High percentage of bone marrow CD8+ tissue-resident-like memory T cells predicts inferior survival in patients with acute myeloid leukemia.

Tissue-resident memory T (TRM) cells infiltrating solid tumors could influence tumor progression and the response to immune therapies. However, the proportion and prognostic value of TRM cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) are unclear. In this study, we used flow cytometry to assay the phenotype of 49 BM samples from patients newly diagnosed with AML (ND-AML). We found that the BM CD8+ effector memory (TEM) cells highly expressed CD69 (CD8+ TRM-like T cells), and their percentage was significantly increased in patients with ND-AML compared with that in healthy individuals (HI). The high percentage of CD8+ TRM-like subset was associated with poor overall survival in our ND-AML cohort. The Kaplan-Meier Plotter database verified a significantly reduced survival rate among patients with high expression of CD8+ TRM-like T cell characteristic genes (CD8A, CD69, and TOX), especially the M4 and M5 subtypes. Phenotypic analysis revealed that the BM CD8+ TRM-like subpopulation exhibited exhausted T cell characteristics, but its high expression of CD27 and CD28 and low expression of CD57 suggested its high proliferative potential. The single-cell proteogenomic dataset confirmed the existence of TRM-like CD8+ T cells in the BM of patients with AML and verified the high expression of immune checkpoints and costimulatory molecules. In conclusion, we found that the accumulation of BM CD8+ TRM-like cells could be an immune-related survival prediction marker for patients with AML.

Single-cell RNA-seq reveals a microenvironment and an exhaustion state of T/NK cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous blood cancer. Effective immunotherapies for AML are hindered by a lack of understanding of the tumor microenvironment (TME). Here, we retrieved published single-cell RNA sequencing data for 128,688 cells derived from 29 bone marrow aspirates, including 21 AML patients and eight healthy donors. We established a global tumor ecosystem including nine main cell types. Myeloid, T, and NK cells were further re-clustered and annotated. Developmental trajectory analysis indicated that exhausted CD8+ T cells might develop via tissue residual memory T cells (TRM) in the AML TME. Significantly higher expression levels of exhaustion molecules in AML TRM cells suggested that these cells were influenced by the TME and entered an exhausted state. Meanwhile, the upregulation of checkpoint molecules and downregulation of granzyme were also observed in AML NK cells, suggesting an exhaustion state. In conclusion, our comprehensive profiling of T/NK subpopulations provides deeper insights into the AML immunosuppressive ecosystem, which is critical for immunotherapies.

Comprehensive analysis of the immune pattern of T cell subsets in chronic myeloid leukemia before and after TKI treatment.

Background: Immunological phenotypes and differentiation statuses commonly decide the T cell function and anti-tumor ability. However, little is known about these alterations in CML patients. Method: Here, we investigated the immunologic phenotypes (CD38/CD69/HLA-DR/CD28/CD57/BTLA/TIGIT/PD-1) of T subsets (TN, TCM, TEM, and TEMRA) in peripheral blood (PB) and bone marrow (BM) from de novo CML patients (DN-CML), patients who achieved a molecular response (MR) and those who failed to achieve an MR (TKI-F) after tyrosine kinase inhibitor (TKI) treatment using multicolor flow cytometry. Results: CD38 or HLA-DR positive PB CD8+TN and TCM cells decreased in the DN-CML patients and this was further decreased in TKI-F patients. Meanwhile, the level of PD-1 elevated in CD8+ TEM and TEMRA cells from PB in all groups. Among BM sample, the level of HLA-DR+CD8+TCM cells significantly decreased in all groups and CD8+TEMRA cells from TKI-F patients exhibited increased level of TIGIT and CD8+ tissue-residual T cells (TRM) from DN-CML patients expressed a higher level of PD-1 and TIGIT. Lastly, we found a significantly decreased proportion of CD86+ dendritic cells (DCs) and an imbalanced CD80/CD86 in the PB and BM of DN-CML patients, which may impair the activation of T cells. Conclusion: In summary, early differentiated TN and TCM cells from CML patients may remain in an inadequate activation state, particularly for TKI-F patients. And effector T cells (TEM, TEMRA and TRM) may be dysfunctional due to the expression of PD-1 and TIGIT in CML patients. Meanwhile, DCs cells exhibited the impairment of costimulatory molecule expression in DN-CML patients. Those factors may jointly contribute to the immune escape in CML patients.

Terminal differentiation of bone marrow NK cells and increased circulation of TIGIT+ NK cells may be related to poor outcome in acute myeloid leukemia.

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 CD56dim CD16+ 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+ CD56dim CD16+ 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.

PD-1 and TIGIT Are Highly Co-Expressed on CD8+ T Cells in AML Patient Bone Marrow.

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.

Characterization of KIR + NKG2A + Eomes- NK-like CD8+ T cells and their decline with age in healthy individuals.

BACKGROUND: KIR+NKG2A + Eomes+ CD8+ T cells, which are preferentially found with a TEMRA (CD45RA + CCR7-) phenotype while having the capacity to rapidly produce IFN-γ in response to innate stimulation (IL-12 and IL-18), 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 senescence-associated 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 TEMRA phenotypes while instead preferentially displaying a more naïve and TCM 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.

Increased Expression of TIGIT/CD57 in Peripheral Blood/Bone Marrow NK Cells in Patients with Chronic Myeloid Leukemia.

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.

Increased PD-1+Tim-3+ exhausted T cells in bone marrow may influence the clinical outcome of patients with AML.

BACKGROUND: Altered expression of T cell immune inhibitory receptors may result in immunosuppression and associate with the poor prognosis of leukemia patients in which the leukemic bone marrow (BM) microenvironment may contribute to such immunosuppression. We found higher numbers of programmed death-1 (PD-1) + exhausted T cells in peripheral blood (PB) from acute myeloid leukemia (AML) patients. To investigate the leukemic BM influence on immunosuppression, we further compared the distributions of PD-1 and T cell immunoglobulin mucin-3 (Tim-3) and the exhausted T cell phenotype in PB and BM from AML patients and characterized their relationship with clinical outcome. METHODS: PB and BM samples from 15 patients with newly diagnosed AML were collected and analyzed for the expression of PD-1, Tim-3, CD244, and CD57 on CD3+, CD4+, and CD8+ T cells by multicolor flow cytometry. RESULTS: The proportions of PD-1 + CD3+ and PD-1 + CD8+ T cells were significantly higher in BM compared with PB. Similarly, higher PD-1 + CD244 + CD3+ and PD-1 + CD244 + CD8+ T cells were found in BM, and an increased tendency for PD-1 + CD244 + CD4+ T cells was also detected in this group. In contrast, increased Tim-3 + CD4+/Tim-3 + CD244 + CD4+ T cells were predominant in BM compared with PB, but there was no statistically significant difference in Tim-3 + CD8+ T cells. Moreover, PD-1 and Tim-3 double-positive CD3+/CD4+/CD8+ T cells were significantly increased in the BM group. In addition, a higher proportion of PD-1 + Tim-3 + CD3+ T cells in the BM and PD-1 + Tim-3 + CD4+ T cells in PB was detected in non-complete remission (NCR) compared with complete remission (CR) patients after first-cycle chemotherapy. CONCLUSIONS: Upregulation of PD-1 and Tim-3 and the exhausted phenotype of CD4+ and CD8+ T cells in the BM of AML patients may contribute to mediating the leukemic immunosuppressive microenvironment, and increased PD-1 + Tim-3+ CD8+ T cells may be related to T cell dysfunction in AML, which may influence clinical outcome.

Age related human T cell subset evolution and senescence.

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.

Memory T cells skew toward terminal differentiation in the CD8+ T cell population in patients with acute myeloid leukemia.

Stem cell memory T (TSCM) and central memory T (TCM) cells can rapidly differentiate into effector memory (TEM) and terminal effector (TEF) T cells, and have the most potential for immunotherapy. In this study, we found that the frequency of TSCM and TCM cells in the CD8+ population dramatically decreased together with increases in TEM and TEF cells, particularly in younger patients with acute myeloid leukemia (AML) (< 60 years). These alterations persisted in patients who achieved complete remission after chemotherapy. The decrease in TSCM and TCM together with the increase in differentiated TEM and TEF subsets in CD8+ T cells may explain the reduced T cell response and subdued anti-leukemia capacity in AML patients.

Higher PD-1 expression concurrent with exhausted CD8+ T cells in patients with de novo acute myeloid leukemia.

OBJECTIVE: To investigate the association between the T cell inhibitory receptor programmed death 1 (PD-1) and T cell exhaustion status in T cells from patients with de novo acute myeloid leukemia (AML) and AML in complete remission (CR). METHODS: Surface expression of PD-1 and the exhaustion and immunosenescence markers CD244 and CD57 on CD3+, CD4+ and CD8+ T cells from peripheral blood samples from 20 newly diagnosed, untreated AML patients and 10 cases with AML in CR was analyzed by flow cytometry. Twenty-three healthy individuals served as control. RESULTS: A significantly higher percentage of PD-1+ cells were found for CD3+ T cells in the de novo AML group compared with healthy controls. In addition, an increased level of PD-1+CD8+ T cells, but not PD-1+CD4+, was found for CD3+ T cells in the de novo AML and AML-CR samples. A higher percentage of CD244+CD4+, CD244+CD8+, CD57+CD4+ and CD57+CD8+ T cells was found in CD3+ T cells in samples from those with de novo AML compared with those from healthy controls. Strong increased PD-1+CD244+ and PD-1+CD57+ co-expression was found for CD4+ and CD8+ T cells in the de novo AML group compared with healthy controls. CONCLUSIONS: We characterized the major T cell defects, including co-expression of PD-1 and CD244, CD57-exhausted T cells in patients with de novo AML, and found a particular influence on CD8+ T cells, suggesting a poor anti-leukemia immune response in these patients.

Re-balance of memory T cell subsets in peripheral blood from patients with CML after TKI treatment.

T cell immune surveillance is considered an important host protection process for inhibiting carcinogenesis. The full capacity of T cell immune surveillance is dependent on T cell homeostasis, particularly for central memory T (TCM) cells and stem cell memory T (TSCM) cells. In this study, distribution of T cell subsets in peripheral blood from 12 patients with chronic myeloid leukemia (CML) and 12 cases with CML in complete remission (CR) was analyzed using a multicolor flow cytometer, and 16 samples from healthy individuals (HIs) served as control. The proportion of CD8+ TSCM and CD4+ and CD8+ TCM cells were lower, while CD4+ effector memory T (TEM) cells and CD4+ and CD8+ terminal effector T (TEF) cells were higher in CML patients compared with HIs. Moreover, the proportion of CD8+CD28- T cells, which were found to have the immune suppressive function, increased in the naive T (TN) cell and TCM subsets in CML patients compared with HIs. Our study reveals that elimination of leukemia cells by treating with tyrosine kinase inhibitors (TKIs) restores the memory T cell distribution from a skewed pattern in CML patients who are under leukemia burden, indicating that leukemia-specific immune responses mediated by T cells might be induced and maintained in CML patients, however, these responsive T cells might gradually become exhausted due to the continued existence of leukemia cells and their environment; therefore, T cell activation using a different approach remains a key point for enhancing global T cell immunity in CML patients, even for those with CR status.