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. 2018 Nov;194(2):259-272.
doi: 10.1111/cei.13162. Epub 2018 Sep 23.

Characterization of eomesodermin and T-bet expression by allostimulated CD8+ T cells of healthy volunteers and kidney transplant patients in relation to graft outcome

A Perez-Gutierrez  1 , D M Metes  1   2 , L Lu  1 , S Hariharan  1   3 , A W Thomson  1   3 , M B Ezzelarab  1
Affiliations

Characterization of eomesodermin and T-bet expression by allostimulated CD8+ T cells of healthy volunteers and kidney transplant patients in relation to graft outcome

A Perez-Gutierrez et al. Clin Exp Immunol. 2018 Nov.

Abstract

Memory T cell (Tmem) responses play a critical role in the outcome of allo-transplantation. While the role of the T-box transcription factor Eomesodermin (Eomes) in the maintenance of antigen-specific Tmem is well studied, little is known about Eomes+ CD8+ T cell responses after transplantation. We evaluated the phenotype and function of allo-reactive Eomes+ CD8+ T cells in healthy volunteers and kidney transplant patients and their relation to transplant outcome. High Eomes expression by steady-state CD8+ T cells correlated with effector and memory phenotype. Following allo-stimulation, the expression of both the T-box proteins Eomes and T-bet by proliferating cells increased significantly, where high expression of Eomes and T-bet correlated with higher incidence of allo-stimulated IFNγ+ TNFα+ CD8+ T cells. In patients with no subsequent rejection, Eomes but not T-bet expression by donor-stimulated CD8+ T cells, increased significantly after transplantation. This was characterized by increased Eomeshi T-bet-/lo and decreased Eomes-/lo T-bethi CD8+ T cell subsets, with no significant changes in the Eomeshi T-bethi CD8+ T cell subset. No upregulation of exhaustion markers programmed-death-1 (PD-1) and cytotoxic-T-lymphocyte-associated-antigen-4 (CTLA4) by donor-stimulated Eomes+ CD8+ T cells was observed. Before transplantation, in patients without rejection, there were higher incidences of Eomeshi T-bet-/lo , and lower incidences of Eomeshi T-bethi and Eomes-/lo T-bethi donor-stimulated CD8+ T cell subsets, compared to those with subsequent rejection. Overall, our findings indicate that high Eomes expression by allo-stimulated T-bet+ CD8+ T cells is associated with enhanced effector function, and that an elevated incidence of donor-stimulated CD8+ T cells co-expressing high levels of Eomes and T-bet before transplantation, may correlate with an increased incidence of acute cellular rejection.

Keywords: Eomesodermin; T-bet; human; kidney transplantation; memory T cells; rejection.

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Figures

Figure 1
Figure 1
Memory and effector phenotype of Eomeshi versus Eomes‐/lo non‐activated CD8+T cells in healthy human volunteers. (a) Memory CD8+T cell subsets were defined based on their differential expression of CD45RA and CCR7: naïve T cells (Tn; CD45RA+CCR7+), central memory T cells (Tcm: CD45RA‐CCR7+), effector memory T cells (Tem: CD45RA‐CCR7‐) and terminally‐differentiated effector memory T cells (Temra; CD45RA+CCR7‐). Dot plots are from one representative individual. Mean values are indicated by horizontal bars (n = 7 individuals). (b) Expression of TNFα, IFNγ and granzyme‐B was assessed after 3‐4 hours of PMA/ionomysin stimulation (n = 7 individuals). Histograms (left) are from one representative individual; results are expressed as percent positive cells. Gray histograms indicate isotype controls. Data from all 7 individuals examined are shown on the right. Wilcoxon‐Mann‐Whitney test; *P < 0.05.
Figure 2
Figure 2
Memory and effector phenotype of allo‐stimulated Eomeshi versus Eomes‐/lo CD8+T cells. (a) CFSE‐labeled purified T cells were co‐cultured with allogeneic stimulators for 5 days. Percent responder cell proliferation was determined by CFSE dilution and Eomes expression by proliferating cells was then evaluated by flow cytometry. The dot plot is from one representative individual. Data from all individuals examined are shown on the right (n = 12). (b) Memory CD8+T cell subsets of proliferating cells were defined based on their differential expression of CD45RA and CCR7 (as in Fig. 1). The dot plots are from one representative individual. Data from 10 individuals examined are shown. (c) Expression of TNFα, IFNγ and granzyme‐B assessed 3‐4 hours after PMA/ionomysin stimulation following coculture with allogeneic stimulators. Histograms (left) are from a representative individual; results are expressed as percent positive cells, gating on proliferating cells. Gray histograms indicate isotype controls. Data from all individuals examined (n = 8) are shown on the right. Wilcoxon‐Mann‐Whitney test; *P < 0.05.
Figure 3
Figure 3
Kinetics of Eomes expression by CD8+T cells following allo‐stimulation in vitro. CFSE‐labeled purified T cells were co‐cultured with allogeneic stimulators for 3, 5 and 7 days. Percent responder cell proliferation determined by CFSE dilution and Eomes expression by proliferating cells over time was then evaluated by flow cytometry. (a) The dot plots are from one representative individual. (b) Percent of proliferation of Eomeshi and Eomes‐/lo CD8+T cells (top) and MFI for Eomes expression (bottom). Combined data from five individuals examined are shown.
Figure 4
Figure 4
T‐bet expression by non‐stimulated and allo‐stimulated Eomeshi and Eomes‐/lo CD8+T cells. (a) T‐bet expression by non‐activated CD8+T cells (left) and proliferating cells after allo‐stimulation (right). Histograms are from one representative individual. Data from multiple normal healthy individuals are shown below (non‐activated; n = 20 and allo‐stimulated; n = 6). (b) Dual expression of IFNγ and TNFα by EomeshiT‐bethi, EomeshiT‐bet‐/lo, Eomes‐/loT‐bethi and Eomes‐/loT‐bet‐/lo CD8+T cell subsets, following coculture with allogeneic stimulators. Dot plots (left and center) are representative of one individual. Data from 6 individuals are shown in the graph (right). Gray histograms indicate isotype controls. Horizontal bar represents the mean. Wilcoxon‐Mann‐Whitney test; *P < 0.05, ****P < 0.0001.
Figure 5
Figure 5
Eomes and T‐bet expression by proliferating CD8+T cells following allo‐stimulation in vitro. Divisions of CFSE‐labeled proliferating cells measured using flowjo software. Mean fluorescence intensity (MFI) of Eomes and T‐bet expression was evaluated for each division. The histogram (left) is from one representative individual coculture. Combined data from 8 individuals examined are shown (left). Wilcoxon‐Mann‐Whitney test; *P s< 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
Pre‐ and post‐tx Eomes and T‐bet expression by donor‐stimulated CD8+T cells of kidney tx recipients with no rejection. (a) After co‐culture of recipient PBMC with donor PBMC, total CD8+T cells were evaluated for Eomes and T‐bet co‐expression. Dot plots are representative of one kidney tx recipient. (b) The percentages of EomeshiT‐bethi, EomeshiT‐bet‐/lo and Eomes‐/loT‐bethi subsets in total CD8+T cells were determined. Results are expressed as percentages of each subset. (c) Percentages of PD‐1+, CTLA4+ and CD57+ Eomeshi CD8+T cells following stimulation with donor cells. Wilcoxon‐Mann‐Whitney test; *P < 0.05, **P < 0.01.
Figure 7
Figure 7
Pre‐tx Eomes and T‐bet expression by donor‐stimulated CD8+T cells of kidney tx recipients with or without subsequent rejection. PBMC were obtained before transplantation from kidney allograft recipients with no subsequent rejection (n = 11), subclinical rejection (SCR; n = 5) and acute cellular rejection (ACR; n = 5). Recipient PBMC were co‐cultured with donor PBMC for 7 days. After coculture, total CD8+T cells were evaluated for (a) Mean fluorescence intensity (MFI) of Eomes and T‐bet expression, and (b) for the incidences of EomeshiT‐bethi, EomeshiT‐bet‐/lo and Eomes‐/loT‐bethi subsets. Color coded dots correspond with the same patients in Table 1. Wilcoxon‐Mann‐Whitney test; *P < 0.05.
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References

    1. Shiao SL, McNiff JM, Pober JS. Memory T cells and their costimulators in human allograft injury. The Journal of Immunology. 2005;175:4886–96. - PubMed
    1. Masopust D, Vezys V, Marzo AL, Lefrancois L. Preferential localization of effector memory cells in nonlymphoid tissue. Science. 2001;291:2413–7. - PubMed
    1. Chalasani G, Dai Z, Konieczny BT, Baddoura FK, Lakkis FG. Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:6175–80. - PMC - PubMed
    1. Yang J, Brook MO, Carvalho‐Gaspar M, et al. Allograft rejection mediated by memory T cells is resistant to regulation. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:19954–9. - PMC - PubMed
    1. Lakkis FG, Sayegh MH. Memory T cells: a hurdle to immunologic tolerance. Journal of the American Society of Nephrology. 2003;14:2402–10. - PubMed

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