Total Body Irradiation Creates a Proinflammatory Milieu That Is Required for Minor, but Not Major, Mismatch Graft-Versus-Host-Disease.

Graft-versus-host disease (GVHD) is a potentially fatal complication following allogeneic bone marrow transplantation (BMT). GVHD is characterized by three phases: (1) recipient tissue injury mediated by the conditioning regimen of irradiation and/or chemotherapy; (2) donor cell priming and activation; and (3) effector destruction of target tissue such as the intestine, liver, skin, and lung. There is some evidence to suggest that the second and third phases are dependent on the first, however, this has not been rigorously tested. Therefore, we developed major and minor mismatch models of GVHD and used immunodeficient recipients that readily accept allografts to analyze the requirement of irradiation conditioning on GVHD initiation and development.

Experimental Design Wild-type (WT) B6 (H-2^b) recipients received major mismatch (Balb/c, H-2^d), minor mismatch (129, H-2^b), or syngeneic (B6) BMTs. Recipients were lethally irradiated, and 24 hours later received an intravenous infusion of 5 × 10⁶ bone marrow cells and 30 × 10⁶ splenocytes, as a source of mature T cells. In adoptive transfer (AT) experiments, B6 Rag1^−/− or B6 Rag2^−/−/Pfp^−/− (perforin deficient) recipients received 30 × 10⁶ splenocytes. Data are represented as mean ± SEM.

In lethally irradiated WT recipients, Balb/c→B6 caused severe GVHD that was fatal in all recipients by 24 days after BMT. 129→B6 led to a milder GVHD, where 50% of recipients survived to 35 days after BMT, and a few survived long-term (>100 days). Both major and minor mismatch GVHD were characterized by 15–25% weight loss, clinical symptoms including decreased activity, hunched posture, ruffled fur, and hair loss, and target organ histopathology. To test the requirement of irradiation conditioning in GVHD, we transferred Balb/c splenocytes into unconditioned B6 Rag1^−/− recipients. No signs of GVHD developed. However, donor T cells were virtually undetectable 5 weeks after AT, indicating graft rejection. To test if this was due to recipient natural killer (NK) cells, a major mismatch AT was performed into B6 Rag2^−/−/Pfp^−/− recipients, which lack fully functional NKs. Balb/c→B6 Rag2^−/−/Pfp^−/− resulted in GVHD, including 10–15% weight loss, clinical symptoms, and target organ histopathology, although the disease was not as severe as that following lethal irradiation of WT recipients, consistent with a facilitative role for conditioning in disease progression. In contrast to the above, a 129 splenocyte AT did not cause GVHD in Rag1^−/− or Rag2^−/−/Pfp^−/− recipients, despite donor T cell engraftment. This suggested that conditioning was required for minor mismatch GVHD. To test this, we sublethally irradiated Rag1^−/− recipients prior to 129 AT. This resulted in GVHD, with 10–15% weight loss, clinical symptoms, and histopathology. At day 7 after AT, serum IFNγ, TNF, and IL-6 were significantly greater in mice that received irradiation and AT (compared to AT alone, p<0.05). Donor cells within spleens of mice that received irradiation and AT had a reduced fraction of Foxp3⁺/CD4⁺ (9.5%±2.3) compared to AT alone (16.8%±1.6, p=0.06), and increased intracellular CD8⁺ IFNγ expression (%IFNγ⁺:47.7±2.4 vs. 41.6±1.8, p<0.05; MFI:15722±2003 vs. MFI:8025±319, p<0.05). To determine whether more alloreactive donor cells were primed after irradiation, an in vivo killing assay was performed with recipient-specific targets. Combined irradiation and AT resulted in 66.3±13.5% killing efficiency while AT only had an 18.9±11.7% killing efficiency.

We conclude that irradiation conditioning is required for minor, but not major mismatch GVHD. The conditioning regimen creates the necessary proinflammatory milieu to prime sufficient numbers of alloreactive cells for GVHD. While this milieu can enhance development of major mismatch GVHD, it is not required for development of disease.

No relevant conflicts of interest to declare.