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 is 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-PD-L1 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-PD-L1 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-PD-L1 leads to clonal expansion of leukemia-specific CD4+ T-cells with the afore-mentioned helper/cytotoxic 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.

The molecular underpinnings of acquired resistance to carboplatin are poorly understood and often inconsistent between in vitro modeling studies. After sequential treatment cycles, multiple isogenic clones reached similar levels of resistance, but significant transcriptional heterogeneity. Gene-expression based virtual synchronization of 26,772 single cells from 2 treatment steps and 4 resistant clones was used to evaluate the activity of Hallmark gene sets in proliferative (P) and quiescent (Q) phases. Two behaviors were associated with resistance: (1) broad repression in the P phase observed in all clones in early resistant steps and (2) prevalent induction in Q phase observed in the late treatment step of one clone. Furthermore, the induction of IFNα response in P phase or Wnt-signaling in Q phase were observed in distinct resistant clones. These observations suggest a model of resistance hysteresis, where functional alterations of the P and Q phase states affect the dynamics of the successive transitions between drug exposure and recovery, and prompts for a precise monitoring of single-cell states to develop more effective schedules for, or combination of, chemotherapy treatments.

BackgroundPhenotypic exhaustion of CD4+ T-cells is a strong negative prognostic factor in acute lymphoblastic leukemia (ALL).1–3 Despite this, PD1/PD-L1 immune checkpoint therapy has shown little activity in this disease setting to date. Factors influencing the responsiveness of the T-cell compartment to checkpoint blockade are unknown.MethodsAn established murine model of BCR-ABL+ ALL was used. Leukemia was established by tail vein injection, and mice were treated with the BCR-ABL tyrosine kinase inhibitor nilotinib with or without PD-L1 mAb therapy. scRNAseq/TCRseq was performed using multiple treatment groups.ResultsTreatment of leukemia-bearing mice with a combination of the BCR-ABL tyrosine kinase inhibitor nilotinib and PD-L1 immune checkpoint blockade led to eradication of leukemia in 70% of treated mice (figure 1). Efficacy was dependent on the presence of CD4+ T-cells, while CD8+ T-cells appeared to play a lesser role. Direct cytotoxicity by CD4+ T-cells was confirmed in live cell-killing assays (figure 2). Mice that were treated with PD-L1 blockade and survived to day 100 were found to have no detectable residual leukemia. They were also protected from leukemia rechallenge, suggesting the elicitation of a memory response. scRNAseq analysis revealed that CD44hi CD4+ T-cells were highly heterogeneous, with regulatory, effector, and stem-like TCF7+ precursor subsets present (figures 3–4). A unique population of CD4+ T-cells was elicited by live leukemia challenge (clusters 6 and 7 in figure 3) but not by vaccination with heat-killed leukemia cells. This subset was characterized by relatively low levels of expression of TCF7, but high levels of expression of Granzyme B, TOX, the effector cytokines IFNγ and TNFα, the inhibitory receptors PD1, TIM3, and LAG3, and the chemokine CCL5 (figure 5). PD-L1 checkpoint blockade was associated with early narrowing of the clonality of this population (figure 6), decreased markers of exhaustion, and more robust synthesis of TNFα.Abstract 321 Figure 1Survival analysis. BCR-ABL+ ALL was established by tail vein injection on day 0. Nilotinib (75 mg/kg) was administered via oral gavage. mAbs targeting PD-L1 with or without depleting antibodies towards CD4 or CD8 were administered via intraperitoneal injection. p-value derived by log-rank analysisAbstract 321 Figure 2Analysis of the increase in the number of dead cells (y-axis) over time (x-axis) from a live killing assay (Incucyte) using splenic CD4+ or CD8+ T-cells from experimental arms as treated in figure 3. Control traces from separate wells with LM138 target cells only are included. Experiments were done using Cytotox NIRAbstract 321 Figure 3(Left) Experimental approach. 5 groups (n=4 mice/group) were treated in parallel with the indicated conditions. CD44hi CD4+ T-cells from the spleen and bone marrow of mice in each group were labelled with oligo-conjugated hashtag antibodies (Biolegend) and CITE-SEQ antibodies towards PD1, TIM3, LAG3, CD25, and TIGIT, prior to FACs-sorting. scRNAseq/TCRseq analysis (10x Genomics) was performed on 5,349 individual cells after multiplet removal. (Right) UMAP plots of all cells combined. Clusters were identified by differential expression of canonical gene productsAbstract 321 Figure 4Feature plots demonstrating expression of canonical gene products projected onto the UMAP plot in figure 3. Antibody derived tags (ADTs; bottom row) indicate expression level of surface proteins profiled using CITESEQ antibodiesAbstract 321 Figure 5Heatmap of select gene product expression levels in exhausted (cluster 6) CD4+ T-cells across treatment conditionsAbstract 321 Figure 6Simpsons diversity index of the TCR repertoire across treatment arms. Lower values indicate relatively decreased clonalityConclusionsPDL1 immune checkpoint blockade is effective at eradicating residual disease in preclinical models of BCR-ABL+ ALL. ALL elicits a unique CD4+ memory/effector subset characterized by the potential for both chemotactic and cytotoxic functions. Leukemia induces early exhaustion of this subset, which is countered by PDL1 blockade. Efforts to extend these observations to human specimens are underway and will be reported.ReferencesHohtari H, Brück O, Blom S, et al. Immune cell constitution in bone marrow microenvironment predicts outcome in adult ALL. Leukemia 2019;33(7):1570–1582. Blaeschke F, Willier S, Stenger D, et al. Leukemia-induced dysfunctional TIM-3. Leukemia 2020;34(10):2607–2620.Liu L, Chang YJ, Xu LP, et al. T cell exhaustion characterized by compromised MHC class I and II restricted cytotoxic activity associates with acute B lymphoblastic leukemia relapse after allogeneic hematopoietic stem cell transplantation. Clin Immunol 2018;190:32–40.