*Purpose: Pre-conditioning using total body irradiation prior to HSCT is a well-established regimen for patients with hematological malignancy. However, irradiation (IR) elicits not only immunosuppressive effects, but also immune activation. The relationship of this IR-induced immune activation with enhanced allo-immune responses and HSCT-complications is not well-studied. The aim is to evaluate, using an in vitro model, the effect of IR pre-conditioning on allo-immune responses, and to develop potential predictive markers.

*Methods: Human PBMCs were obtained from healthy volunteers. Different stimulator PBMC samples (n=9, HLA-A2+, HLA-B8-) were irradiated (40Gy) and cultured for 48hr to mimic pre-conditioning (iS). Secreted pro-inflammatory cytokines in medium were analyzed by ELISA (TNFa, IFNg, IL-2). Immune cells were studied using FACS (CD3, CD4, CD8, CD11c, CD14, CD19, CD56, HLA-ABC, HLA-DR). After 48hr, HLA-A2-, -B8+ mismatched PBMC from a single responder (R) were co-cultured with each of the iS samples by adding R to either the irradiated iS PBMC and their conditioned media (CM) or only to the CM. R proliferation to each stimulator/condition was assessed at day 5 using ³H-thymidine incorporation.

*Results: Co-culture with pre-conditioned iS resulted in R proliferation, but ³H-thymidine incorporation differed significantly by iS samples. While an increase in TNFa secretion was detected in iS CM, CM alone did not induce R proliferation, suggesting that immune cells but not soluble factors induced the alloproliferation. FACS analysis showed that only 20% of iS PBMC are viable 48h after IR, with CD14⁺ monocytes as the most radioresistant and CD56⁺ NK cells as the most radiosensitive cell subsets.

iS PBMC with higher %HLA-DR⁺ CD8⁺ T cells and higher HLA-ABC expressing CD19⁺ B cells at baseline (before IR) induced significantly more R proliferation.

*Conclusions: The effect of IR pre-conditioning on alloproliferative responses can be evaluated in vitro. In this system, higher % HLA-DR⁺ CD8⁺ T cells, and HLA-ABC high-expressing CD19⁺ B cells in baseline PBMCs (pre-IR) are associated with increased alloproliferation, suggesting that they might be considered as potential markers to predict allo-immune responses after IR pre-conditioning for transplant.

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