Abstract Acute myeloid leukemia (AML) is a heterogeneous malignancy of the blood primarily treated with intensive chemotherapy. The allogeneic T cell anti-leukemic activity via donor lymphocyte infusions and stem cell transplantation suggests a potential role for checkpoint blockade therapy in AML. While clinical trials employing these treatments have fallen short of expected results, a deeper exploration into the functional states of T cells in AML could bridge this knowledge gap. In this study, we analyzed the polyfunctional activity of T cells in a cohort of relapsed/refractory (RelRef) AML patients treated on the clinical trial (ClinicalTrials.gov identifier: NCT02397720) of combination therapy using azacitidine and nivolumab (Aza/Nivo). We utilized the single cell polyfunctional multiplexed immune assay IsoPlexis to evaluate the CD4 and CD8 T cells in peripheral blood and bone marrow samples collected before and after immunotherapy. This revealed at a pseudobulk level that the CD4 T cells exhibited higher functional activity post-immunotherapy (post-IO), suggesting that CD4-directed therapies may play a role in RelRef AML. Additional single-cell analysis revealed significant differences in baseline polyfunctionality in bone marrows of responders as compared to non-responders for both CD4 and CD8 T cells. Overall, this study highlights the impact of polyfunctional assessment in understanding CD4 and CD8 dynamics in contexts of therapy in AML.