Current induction therapy using thymoglobulin (ATG) results in nonspecific T cell depletion. We have generated a polyclonal rabbit anti-human leukocyte globulin (RALG) by immunizing rabbits with activated lymphocytes and monocytes to increase specificity to cells most responsible for graft rejection. Flow cytometry verified specificity of RALG in comparison with ATG in targeting major CD markers including adhesion and co-stimulatory molecules. To assess the immunomodulatory capacities of RALG, purified human CD4 and CD8 cells were treated with RALG for 24, 48, and 72 hours, then analyzed by RT-PCR evaluating transcripts for apoptosis/survival (Bcl-2, Bax, PDCD-1, PDCD-1 L1, PDCD-1 L2, FAS, FAS-L, caspase-3), costimulation (CD154, CD40, CD80, CTLA-4, ICOS, OX40, GITR), cytokines (IFN-gamma, TNF-alpha, IL-10, IL-2R), and regulatory molecules (Foxp3, SOCS-1, SOCS-3, T-box 21, NF-kb, JAK3). The culture supernatants collected from RALG treated CD4 and CD8 cells were analyzed by multiplex array to detect IL-2, IL-4, IL-10, IFN-gamma, IP-10, and CCL5. Treated cells were tested in mixed lymphocyte reactions (MLR) to determine the inhibition of allo-specific T cell proliferation. RALG treated CD4 cells were also tested in MLR to assess the effects in inhibiting CD4 cell proliferation in response to alloantigens. We found that the RALG had higher affinity for surface proteins more commonly associated with activated T cells (CD58, CD28, CD154) and monocytes (CD80, CD86, HLA). Compared to a rabbit Ig control, CD4 and CD8 cells remaining after incubation with RALG demonstrated dramatic upregulation of gene transcripts for cell apoptosis and survival. A regulatory phenotype transcriptome including CTLA-4, Foxp3, GITR, OX40, and IL-2R was dramatically upregulated following treatment. Gene transcripts for cytokines and their regulatory molecules were also upregulated. Supernatants from antibody treated cells demonstrated increased production of cytokines and chemokines. This culture supernatant also suppressed lymphocyte proliferation in response to alloantigens. RALG treated CD4 cells not only showed reduced proliferation in response to alloantigens, but also suppressed proliferation of untreated CD4 cells in response to alloantigens. This study demonstrates effects of a novel RALG preparation in modulating purified human T cells. This preparation specifically targets molecules of interest in the allotransplant setting and provides a mechanistic basis for potential application as immunosuppression.

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