Tumor‐infiltrating gamma delta T‐cells reveal exhausted subsets with remarkable heterogeneity in colorectal cancer
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Abstract The γδT‐cells recognize infected or transformed cells. However, unlike αβT‐cells, γδT‐cells are innate‐like immune cells, with no major histocompatibility complex restriction requirements. γδT‐cells are the main population of intestinal intraepithelial lymphocytes (IELs) and are associated with the antitumor immune response, particularly in colorectal cancer (CRC). Although CD8 + T‐cells exhibit dysfunction and even exhaustion in the tumor microenvironment (TME), which contributes to tumor immune escape, whether the same applies to tumor‐infiltrating (TI)‐γδT‐cells is not completely understood. Here, we sought to investigate the expression pattern of inhibitory receptors and functional state of TI‐γδT‐cells, and reveal the features of exhausted TI‐γδT‐cells in the CRC TME. We demonstrated that TI‐γδT‐cells exhibited exhaustion phenotypes and displayed more severe functional exhaustion than TI‐CD8 + T‐cells or NK‐cells in the TME of CRC. In addition, scRNA‐seq analysis of TI‐γδT‐cells revealed three exhausted subsets with remarkable heterogeneity. The presence of three heterogeneous exhausted γδT‐cell (Tex) populations, including Tex prog , Tex tran and Tex term were further confirmed by flow cytometry, on the basis of PD‐1 and TIM‐3 expression. Finally, we revealed that c‐Maf not only contributed to γδT‐cell exhaustion via upregulation of inhibitory receptors, but also involved in the exhaustion of CD8 + T and NK‐cells. c‐Maf may also be an important contributor to γδT‐cell exhaustion in CRC patients. These findings indicated that TI‐γδT‐cells exhibit phenotypic and functional exhaustion in the CRC TME. The revealed features of exhausted TI‐γδT‐cells may provide help for understanding the mechanisms and the association of γδT‐cell exhaustion with tumor development and pathogenesis.Keywords:
Intraepithelial lymphocyte
Abstract Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.
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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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Objective To study mechanism of graft versus host disease (GVHD) induced by Cyclosporine A (CsA) following autologous bone marrow transplantation. Methods The intraepithelial lymphocytes (IEL) were isolated from colon (major target organ of sGVHD), the phenotype of the cells and expression of cytokines were identified by cytometry and RT PCR. Results The CD4 +CD8 -αβ T cell increased and CD4 -CD8 - (double negative, DN) αβ T cell decreased in IEL from mice with sGVHD compared to the control ( P 0.01). The ratio of CD4 +CD8 -/CD4 -CD8 -αβ T cell enhanced from 1.0 1.5 to 13 25. Meanwhile, there was an enhanced level of cytokines IL 12,IFN γ and TNF α in IEL. The disregulation of CD4 + CD8 -/CD4 -CD8 -αβ T cells and cytokine increase in IEL begans at three weeks after CsA treatment. But there is no change for γδ T cells. Conclusion That equilibrium of CD4 +CD8 - and CD4 -CD8 -αβ T cells in colon IEL is important for stability of colon function. The reason that CsA induced sGVHD may be explained by interfering the balance of CD4 +CD8 -/CD4 -CD8 -αβ T cells in colon IEL. [
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TCR alpha beta+ intestinal intraepithelial lymphocytes (IEL) can express either the typical CD8 alpha beta heterodimer or an unusual CD8 alpha alpha homodimer. Both types of CD8+ IEL require class I molecules for their differentiation, since they are absent in beta2m-/- mice. To gain insight into the role of class I molecules in forming TCR alpha beta+ CD8+ IEL populations, we have analyzed the IEL in mice deficient for either TAP, beta 2m, CD1, or K and D. We find that K-/-D-/- mice have TCR alpha beta+ CD8 alpha alpha+ IEL, although they are deficient for TCR alpha beta+ CD8 alpha beta+ cells. This indicates that at least some TCR alpha beta+ CD8 alpha alpha+ IEL require only nonclassical class I molecules for their development. Surprisingly, the TCR alpha beta+ CD8 alpha alpha+ 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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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.
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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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Objective To observe the dynamics of intestinal intraepithelial lymphocyte subsets in mice intranasaly immunized with soluble tachyzoites antigen(STAg) in association with propolis and gamma interferon(IFN-γ).Methods Ninety-six female BALB/c mice were randomly divided into immunized and control group and immunized with 2 doses of STAg plus propolis and IFN-γ against Toxoplasma gondii and phosphate buffer,respectively,at an interval of 2 weeks.The mice were killed 1,2,3,4,6,8,10,and 12 weeks after the 2nd dose,respectively,and intestinal intraepithelial lymphocytes(IEL) were isolated and counted.Percentage of CD4+ and CD8+ T cells was determined by immunocytochemistry.ResultsThe number of IEL of the mice in immunized group reached a peak value at week 2(2.27×105)and was significantly higher than that of in the control group during weeks 1-4(F=8.42,P0.01).The numbers of CD4+ and CD8+ T cells were both higher than those of the control.CD8+ T cells was significantly higher at weeks 1,2,3,4,and 6 than those in the control group(F=6.38,P0.05),while CD4+ T cells in immunized group increased significantly at week 2 and 3(F=5.64,P0.05).The ratio of CD4+/CD8+T cells was higher at week 1 and 2(F=5.74,P0.05).Conclusion The immunization with STAg in association with propolis and IFN-γ by intranasal drip can effectively induce immune responses of IEL.
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Abstract In conventional mice, the T cell receptor (TCR)αβ + CD8αα + and CD8αβ + subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy‐Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180 : 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ + IEL populations from the small intestine of individual germ‐free mice that contain ten times less TCRαβ + T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ‐free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ + IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10 3 cells up to 1.2 × 10 6 cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.
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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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Objective To investigate the variation of T lymphocyte subsets in peripheral blood of the patients with tumor and their clinical significance.Methods The T lymphocyte subsets in peripheral blood from patients with tumor n = 27 and healthy controls n = 25 were detected with flow cytometry.Results The CD3+ T cells,CD4+ T cells and CD4+/CD8+ ratio in patients with tumor were significantly lower than that in the control group.Comparied with the control group the CD8+ T cells in patients with tumor were significantly higher P 0.01 .Conclusion The immunological function of patients with tumor was decreased.The flow cytometry is a rapid,sensitive and accurate measure to detect the immunological function of patients with tumor.It can play an important role in evaluating the curative effect and prognosis of the disease.
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