Combination therapy with nilotinib and PDL1 blockade reverses CD4 T cell dysfunction and prevents relapse in acute B cell leukemia

Patients with acute lymphoblastic leukemia have experienced significantly improved outcomes due to the advent of chimeric antigen receptor T-cells and bispecific T-cell engagers, although a proportion of patients still relapse despite these advances. T cell exhaustion has been recently suggested to be an important driver of relapse in these patients. Indeed, phenotypic exhaustion of CD4+ T-cells are predictive of relapse and poor overall survival in B-ALL. Thus, therapies that counter T cell exhaustion, such as immune checkpoint blockade, may improve leukemia immunosurveillance and prevent relapse. Here, we used a murine model of Ph+ B-ALL as well as human bone marrow biopsy samples to assess the fundamental nature of CD4+ T-cell exhaustion and the preclinical therapeutic potential for combining anti-PDL1 based checkpoint blockade with tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein. scRNA-seq analysis revealed that B-ALL induces a unique subset of CD4+ T-cells with both cytotoxic and helper functions. Combination treatment with the tyrosine kinase inhibitor nilotinib and anti-PDL1 dramatically improves long-term survival of leukemic mice. Depletion of CD4 T cells prior to therapy completely abrogates the survival benefit, implicating CD4 T cells as key drivers of the protective anti-leukemia immune response. Indeed, treatment with anti-PDL1 leads to clonal expansion of leukemia-specific CD4 T cells with the afore-mentioned cytotoxic/helper phenotype, as well as reduced expression of exhaustion markers. These findings support efforts to utilize PD1/PD-L1 checkpoint blockade in clinical trials and highlight the importance of CD4+ T-cell dysfunction in limiting the endogenous anti-leukemia response. For the mouse study, C57BL/6 mice were injected with leukemia cell line (LM138) cells via tail vein, then treated with nilotinib 5 days/week for 2 weeks. Some mice were treated with anti-mouse PDL1 on days 14, 16 and 18 post-leukemia establishment. For the CD4 and CD8 T cell depletion studies, mice were treated with anti-CD4 or anti-CD8 depleting antibodies on days 7, 9 and 11 post-leukemia establishment. Sorted CD44+CD4+ T cells from the indicated experimental arms were labeled with arm-specific hashtag oligo (HTO) antibodies, as well as CITE-Seq antibody derived tags (ADT) antibodies to CD25, OX40, TIGIT, PD1, Tim3, LAG3. Cells were captured and single cell sequencing for gene expression (GEX), sample-specific and protein feature barcodes, and T-cell receptor (TCR) repertoires was performed using the 10X Genomics Chromium Next GEM Single Cell 5' (ver. 1.1) reagents, resulting in three (GEX, HTO/ADT, and TCR) libraries. For the human study, leukemia blasts, T cells (CD45+CD3+), and other immune cells (CD45+CD3-CD10-) were sorted from bone marrow biopsy samples from five leukemia patients. Cells from each patient were labeled with different HTO antibodies. Cells were captured and single cell sequencing for gene expression, sample-specific feature barcodes, and T-cell receptor repertoires was performed using the 10X Genomics Chromium Next GEM Single Cell 5' (ver. 1.1) reagents, resulting in three (GEX, HTO, and TCR) libraries.