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Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.

Adaptive tolerance is the physiologic down-regulation of T cell responsiveness in the face of persistent antigenic stimulation. In this study, we examined the role of CTLA-4 in this process using CTLA-4-deficient and wild-type TCR transgenic, Rag2(-/-), CD4(+) T cells transferred into a T cell-deficient, Ag-expressing host. Surprisingly, we found that the tuning process of adoptively transferred T cells could be induced and the hyporesponsive state maintained in the absence of CTLA-4. Furthermore, movement to a deeper state of anergy following restimulation in vivo in a second Ag-bearing host was also unaffected. In contrast, CTLA-4 profoundly inhibited late T cell expansion in vivo following both primary and secondary transfers, and curtailed IL-2 and IFN-gamma production. Removal of this braking function in CTLA-4-deficient mice following Ag stimulation may explain their lymphoproliferative dysregulation.

TCR affinity and ligand off-rate have both been found to influence the degree of T cell activation by peptide-MHC. A report in this issue of Immunity finds that ligand off-rate does not correlate with antigen-specific peripheral T cell expansion. Moreover, the data point to the surprising notion that there exist TCR affinity thresholds and, once attained, T cells with higher affinity receptors have no competitive advantage.

Helper T cell responses that focus the TCR repertoire of responding clones provide experimental access to the mechanisms of clonal selection in vivo. Using TCRbeta chain animals, we directly evaluate the extent of TCRalpha CDR3 diversity and the pMHCII binding attributes of individual antigen-specific Th cells. Here, we demonstrate that dominant clonotypes, as defined by TCR junctional sequence similarities, are surprisingly diverse at the level of pMHCII binding properties, before and after antigen exposure. During an immune response, we can detect and quantify the selective loss of antigen-specific clonotypes that express lower-affinity TCR. This affinity threshold selection is followed by the unbiased propagation of preferred clonotypes regardless of TCR-pMHCII half-lives or affinity. Thus, an affinity threshold mechanism discriminates Th clones with TCR of best fit and propagates clonal diversity without promoting autoreactivity.

A polyepitope DNA vaccine has the potential to generate protective immune responses to a range of antigens in a single construct. We investigated whether it was possible to obtain responses to individual epitopes from different antigens, directly linked in a string, and whether the response to a given epitope was enhanced by adjacent epitopes within the construct. A polyepitope plasmid was created, which included three Th epitopes (influenza haemagglutinin, moth cytochrome c and ovalbumin), a Tc epitope (ovalbumin) and two B cell epitopes (haemagglutinin and ovalbumin). Mice were immunized with DNA by using a gene gun. Responses to the polyepitope DNA vaccine were compared with those to DNA vaccine comprising only the haemagglutinin Th and B epitopes (HAT(h)B) or with responses to the recombinant protein. These experiments showed that the polyepitope DNA vaccine induced greater antigen-specific responses to HAT(h)B peptide than the HAT(h)B DNA vaccine. Antigen-specific in vivo cytotoxic responses following polyepitope DNA vaccination were also clearly demonstrable. We conclude that a 'naked DNA' polyepitope vaccine generates specific responses to constituent epitopes and that adjacent irrelevant epitopes may enhance these responses.

Wiskott-Aldrich syndrome protein (WASP)-deficient T cells exhibit defects in IL-2 production that are widely believed to stem from primary defects in actin remodeling and immune synapse formation. Surprisingly, however, we find that WASP-deficient T cells responding to Ag-specific APCs polymerize actin and organize talin and PKC theta normally, forming an immune synapse that is stable for at least 3 h. At low doses of peptide, WASP-deficient T cells show less efficient talin and PKC theta polarization. Thus, although WASP may facilitate immune synapse formation at low peptide concentrations, WASP is not required for this process. Defects in IL-2 production are observed even under conditions in which immune synapse formation proceeds normally, suggesting that the role of WASP in regulating IL-2 production is independent of its role in immune synapse formation.

Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 processing using conformation sensitive monoclonal antibodies. This method proved to identify deficient mitochondrial apoptosis signaling in leukemia cells overexpressing Bcl-2 by a pattern of apoptosis resistance, deficient cytochrome c reduction and partial processing of caspase-3. In primary leukemia cells, reduction of cytochrome c and caspase-3 activation was induced by treatment with anticancer drugs in vitro. In leukemia cells of a patient with resistant disease, a pattern of deficient apoptosis signaling as in Bcl-2 transfected cells was observed, suggesting that deficient mitochondrial signaling contributed to the clinical phenotype of drug resistance in this patient. Flow cytometric analysis of mitochondrial apoptosis signaling may provide a useful tool for the prediction of drug resistance and treatment failure in primary leukemia., (Copyright 2004 Kluwer Academic Publishers)

T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) theta clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKCtheta raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKCtheta clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4(+) T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4(+)CD25(+) regulatory T cells (T(reg)) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T(reg). In this study, we demonstrate that after a primary Ag challenge, T(reg) proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T(reg) after primary Ag challenge augmented the total suppressive function of T(reg) against TCR repertoire modification but not against the proliferation of memory CD4(+) T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T(reg), which would be crucial for limiting excess primary and memory CD4(+) T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T(reg) is an ideal strategy for the clinical use of T(reg).

We and others have previously reported in an in vivo rat colon cancer cell model that cell death precedes and is necessary for the development of a specific antitumoral immune response. To sensitize colon cancer cells to death, we depleted cytochrome c by stable transfection with an antisense construct. Cytochrome c depletion sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli. This increased sensitization to a necrosis-like cell death may be related to a decrease in cellular ATP levels and an increase in reactive oxygen species production caused by cytochrome c depletion. In vivo, depletion of cytochrome c decreases the tumorigenicity of colon cancer cells in syngeneic rats without influencing their growth in immune-deficient animals. Furthermore, decreased expression of cytochrome c in tumor cells facilitates in vivo "necrotic" cell death and the induction of a specific immune response. These results delineate a novel strategy to sensitize colon cancer cells to chemotherapy and to increase their immunogenicity in immuno-competent hosts.

Thymocyte maturation in the thymus is controlled by stromal and humoral components. Among the humoral regulators locally produced glucocorticoids (GCs) seem to have a key role in the positive selection of thymocytes. Our previous studies have shown that the administration of GCs or the stimulation through the CD3 complex can induce apoptosis of double positive (DP) cells, but the combined presence of these stimuli induces positive selection. In this work our aim was to investigate the effects of antigen exposure and synthetic GC hormone (dexamethasone, DX) administration on the selection processes of DP cells in TcR transgenic mice. In our model, AND-pigeon cytochrome c (PCC)-specific I-E(k) (MHC-II) restricted Vbeta3, Valpha11 TcR expressing transgenic mice were treated with PCC, with high or low dose DX, or with PCC and DX together, followed by the analysis of total thymocyte numbers, thymocyte composition, with regard to their CD69, Vbeta3 and Annexin V expression. The administration of PCC and/or DX for 2 days resulted in a decreased DP cell number and a significantly increased CD4 SP cell ratio. However, in both cases the total thymocyte numbers decreased. CD69 expression increased on both DP and CD4 SP cells after PCC and/or DX treatments. We found that after DX or combined treatment, the percentage of Annexin V positive cells increased. The ratio of Vbeta3 TcR bearing DP thymocytes showed no change after DX or PCC administrations alone, but it decreased significantly after combined treatment. MHC-II bound PCC peptides in the presence of GCs enhanced the maturation of Vbeta3+ DP cells into CD4 SP stage, therefore, the Vbeta3- cells remained mostly in the DP immature stage. These data indicate that both antigen and low dose GC alone are capable of inducing positive selection of DP cells, but together they gave a stronger effect in promoting positive selection. From these we conclude that GCs influence the maturation and selection processes of thymocytes.

T cells acquire immune functions during expansion and differentiation in the thymus. Mature T cells respond to peptide antigens (Ag) derived from foreign proteins when these peptide Ag are presented on the self major histocompatibility complex (MHC) molecules but not on allo-MHC. This is termed self-MHC restriction. On the other hand, T cells do not induce aggressive responses to self Ag (self-tolerance). Self-MHC restriction and self-tolerance are not genetically determined but acquired a posteriori by positive and negative selection in the thymus in harmony with the functional maturation. Allogeneic bone marrow (BM) chimera systems have been a useful strategy to elucidate mechanisms underlying positive and negative selection. In this communication, the contribution of BM chimera systems to the investigation of the world of T-ology is discussed.

Oral tolerance is the result of a complex immunoregulatory strategy used by the gut and its associated lymphoid tissues to render the peripheral immune system unresponsive to nonpathogenic proteins, such as food or commensal bacteria. The mechanism of oral tolerance induction and maintenance is not well understood. We have previously shown that the chemokine, CC chemokine ligand 2 (CCL2), is important for the induction and maintenance of oral tolerance. To address the role CCL2 plays in oral tolerance, we used both CCL2(-/-) and CCR2(-/-) mice. Cells from the spleen, mesenteric lymph nodes, and peripheral lymph nodes of CCL2(-/-) and CCR2(-/-) mice fed high doses of OVA showed robust proliferative responses compared with cells from Ag-fed wild-type mice. CCL2(-/-) and CCR2(-/-) mice also produced high amounts of Th1 cytokines such as IL-2 and IFN-gamma and very low amounts of IL-4 and IL-10. The ability of APCs from the gut of CCL2(-/-) and CCR2(-/-) OVA-fed mice to stimulate an indicator T cell line was evaluated. APCs from the Peyer's patch of OVA-fed knockout animals could induce a T cell response measured by an increase in proliferation and generation of IL-12 and IFN-gamma with a concomitant reduction of TGF-beta compared with wild-type controls that did not induce a Th1 response. These data indicate that CCL2 and signaling through its receptor CCR2 is critical for the induction of oral tolerance by regulating Ag presentation leading to a disruption in the balance of inflammatory and regulatory cytokines.