Human memory T cells from the bone marrow are resting and maintain long-lasting systemic memory

Abstract

In the bone marrow, a population of memory T cells has been described that promotes efficient secondary immune responses and has been considered to be preactivated, owing to its expression of CD69 and CD25. Here we show that human bone marrow professional memory T cells are not activated but are resting in terms of proliferation, transcription, and mobility. They are in the G0 phase of the cell cycle, and their transcriptome is that of resting T cells. The repertoire of CD4(+) bone marrow memory T cells compared with CD4(+) memory T cells from the blood is significantly enriched for T cells specific for cytomegalovirus-pp65 (immunodominant protein), tetanus toxoid, measles, mumps, and rubella. It is not enriched for vaccinia virus and Candida albicans-MP65 (immunodominant protein), typical pathogens of skin and/or mucosa. CD4(+) memory T cells specific for measles are maintained nearly exclusively in the bone marrow. Thus, CD4(+) memory T cells from the bone marrow provide long-term memory for systemic pathogens.

Keywords: antigen-specific response; polyfunctional; short- and long-term memory.

Fig. 1.

Memory T cells in human bone marrow and peripheral blood. (A and B) Frequencies of CD45RO⁺CD45RA⁻ memory CD4⁺ and CD8⁺ T cells (A) and frequencies of CD4⁺ and CD8⁺ T cells (B) from paired bone marrow (BM) and peripheral blood (PB) samples. (C) Absolute numbers were calculated according to the frequencies of memory CD4⁺ and CD8⁺ T cells (A) and of CD4⁺ and CD8⁺ T cells (B) and according to the estimated numbers of T cells within BM and PB of healthy young adults (18, 19). (D) Memory CD4⁺ and CD8⁺ T cells of BM express CD69. A representative plot of overlaid histograms of CD69 expression on memory CD4⁺ or CD8⁺ T cells of BM and PB, and the average frequencies and absolute numbers of CD69⁺ memory CD4⁺ and CD8⁺ T cells are shown. In A–D, n = 12; P values were obtained as described in SI Materials and Methods. (E) CD137 expression on CD69⁺ and CD69⁻ memory CD4⁺ and CD8⁺ T cells from BM and PB samples. Data shown are representative of four separate experiments. (F) Average frequencies of CD25⁺ cells among CD69⁺ or CD69⁻ memory CD4⁺ T cells of five paired BM and PB samples. Error bars represent SEM. (G) CD127 and FOXP3 expression on CD69⁺CD25⁺ and CD69⁻CD25⁺ memory CD4⁺ T cells of BM and on CD25^hiCD69⁻ and CD25⁻CD69⁻ memory CD4⁺ T cells of PB. Data shown are representative of five separate experiments.

Fig. 2.

Cell-cycle status of ex vivo human memory CD4⁺ and CD8⁺ T cells from BM and PB. (A) Frequencies of Ki67⁺ memory CD4⁺ and CD8⁺ T cells in seven paired BM and PB samples. P values were obtained as described in SI Materials and Methods. (B) Sample flow cytometry expression profiles showing the counterstainings for Ki67 and CD69 expression on memory CD4⁺ and CD8⁺ T cells. (C) Frequencies of ex vivo (n = 6) and 72-h anti-CD3/CD28 activated (n = 2) memory CD4⁺ and CD8⁺ T cells in the cell-cycle S+G₂/M phase. Error bars represent SEM.

Fig. 3.

Global resting gene expression profiles of ex vivo memory CD4⁺ T cells from BM and PB. Transcriptomes of memory CD4⁺ T-cell subsets from four paired BM and PB samples were compared with transcriptomes of CD4⁺ memory T cells from PB of eight unrelated donors, directly after isolation (ex vivo) or after stimulation for 3 h with PMA/ionomycin. Differentially expressed genes (7,383) were hierarchically clustered and displayed. The normalized induction (red) or repression (blue) is shown for each gene. Transcriptomes of the following samples are shown: from paired ex vivo BM and PB samples, BM CD69⁺, BM CD69⁻, and PB CD69⁻; from PB samples of unrelated donors, PB CD69⁻ unstimulated (unst) or restimulated with PMA/ionomycin (rest).

Fig. 4.

Comparison of antigen specificity and diversity of memory CD4⁺ T cells from paired BM and PB samples. Mononuclear cells isolated from four to seven paired BM and PB samples were stimulated with the indicated antigens, and the induced cytokine production (IFN-γ, IL-2, or TNF-α) in memory CD4⁺ T cells was examined according to CD154 expression. For each subpopulation, the background (as detected in the anti-CD28 stimulated but otherwise equally treated control samples) was subtracted. (A) Frequencies and (B) estimated absolute numbers of antigen-specific CD154⁺cytokine⁺ (total cytokine-producing) cells are shown. Absolute numbers of antigen-specific memory CD4⁺ T cells were calculated using the frequency of these cells and the estimated numbers of T cells within BM and PB of healthy young adults (18, 19). Symbols in gray indicate frequencies under reliable detection limit (10⁻⁴ of memory CD4⁺ T cells) or estimated cell numbers calculated according to such frequencies. P values were obtained as described in SI Materials and Methods.