Novel Immunosuppressive Approach Attenuates Severe Aplastic Anemia in Mouse Lymphocyte Infusion AA Models

Aplastic Anemia (AA) is an immune-mediated and life-threatening form of acquired bone marrow failure. AA ranges from moderate to severe AA, and is characterized by development and expansion of self-reactive effector T cells, which cause apoptosis of mature blood cells, progenitors and hematopoietic stem cells (HSCs). Current treatments for AA, which are not always effective or feasible, include immunosuppressive therapy (IST) and allogeneic HLA-identical sibling or well-matched unrelated donor BM transplant. Because the self-antigens triggering AA remain to be identified, mouse lymphocyte infusion models of AA with striking similarities to human AA have been developed utilizing alloantigen recognition. The AA in these models is induced by infusing lymph node cells (LNCs) from C57BL/6J mice into MHC partially mismatched F1 hybrid B6D2F1 or CByB6F1 recipients, or into minor-H antigen mismatched C.B10 recipients. The host mice develop SAA without any clinical signs of generalized GVHD, and characterized by BM infiltration and oligoclonal expansion of donor effector T cells, apoptosis of all host BM cells, severe BM aplasia and death within 3-5 weeks after LNC infusion. These preclinical mouse models represent a very useful in vivo system for testing new therapeutic approaches to treat and manage SAA. Activation of self-reactive T cells in human AA and alloreactive donor T cells in mouse AA models involves interaction of T cells with dendritic cells (DCs) as professional APCs. DCs express β2 integrin CD11b/CD18 (Mac-1), which plays an important role in inflammation, cell-mediated killing and cell activation. Notably, Mac-1 expressed on DCs is inactive and is not activated on contact with T cells. More importantly, activation of Mac-1 on DCs by Mg2+ treatment significantly reduces their T cell-activating capacity, and active CD11b represses DC cross-priming of cytotoxic T cells. Thus, activation of Mac-1 on DCs represents a potential new immunosuppressive strategy for reducing pathological T cell responses in AA. Dr. Gupta has identified novel Mac-1 agonists, termed Leukadherins (LAs1-3) that bind to and activate Mac-1. Multiple lines of experimental evidence generated by Dr. Gupta’s and Dr. Jurecic’s groups have shown that LAs have potent anti-inflammatory and immunosuppressive properties. For example, treatment with LA1 is safely and effectively reducing the onset and severity of Experimental Autoimmune Encephalomyelitis (EAE) in mice, induced by Myelin Oligodendrocyte Glycoprotein (MOG). Moreover, in EAE mice LA1 efficiently decreased the activation of myelin-reactive T cells and their IFN-γ production. We hypothesized that by activating Mac-1 on DCs LAs could effectively (a) reduce T cell-activating capacity of DCs and attenuate allo-reactive T cell responses, and (b) reduce severity of AA in mouse models. Indeed, in mixed lymphocyte reaction, which depends on stimulation of allogeneic T cells by DCs, LA1 significantly suppressed proliferation of lymph node T cells from C57BL6/J mice in the presence of irradiated splenocytes from allogeneic DBA/2J mice. SAA was induced in B6D2F1 mice by tail vein injection of 5 x 10e7 LNCs from C57BL/6J mice. The untreated AA mice died within 21 days of LNC infusion and exhibited (a) severe BM aplasia, (b) ~5-fold expansion of CD4⁺ T cells and >20-fold expansion of CD8⁺ T cells in comparison to Control B6D2F1 mice, and (c) severe depletion of HSCs (LSK CD150⁺ CD48^- BM cells); Multipotent progenitors (MPPs, LSK CD150^- CD48^- BM cells); and Hematopoietic progenitors (HPC-1, LSK CD150^- CD48⁺ cells; HPC-2, LSK CD150⁺ CD48⁺ cells). In contrast, AA mice treated IP with 1 mg/kg/day of LA1 for 7 or 21 days after LNC infusion exhibited (a) mild BM aplasia and improved BM cellularity, (b) significantly reduced expansion of CD4 (~2-fold) and CD8 (~12-fold) T cells in the BM, and (c) significantly improved frequency and total numbers of HSCs and progenitors in comparison to untreated AA mice. More importantly, treatment of AA mice with LA1 for 21 days resulted in 40-50% of AA mice surviving for more than 7 weeks after LNC infusion. These results demonstrate that treatment with LA1 can safely convert SAA into a moderate disease in preclinical mouse AA models and provide a platform for testing of LAs as new alternative or adjuvant therapy to manage ongoing AA in patients who (1) are not responding to IST and are not candidates for BMT, and/or (2) are undergoing IST and awaiting BMT.