Activation-driven T cell death. II. Quantitative differences alone distinguish stimuli triggering nontransformed T cell proliferation or death.

Clonal deletion is the major mechanism by which T cell tolerance is achieved in vivo. The process of activation-driven cell death, originally characterized with T cell hybridomas, likely represents the mechanism of clonal deletion because it shares a number of properties with the in vivo process, especially the ability to be triggered in an Ag-specific manner, the cell-autonomous nature of the response, and its sensitivity to the drug cyclosporin A. We now have extended our analysis of activation-driven cell death to clonal populations of nontransformed T cells. Activation-driven cell death can be induced in nontransformed T lymphocytes by combinations of mitogenic stimuli. In particular, two mitogenic stimuli at high dose, one a lymphokine and the other delivered via the TCR or another activation structure, are required to induce activation-driven cell death. Activation-driven cell death is an active cell suicide process with attributes typical of physiological cell death, including early nuclear disintegration and a requirement for macromolecular synthesis, and is distinct from death by factor deprivation. Susceptibility to the induction of cell death by antigenic or activating stimulation is a common aspect of most T cells and is consistent with observations that clonal deletion can occur throughout T cell ontogeny. Most importantly, the alternative cellular responses of cell death and cell proliferation in nontransformed T cells appear to be triggered solely as a function of quantitative differences in the doses of identical stimuli. This can be viewed as a dose-dependent switch that determines cell fate. Developmental regulation of this switch may explain the processes of positive and negative selection during T cell ontogeny and also provide a mechanistic rationale for a strategy of selective anti-tumor therapy.     

Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex.

The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.     

Specific immunotherapy by genetically engineered APCs: the "guided missile" strategy

We tested the hypothesis that APCs genetically engineered to present an Ag and to express Fas ligand (FasL) simultaneously can target and eliminate Ag-specific T cells. Transgenic T cells specific for influenza hemagglutinin (HA) were used as targets. We prepared recombinant vaccinia virus vectors (VVV) to transfer the gene constructs individually or simultaneously into APCs. We prevented unwanted viral replication by attenuating the VVVs with psoralen-UV light treatment. For presentation of the HA Ag, APCs were transduced with cDNA for HA flanked by sequences of the lysosome-associated membrane protein that direct efficient processing and presentation of the Ag by APCs. As a "warhead" for the APCs, we transduced them with the gene for FasL, which induces apoptosis of Fas-expressing activated T cells. To protect the transduced APCs from self-destruction by FasL, we transferred cDNA for a truncated form of Fas-associated death domain, which inhibits Fas-mediated cell death. Our results show that the engineered APCs effectively expressed the genes of interest. APCs transduced with VVV carrying all three gene constructs specifically killed HA-transgenic T cells in culture. Coculture with T cells specific for an unrelated Ag (OVA) had no significant effect. Our in vitro findings show that APCs can be genetically engineered to target and kill Ag-specific T cells and represent a promising novel strategy for the specific treatment of autoimmune diseases.     

Apoptotic cells induce tolerance by generating helpless CD8+ T cells that produce TRAIL

The decision to generate a productive immune response or immune tolerance following pathogenic insult often depends on the context in which T cells first encounter Ag. The presence of apoptotic cells favors the induction of tolerance, whereas immune responses generated with necrotic cells promote immunity. We have examined the tolerance induced by injection of apoptotic cells, a system in which cross-presentation of Ag associated with the dead cells induces CD8+ regulatory (or suppressor) T cells. We observed that haptenated apoptotic cells induced CD8+ suppressor T cells without priming CD4+ T cells for immunity. These CD8+ T cells transferred unresponsiveness to naive recipients. In contrast, haptenated necrotic cells stimulated immunity, but induced CD8+ suppressor T cells when CD4+ T cells were absent. We further found that CD8+ T cells induced by these treatments displayed a "helpless CTL" phenotype and suppress the immune response by producing TRAIL. Animals deficient in TRAIL were resistant to tolerance induction by apoptotic cells. Thus, the outcome of an immune response taking place in the presence of cell death can be determined by the presence of CD4+-mediated Th cell function.     

Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood.

The Fas Ag is a newly defined cell-surface molecule that may mediate apoptosis. The antibody against Fas Ag can induce the apoptotic cell death in cell lines expressing this Ag. PBL subpopulations at various ages were here examined for Fas expression by two-or three-color flow-cytometric analyses using anti-Fas mAb. It was found that Fas Ag was appreciably detected on a proportion of T and B cells, whereas its expression was absent for NK cells. For CD4+ and CD8+ T cells, Fas Ag was expressed preferentially on CD45RO+ (memory or previously activated) populations, but not on CD45RO- naive ones. TCR-gamma/delta+ T cells, especially their CD45RO+ subsets, also expressed Fas Ag. Expectably, neonatal T cell subpopulations, most of which had the naive (CD45RO-) phenotype, expressed little Fas Ag. Fas-expressing B cells dominated in surface(s) IgD- populations, but neonatal B cells as well as adult sIgD+ B cells had little Fas Ag. The Fas Ag was inducible after in vitro mitogenic stimulation of naive T and B cells from neonatal blood. These observations suggested that expression of Fas Ag on T and B cells in the peripheral blood might reflect their in vivo Ag-activated status. In contrast to Fas-expressing cultured cell lines, however, viability of in vitro stimulated T and B cells as well as freshly isolated CD45RO+ T cells was not significantly changed after the treatment with anti-Fas mAb, indicating that additional cellular conditions to Fas expression might be required for anti-Fas-induced cell death.     

The ability of cultured Langerhans cells to process and present protein antigens is MHC-dependent.

There is controversy regarding the ability of short term (2 to 3 days) cultured epidermal Langerhans cells (cLC) to process and present intact protein Ag to primed T cells. Some studies have shown that cLC are potent APC for both haptens and intact protein Ag, whereas in others cLC have been unable to process and present intact protein Ag. In an attempt to resolve this controversy, we tested the ability of Langerhans cells from several strains of mice to process and present intact protein Ag to T cell clones and T cell hybridomas. We found that both cLC and freshly prepared Langerhans cells from various Iak mice, including BALB.k mice, process and present intact protein antigens (i.e., hen egg lysozyme, cytochrome c, and OVA) to T cells. These functions are retained in cLC cultured for 7 days. In contrast, cLC from Iad mice do not process intact protein Ag, such as hen egg lysozyme and myoglobin, although they can present relevant peptides to specific T cells and are potent stimulators of allogeneic responses. Furthermore, cLC from (Iak x Iad)F1 mice process and present intact protein Ag to Iak-restricted T cells, but not to Iad-restricted T cells. Although cLC that processed and presented intact protein Ag to T cells exhibited enhanced class II MHC expression, they were, on a per cell basis, somewhat less efficient than were fresh Langerhans cells. Finally, we found that if Iad Langerhans cells are pulsed with intact protein Ag and then cultured for 3 days, they are then fully capable of inducing Ag- and MHC-specific T cell proliferation.     

Minor lymphocyte stimulatory antigen-bearing stimulator cells require lipopolysaccharide activation to induce programmed cell death in T cell hybridomas.

T cell hybridomas respond to conventional peptide Ag associated with self major histocompatibility restriction elements, as well as to alloantigens, activating lectins, and stimulatory forms of mAb by producing lymphokines and undergoing programmed cell death (PCD). We show here that the level of PCD and IL-2 production correlate well in responses to CD3 or allostimulation. The response to minor lymphocyte-stimulatory (Mls) Ag, members of the family of endogenous superantigens, however, are marked by divergence in the levels of the PCD and lymphokine responses. Specifically, PCD in response to Mls activation is achieved poorly despite vigorous IL-2 production. B lymphoma cell stimulators induced PCD in alloreactive T cell hybridomas but not in Mls-reactive T cell hybridomas. This suggests that the absence of PCD in the Mls response is a function of superantigen recognition rather than the stimulator cell type. LPS-preactivated Mls+ stimulators, either splenic B or B lymphoma cells, are shown to trigger PCD in the T cell hybridomas. These results imply that T cell interaction with Mls presented by untreated stimulator cells is not sufficient for induction of PCD and thus is distinct from interactions with conventional Ag.     

The role of TCR engagement and activation-induced cell death in sepsis-induced T cell apoptosis

Sepsis induces extensive apoptosis in T and B cells suggesting that the loss of immune effector cells could be one explanation for the profound immunosuppression observed in this disorder. Unfortunately, the mechanisms responsible for lymphocyte apoptosis in sepsis remain unknown. In T cells, apoptosis can occur through activation-induced cell death (AICD) in which engagement of the Ag receptors by cognate Ag or polyclonal activators such as bacteria-derived superantigens induces activation, proliferation, and apoptosis. We examined whether proliferation and AICD are necessary for apoptotic cell death in sepsis using normal and TCR transgenic mice. Results show that although sepsis resulted in activation of a small percentage of T cells, no proliferation was detected during the first 48 h following onset, a time when extensive apoptosis is observed. We also observed that T cells do not enter the cell cycle, and stimulation via the TCR in TCR transgenic animals does not enhance or decrease cell death in sepsis. Interestingly, T cells recovered from septic mice retained their ability to proliferate and synthesize cytokines albeit at reduced levels. With the exception of IL-10, which was increased in lymphocytes from mice with sepsis, sepsis caused a decrease in the production of both proinflammatory and anti-inflammatory cytokines. We conclude that lymphocyte apoptosis in sepsis does not require proliferation, TCR engagement, or AICD. Thus the immunosuppression observed in sepsis cannot be the result of T cell deletion via the TCR.     

Characterization of antigen processing and presentation by resting B lymphocytes

The production of antibody to a thymus-dependent Ag requires cooperation between the B cell and an Ag-specific Th cell. MHC restriction of this interaction implies that the Th cell recognizes Ag on the B cell surface in the context of MHC molecules and that the Ag-specific B cell gets help by acting as an APC for the Th cell. However, a number of studies have suggested that normal resting B cells are ineffective as APC, implying that the B cell must leave the resting state before it can interact specifically with a Th cell. Other studies, including our own with rabbit globulin-specific mouse T cell lines and hybridomas, show that certain T cell lines can be efficiently stimulated by normal resting B cells. One possible explanation for the above contradiction is that our B cells have become activated before presentation. Here we show that presentation by size-selected small B cells is not the result of nonspecific activation signals generated by the T cells or components of the medium. Also, although LPS activation does increase the efficiency of presentation by small B cells, use of large cells in place of small cells or preincubation of resting B cells with mitogenic doses of anti-Ig does not. Another possibility that we considered was that small B cells are unable to process Ag and that we had selected T cell lines that were capable of recognizing native Ag on the B cell surface. In the majority of cases, experiments with B cell lines and macrophages have shown that Ag presentation requires Ag processing, a sequence of events that includes internalization of Ag into an acid compartment, denaturation or digestion of Ag into fragments, and its return to the cell surface in the context of class II MHC molecules. The experiments reported here show that our T cell lines require an Ag processing step and that small resting B cells, like other APC, process Ag before presenting it to T cells. Specifically, we show that an incubation of 2 to 4 h is required after the Ag pulse before Ag presentation becomes resistant to irradiation. Shortly after the pulse, the Ag enters a pronase-resistant compartment. Although efficient Ag presentation requires initial binding to membrane Ig, Ag is no longer associated with membrane Ig at the time of presentation and is not presented in its intact form, because removal of membrane Ig by goat anti-Ig blocks presentation before but not after the Ag pulse.     

Primed T cells are more resistant to Fas-mediated activation-induced cell death than naive T cells.

Memory T cells respond in several functionally different ways from naive T cells and thus function as efficient effector cells. In this study we showed that primed T cells were more resistant to Fas-mediated activation-induced cell death (AICD) than naive T cells using OVA-specific TCR transgenic DO10 mice and Fas-deficient DO10 lpr/lpr mice. We found that apoptosis was efficiently induced in activated naive T cells at 48 and 72 h after Ag restimulation (OVA peptide; 0.3 and 3 microM), whereas apoptosis was not significantly increased in activated primed T cells at 24-72 h after Ag restimulation. We further showed that the resistance to AICD in primed T cells was due to the decreased sensitivity to apoptosis induced by Fas-mediated signals, but TCR-mediated signaling equally activated both naive and primed T cells to induce Fas and Fas ligand expressions. Furthermore, we demonstrated that primed T cells expressed higher levels of Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, at 24-48 h after Ag restimulation than naive T cells. In addition, Bcl-2 expression was equally observed between activated naive and primed T cells after Ag restimulation. Thus, these results indicate that naive T cells are sensitive to Fas-mediated AICD and are easily deleted by Ag restimulation, while primed/memory T cells express higher levels of FLIP after Ag restimulation, are resistant to Fas-mediated AICD, and thus function as efficient effector cells for a longer period.     

Presentation of autoantigen by human T cells.

Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag.     

In situ beta cell death promotes priming of diabetogenic CD8 T lymphocytes.

CTLs are important mediators of pancreatic beta cell destruction in the nonobese diabetic mouse model of type 1 diabetes. Cross-presentation of Ag is one means of priming CTLs. The death of Ag-bearing cells has been implicated in facilitating this mode of priming. The role of beta cell death in facilitating the onset of spontaneous autoimmune diabetes is unknown. Here, we used an adoptive transfer system to determine the time course of islet-derived Ag presentation to naive beta cell-specific CD8 T cells in nonobese diabetic mice and to test the hypothesis that beta cell death enhances the presentation of beta cell autoantigen. We have determined that beta cell death enhances autoantigen presentation. Priming of diabetogenic CD8 T cells in the pancreatic lymph nodes was negligible before 4 wk, progressively increased until 8 wk of age, and was not influenced by gender. Administration of multiple low doses of the beta cell toxin streptozotocin augmented in situ beta cell apoptosis and accelerated the onset and magnitude of autoantigen presentation to naive CD8 T cells. Increasing doses of streptozotocin resulted in both increased pancreatic beta cell death and significantly enhanced T cell priming. These results indicate that in situ beta cell death facilitates autoantigen-specific CD8 T cell priming and can contribute to both the initiation and the ongoing amplification of an autoimmune response.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Antigen persistence is required for dendritic cell licensing and CD8+ T cell cross-priming

It has been demonstrated that CD4(+) T cells require Ag persistence to achieve effective priming, whereas CD8(+) T cells are on "autopilot" after only a brief exposure. This finding presents a disturbing conundrum as it does not account for situations in which CD8(+) T cells require CD4(+) T cell help. We used a physiologic in vivo model to study the requirement of Ag persistence for the cross-priming of minor histocompatibility Ag-specific CD8(+) T cells. We report inefficient cross-priming in situations in which male cells are rapidly cleared. Strikingly, the failure to achieve robust CD8(+) T cell activation is not due to a problem with cross-presentation. In fact, by providing "extra help" in the form of dendritic cells (DCs) loaded with MHC class II peptide, it was possible to achieve robust activation of CD8(+) T cells. Our data suggest that the "licensing" of cross-presenting DCs does not occur during their initial encounter with CD4(+) T cells, thus accounting for the requirement for Ag persistence and suggesting that DCs make multiple interactions with CD8(+) T cells during the priming phase. These findings imply that long-lived Ag is critical for efficient vaccination protocols in which the CD8(+) T cell response is helper-dependent.     

T cell surface molecules regulating noncognate B lymphocyte activation. Role of CD2 and LFA-1.

A central event in humoral responses is the Ag-mediated interaction of Th cells and B cells. This interaction leads to the activation of both cell types and results in cytokine secretion by the T cells and proliferation and secretion of Ig by the B cells. The proliferative and differentiative responses of B cells are dependent on contact-mediated signals and cytokines provided by the activated Th cells. Although the role of cytokines in B cell activation and differentiation is understood, the nature of the signals delivered by the activated Th cells and the molecules involved in this process are not known. In this study we have examined Ag-mediated "cognate" T-B cell interactions as well as B cell activation induced by contact with preactivated and fixed Th lymphocytes. Our results indicate that both the T cell surface molecules lymphocyte function associated Ag-1 and CD2 are important in the activation of T cells by Ag presented by B lymphocytes. This indicates that B cells have similar characteristics as other APC. However, once the T cells are activated, contact-mediated stimulation of resting B lymphocytes (the noncognate phase) is dependent on CD2 but not lymphocyte function associated Ag-1. Two lines of evidence indicate this; first, it is inhibited by blocking of CD2 on the T cells and, second, such stimulation is not efficiently mediated by a CD2- Th cell line. Thus, CD2 plays an obligatory role at several discrete stages of T cell-mediated activation of resting B lymphocytes.     

Mechanisms of immune memory. T cell activation and CD3 phosphorylation correlates with Ta1 (CDw26) expression

The Ta1 (CDw26) Ag distinguishes a subset of circulating T lymphocytes that is the major population proliferating to recall Ag challenge. Unlike receptors for growth factors such as IL-2 and transferrin, the Ta1 Ag is present on T cell lines and clones irrespective of cell cycle. The appearance of Ta1 on T cells that respond to recall Ag allowed us to investigate activation requirements that may be associated with T cell immune memory. Ta1+ peripheral blood T cells were induced to proliferate by mAb recognizing either the invariant chains of the TCR, or by pairs of mitogenic antibodies directed to the CD2 molecule. In contrast, Ta1- cells were not stimulated by these antibodies. In addition, Ta1-cells did not proliferate maximally after addition of the phorbol ester PMA in combination with the calcium ionophore Ionomycin, suggesting that the intracellular targets of these agents may not be fully active. Anti-CD3-induced elevation of intracellular calcium levels was equivalent in the two subpopulations, suggesting that calcium mobilization mechanisms were intact. In contrast, PMA-induced phosphorylation of TCR CD3 chains was significantly greater in Ta1+ cells as compared to Ta1- T cells. Taken together, our results indicate that Ta1 expression, which is associated with T cell activation and memory, may be causally related to TCR and CD2-mediated activation mechanisms. The PMA inducible TCR phosphorylation in Ta1+ memory cells associated with their increased ability to proliferate after CD3/TCR or CD2 stimulation suggests that intracellular phosphorylation events may be causally associated with T cell immune memory.     

HIV-1 transmission and function of virus-infected monocytes/macrophages.

Monocyte/macrophages (MM) were isolated from HIV-1 seronegative individuals, infected with HIV-1 and examined for their ability to infect autologous T lymphocytes with and without concomitant presentation of exogenous Ag. HIV-1-infected MM presented tetanus toxin (TT) and streptokinase to T cells (as measured by [3H]thymidine incorporation) comparable to presentation by uninfected MM. In these studies, it was observed that HIV-1-infected MM without additional exogenous Ag stimulated autologous T lymphocytes, however, to a lesser degree than with TT and streptokinase. Virus production in T cells appeared to be relative to the degree of stimulation with the highest levels of stimulation and infection observed when T cells were exposed to HIV-1-infected TT-presenting MM. Studies were carried out to examine some of the restricting elements in MM-mediated infection of T lymphocytes with and without TT presentation. Antibodies to CD4, as well as soluble immunopurified gp120, blocked cell-mediated infection indicating that infection of T cells was through the CD4 molecule as has been demonstrated with cell-free virus. In addition, soluble gp120 inhibited Ag presentation by HIV-1-infected and uninfected MM. mAb to MHC class II Ag HLA-DR and -DP blocked T cell infection by HIV-1-infected MM with and without presentation of TT. No effect was observed with mAb to MHC class I Ag. These results indicate that virus transmission to T lymphocytes can be mediated by HIV-1-infected MM and that these cells maintain their function as APC. Activation of T cells appears to be important in the process of T cell infection in this system inasmuch as antibodies that block Ag presentation and thus a T cell proliferative signal inhibit infection.     

Defective antigen presentation by human melanoma cell lines cultured from advanced, but not biologically early, disease

The present studies were undertaken to characterize Ag presentation by cultured human melanoma cell lines. Cell lines established from "biologically early" lesions of malignant melanoma were able to present the soluble Ag tetanus toxoid (TT) to autologous and HLA-DR-matched allogeneic, TT-immune T cell clones. Proliferation of T cell clones in response to Ag presented by primary melanoma peaked on day 2 of culture with Ag. Ag presentation was blocked by pretreatment of TT-pulsed and fixed melanoma cells with mAb against HLA-DR, but not HLA-DQ, HLA-DP, or HLA-ABC. Ag processing and presentation were inhibited by treating the melanoma cells with ammonium chloride. In parallel with previous findings from this laboratory demonstrating the inability of cell lines cultured from "advanced" primary or metastatic melanoma to induce autologous T cell proliferation, such cell lines also failed to present this exogenous Ag despite the presence of cell-surface HLA-class II molecules. Thus, in contrast to the finding in biologically early melanoma, none of the multiple TT-immune, T cell clones from autologous patients or HLA-DR matched donors was able to respond to TT presented by melanoma cells cultured from advanced disease. Co-incubation studies revealed that metastatic melanoma cells did not secrete inhibitory substances during the APC assay, however, they were able to process TT, rendering it "immunogenic" in the presence of fixed, autologous non-T cells. When fixed, autologous melanoma cells were assayed for their ability to present processed Ag; fixed cells of early but not advanced disease were able to present Ag in this setting, indicating that the presenting limb becomes flawed in the evolution of the metastatic phenotype. Finally, studies of chloroquine inhibition of the capacity of melanoma cells derived from early primary disease to stimulate autologous peripheral blood T cells suggest that such cells process and present tumor-associated Ag in the same fashion as the "model" Ag TT.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. II. Chronic antigen presentation overrules antigen-presenting B cell activation

Ag presentation in the absence of danger signals and Ag persistence are the inductive processes of peripheral T cell tolerization proposed so far. Nevertheless, it has never been definitively shown that chronic Ag presentation per se can induce T cell tolerance independent of the state of activation of APCs. In the present work, we investigated whether chronic Ag presentation by either resting or activated B cells can induce tolerance of peripheral Ag-specific T cells. We show that CD4(+) T cells that re-encounter the Ag for a prolonged period, presented either by resting or activated Ag-presenting B cells, become nonfunctional and lose any autoimmune reactivity. Thus, when the main APCs are B cells, the major mechanism responsible for peripheral T cell tolerization is persistent Ag exposure, independent of the B cell activation state.     

Invariant NKT cells rapidly activated via immunization with diverse contact antigens collaborate in vitro with B-1 cells to initiate contact sensitivity

In cutaneous contact sensitivity there is an early elicited innate cascade of complement, mast cells, and platelets activated via IgM Abs. This response is required to initiate the elicitation of acquired classical contact sensitivity by leading to local recruitment of effector T cells. We recently performed in vivo experiments showing that collaboration is required between innate-like invariant Valpha14+ NKT cells (iNKT) and the innate-like B-1 B cell subset to induce this initiation process. Contact sensitization triggers iNKT cells to produce IL-4 to coactivate the B-1 cells along with specific Ag for production of the initiating IgM Abs. We now describe in vitro collaboration of iNKT and B-1 cells. Normal peritoneal B-1 cells, incubated in vitro with soluble Ag, and with 1-h in vivo immune iNKT cells producing IL-4, are activated to mediate the contact sensitivity-initiation cascade. The three components of this process can be activated by different Ag. Thus, 1-h iNKT cell activation, B-1 cell stimulation, and generation of immune effector T cells can be induced by sensitization with three different Ag to respectively generate IL-4 and Ag-specific IgM Abs, to recruit the Ag-specific effector T cells. These findings have relevance to allergic and autoimmune diseases in which infections can trigger exacerbation of T cell responses to allergens or to autoantigens.