Session: 614. Acute Lymphoblastic Leukemia: Therapy, excluding Transplantation: Poster I
Hematology Disease Topics & Pathways:
Biological, antibodies, CAR-Ts, Therapies, immunotherapy, Clinically relevant, immune mechanism, inflammation
To study the impact of adiposity on T-cell function, CD4+ and CD8+ T-cells were purified from the spleens of C57BL/6 mice and activated with PMA/Ionomycin for 72 hours in unconditioned media (UCM), bone marrow stromal-cell conditioned media (SCM), and adipocyte-conditioned media (ACM) followed by flow cytometry analysis for surface marker expression, cytokine production, and the induction of cytolytic mediators. Interestingly, T-cells activated in ACM, but not UCM or SCM, showed an attenuated phenotype highlighted by decreased CD44 and PD-1 expression, diminished cytokine production (IFN-γ/TNF-α) and reduced induction of cytolytic mediators (granzyme B/perforin). These observations were also true in obese, relative to lean, patients with B-ALL where we found that T-cells purified from the peripheral blood mononuclear cells (PBMCs) failed to produce significant levels of TNF-α when stimulated with PMA/Ionomycin. In all, these results demonstrate that adipocyte-secreted factors directly compromise the function of endogenous T-cells, which phenocopies T-cell defects observed in obese relative to lean pediatric patients with B-ALL.
We next assessed the impact of adiposity on malignant cells by culturing human B-ALL cell lines in the conditioned mediums described above and performed flow cytometric analysis to assess their surface expression of the B-cell lineage antigen CD19 and proteins that modulate immunity. In addition to being a marker for B-cells, CD19 is the primary target of the T-cell based immunotherapies Blinatumomab and CAR T-cells directed against B-ALL cells. Surprisingly, when human B-ALL cells were co-cultured with adipocytes, every cell line tested (n=6) exhibited lower surface CD19 expression with 5 out 6 reaching statistical significance. Furthermore, adipocyte-secreted factors alone were sufficient to reduce CD19 surface levels on B-ALL cells in 2 of the 6 cell lines tested. Human B-ALL cells cultured in ACM, but not UCM or ACM, also upregulated their surface expression of the immunoinhibitory proteins HVEM, PD-L1, and PD-L2. These results demonstrate that adipocytes directly induce the downregulation of CD19 on human B-ALLs and increase their immune evasive capacity.
Given these observations, we hypothesized that adipocyte-secreted factors would compromise T-cell-based immunotherapies targeting CD19-expressing B-ALL cells. To this end, primary human T-cells were engineered to express a CD19-directed chimeric antigen receptor (CAR). CAR T-cells and human B-ALL cells were separately pre-treated for 24 hours in UCM, SCM or ACM followed by co-culture for cytolytic analysis using Annexin-V/PI staining. Adipocyte-secreted factors significantly inhibited CAR T-cell mediated killing of CD19-expressing B-ALL cells at 4 hours. In addition to CAR T-cells, we tested the leukemia killing efficacy of the bispecific T-cell engager, Blinatumomab. After 3 days of culture, we found that Blinatumomab significantly increased the killing capacity of endogenous T-cells with 60-80% of B-ALL cells being killed after 3 days of culture in UCM and SCM. In contrast, we found that ACM significantly compromised the efficacy Blinatumomab with only 30% of B-ALL cells being killed over 3 days when co-cultured with human T-cells.
Our pre-clinical data highlights the negative impact of an adipose-rich microenvironment on T-cell function and B-ALL immunogenicity, which subsequently compromises the efficacy of multiple classes of immunotherapies targeting CD19.
Disclosures: No relevant conflicts of interest to declare.
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