T cells in multiple myeloma display features of exhaustion and senescence at the tumor site

Abstract

Background: Multiple myeloma is an incurable plasma cell malignancy that is mostly restricted to the bone marrow. Cancer-induced dysfunction of cytotoxic T cells at the tumor site may be responsible for immune evasion and therapeutical failure of immunotherapies. Therefore, enhanced knowledge about the actual status of T cells in myeloma bone marrow is urgently needed. Here, we assessed the expression of inhibitory molecules PD-1, CTLA-4, 2B4, CD160, senescence marker CD57, and CD28 on T cells of naive and treated myeloma patients in the bone marrow and peripheral blood and collected data on T cell subset distribution in both compartments. In addition, T cell function concerning proliferation and expression of T-bet, IL-2, IFNγ, and CD107a was investigated after in vitro stimulation by CD3/CD28. Finally, data was compared to healthy, age-matched donor T cells from both compartments.

Methods: Multicolor flow cytometry was utilized for the analyses of surface molecules, intracellular staining of cytokines was also performed by flow cytometry, and proliferation was assessed by ³H-thymidine incorporation. Statistical analyses were performed utilizing unpaired T test and Mann-Whitney U test.

Results: We observed enhanced T cell exhaustion and senescence especially at the tumor site. CD8+ T cells expressed several molecules associated with T cell exhaustion (PD-1, CTLA-4, 2B4, CD160) and T cell senescence (CD57, lack of CD28). This phenotype was associated with lower proliferative capacity and impaired function. Despite a high expression of the transcription factor T-bet, CD8+ T cells from the tumor site failed to produce IFNγ after CD3/CD28 in vitro restimulation and displayed a reduced ability to degranulate in response to T cell stimuli. Notably, the percentage of senescent CD57+CD28- CD8+ T cells was significantly lower in treated myeloma patients when compared to untreated patients.

Conclusions: T cells from the bone marrow of myeloma patients were more severely impaired than peripheral T cells. While our data suggest that terminally differentiated cells are preferentially deleted by therapy, immune-checkpoint molecules were still present on T cells supporting the potential of checkpoint inhibitors to reactivate T cells in myeloma patients in combination therapies. However, additional avenues to restore anti-myeloma T cell responses are urgently needed.

Keywords: Bone marrow; Immune-checkpoint molecules; Multiple myeloma; T cell exhaustion; T cell senescence.

Fig. 1

Accumulation of an activated CD8+ T cell population in the BM of myeloma patients. a The dot plots show the gating strategy as well as the proportion of human CD8+ T cell subsets of a representative healthy and myeloma BM sample. The numbers indicate the percentage of naive (Tn: CD45RA+CD62L+), central-memory (Tcm: CD45RA-CD62L+), effector memory (Tem: CD45RA−CD62L−) and effector (Temra: CD45RA+CD62L−) CD8+ T cells. b Pie charts summarize the median proportion of Tn, Tcm, Tem, and Temra in PB and BM for 12 healthy and 16 myeloma samples. c Percentages of PD-1 expressing CD8+ T cell subsets (Tn, Tcm, Tem, and Temra) of healthy, age-matched controls and myeloma patients were assessed by flow cytometry (n = 11; median with interquartile range (IQR) is shown). Significance levels *p < 0.05; **p < 0.01

Fig. 2

CD8+ T cells of myeloma patients show functional defects in proliferation and cytotoxicity. a Proliferation was assessed by ³H-thymidine incorporation after stimulating the cells for 72 h with anti-CD3/anti-CD28 antibodies. The proliferative index (³H-thymidine incorporation of stimulated T cells divided by ³H incorporation of unstimulated cells) is shown for BM CD8+ T cells from myeloma patients (n = 12) and from healthy controls (n = 6). b T-bet and c IFNγ expression was assessed by intracytoplasmic staining of total CD8+ BMMCs before (0 h) and after stimulation (24 h) of the cells (n = 7; median with IQR is shown). d T-bet and IFNγ expression on different T cell subtypes (CD8+ CD45RA+ or CD45RA−) was assessed by intracytoplasmic staining before (0 h) and after (24 h) stimulation with CD3/CD28 antibodies (n = 7; mean ± SEM is shown). e Surface levels of CD107a were detected by flow cytometry before (0 h) and after stimulation (24 h) (n = 3; median with IQR is shown). Significance levels: *p < 0.05; **p < 0.01; n.s. not significant

Fig. 3

Accumulation of highly activated but senescent CD8+ T cells in the BM of myeloma patients. The expression of CD28 and CD57 was assessed on CD8+ T cells from patients with multiple myeloma and healthy, age-matched controls. Percentages of a CD8+CD28− T cells or b CD8+CD57+ T cells in healthy (n = 12) and myeloma samples (n = 12). c Percentages of CD8+CD57+CD28− T cells compared to CD8+CD57+CD28+ T cells (n = 10). d Concomitant expression of CD57 and PD-1 on CD8+ T cells of the BM of healthy and diseased (n = 6–8). Median with IQR is shown. Significance levels *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant

Fig. 4

Differential expression of T cell markers in BM of newly diagnosed and refractory myeloma patients. a Percentage of cell surface expression of exhaustion markers PD1, CTLA-4, CD160, 2B4 was quantified on CD8+ effector T cells from patients with newly diagnosed myeloma (MM naive) and treated myeloma (MM Tx) compared to healthy donors. b Similar, expression of senescent markers CD57 and CD28 was investigated by flow cytometry. (Healthy n = 7–12; myeloma newly diagnosed n = 7–11; treated myeloma n = 5–6; median with IQR is shown). Significance levels *p < 0.05; **p < 0.01; n.s. not significant

Fig. 5

Proliferation of T cells in myeloma BM expressing CD57. Proliferation was determined by measuring dilution of CFSE using flow cytometry. Representative plots are shown for CD8+ T cells of (a) a healthy person, b a naive, and (c) a treated myeloma patient. The graphs display the results of independent experiments performed on samples from a four healthy persons, b seven chemo-naive, and c three treated patients (who were off IMiD-containing therapy for at least 6 months), showing the percentage of CD8+ T cells within each cell division. On the x-axis, the number of cell divisions according to CFSE dilution is indicated (G0 undivided; G1-3 1–3 cell divisions; significance levels *p < 0.05; **p < 0.001)