Costimulatory Blockade (CSB) during Mixed Lymphocyte Reaction (MLR) Prevents IL27 Upregulation and Signalling.

Numerous methods for selectively depleting or manipulating alloantigen (alloAg) specific CD4 T cells (CD4 T) are being studied for their potential in improving transplant outcomes by limiting GVHD or graft rejection. Although some methods target specific molecules (e.g. costimulatory receptors and ligands, activation antigens), the effects of these methods on “off-target” CD4 T cellular pathways and functions as well as effects on other PBMC, direct or indirect, have not been well characterized. Such effects may impact significantly on clinical efficacy and/or may shed light on the mechanism of action of manipulated PBMC in a complex in vivo milieu. To understand the molecular impact on bystander PBMC of inducing alloAg specific CD4 T anergy by CSB via disruption of the positive costimulatory signal between B7.1 and B7.2 on antigen presenting cells (APC) and CD28 on CD4 T by humanized anti B7.1/B7.2 monoclonal antibodies (MoAbs), we have used global gene expression profiling (GEP). Mimicking our ex vivo clinical anergization protocol, we used PBMC obtained from fully-HLA mismatched healthy volunteers (n=12 pairs) to perform an ex vivo primary MLR +/− anti B7.1/B7.2 MoAbs. CD28:B7 CSB inhibited mean proliferation by 73% after 72 hours of MLR. GEP was performed using Affymetrix hu133 plus2 chips on monocytes (Mo), CD4 and CD8 T cells, and B and NK cells purified from the MLR. Differentially expressed genes between cells from MLR +/− CSB were determined by SAM and EBAM analysis. Despite the low published frequency (1–10%) of alloAg specific CD4 T, we could detect global gene expression variance between CD4 T isolated from MLR +/− CSB (P≤0.05) suggesting effects on non-alloAg specific CD4 T. Use of the Ingenuity pathway analyzer to classify these differentially expressed genes by specific cellular pathways showed most were involved in cell proliferation and differentiation. Differences in IL27 downstream signaling molecules (DSM) in Mo and CD4 T were pronounced. IL27 is a member of IL12 cytokine family produced by APC and is a heterodimer of p28 and EBV-Induced gene 3 (EBI3). The IL27 receptor (IL27R) WSX1 is expressed on CD4 T. IL27 regulates adaptive immunity by controlling T cell proliferation, Th1 cell differentiation and IFNγ synthesis. Both p28 and EBI3 transcriptional and translational levels were decreased in Mo from MLR + CSB. Expression of STAT3, an IL27 pathway DSM was also decreased. After CD28:B7 CSB, CD4 T exhibited decreased expression of the IL27R and also inactivation of IL27 DSM including pSTAT1 and NFκB at both the transcriptional and translational levels. Consistent with a negative effect on Th1 differentiation, intracytoplasmic cytokine (ICC) analysis showed decreased expression of type 1 cytokines IL2 and IFNγ in CD4 T from MLR + CSB. ICC also showed decreased expression of the type 1 cytokine IL15 and increased expression of the type 2 cytokine IL10 in Mo from MLR + CSB. These results suggest that CD28:B7 signaling during MLR is required for Mo production of IL27. Decreased expression of IL27 and its DSMs pSTAT1 and NFκB1 after CSB with antiB7 MoAbs may contribute to CD4 T alloAg anergy induction by suppressing effector cytokines and Th1 cell differentiation. The observation that CD28:B7 modulates IL27 and IL27R emphasizes that targeted therapies used in the complex environment of human PBMC may have effects not predicted by in vitro clonal systems. Such effects may be important in the functional outcome of the intervention.