T cells are fundamental effector cells against viruses and cancers that can be divided into different subsets based on their long-term immune protection and immediate immune response effects. The percentage and absolute number of these subsets change with ageing, which leads to a reduced immune response in older individuals. Stem cell memory T cells (TSCM) represent a small population of memory T cells with enhanced proliferation and differentiation properties that are endowed with high potential for maintaining T cell homeostasis. However, whether these cells change with ageing and gender remains unknown. Here, we assayed the distribution of TSCM and other T cell subsets in peripheral blood from 92 healthy subjects (44 females and 48 males) ranging from 3 to 88 years old by flow cytometry. We found that CD4+ and CD8+ TSCM in the circulation have relatively stable frequencies, and the absolute number of CD8+ TSCM decreased with age; however, the ratio of TSCM to the CD4+ or CD8+ naïve population increased with age. Unlike the obvious changes in other T cell subsets with age and gender, the stable level of TSCM in peripheral blood may support their capacity for sustaining long-term immunological memory, while their importance may increase together with ageing.

Despite the great success of immune-checkpoint inhibitor (ICI) treatment for multiple cancers, evidence for the clinical use of ICIs in acute myeloid leukemia (AML) remains inadequate. Further exploration of the causes of immune evasion in the bone marrow (BM) environment, the primary leukemia site, and peripheral blood (PB) and understanding how T cells are affected by AML induction chemotherapy or the influence of age may help to select patients who may benefit from ICI treatment. In this study, we comprehensively compared the distribution of PD-1 and TIGIT, two of the most well-studied IC proteins, in PB and BM T cells from AML patients at the stages of initial diagnosis, complete remission (CR), and relapse-refractory (R/R) disease after chemotherapy. Our results show that PD-1 was generally expressed higher in PB and BM T cells from de novo (DN) and R/R patients, while it was partially recovered in CR patients. The expression of TIGIT was increased in the BM of CD8+ T cells from DN and R/R patients, but it did not recover with CR. In addition, according to age correlation analysis, we found that elderly AML patients possess an even higher percentage of PD-1 and TIGIT single-positive CD8+ T cells in PB and BM, which indicate greater impairment of T cell function in elderly patients. In addition, we found that both DN and R/R patients accumulate a higher frequency of PD-1+ and TIGIT+ CD8+ T cells in BM than in corresponding PB, indicating that a more immunosuppressive microenvironment in leukemia BM may promote disease progression. Collectively, our study may help guide the combined use of anti-PD-1 and anti-TIGIT antibodies for treating elderly AML patients and pave the way for the exploration of strategies for reviving the immunosuppressive BM microenvironment to improve the survival of AML patients.

Thymocyte selection-associated HMG box (TOX) is a transcription factor that belongs to the high mobility group box (HMG-box) superfamily, which includes four subfamily members: TOX, TOX2, TOX3, and TOX4. TOX is related to the formation of multiple malignancies and contributes to CD8+ T cell exhaustion in solid tumors. However, little is known about the role of TOX genes in hematological malignancies. In this study, we explored the prognostic value of TOX genes from 40 patients with de novo acute myeloid leukemia (AML) by quantitative real-time PCR (qRT-PCR) in a training cohort and validated the results using transcriptome data from 167 de novo AML patients from the Cancer Genome Atlas (TCGA) database. In the training cohort, higher expression of TOX and TOX4 was detected in the AML samples, whereas lower TOX3 expression was found. Moreover, both the training and validation results indicated that higher TOX2, TOX3, and TOX4 expression of AML patients (3-year OS: 0% vs. 37%, P = 0.036; 3-year OS: 4% vs. 61%, P < 0.001; 3-year OS: 0% vs. 32%, P = 0.010) and the AML patients with highly co-expressed TOX, TOX2, TOX4 genes (3-year OS: 0% vs. 25% vs. 75%, P = 0.001) were associated with poor overall survival (OS). Interestingly, TOX2 was positively correlated with CTLA-4, PD-1, TIGIT, and PDL-2 (rs = 0.43, P = 0.006; rs = 0.43, P = 0.006; rs = 0.56, P < 0.001; rs = 0.54, P < 0.001). In conclusion, higher expression of TOX genes was associated with poor OS for AML patients, which was related to the up-regulation of immune checkpoint genes. These data might provide novel predictors for AML outcome and direction for further investigation of the possibility of using TOX genes in novel targeted therapies for AML.

The antitumor activity of NK cells in patients with chronic myeloid leukemia (CML) is inhibited by the leukemia microenvironment. Recent studies have identified that the expression of TIGIT, CD57, and KLRG1 is related to the function, maturation, and antitumor capabilities of NK cells. However, the characteristics of the expression of these genes in the peripheral blood (PB) and bone marrow (BM) from patients with CML remain unknown. In this study, we used multicolor flow cytometry to assay the quantity and phenotypic changes of NK cells in PB and BM from de novo CML (DN-CML) and CML patients acquiring molecular response (MR-CML). We found that the expression of TIGIT, which inhibits NK cell function, is increased on CD56+ and CD56dim NK cells in DN-CML PB compared with those in healthy individuals (HIs), and it is restored to normal in patients who achieve MR. We also found that the expression of CD57 on NK cells was approximately the same level in PB and BM from DN-CML patients, while decreased CD57 expression was found on CD56+ and CD56dim NK cells in HI BM compared with PB. Additionally, those two subsets were significantly increased in DN-CML BM compared to HI BM. The expression of CD57 correlates with replicative senescence and maturity for human NK cells; therefore, the increase in TIGIT on PB NK cells together with an increase in CD57 on BM NK cells may explain the subdued NK cell antileukemia capacity and proliferative ability in DN-CML patients. These results indicate that reversing the immune suppression of PB NK cells by blocking TIGIT while improving the proliferation of BM NK cells via targeting CD57 may be more effective in removing tumor cells.

Background: KIR+NKG2A + Eomes+ CD8+ T cells, which are preferentially found with a T EMRA (CD45RA + CCR7−) phenotype while having the capacity to rapidly produce IFN-γ in response to innate stimulation , have been demonstrated to exist in human cord blood and the adult blood circulation. This highly responsive T-cell type was termed NK-like CD8+ T cells due to their capability to act in an innate immune fashion in mice similar to NK cells. However, KIR+NKG2A + CD8+ T cells that are Eomes− represent a small proportion of unconventional T cells that have not been described until now. Methods:We compare the distribution of the memory phenotypes and senescenceassociated markers of two T-cell subsets by multicolor flow cytometry in 10 cord blood samples and 105 healthy individuals (HIs) ranging from 6 to 84 years of age.Results: We found that the Eomes+ population has a higher differentiation degree than the Eomes− population. T cells in the Eomes− subset show proportionally less T EMRA phenotypes while instead preferentially displaying a more naïve and T CM phenotype. Furthermore, the Eomes− population was shown to linearly decrease with age, while the Eomes+ population exhibited more senescence-associated characteristics, such as CD57 expression and loss of CD28. Conclusion:Overall, the KIR+NKG2A + Eomes− CD8+ T-cell population shares similar characteristics with the Eomes+ population, although with a lower degree of differentiation, lower senescence marker expression, and a proportional decrease with age. Thus, we suspect that KIR+NKG2A + Eomes-CD8+ T cells may represent a less differentiated stage of the NK-like CD8+ T-cell subset.

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Aim In order to further understand the feature of natural killer cell (NK) dysfunction in acute myeloid leukemia (AML), The distribution of NK cell subset the expression of the inhibitory receptors immunoglobulin and ITIM domain (TIGIT), killer cell lectin‐like receptor (KLRG1), and the expression of maturation marker CD57 in NK cell subsets and their correlation with patient outcomes were analyzed in this study. Methods We collected peripheral blood (PB) and bone marrow (BM) samples from de novo AML (AML‐DN) patients, patients who achieved complete remission after chemotherapy (AML‐CR), and healthy individuals. An eight‐color flow cytometry panel was used to identify different NK subsets and their expression of TIGIT, CD57 and KLRG1. Results Decreased percentage of CD56dimCD16+ NK cells was found only in the PB of AML‐DN and AML‐CR patients but not in the BM. The expression frequency of TIGIT and KLRG1 was elevated on NK cells from the PB of AML‐DN patients, while it was recovered in AML‐CR patients. Moreover, a higher percentage of CD57+CD56dimCD16+ NK cells, representing a terminally differentiated NK subset with strong cytotoxic capacity but defective replication potential, was detected in the BM of AML‐DN patients and predicted sub‐optimal survival for patients. Conclusion The results indicated that the NK cell subsets in the PB of AML patients had an exhaustion phenotype, while the BM NK cells had a terminally differentiated phenotype, which correlated with short survival for AML patients.

Immune dysfunction in patients with multiple myeloma (MM) includes TGF-β induced dendritic cell dysfunction, regulatory T cell (Tregs)/Th17 cell imbalance, accumulation of Tregs and myeloid derived suppressor cells. These tumor-induced dysfunctions may contribute to immune escape and even suppress immune cells introduced in adoptive cellular immunotherapy. CD28 independent T cells of the effector memory (TEM) and CD45RA+ effector memory (TEMRA) population were shown to accumulate with age and contribute to the immunosuppressive tumor microenvironment in several solid tumors and hematological cancers. Especially in the CD8+ population, the loss of CD28 is associated with high cytotoxicity and regulatory function while showing high diversity and defective antigen-induced proliferation. In the CD4 subset, regulatory and senescent T cells were studied extensively, while in the CD8 positive subset their heterogeneity is still not clearly defined. Their potential immunosuppressive role and distribution in healthy individuals (HI) as well as patients with multiple myeloma (MM) remains to be observed. Furthermore, a recently characterized CD8+CD28- NK-like T cell subset showing expression of NK-related inhibitory receptors and TCR independent effector function is potentially of interest in the progression of MM. In the present study, we compared the changes of distribution of CD8+CD25+ and CD8+FOXP3+ regulatory T cells (Treg), CD28-CD57+ senescent T cells (Tsen), and CD8+KIR/NK2GA+EOMES+ NK-like T in peripheral blood (PB) between HI and patients with MM by multicolour flow cytometry (Gating strategy shown in Figure 1A). When comparing 35 HIs (Median age is 54) and 14 MM patients (Median age is 52), it was shown that there is no significant change in the proportion of senescent T cells in CD8 (P = 0.2452), TEM/CD8 (P = 0.1686) and TEMRA/CD8 (P = 0.4861) between HIs and the MM group, while both the CD25+FOXP3+ regulatory T cells of the CD4 population (P = 0.0031) and CD28-FOXP3+ regulatory T cells of the CD8 population (P = 0.0014) were shown to increase. There is no significant difference in the percentages of KIR/NK2GA+EOMES+ in the CD8 T cell and TEMRA/CD8 T cell population between HIs and the MM group. Remarkably, although there was no overall increase in senescent T cell in MM patients, senescent CD8+NK2GA+EOMES+ NK-like T cells increased in MM patients in comparison to HIs (P = 0.0068) (Figure 1B). In conclusion, the increase of regulatory T cells of both the CD4 and CD8 population as well as the increase of senescent NK-like T cells in the CD8 population potentially contributes to cancer progression through creation of suppressive microenvironments. Moreover, we found that regulatory CD8 T cells and CD8 NK-like T cells only contribute to a small part of the overall CD28- senescent T cell pool. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.