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Retinoic acid-related orphan receptor (ROR)γt(+) TCRαβ(+) cells expressing IL-17, termed Th17 cells, are most abundant in the intestinal lamina propria. Symbiotic microbiota are required for the generation of Th17 cells, but the requirement for microbiota-derived Ag is not documented. In this study, we show that normal numbers of Th17 cells develop in the intestine of mice that express a single TCR in the absence of cognate Ag, whereas the microbiota remains essential for their development. However, such mice, or mice monocolonized with the Th17-inducing segmented filamentous bacteria, fail to induce normal numbers of Foxp3(+) RORγt(+) T cells, the regulatory counterpart of IL-17(+)RORγt(+) T cells. These results demonstrate that a complex microbiota and cognate Ag are required to generate a properly regulated set of RORγt(+) T cells and Th17 cells.

IL-15 drives chronic inflammation in several human diseases. We have recently shown that IL-15 inhibits the immunosuppressive effects of TGF-beta through blockage of the Smad3-signaling pathway. Data pointing to reciprocal interactions between TGF-beta and CD4(+) regulatory T cells led us to investigate the impact of IL-15 on the de novo generation and function of regulatory T cells in humans. Our data indicate that IL-15 does not counteract, but rather promotes the effect of TGF-beta on the de novo generation of regulatory T cells (Treg). Thus, in the presence of TGF-beta, IL-15 enhanced the acquisition of regulatory functions by CD4(+)CD25(-) T cells stimulated by anti-CD3 and anti-CD28 Abs. In contrast, IL-15 impaired the functions of Tregs by acting on effector CD4 and CD8 T cells. Accordingly, in the presence of IL-15, proliferation and IFN-gamma production by peripheral CD4 and CD8 T cells could not be efficiently inhibited by Tregs. IL-15-induced resistance of effector T cells to Tregs resulted from activation of the PI3K signaling pathway but did not involve the rescue of effector T cells from apoptosis. Altogether, these data point to the ambiguous role of IL-15 in the control of Treg functions. This dual role may be instrumental to mount rapid but transient proinflammatory immune responses against pathogens but may become deleterious in situations associated with protracted IL-15 over-expression.

V delta 3 usage and combinatorial expression of V gamma and V delta regions was studied on peripheral T cells with a novel V delta 3-specific mAb (p11.10b), generated against a soluble V gamma 9V delta 3 TCR. V delta 3+ cells represented the vast majority of V delta 1/V delta 2- gamma delta T cells within peripheral blood and mucosal lymphocytes. No preferential V gamma region expression was noted within V delta 3+ cells, but the frequency of V gamma 9+ cells was significantly lower among V delta 3+ than among V delta 1+ or V delta 2+ PBL. Phenotypic analysis of cultured V delta 3+ cells sorted with p11.10b mAb revealed the presence of T lymphocytes with unusual phenotypes. First, cells carrying two distinct surface TCR delta-chains, recognized by both V delta 1- and V delta 3-specific mAbs, were detected in most T cell lines, though at frequencies much lower than that of dual gamma expressors, indicating that allelic exclusion of delta genes is more tightly regulated than that of gamma genes. Moreover, a significant fraction of V delta 3+ cells were recognized by C beta- but not C delta-specific mAbs. Molecular analysis of V delta 3+C beta+ clones revealed the presence of V delta 3J alpha C alpha transcripts in all of them. Given the peculiar location of the V delta 3 gene between the delta Rec/psi J alpha elements, those observations formally demonstrate that activation of rearrangements with J alpha elements is not necessarily preceded by a delta Rec/psi J alpha-mediated deletion of the delta locus on the same chromosome.

Monoclonal antibodies reactive with T cells, T cell subsets, B cells, monocytes, and natural killer cells were used to characterize the nature of mucosal lymphocytes in the human small intestine by application of the immunoperoxidase technique to tissue sections for light and electron microscopic examination. In addition, for comparison, peripheral blood mononuclear cells (PBL) were studied by immunoelectron microscopy. Most of the intraepithelial lymphocytes (IEL) were T cells (Leu-1+, T3+) and expressed the phenotype associated with cytotoxic/suppressor T cells (Leu-2a+, T8+). In contrast, a majority of T lymphocytes in the lamina propria expressed the phenotype associated with helper/inducer T cells (Leu-3a+, T4+). These observations confirm and extend the findings previously reported. In addition, a small number of cells in the lamina propria with the ultrastructural features of macrophages were found to react with anti-Leu-3a and anti-T4 antibodies. Although many IEL contained cytoplasmic granules and had ultrastructural features similar to those of circulating granular lymphocytes, none of these cells reacted with anti-Leu-7 (HNK-1), anti-T10, or anti-M1 antibodies. This suggests that IEL may not be related to circulating large granular lymphocytes, which are Leu-7+, T10+, M1+ and are associated with natural killer activity. Not only Leu-7+ PBL, but T8+, T4+, or T3+ mucosal lymphocytes or PBL also may contain cytoplasmic granules. Therefore, the cytoplasmic granules are not restricted to one cell type, in particular, to Leu-7+ cells.

A monoclonal antibody, RGL-1, was produced by fusion of NSI myeloma cells with spleen cells of a mouse immunized with isolated rat intraepithelial lymphocytes (IEL). SDS-PAGE analysis revealed that RGL-1 precipitated two major noncovalently bound chains of about m.w. 100,000 and 125,000, and a minor component of m.w. 200,000. Examination of both tissue sections and isolated cells indicated that RGL-1 stained the majority of the lamina propria lymphocytes and IEL but only very few cells (less than 2%) in the lymphoid organs and small numbers of lymphocytes in other mucosae. In the small intestine, RGL-1 stained lymphocytes with the helper (W3/25) as well as the cytotoxic/suppressor (OX8) phenotype. The antibody reacted with 95% of the granular IEL but with less than 0.1% of the blood large granular lymphocytes. Although mature IgA plasma cells in the lamina propria were RGL-1-, some large IgA-containing cells were weakly positive. In the gut-associated lymphoid tissues (GALT), studies combining immunofluorescence and autoradiography indicated that 56 and 73% of rapidly dividing cells of mesenteric lymph nodes and of thoracic duct lymph (TDL) stained with RGL-1, respectively. In addition, 90 to 100% of the IgA-containing blasts of MLN and 75% of those of TDL were labeled by RGL-1. In contrast, rapidly dividing cells of spleen and of peripheral lymph nodes did not stain with RGL-1. Because RGL-1 can be demonstrated on both intestinal lymphocytes and their immediate precursors in the GALT, its expression may be related to the homing of lymphocytes into the gut mucosa.

We have demonstrated that although intestinal epithelial cells in fetuses and young rats do not express Ia antigens, in adult rats intestinal epithelial cells do express Ia antigens, as indicated by immunoperoxidase staining with monoclonal antibodies. Ia expression by intestinal epithelial cells appeared to be related to an increase in the number of intraepithelial lymphocytes (IEL). Most of the IEL were T cells and expressed the phenotype associated with cytotoxic/suppressor T cells, and a large number contained cytoplasmic granules. To directly study a possible modulating effect of IEL on intestinal epithelium, an Ia-negative intestinal epithelial cell line (IEC 17) of rat origin was cultured in the presence of supernatants obtained from Con A- or PHA-stimulated lymphocytes. IEL, as well as spleen cells but not bone marrow cells, were able to secrete a factor(s) capable of inducing Ia antigens on IEC 17 cells, as judged by immunoperoxidase staining and radioimmunoassay. Ia-positive IEC 17 cells were detectable after 12 hr and maximum Ia expression was obtained by 48-hr incubation. Persistence of Ia expression by intestinal epithelial cells required the continued presence of Ia-inducing factor in the medium. Lymphocyte proliferation was not essential for the secretion of the Ia-inducing factor(s). The characteristics and the kinetics of secretion of the Ia-inducing factor were similar to that of an interferon-like activity, but not of interleukin 2. Con A-induced supernatants from IEL and spleen cells were also capable of suppressing the growth of IEC 17 cells. The results of this study indicate that IEL, because of their close association with intestinal epithelial cells, may be involved in modulating a variety of epithelial cell functions, including the expression of Ia antigens. This leads us to speculate that Ia-positive epithelial cells, like Ia-positive macrophages and dendritic cells, may be involved in antigen presentation to T lymphocytes.