Allotransplantation Is Associated With Exacerbation of CD8 T-Cell Senescence: The Particular Place of the Innate CD8 T-Cell Component

Abstract

Immunosenescence is a physiological process that is associated with changes in the immune system, particularly among CD8 T-cells. Recent studies have hypothesized that senescent CD8 T-cells are produced with chronologic age by chronic stimulation, leading to the acquisition of hallmarks of innate-like T-cells. While conventional CD8 T-cells are quite well characterized, CD8 T-cells sharing features of NK cells and memory CD8 T-cells, are a newly described immune cell population. They can be distinguished from conventional CD8 T-cells by their combined expression of panKIR/NKG2A and Eomesodermin (E), a unique phenotype closely associated with IFN-γ production in response to innate stimulation. Here, we first provided new evidence in favor of the innate character of panKIR/NKG2A(+) E(+) CD8 T-cells in normal subjects, documenting their position at an intermediate level in the innateness gradient in terms of both innate IFN-γ production and diminished mitochondrial mass. We also revealed that CD8 E(+) panKIR/NKG2A(+) T-cells, hereafter referred to as Innate E(+) CD8 T-cells, exhibit increased senescent (CD27(-) CD28(-)) phenotype, compared to their conventional memory counterparts. Surprisingly, this phenomenon was not dependent on age. Given that inflammation related to chronic viral infection is known to induce NK-like marker expression and a senescence phenotype among CD8 T-cells, we hypothesized that innate E(+) CD8 T-cells will be preferentially associated with exacerbated cellular senescence in response to chronic alloantigen exposure or CMV infection. Accordingly, in a pilot cohort of stable kidney allotransplant recipients, we observed an increased frequency of the Innate E(+) CD8 T-cell subset, together with an exacerbated senescent phenotype. Importantly, this phenotype cannot be explained by age alone, in clear contrast to their conventional memory counterparts. The senescent phenotype in CD8 T-cells was further increased in cytomegalovirus (CMV) positive serology transplant recipients, suggesting that transplantation and CMV, rather than aging by itself, may promote an exacerbated senescent phenotype of innate CD8 T-cells. In conclusion, we proposed that kidney transplantation, via the setting of inflammatory stimuli of alloantigen exposure and CMV infection, may exogenously age the CD8 T-cell compartment, especially its innate component. The physiopathological consequences of this change in the immune system remain to be elucidated.

Keywords: NK-like T-cells; allotransplantation; immune memory; innate memory CD8(+) T-cells; innateness gradient; senescence.

Figure 1

Innate T-cells in humans: current state of knowledge. (A, B) Innate T-cells are memory phenotype CD8 T-cells with innate functions. In humans, as their conventional memory counterparts (panel A, left), innate CD8 T-cells (panel A, middle) express TCRαβ and CD8, and have memory characteristics, as attested notably by Eomes expression and EMRA phenotype (CD45RA(+) CCR7(-)). They also share with NK cells some innate immune receptors such as KIR (KIR2D and KIR3DL1/2), NKG2A, CD122 and CD16. Co-expression of Eomes and KIR/NKG2A brings together the major fraction of CD8 T-cells able to free lytic granules in response to CD16 stimulation with antibodies (ADCC, antibody-dependent cellular cytotoxicity) and to produce IFN-γ upon innate-like stimulation by IL-12/IL-18 [(24) panel B]. As a result, CD8 T-cells co-expressing Eomes and KIR/NKG2A are identified as innate CD8 T-cells. A representative dot plot graph of Eomes(+) KIR/NKG2A(+) cells among CD8(+)CD3(+) PBMC from a healthy adult analyzed by flow cytometry is shown in panel A (right). (C) Ontogeny of innate CD8 T-cells. Innate CD8 T-cells have been evidenced in human cord blood, and like other fetal immune cells, they may be able to persist in adulthood. In mice, it is well-documented that they are produced in the thymus not only during the first stages of life but throughout a lifetime. With ageing, naive cells from the periphery differentiate into T-cell expressing innate features without antigen-stimulation. (D, E) Innate CD8 T-cells are part of the innateness gradient. PBMC from healthy donors were stained after 24h stimulation in vitro with IL-12/18 (D) or ex vivo (E), and analyzed by flow cytometry for innate-induced IFN-γ expression and mitochondrial mass (MTDR), respectively. Each point represents one healthy adult donor. A representative histogram is shown for each cell population. For detailed gating strategy, see Figure S1 .

Figure 2

Hallmarks of innateness gradient. The more T-cells are close to innate cells like the NK-cells, the more they display the following hallmarks: 1/Transcriptomic signature with transcription factors expression such as PLZF, Eomes or T-bet. 2/Low TCR/CD3 sensitivity and low proliferation induced by TCR stimulation. 3/Metabolic changes with lower mitochondrial mass, lower ROS production and the Warburg effect. 4/IL-15 sensitivity, with increased CD122 expression. 5/A higher transcription rate of preformed cytokine mRNA prior to stimulation. 6/Effector functions independent from TCR stimulation such as ADCC and IFN-γ production after IL-12/IL-18 stimulation. Adapted from Gutierrez-Arcelys et al. (45).

Figure 3

Senescence of CD8 T-cells is associated with phenotypic and metabolic changes. (A) Gradient of activation or function marker expression from naive (early differentiated) to senescent (terminal differentiated) cells. (B) Age-related metabolic changes of CD8 T-cells. (C) Events and signalling factors leading to the loss of TCR sensitivity that accumulate with age. ECAR, extracellular acidification rate; FA, fatty acid; NKR, NK receptors; sMAC, Sesn-MAPK activation Complex.

Figure 4

PanKIR/NKG2A(+) Eomes(+) and panKIR/NKG2A(+) Eomes(-) CD8 T-cells share innate functions and a senescent/inflammaging-like signature. Flow cytometry analysis of TCRαβ(+) CD8(+) circulating live lymphocytes from healthy donors. (A) CD8 T-cells expressing panKIR/NKG2A or Eomes markers preferentially harbor a senescent signature and a terminal effector phenotype. The frequencies of senescent (CD27(-) CD28(-)) (n=14), EMRA (CD45RA(+) CCR7(-)) (n=13) and perforin(high)-expressing cells (n=22) among CD8 T-cells expressing panKIR/NKG2A (upper panel) or Eomes (lower panel) were analyzed. (B) panKIR/NKG2A and Eomes markers define two innate CD8 T-cell subsets besides their conventional memory and naive counterparts: evidence for an innateness gradient. CD122-expressing cells (n=7) ex vivo (left panel) and IFN-γ-producing cells (n=17) (right panel) frequencies after 48h stimulation with IL-12/IL-18 among the four CD8 T-cell subpopulations defined by panKIR/NKG2A and Eomes markers. Representative plots are shown in Figure S2 . (C) Innate CD8 T-cell subsets are enriched in senescent/terminal effector cells. Frequencies of senescent (CD27(-)CD28(-)) cells (n=14) (left panel), EMRA cells (n=13) (middle panel) and perforin(high)-expressing cells (n=22) (right panel) among the four CD8 T-cell subpopulations defined by panKIR/NKG2A and Eomes markers. Differences between E(-)K(-) and the other three cell populations, although significant, are not shown. Data are presented as mean ± SEM. Two-tailed Wilcoxon non-parametric test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. For detailed gating strategy, see Figure S1 .

Figure 5

Chronic allo-antigenic stimulation promotes the generation of innate CD8 T-cell subsets and a senescent/inflammaging signature. (A) Frequencies of innate CD8 T-cell subpopulations are selectively increased in long-term kidney allograft recipients. Frequencies of CD8 T-cell subpopulations according to panKIR/NKG2A and/or Eomes expression among TCRαβ(+) CD8(+) live lymphocytes in healthy donors (HD, n=22) and long-term kidney allograft recipients (AlloTx, n=47). Representative plots are shown for one HD and one AlloTx recipient. (B–D) Senescent, EMRA and perforin(high) CD8 T-cell frequencies are increased in long-term kidney allograft recipients. Frequencies of senescent (CD27(-)CD28(-)) cells (HD n=15, AlloTx n=35) (A), EMRA (CD45RA(+) CCR7(-); HD n=13, AlloTx n=8) (B) and perforin(high)- expressing cells (HD n=22, AlloTx n=45) (C) in CD8 T-cell subpopulations. In the Conv E(-) cell pool from AlloTx patients, the increased frequency of EMRA cells ( Figures 5 and S4 ) could explain the relatively high frequency of senescent cells (19% vs 12% in healthy controls). Representative plots are shown for one HD and one AlloTx recipient in Figure S2 . Each dot represents one HD or AlloTx recipient. Two-tailed Mann-Whitney non-parametric test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. For detailed gating strategy, see Figure S1 .

Figure 6

Frequencies of senescent and perforin(high) CD8 T-cells are increased in long-term kidney allograft with a CMV positive serology. (A) Frequencies of CD8 T-cell subpopulations according to panKIR/NKG2A and/or Eomes expression among TCRαβ(+) CD8(+) live lymphocytes in CMV(-) (n=17) or CMV(+) (n=26) long-term kidney allograft (AlloTx) recipients. (B) Senescent (CD27(-)CD28(-)) cells (CMV(-) n=17; CMV(+) n=16) and (C) perforin(high)-expressing cells (CMV(-) n=16; CMV(+) n=25) in CD8 T-cell subpopulations in AlloTx recipients. Two-tailed Mann-Whitney non-parametric test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.