CFSE dilution profiles. Analysis of CD4(+) T cell proliferation in vivo using BrdU labeling similarly failed to identify a hyperproliferative phenotype in T cells lacking IL-16. These data demonstrate that pro-IL-16 mRNA and protein expression are dynamically regulated during CD4(+) T cell activation by a calcineurin-dependent mechanism, and that pro-IL-16 might influence T cell cycle regulation, although not in a dominant manner.     

Defective clonogenic potential of CD8+ T lymphocytes in patients with AIDS. Expansion in vivo of a nonclonogenic CD3+CD8+DR+CD25- T cell population

This study examines the potential mechanism(s) responsible for the defective clonability of CD8+ T lymphocytes in patients with AIDS. By the combined use of one- and two-color fluorescence cytofluorometry we have shown an increase in the number of circulating DR+ cells due to the expression of DR on a relatively large proportion of T lymphocytes (one-third of CD3+ cells), the majority of them belonging to the CD8+ subset. In addition, the majority of CD8+DR+ cells in AIDS patients did not express CD25 Ag (the receptor for IL-2), a surface marker generally expressed on normal activated T lymphocytes. Sorted CD8+DR+ and CD8+DR- cell populations were analyzed comparatively for their ability to proliferate in response to different stimuli, including anti-CD3, anti-CD2, alone or in combination with anti-CD28 mAb and mitogens such as PHA, alone or in combination with PMA. We have demonstrated that CD8+DR+ cells were severely defective in their proliferative response to triggering via these major pathways of T cell activation even when an exogenous source of IL-2 or IL-4 was added to the microcultures 24 h after initiating the cultures. In contrast, CD8+DR- cells showed a significant proliferation in response to the different stimuli and the proliferative response was strongly enhanced by the addition of IL-2 or IL-4. At the end of the stimulation period CD8+DR+ and CD8+DR- proliferating populations were analyzed for CD25 Ag expression. Only 1 to 10% of CD8+DR+ cells expressed CD25 antigen compared with 40 to 50% of CD8+DR- cells. The proliferative defect of CD8+DR+ cells was further confirmed in experiments performed at the clonal level. The analysis of the frequency of proliferating T lymphocyte-precursors in both CD8+DR+ and CD8+DR- subsets showed that the defective clonogenic potential of CD8+ cells in AIDS patients could be in large part ascribed to CD8+DR+ cells. Five percent of CD8+DR+ cells showed a clonogenic potential compared to the 25% of CD8+DR- cells. Finally, we analyzed the surface expression of VLA-2 Ag, a marker of a chronic state of T cell activation, on circulating T lymphocytes. We have shown that a large proportion of CD3+DR+CD25- cells (50 to 80% in the different patients with AIDS analyzed) expressed VLA-2 Ag.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential responses of invariant V alpha 24J alpha Q T cells and MHC class II-restricted CD4+ T cells to dexamethasone.

NK T cells are a T cell subset in the human that express an invariant alpha-chain (V alpha 24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on V alpha 24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of V alpha 24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of V alpha 24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated V alpha 24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The V alpha 24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of V alpha 24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the V alpha 24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between V alpha 24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.     

The Gads (GrpL) adaptor protein regulates T cell homeostasis

Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4(+) and CD8(+) T cells are markedly different. Gads(-/-) CD4(+) T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads(-/-) CD4(+) T cells continued to proliferate at a higher rate than wild-type CD4(+) T cells, demonstrating a defect in homeostatic proliferation. Gads(-/-) CD8(+) T cells had a memory-like phenotype, produced IFN-gamma in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads(-/-) T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads(-/-) CD4(+) T cells, but not CD8(+) T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid turnover of Gads(-/-) CD4(+) T cells is due to a defect in cell survival. The intracellular signaling pathways that regulate homeostasis in CD4(+) and CD8(+) T cells are clearly different, and the Gads adaptor protein is critical for homeostasis of CD4(+) T cells.     

Identification of the anti-CD3-unresponsive subpopulation of CD4+, CD45RA+ peripheral T lymphocytes.

The majority of peripheral CD4+ T lymphocytes proliferate in vitro in response to anti-CD3 in presence of autologous APC. The present study describes a subpopulation of CD4+ T cells that cannot be activated and progress into cell cycle by stimulation with anti-CD3 plus APC or with mitogenic combinations of anti-CD2. The in vitro responses of these anti-CD3-unresponsive CD4+ T cells were investigated with a panel of mAb to CD2, CD3, and CD28, and found to be similar to those previously observed for mature thymocytes: only the combination of anti-CD2 plus anti-CD28 produced cell proliferation. Anti-CD3-unresponsive T cells were CD45RA+, but represented only 14 to 22% of the CD4+, CD45RA+ T cell population. Activation with anti-CD2 plus anti-CD28 mAb resulted in major changes in the cell surface phenotype and functional properties: a loss of CD45RA+ occurred and an increased expression of CD45RO, CD29, and CD58 (LFA3), as well as a gain in responsiveness to anti-CD3 and anti-CD2. This change in CD45 phenotype from CD45RA to CD45RO occurs in both the anti-CD3-responsive and in the anti-CD3-unresponsive subsets of the CD45RA+, CD4+ cells after cell proliferation. The anti-CD3-unresponsive subset may represent a pool of not yet fully differentiated peripheral T cells. The acquisition of anti-CD3 responsiveness could occur as a consequence of Ag priming or by an Ag-independent mechanism. Involvement of the CD28 Ag in this process is suggested from the present study.     

Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3

The capacity of the monoclonal antibodies (Mab) 64.1 and OKT3 directed at CD3 molecules to induce T4 cell proliferation and interleukin 2 (IL 2) production was examined. Each was tested in soluble form or was immobilized by adhering it to the wells of plastic microtiter wells. Soluble anti-CD3 did not induce proliferation of accessory cell (AC)-depleted T4 cells. In contrast, immobilized anti-CD3 induced T4 cell IL 2 production and proliferation in the complete absence of AC. When T4 cells were stimulated with high density immobilized anti-CD3, responses did not require AC, IL 2, or Mab directed at the Tp44 molecule (9.3). In contrast, responses stimulated by lower densities of immobilized anti-CD3 were enhanced by IL 2, AC, and 9.3, and with even lower densities of immobilized anti-CD3 proliferation, required these additional signals. A variety of other immobilized Mab directed at T cell surface proteins including class I major histocompatibility complex encoded gene products, CD2, CD5, 4F2, and Tp44, did not induce proliferation even in the presence of IL 2. Anti-CD4 Mab (66.1) inhibited immobilized anti-CD3-stimulated T4 cell responses, with a greater degree of inhibition noted when lower densities of immobilized anti-CD3 were used to stimulate T4 cells. The data demonstrate that stimulation of T4 cells by anti-CD3 is completely AC independent when the antibody is immobilized onto a surface. Furthermore, the results indicate that maximal stimulation requires multiple interactions with anti-CD3 without internalization of the CD3 molecule. The observation that additional signals are required to support T4 cell proliferation when the density of immobilized anti-CD3 is diminished suggests that these are necessary only when insufficient interactions with the CD3 molecule have occurred to transmit a maximal activation signal to the cell. Finally, the results indicate that anti-CD4 provides a direct inhibitory signal to the T4 cell, the effect of which is inversely proportional to the intensity of the activation signal.     

Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells

Microbial superantigens can alter host immunity through aberrant activation and subsequent anergy of responding naive T cells. We show here that the superantigen, staphylococcal enterotoxin B (SEB), directly induces tolerance in memory CD4 T cells. Murine naive and memory CD4(+) T cells were labeled with the fluorescent dye CFSE and the cells were exposed to SEB before they were cultured with specific peptide antigen. Memory, but not naive, T cells became anergic and did not respond to their cognate peptide antigen. The extent and duration of T cell receptor (TCR) clustering was similar to promote naive T cell activation and memory T cell anergy, suggesting similar TCR-SEB interactions led to distinct intracellular signaling processes in the two cell types. Like SEB, soluble anti-CD3 mAb does not stimulate memory cell proliferation. However, unlike SEB, soluble anti-CD3 mAbs did not induce anergy to cognate peptide. Anergy was directly visualized in vivo. CD4(+) memory T cells were identified in mice that had been administered SEB. The cells failed to proliferate in response to subsequent immunization with their cognate recall antigen. Hence, one mode of pathogen survival is the modulation of host immunity through selective elimination of memory T cell responses.