Deciphering the T-cell differentiation landscape in patients with Acute Myeloid Leukemia

The role of T cells in chemotherapy response and maintenance of remissionin acute myeloid leukemia (AML) is not fully understood. In solid tumorsand chronic infections, exhaustion is a multistep process ranging from lessdifferentiated progenitor exhausted (Tpex) to intermediate and terminallyexhausted T cells (Beltra et al. 2020). High frequencies of Tpex correlatewith response to immune-checkpoint blockade in solid tumors (Miller et al.2019). In AML, where the backbone of treatment is chemotherapy, the roleof dysfunctional T-cell subsets has yet to be elucidated. In our study we useddifferent cohorts of AML patients to study the immunologic T-celllandscape. Particularly, we performed an exploratory flow cytometryanalysis on samples collected at baseline and at response assessment frompatients treated with chemotherapy. Through high dimensional flow-cytometry analysis we identified a CD8+ CD28+ PD1+subset increased atbaseline and after treatment in responder patients vs non-responders. Tofurther investigate these results, we applied the same approach to analyzesamples from a cohort of relapsed/refractory AML patients treated with highdose cytarabine (HIDAC) plus pembrolizumab. In these cohort, patientswith a higher frequency of CD8+CD45RA-CD27+/intCD28+PD1+TCF1+(progenitor-exhausted like) were more likely to respond to therapy. Next,we decided to better characterize the identified subset and study therelationships with the others CD8+subpopulations by using 5' VDJ single-cell RNA sequencing. Studying the different gene signatures in our dataset,we identified 5 main CD8+ clusters: Naive, Tpex, Effectors, Terminallyexhausted 1 (Term_exh1) and Terminally exhausted 2 (Term_exh2).Comparing these subsets in responders and non-responders, Tpex weregreatly increased in responders compared to non-responders at bothtimepoints (baseline and response assessment). Conversely, Term_exh2cells were more abundant in non-responders. Of note, the two mostupregulated genes in Tpex were GZMK and IL-7R. Next, we measured themagnitude of clonal expansion in antigen-experienced CD8+ T cells inresponders and non responders. The most clonally expanded subsets wereTpex and Effectors in responders and Term_exh2 in non-respondersrevealing a strong relationship between abundance and clonal expansion ofthe CD8+ T-cell subsets. Our scRNAseq results were then confirmed at theprotein level with spectral flow-cytometry and reproduced by manual gatingof the GZMK+CD127+subset which was significantly enriched (p < 0.01)in responders vs non-responders. Notable, patients with a higher-than-median frequency of GZMK+CD127+CD8+ T cells experienced significantly(p<0.02) prolonged overall survival after therapy. These findings show thatimproving our understanding of the immune microenvironment in AML iscritical for the rational integration of novel treatment strategies that seek toincrease the response rate and/or maintain remission. We identifiedGZMK+IL7R+ CD8+cells as a distinct entity in the early differentiated CD8+memory T cell pool that is clonally expanded and more abundant inresponders compared to non-responders. This subset has a stem-likesignature and may be associated with longer in vivo CD8+ T cell persistenceand long-term AML control. An in-depth functional characterization within vitro experiments and in vivo mouse models is currently ongoing.