L-Asparaginase Treatment Induces Reversible Immunoregulatory and Immunosuppressive Effects in Non-Malignant B Cells in a Model of T-Cell Dependent B Cell Activation

Introduction: Metabolic re-programming of immune cells including B cells is a prerequisite for their differentiation and functional adaptation during immune responses. Here, the production of building blocks for nucleic acid and protein synthesis is as important as energy generation. While our understanding of immunometabolic processes has emerged in recent years, targeting of amino acid availability and metabolism by L-asparaginase has been exploited therapeutically for decades in acute lymphoblastic B cell leukemia (B-ALL) treatment based on their deficiency to synthesize asparagine. Interestingly, L-asparaginase has recently been reported to exert glutaminolysis activity also, and the non-essential amino acid glutamine is key for immune cell biology and function. However, the immunologic and metabolic consequences of L-asparagine exposed non-malignant B cells are ill-defined.

Here, in an in-vitro model of T cell dependent CD40-mediated B cell activation, we characterized the biology, differentiation and functional consequences of activated physiologic B cells upon L-asparaginase treatment. We also dissect the individual and combinatorial effects of glutamine and asparagine in rescue experiments.

Methods: B cells were isolated from healthy donors and activated with soluble CD40L-multimers under increasing concentrations of L-asparaginase (0.01-10 U/ml). After five days of activation, CD40-activated B cells were analyzed for proliferation, homotypic cluster formation, immune-phenotypes and metabolic programs via extracellular flux experiments (Agilent Seahorse). APC-function was addressed in allogeneic mixed lymphocyte reactions with untreated T cells and L-asparaginase pre-treated, washed CD40-activated B cells. Rescue experiments were performed by asparagine and glutamine supplementation in various concentrations and combinations.

Results: L-asparaginase significantly reduced cell numbers and homotypic cluster formation of CD40-activated B cells in a dose dependent manner. Reduced cell numbers did not result from increased apoptosis but from reduced proliferation rates and were already seen in clinically relevant L-asparaginase concentrations. In addition to lower cell proliferation, impaired cluster formation was also linked to reduced LFA-1 expression on CD40-activated B cells. Single-cell level analyses of cell size and granularity which demonstrated smaller and low-granulated B cells under L-asparaginase treatment, pointed to changes in metabolic programs. Indeed, extracellular flux analyses demonstrated significantly downregulated measures of both glycolysis (ECAR) and oxidative phosphorylation (OCR) and their respective reserve capacities. L-asparaginase reduced cell surface marker expression of B cell activation and APC-function (CD80, CD86, HLA-DR), as well as the formation of a B cell subpopulation with an immunoregulatory phenotype (CD24+CD38+CD27+) and increased interleukin-10 secretion and TGF-beta expression. Reduced APC-phenotypes were functionally validated in allogeneic mixed lymphocyte reactions showing significantly reduced T cell activation and proliferation by L-asparaginase pre-treated CD40-B cells. Supplementation of culture medium with increasing concentrations of asparagine restored B cell proliferation and cluster formation as well as their APC-function. Interestingly, glutamine substitution was not only sufficient but slightly superior to rescue L-asparagine mediated suppressive effects.

Conclusions: Our study demonstrates profound immunomodulatory and immunosuppressive effects of L-asparaginase on physiologic activated B cells. Moreover, our data suggest that L-asparaginase induced induction of regulatory B cell formation might be an important additional mechanism of action, as well as its glutaminolysis activity. This work might hint to a putative therapeutic use of L-asparaginase in B-cell mediated autoimmune settings as the observed effects were already seen in low concentrations of L-asparaginase compared to those used for B-ALL treatment. Thus, further studies on this indication are warranted.