The Human Bone Marrow May Offer an IL-15-Dependent Survival Niche for EOMES+ Tr1-Like Cells

Abstract

Maintenance of memory T-cells in the bone marrow and systemically depends on the homeostatic cytokines IL-7 and IL-15. An immunological memory may also exist for regulatory T-cells. EOMES⁺type-1 regulatory (Tr1)-like cells have a rapid in vivo turnover, but whether they are short-lived effector cells or are maintained long-term has not been investigated. EOMES⁺Tr1-like cells expressing GzmK were enriched among CD69⁺Ki67^-T-cells in the bone marrow of healthy donors, suggesting that they became quiescent and bone marrow-resident. Conversely, CD4⁺GzmB⁺ effector T-cells were excluded from the bone marrow-resident fraction. The dichotomy between GzmK⁺ and GzmB⁺T-cells was observed both in healthy individuals and in multiple sclerosis patients, and also among CD8⁺T-cells. Intriguingly, bone marrow-resident CD4⁺ memory T-cells expressed increased levels of IL-7Rα, while EOMES⁺Tr1-like cells were consistently IL-7Rα^lo. However, EOMES⁺Tr1-like cells expressed the IL-2/15Rβ chain, and the latter was induced upon forced expression of EOMES in primary human CD4⁺ T-cells. Finally, IL-15 rescued EOMES⁺Tr1-enriched populations from death by neglect but was not required for CD4⁺ memory T-cell survival. These findings suggest that the bone marrow may provide a survival niche for EOMES⁺Tr1-like cells. The different IL-7 and IL-15 receptor expression patterns of CD4⁺ memory T-cells and EOMES⁺Tr1-like cells suggest furthermore that they compete for different homeostatic niches.

Keywords: CD69; bone marrow; memory T lymphocytes; regulatory T‐cells.

FIGURE 1

EOMES⁺ and FOXP3⁺CD4⁺T‐cells in the bone marrow upregulate CD69 and become quiescent. (A) Percentages of CD69⁺ cells among CD4⁺ and CD8⁺T‐cells in the blood (PB, n = 11) and in the bone marrow (BM, n = 5). (B) Percentages of Ki67⁺ cells among CD69⁺ and CD69⁻CD4⁺T‐cells in the blood and the bone marrow. (C) Upper pie charts illustrate the contributions of naïve, central memory, and effector memory cells as well as FOXP3⁺ and EOMES⁺ cells to the CD4⁺T‐cell compartment in the blood and the bone marrow. Lower pie charts show the contributions of the same subsets to the CD69⁺ and CD69⁻ compartments in the bone marrow. Statistical tendencies (p‐values >0.05) are reported as numbers in the pie charts. (D) Ki67 expression in the CD4⁺T‐cell subsets analyzed in (C) in peripheral blood and in the CD69⁺ and CD69⁻ fractions in the bone marrow.

FIGURE 2

Bone marrow‐resident CD4⁺ and CD8⁺T‐cells are strongly enriched for GzmK‐expressing subsets, whereas GzmB⁺ effector CTL are largely excluded. (A) Percentages of CD69⁺ cells among CD4⁺EOMES⁺T‐cell subsets in the blood and in the bone marrow (BM). (B) Frequencies of CD4⁺EOMES⁺T‐cell subsets among CD69⁺ and CD69⁻CD4⁺T‐cells in the bone marrow. (C) Ki67 expression in CD4⁺ EOMES⁺T‐cell subsets in peripheral blood and the CD69⁺ and CD69⁻ fractions in the bone marrow. (D) The upper pie charts show the contributions of EOMES⁺ subsets classified according to the expression of GzmK and/or GzmB, as well as EOMES⁻ cells, to the CD8⁺T‐cell compartments in the blood and the bone marrow. Lower pie charts show the contributions of the same subsets to the CD69⁺ and CD69⁻ compartments in the bone marrow. (E) Percentages of CD69⁺ cells among CD8⁺EOMES⁺T‐cell subsets in the blood and the bone marrow. (F) Ki67 expression in CD8⁺EOMES⁺T‐cell subsets in peripheral blood and in the CD69⁺ and CD69⁻ fractions in the bone marrow. The left panel shows CD8⁺EOMES⁺T‐cell subsets expressing GzmK and/or GzmB (DP: double‐positive: GzmK⁺GzmB⁺), the right panel CD8⁺EOMES⁺GzmK⁺GzmB⁻T‐cell cells stratified according to IL‐7Rα expression. (G) Percentages of GzmK⁺ and GzmB⁺T‐cell subsets in paired samples of peripheral blood (PB), and in the CD69⁺ and CD69⁻ fractions of the bone marrow (BM) in four healthy donors (HD) and in five multiple sclerosis patients (MS). Upper histograms show CD4⁺T‐cells and lower histograms CD8⁺T‐cells.

FIGURE 3

IL‐7R^loEOMES⁺Tr1‐like cells express high levels of IL‐2/15Rβ and are rescued from death by neglect by IL‐15. (A) IL‐7Rα expression levels on CD4⁺T‐cell subsets in the CD69⁺ and CD69⁻ compartments of the bone marrow. The upper panel shows naïve, central, and effector memory cells as well as FOXP3⁺Treg and the lower panel shows CD4⁺EOMES⁺GzmK⁺ subsets. Representative histogram overlays are shown in Figure S4A. (B) Naive CD4⁺T‐cells were activated with anti‐CD3 and anti‐CD28 antibodies, IL‐2 and IL‐12, and transduced with lentiviral vectors coding for GFP (Mock) or for GFP and Eomesodermin (Eomes). Left: Representative Dot plots of cells analyzed for EOMES and CD122 (IL‐2/15Rβ) expression. Mean percentage of CD122 expression (n = 6). (C) Percentages of CD122⁺ cells in the indicated T‐cell subsets. The gating strategy is shown in Figure S5. (D) CD4⁺T‐cell subsets were enriched by cell sorting according to the gating strategy reported in Figure S6. They were cultured for 4 days in the absence or presence of IL‐15, and the mean percentage of viable cells was reported (n = 4). (E) Pre‐Tr1/Tr1‐cell‐enriched populations and CD4⁺CCR5⁻CD27⁺ Tconv control cells were cultured for 4 days with IL‐15, and GzmK expression of viable cells was analyzed.