Oligoclonality of Rat Intestinal Intraepithelial T Lymphocytes: Overlapping TCR β-Chain Repertoires in the CD4 Single-Positive and CD4/CD8 Double-Positive Subsets
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Abstract Previous studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR β-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR β-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8αα and CD8αβ single-positive (SP) IELs used most Vβ genes, but relative Vβ usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vβ17 and Vβ19; these two genes accounted collectively for more than half the Vβ repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping β-chain repertoires. In conclusion, our results show that rat TCR-αβ+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vβ usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.Keywords:
Intraepithelial lymphocyte
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Abstract Age‐related changes in T cell subsets were examined in intestinal intraepithelial lymphocytes (i‐IEL), which contain unique T cells differentiating extrathymically. In 2‐month‐old mice bred under conventional condition, i‐IEL consisted of a large number of CD4 − CD8α/α + cells bearing either T cell receptor (TcR)α/β or TcRγ/δ and only a few CD4 + CD8α − cells. In aged mice (6 months old and 24 months old), unique CD4 + CD8α/α + i‐IEL bearing TcRα/β increased in number and conversely the proportion of TcRγ/δ + i‐IEL was decreased. Such an increase in number of CD4 + CD8α/α + cells was detected in i‐IEL from aged (14‐months old) nude mice, but not in aged (14 months old) germ‐free mice, suggesting that a significant fraction of TcRα/β T cells such as CD4 + CD8α + i‐IEL can develop along an extrathymic pathway under the influence of intestinal microflora with age.
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Although much has been learned about CD8 structure-function properties, it has so far not been tested whether the nature of the TCR is sufficient to transfer the property of CD8 dependence versus non-dependence to CD8+ cytotoxic T lymphocytes (CTL) and their precursors differentiating in T cell receptor (TCR)-transgenic (Tg) mice. In the present study, we compared the characteristics of dependence on CD8 for stimulation of CTL precursors and antigen-specific cytolysis by CD8+ T cells from two TCR-Tg mice expressing respectively the TCR (Tg) from a “CD8-dependent” and from a “CD8-independent” CTL clone, which were both reactive against the H-2Kb alloantigen and originated from H-2k mice. The results indicate that the property of the Tg+CD8+ cells from H-2k TCR-Tg mice corresponds to that of the CTL clone of origin, demonstrating that it is linked to the nature of the TCR. Consistent with this property, Tg+CD4+ cells could also differentiate into H-2Kb-specific CTL when originating from the “CD8-independent”, but not from the “CD8-dependent” Tg-TCR. The influence of the property of “CD8 dependence” on negative selection occurring in TCR-Tg H-2klb mice was apparent at two levels: (i) in the thymus, the extent of deletion was much more pronounced for the “CD8-independent” TCR-Tg mice; (ii) in the periphery, Tg+(hi) cells with low to negative CD8 expression were present for the “CD8-dependent” Tg-TCR, whereas only Tg+CD4−CD8− cells with low surface Tg-TCR and CD3 expression were found for the “CD8-independent” Tg-TCR, indicating that Tg+CD4−CD8− cells are susceptible to tolerance induction involving TCR/CD3 surface down-modulation. Furthermore, different in vitro conditions led to H-2Kb-induced stimulation of Tg+CD4−CD8− cells to differentiate into CTL detected in an anti-TCR clonotypic monoclonal antibody redirected cytolysis assay. Culture in interleukin-2 of H-2klb Tg+CD4−CD8− cells was sufficient to induce CTL activity in the “CD8-independent” model, whereas stimulation with cells which overexpressed H-2Kb was required in addition to interleukin-2 to induce CTL differentiation in the “CD8-dependent” model. These data suggest that peripheral Tg+CD4−CD8− cells present in a situation of in vivo tolerance to H-2Kb can still be triggered by H-2Kb with a sensitivity correlated with the degree of CD8 dependence.
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Abstract In mice, the majority of Tcells expressing the γ/δ Tcell receptor (TcR) are found at mucosal surfaces, especially the intestinal epithelium. Here we show that in vitro , the majority of TcR γ/δ + intraepithelial lymphocytes, but not TcR α/β + intraepithelial lymphocytes, undergo rapid and selective programmed cell death by apoptosis.
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Rearrangement of both the beta and gamma chain T cell receptor (TcR) genes was detected in intestinal intraepithelial lymphocytes (IEL) from normal euthymic rats. Flow cytometric analyses showed that about 73% of the IEL were CD3+ (1F4) and that 67% were TcR alpha/beta+ (R73). About 5% of the IEL were found to be CD3+, TcR alpha/beta- in double-labeling experiments suggesting that a small fraction of IEL in the rat express the alternative TcR gamma/delta. More than 70% of the IEL were granular implying that many CD3+ IEL are granular. In IEL from athymic nude rats no rearrangement of either the TcR beta or gamma chain genes or surface expression of CD3 or TcR alpha/beta was detected despite the fact that about 95% of the cells were granular and morphologically similar to those in normal rats. Taken together our data suggest that the majority of IEL in the rat express the conventional TcR alpha/beta and that TcR-bearing cells in the gut epithelium are thymus dependent.
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CD4(-)CD8(-) thymocytes expressing a transgenic T cell receptor (TCR) alpha chain have decreased capacity to give rise to CD4(+)CD8(+) thymocytes when compared with wild-type thymocytes. This inefficient CD4(-)CD8(-) to CD4(+)CD8(+) maturation is mediated by the transgenic TCR alpha chain pairing with endogenous TCR beta chain but not with endogenous TCR gamma chain. Comparison between TCR alpha chain-transgenic mice with or without a functional pre-TCR alpha (pT alpha ) chain reveals that the formation of transgenic alpha/endogenous beta TCR on CD4(-)CD8(-) thymocytes inhibits the formation of pre-TCR, but at the same time mediates CD4(-)CD8(-) to CD4(+)CD8(+) maturation in the absence of pre-TCR, albeit inefficiently. These results indicate that alpha beta TCR and pre-TCR provide different signals for thymocyte development. They also suggest that the precise regulation of the sequential rearrangements of TCR beta and alpha loci and the cellular expansion induced by the pre-TCR may both be evolved to ensure the efficient generation of mature alpha beta T cells.
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Abstract TCRαβ+ intestinal intraepithelial lymphocytes (IEL) can express either the typical CD8αβ heterodimer or an unusual CD8αα homodimer. Both types of CD8+ IEL require class I molecules for their differentiation, since they are absent in β2m−/− mice. To gain insight into the role of class I molecules in forming TCRαβ+ CD8+ IEL populations, we have analyzed the IEL in mice deficient for either TAP, β2m, CD1, or K and D. We find that K−/−D−/− mice have TCRαβ+ CD8αα+ IEL, although they are deficient for TCRαβ+ CD8αβ+ cells. This indicates that at least some TCRαβ+ CD8αα+ IEL require only nonclassical class I molecules for their development. Surprisingly, the TCRαβ+ CD8αα+ IEL are significantly increased in K−/−D−/− mice, suggesting a complex interaction between CD8+ IEL and class I molecules that might include direct or indirect negative regulation by K and D, as well as positive effects mediated by nonclassical class I molecules.
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We have used two-colour flow cytometry to examine the heterogeneity of intraepithelial lymphocytes (IEL) from mouse small intestine. We have confirmed the predominance of CD3+ Thy 1- CD8+ IEL and show that a substantial but variable proportion of CD8+ IEL does not express the alpha beta T-cell receptor (TcR) for antigen. Simultaneous analysis of the co-expression of the alpha and beta chains of the CD8 heterodimer and of the alpha beta TcR revealed three populations of CD8+IEL. The first of these expressed both CD8 alpha and beta chains and had normal expression of V beta families and so represented conventional CD8+ alpha beta TcR+ T cells. The second population comprised alpha beta TcR- T cells (presumed gamma delta TcR+) which expressed only the alpha chain of the CD8 molecule. Finally, we identified a second, unique population of alpha beta TcR+ CD8+ IEL which were also CD8 beta-. Gamma delta + IEL predominated in mice aged less than 8 weeks, but there was a rapid increase in both populations of alpha beta TcR+ CD8+ IEL in older mice. CD8+ IEL were similar to peripheral CD8+ T cells in having high expression of the CD45RB molecule, but CD4+ IEL had generally lower expression of CD45RB than their peripheral counterparts, despite having normal expression of TcR. These findings emphasize the heterogeneity of IEL and underline the need to study phenotypically defined populations.
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Abstract The murine intestinal epithelium contains T cell receptor (TcR) γδ‐bearing T cells in high frequency. In the present report, we showed that TcR γδ‐bearing intestinal intraepithelial lymphocytes (IEL) from C3H/He (H‐2 k ) mice can be divided into two subpopulations based on TcR expression level; a subpopulation with a remarkably high level of TcR expression and a subpopulation with a moderate level of TcR expression, designated as TcR γδ hi IEL and TcR γδ mod IEL, respectively. In flow cytometric analysis, the TcR γδ hi IEL expressed a high level of TcR (mean fluorescence channel 518) when compared with the TcR level of TcR γδ + T cells in other lymphoid organs (mean fluorescence channel 88). The TcR γδ hi IEL were detected in IEL from mice of H‐2 k and H‐2 k/b haplotypes but not in H‐2 b and H‐2 d haplotypes. V δ 4, which was reported to be frequently expressed in IEL of H‐2 k mice, was preferentially expressed by TcR γδ hi IEL. These results suggested that the existence of the TcR γδ hi population is related to the high frequency of V δ 4 in H‐2 k mice.
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