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Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4CD25T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4T cells to a secondary irradiated recipient, regulatory CD25Foxp3and CD25Foxp3?CD4T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFN?-dependent mechanism. Moreover, in vivotransfer of tolerance can be abrogated by blocking IFN? or IDO, and anti-IFN? reduces the survivalexpansion of alloantigen-induced regulatory Foxp3CD4T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4CD25T cells and the graft endothelial cells in this local immune privilege, and a key role for IFN? and IDO in this process.

We present evidence that human peripheral blood lymphocytes, free of contaminating monocytes, rapidly produce high levels of tumor necrosis factor (TNF) when stimulated with phorbol diester and calcium ionophore, and lower but significant levels of TNF when stimulated with mitogens. These two types of inducers act preferentially on T cells, both CD4+ and CD8+. NK cells produce TNF only when stimulated with phorbol diester and calcium ionophore, and they do so at a much lower level than T cells. The procedures used in the purification of lymphocytes and the differential ability to respond to various inducers allow us to exclude that monocytes or basophils contaminating the lymphocyte preparation participate in the production of TNF. In particular, LPS, a potent inducer of TNF production from monocytes, is unable to induce significant levels of TNF in the lymphocyte preparations. The TNF produced by lymphocytes has antigenic, physicochemical, and biochemical characteristics identical to those of the TNF produced by myeloid cell lines or monocytes upon stimulation with LPS. LT is also produced by lymphocyte preparations. Production of TNF and LT proteins in response to the different inducers is paralleled by accumulation of cytoplasmic TNF and LT mRNA. Both at mRNA and at protein levels, stimulation of T lymphocytes with phorbol diester and calcium ionophore preferentially induces TNF, whereas mitogen stimulation preferentially induces LT. Our data suggest that the TNF and LT genes, two closely linked genes encoding two partially homologous proteins with almost identical biological functions, are independently regulated in lymphocytes.

In a model of heart allograft rejection in adult congeneic rats mismatched for both class I and class II MHC molecules, we analyzed the TCR β chain repertoire of T cells infiltrating rejected allografts [graft-infiltrating T cells (GITC)]. Although all BV families were used by GITC, oligoclonal expansions reflected by an altered distribution of TCR β chain CDR3 lengths were detected throughout the rejection process. Interestingly, expansions involving TCR β chains with common length and BV usage were recurrently found within distinct individuals at late stages of rejection in vivo and in vitro mixed lymphocyte culture between donor and naive recipient cells. Sequence analysis of the CD3 regions within recurrent TCR β chains comprising either BV2 or BV13 gene segments demonstrated a complete sequence identity between BV2-BJ2S3 junctions derived from GITC in all individuals tested and the presence of conserved amino acids at constrained CDR3 positions within GITC BV13⁺ junctions derived from most individuals. These results suggest the existence of several major alloantigens responsible for expansion of T cell clones bearing a 'public' β chain rearrangement within rejected allografts. The demonstration that such clones are also expanded during in vitro mixed lymphocyte reactions provides an experimental approach which might allow molecular characterization of the above major alloantigens and their possible in vivo targeting.

We report evidence that FcR(CD16) on human NK cells are signal-transducing molecules that, upon ligand binding, induce transcription of genes encoding surface activation molecules [IL-2-R(CD25)] and cytokines (IFN-gamma and TNF) relevant to NK cell biology and functions. Homogeneous NK and T cell populations purified from short-term bulk cultures of PBMC with irradiated B lymphoblastoid cell lines were cultured in the presence of FcR ligands (particulate immune complexes or immobilized anti-CD16 antibodies) alone or with rIL-2. Upon 18 h of stimulation, NK cells express Tac, TfR, and 4F2 antigens and produce IFN-gamma and TNF; both effects are synergistically enhanced in the presence of rIL-2, which is itself ineffective. Treatment of NK cells with FcR(CD16) ligands induces accumulation of mRNA for IFN-gamma and TNF and, to a lesser extent, IL-2-R with fast kinetics also in the absence of de novo protein synthesis. rIL-2 and FcR(CD16) ligands synergize to induce mRNA accumulation. mRNA accumulation and transcription of TNF and IFN-gamma genes induced by FcR(CD16) ligands are greater than those induced by rIL-2, and the reverse is true for IL-2-R. The two stimuli do not synergize at the transcriptional level. These observations indicate that the mechanisms through which FcR(CD16) ligands and rIL-2 induce NK cell activation are, in part, distinct. Both operate at the transcriptional level, although other mechanisms are probably induced by the FcR ligand stimulus per se or in combination with other lymphokines and synergize at a post-transcriptional or translational level to enhance NK cell activation.

We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.

In this study, we present evidence that interaction of Fc gamma R(CD16) with ligands (immune complexes or anti-CD16 antibodies) induces a rapid rise in [Ca2+]i and fast production of both inositol 1,4,5 triphosphate (IP3) and IP4 in homogeneous NK cell preparations. Part of the initial [Ca2+]i rise observed upon stimulation of NK cells with either anti-CD16 antibodies alone or after their crosslinking at the cell membrane depends on Ca2+ mobilization from intracellular stores, but sustained [Ca2+]i levels are maintained, after the initial spike, through influx of extracellular Ca2+. The [Ca2+]i rise is mediated, at least in part, by increases in IP3 after receptor-induced hydrolysis of membrane polyphosphoinositides (PPI). The role of extracellular Ca2+ in Fc gamma R(CD16)-dependent induction of lymphokine gene expression has been tested by evaluating production, mRNA accumulation and transcription of IFN-gamma and TNF in NK cells stimulated with Fc gamma R(CD16) ligands and/or rIL-2 in the presence of EGTA. Under these conditions, accumulation and transcription of both IFN-gamma and TNF mRNA induced by CD16 ligands, but not that induced by rIL-2, is completely abolished and neither cytokine can be detected at significant levels in the supernatant fluids of cells so treated. These data confirm that NK cell activation by specific ligands occurs through mechanisms distinct from those induced by IL-2, and indicate that extracellular Ca2+ represents a stringent requirement for cytokine production induced in NK cells through specific (Fc gamma R) stimulation. Our data also indicate that the [Ca2+]i rise induced upon Fc gamma R(CD16) crosslinking, though necessary, is not sufficient per se to induce activation of lymphokine genes, compatible with the hypothesis that Fc gamma R(CD16) crosslinking generates additional transducing signals that synergize with IL-2 to maximally activate NK cells.