Bone Marrow CD8 T Cells Express High Frequency of PD-1 and Exhibit Reduced Anti-Leukemia Response in AML Patients

Acute myeloid leukemia (AML) is a devastating blood cancer with 5- year survival of only 25%. Optimizing anti-leukemia immune response to improve clinical outcome is a promising strategy for novel effective leukemia therapeutics. However due to limited accessibility, the majority of studies have been restricted to evaluate samples from peripheral blood. The immune response in bone marrow of AML is poorly understood. In this study, we aim to investigate the T cell immune response in bone marrow samples derived from AML patients.

Samples collected from a cohort of patients with newly diagnosed AML (n=19) were used in this study. Mononuclear cells of bone marrow and peripheral blood were isolated from clinical samples before multi-channel flow cytometry analysis. We observed a comparable frequency of CD3+ T cells among lymphocytes in bone marrow and peripheral blood. Similarly there is no difference of CD4, CD8 T cell frequency, or CD4/CD8 ratio. However, when T cell subsets for each differentiation stage were analyzed based on the expression of CD45RA and CCR7, we found significantly increased effector memory T cells (CD45RA^-CCR7^-) among CD8 T cells in bone marrow compared with that in peripheral blood (43.88±5.012% vs. 35.15±5.104%, P =0.0088). Whereas the percentage of terminally differentiated effector cells (CD45RA⁺CCR7^-) was significantly lower in bone marrow (20.36±5.025% vs. 28±5.947%, P =0.0481). No difference was found in the frequency of naive (CD45RA⁺CCR7⁺) and central memory (CD45RA^-CCR7⁺) subsets.

Several studies including ours have demonstrated an involvement of the inhibitory receptors PD-1, TIM-3 and TIGIT in AML progression. Most of the studies were performed using clinical samples from peripheral blood. To evaluate whether the effect of inhibitory pathways is different between bone marrow and peripheral blood, we assessed the expression of these inhibitory receptors on T cells derived from both sites. We observed comparable expression of TIGIT and TIM-3 in bone marrow and peripheral blood. The frequency of PD-1 expression on CD8 T cells was however significantly higher in bone marrow (39.55±3.809% vs. 32.8±3.512%, P =0.0058). We further dissected the intracellular expression of Eomesodermin (Eomes) and T-bet, the two T box transcription factors that are crucial in regulating T cell function. We found that the frequency of Eomes⁺T-bet^int CD8 T cells was significantly higher in bone marrow than in peripheral blood (17.01±1.843% vs. 14.95±1.682%, P =0.0405), indicating a predominance for late stage of T cell exhaustion status in bone marrow. This result suggests a relatively more suppressive environment in bone marrow compared with that of peripheral blood.

We next examined the functional status of CD8 T cells. Intracellular IFN-γ and TNF-α production was assessed upon in vitro stimulation with anti-CD3/CD28. Intriguingly no significant difference between bone marrow and peripheral blood was detected. Consistently we observed no difference between the two sites in term of the expression of Ki67, Granzyme B, and Perforin, suggesting a comparable proliferation and killing capacity of CD8 T cells between bone marrow and peripheral blood. We further investigated the function of leukemia-reactive CD8 T cells by testing T cell response against a WT-1 peptide. WT-1 is a well known tumor-associated antigen, in which HLA-A*0201 restricted WT-1_126-134 is one of the mostly studied epitope in AML. Purified CD8 T cells derived from bone marrow or blood of newly diagnosed HLA-A*0201 AML patients were co-cultured with T2 cells (used as antigen presenting cells) pulsed with WT-1_126-134. We found a significantly lower production of IFN- γ by CD8 T cells from bone marrow compared with that from peripheral blood (0.4325±0.1612% vs, 0.7425±0.2809%, P =0.0472). These data demonstrate that although the total CD8 T cells in bone marrow remain functional, the leukemia-reactive CD8 T cells are less functional in bone marrow compared with that in peripheral blood.

Collectively, our data demonstrate that bone marrow CD8T cells from AML patients express higher frequency of PD-1 and exhibit reduced anti-leukemia response. Our study suggests that bone marrow is a unique anatomical location fostering negative immune response in AML, thus targeting the negative regulatory pathways in bone marrow is of great potential to develop novel effective leukemia treatment.