Effective Control of Mismatched Alloreactivity Via Ex Vivo Alloantigen-Specific Co-Stimulatory Blockade Does Not Significantly Impact Pathogen-Specific Immunity.
Authors: Davies Jeff K. , Gorgun Gullu , Nadler Lee M. , Guinan Eva C. 
Published: October 2019

Abstract
Control of alloreactivity without loss of immunity to pathogens and tumor antigens remains the major challenge in allogeneic hematopoietic stem cell transplantation (AHSCT). One platform for achieving this objective has been based upon the methodologies of stimulating donor T cells with recipient alloantigens and destroying, removing, and/or inactivating alloreactive donor T-cells. We have previously reported a clinical trial employing ex vivo inactivation of alloreactive donor T cells via CTLA-4 immunoglobulin-mediated co-stimulatory blockade of CD28 signaling, which allowed large doses of HLA-mismatched T cells to be infused at the time of AHSCT without excess graft-versus-host disease (GvHD) and with no clinically significant late viral infections in surviving patients. We have now refined this approach, exposing donor peripheral blood mononuclear cells (PBMCs) to irradiated stimulator PBMCs in the presence of clinical grade humanized anti-B7.1 and anti-B7.2 monoclonal antibodies, blocking the delivery of positive co-stimulatory signals to (predominantly CD4+) alloantigen-specific donor T cells. This strategy reduces proliferative responses to irradiated stimulator PBMCs to less than 1% of those seen with untreated cultured responder cells in fully HLA mismatched healthy volunteer donor pairs. The proliferative capacity of responder cells to mitogenic anti-CD3 and -CD28 antibodies following alloantigen-specific co-stimulatory blockade is not impaired. In order to demonstrate the retention of antigen-specific responses in donor CD4+ cells following recipient alloantigen-specific co-stimulatory blockade, we used a highly sensitive 5-color flow cytometric intracellular cytokine secretion assay following stimulation of PBMCs with virus-infected cell lysates. Donors in whom viral lysate-specific CD4+ cells could be detected in untreated PBMCs retain approximately two-thirds of VZV, HSV and CMV-specific Th1 cytokine+ (IFN-γ/IL2) CD4+ cells after anti-B-7 mediated alloantigen-specific co-stimulatory blockade. Th1 cytokine responses to the superantigen staphylococcal enterotoxin B are preserved in all donors. Furthermore, proliferative responses to CMV viral lysate are retained after effective allospecific co-stimulatory blockade in the majority of donors in whom CMV-specific Th1 cytokine+ CD4+ T cells could be detected in untreated PBMCs cultured in parallel. These data demonstrate that HLA-mismatched alloantigen-specific co-stimulatory blockade effectively reduces proliferative responses to alloantigens whilst retaining pathogen-specific CD4+ cells. Moreover, the capacity to secrete cytokines of these pathogen-specific T cells supports the hypothesis that they will be functional when adoptively transferred. An ongoing multicentre proof-of concept dose escalation study is underway in which this strategy is utilized to generate HLA mismatched donor T cells which are administered after haploidentical AHSCT to augment immune recovery without GvHD. Furthermore, the application of highly sensitive assays to quantify pathogen-specific immunity in donor T cells after alloantigen-specific co-stimulatory blockade may allow donor and recipient-specific T cell dosing strategies to be applied to minimize treatment-associated toxicity.