Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets.

CD1d-restricted invariant NKT (iNKT) cells are immunoregulatory cells whose loss exacerbates diabetes in nonobese diabetic (NOD) female mice. Here, we show that the relative numbers of iNKT cells from the pancreatic islets of NOD mice decrease at the time of conversion from peri-insulitis to invasive insulitis and diabetes. Conversely, NOD male mice who have a low incidence of diabetes showed an increased frequency of iNKT cells. Moreover, administration of alpha-galactosylceramide, a potent activating ligand presented by CD1d, ameliorated the development of diabetes in NOD female mice and resulted in the accumulation of iNKT cells and myeloid dendritic cells (DC) in pancreatic lymph nodes (PLN), but not in inguinal lymph nodes. Strikingly, injection of NOD female mice with myeloid DC isolated from the PLN, but not those from the inguinal lymph nodes, completely prevented diabetes. Thus, the immunoregulatory role of iNKT cells is manifested by the recruitment of tolerogenic myeloid DC to the PLN and the inhibition of ongoing autoimmune inflammation.

Decreases in interleukin-4 secretion by invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells in peripheral blood of patientswith relapsing-remitting multiple sclerosis.

The cytokine profile of invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells from patients with multiple sclerosis (MS) was compared with that of healthy controls. CD4(-)CD8(-)V alpha 24(+) T cells from the peripheral blood of 12 patients with relapsing-remitting MS (RR-MS), 5 patients with progressive MS (CP-MS), and 9 control individuals were directly sorted into single wells and expanded in vitro for analysis of IL-4 and IFN-gamma secretion; 315 V alpha 24J alpha Q T cell clones were generated and their T cell receptor (TCR) sequenced. T cell functionality was determined by examining cytokine secretion upon TCR cross-linking. RR-MS patients exhibited lower frequencies of IL-4 secreting CD4(-)CD8(-)V alpha 24J alpha Q T cell clones than patients with CP-MS and controls. No differences in IFN-gamma secretion were observed between the groups. An IL-4 positive cytokine profile could be correlated to the cloning efficiency of the V alpha 24J alpha Q T cells. We conclude that alterations in cytokine secretion patterns of CD4(-)CD8(-)V alpha 24J alpha Q T cells may influence the immune system and thus contribute to relapsing-remitting MS.

CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolytic activity of V alpha 24J alpha Q T cells: a feedback mechanism for immune regulation.

The precise immunologic functions of CD1d-restricted, CD161+ AV24AJ18 (Valpha24JalphaQ) T cells are not well defined, although production of IL-4 has been suggested as important for priming Th2 responses. However, activation of human Valpha24JalphaQ T cell clones by anti-CD3 resulted in the secretion of multiple cytokines notably important for the recruitment and differentiation of myeloid dendritic cells. Specific activation of Valpha24JalphaQ T cells was CD1d restricted. Expression of CD1d was found on monocyte-derived dendritic cells in vitro, and immunohistochemical staining directly revealed CD1d preferentially expressed on dendritic cells in the paracortical T cell zones of lymph nodes. Moreover, myeloid dendritic cells both activated Valpha24JalphaQ T cells and were susceptible to lysis by these same regulatory T cells. Because myeloid dendritic cells are a major source of IL-12 and control Th1 cell differentiation, their elimination by lysis is a mechanism for limiting the generation of Th1 cells and thus regulating Th1/Th2 responses.

A novel population of B7-1+ T cells producing intracellular IL-4 is decreased in patients with multiple sclerosis.

We identified a novel population of human T cells, studied directly ex vivo, that co-express surface B7-1 and intracellular IL-4. These peripheral blood B7-1+/CD4+ T cells expressed cell surface molecules associated with differentiation including CD45RO and MHC class II, yet were CD69(-) and CD25(-). In short-term cultures, T cell receptor (TCR) cross-linking induced further IL-4 production with little IFN-gamma or TNF-alpha. In marked contrast, CD4+ T cells negative for B7-1 expressed intracellular IFN-gamma and high amounts of TNF-alpha but little IL-4 upon TCR cross-linking. The CD4+/B7-1+/IL-4-expressing T cells were of polyclonal origin based on their diverse TCR repertoire. To explore the biological significance of this B7-1+/IL-4+ T cell population and to assess its potential regulatory role in autoimmune disease, we examined whether these T cells isolated ex vivo were altered in subjects with multiple sclerosis (MS). While the frequency of B7-1+ T cells was enhanced in patients with MS as compared to normal subjects, there was a significant diminution of B7-1+/IL-4+ T cells in the patients. The decrease in these IL-4-producing T cells in patients with autoimmune disease is consistent with a possible role as immunoregulatory T cells.

T cell autoreactivity to proinsulin epitopes in diabetic patients and healthy subjects.

We investigated the immune response to proinsulin, a potential autoantigen in IDDM secreted exclusively by pancreatic beta-cells. A total of 2,142 short-term cell lines were generated from 19 individuals; seven IDDM patients at the disease onset and 12 control subjects. No increase in the frequency of proinsulin reactive cells was observed in the IDDM group. To define proinsulin epitopes, proliferative responses of proinsulin-specific lines were examined against 10 overlapping 15 amino acid peptides encompassing the human proinsulin sequence. The predominant immune response was directed against the proinsulin p35-50 peptide located in the (C) connecting peptide between the alpha- and beta-chain of insulin. Recognition of the proinsulin p35-50 peptide could be shown by generating specific T cell clones against the peptide. However, unlike responses to other tissue-specific autoantigens there were only low proliferative responses to proinsulin as measured by 3H-thymidine incorporation. This low reactivity may be partially explained by the location of the p35-50 peptide in the C-peptide which is released into the circulation and therefore, may induce a clonal anergy of T reactive cells. However, the significantly higher 3H-thymidine incorporation after CD3-CD28 triggering showed that peptide specific T cells were capable of a significant response with a stronger TCR signal.

Autoantigen recognition by human CD8 T cell clones: enhanced agonist response induced by altered peptide ligands.

Determination of immunodominant epitopes of MHC class I-restricted self-Ags and elucidation of TCR contact residues is of potential importance in providing a means of manipulating the immune response to self-Ags in human autoimmune diseases. A computer algorithm was used to examine the sequences of the two major encephalitogenic proteins of myelin, MBP and PLP, for HLA-A2 binding motifs. Thirty-eight peptides with HLA-A2.1 binding motifs were synthesized and their binding to HLA-A2.1 was measured. A panel of HLA-A2-restricted T cell clones directed against the PLPp80-88 epitope, which exceeded the binding affinity of the other myelin-peptides tested by at least one order of magnitude, was generated. Using a set of analogue peptides with single amino acid substitutions, we detected a distinct pattern of TCR contact residues for each clone. Surprisingly, modification of different presumed TCR contact residues generated superagonist peptides, which are defined as peptides with equal or lower MHC binding affinity to HLA-A2 that induce half-maximal effector responses at 100-fold lower concentrations than the original peptide. These agonist peptides could drive cytotoxic T cell clones to proliferate, secrete cytokines, and clonally expand at concentrations at which the native peptide induced only cytotoxic responses. The proliferation induced by the superagonist peptides gives additional evidence that the clonal expansion of CD8 T cell clones may in part be regulated on the level of Ag recognition by the TCR.

Regulation of the mRNA expression for tumor necrosis factor-alpha in rat liver macrophages.

Kupffer cells are known to produce tumor necrosis factor-alpha upon stimulation with endotoxin or viruses. This tumor necrosis factor-alpha synthesis is suppressed by prostaglandin E2 or dexamethasone. Using Northern blotting and reverse transcriptase-polymerase chain reaction, it is demonstrated that endotoxin-induced tumor necrosis factor-alpha synthesis is blocked by prostaglandin E2 or dibutyryl 3':5'-cyclic adenosine monophosphate on the transcriptional level. Tumor necrosis factor-alpha itself suppressed endotoxin-evoked tumor necrosis factor-alpha mRNA expression when given in a narrow time interval with lipopolysaccharide. Interleukin-10 of human or mouse origin also inhibited the synthesis of tumor necrosis factor-alpha mRNA and protein when given more than 2 h prior to the endotoxin challenge. The suppressive effect of prostaglandin E2 lasted for more than 36 h while IL-10 blocked tumor necrosis factor-alpha production for barely 24 h. Dexamethasone reduced the endotoxin-induced tumor necrosis factor-alpha mRNA formation by approximately 50% only, although it led to nearly complete inhibition of the synthesis of the mature protein. Taken together with reverse transcriptase-polymerase chain reaction data revealing significant amounts of tumor necrosis factor-alpha mRNA in resting Kupffer cells, an additional posttranscriptional regulation of tumor necrosis factor-alpha synthesis has to be assumed. Tumor necrosis factor-alpha mRNA was not induced by interferon-gamma, interleukin-1 beta or interleukin-6 (the latter two cytokines are also synthesized by Kupffer cells), but a 24-h prestimulation of liver macrophages with interferon-gamma or phorbol ester had a modest priming effect.(ABSTRACT TRUNCATED AT 250 WORDS)