Lack of “Bystander” Activation and Evidence for Competition for Resources among Gvh-Reactive CD4 T Cells

Previous studies have shown that essentially all donor CD4 T cells adopt the activated/memory phenotype in the setting of GVHD induced in freshly conditioned hosts. However, it is unknown whether this activation is all antigen-driven or might reflect lymphopenia-induced activation and/or “bystander” activation by the abundant pro-inflammatory stimuli present in the GVHD environment. We compared the activation and accumulation of GVH-reactive CD4 T cells specific for recipient superantigens, minor (mHA) and MHC antigens with that of GVH non-reactive TCR transgenic CD4 T cells in lethally irradiated BMT recipients across a full haplotype barrier (B6→B6D2F1). Wild-type B6D2F1 or transgenic B6D2F1 mice expressing membrane-bound chicken ovalbumin (OVA-B6D2F1) received lethal total body irradiation (TBI), B6 bone marrow cells (BMC) to prevent death from aplasia and a donor lymphocyte inoculum of 10⁶ OTII CD4 T cells (specific for OVA peptide 323-339/IA^b, representing a mHA) and 10⁷ polyclonal B6 splenocytes, to preserve the strong polyclonal T cell response. On days 3 and 6 post-transplant, evidence for activation of OTII CD4 T cells was detected only in OVA-B6D2F1 recipients and not in wild-type recipients, as shown by upregulation of CD44 (average median fluorescence intensity 250 vs 92; p<0.05), CD25 (average 55% vs 10% positive cells; p<0.05) and CD69 (average 34% vs 16% positive cells; p<0.05). Absolute numbers of OTII CD4 T cells in the spleen were 5.5 times greater in the OVA-B6D2F1 than in the wild-type recipients (mean±SD of 1.0±0.4×10⁵ vs 0.18±0.07×10⁵; p<0.05). In contrast, Vβ6 CD4 T cells (recognizing a superantigen expressed by DBA/2) became activated in both groups, with similar increases in CD44, CD25 and CD69 expression. However, numbers of these cells were 3.6 times greater in wild-type B6D2F1 than in OVA-B6D2F1 recipients (mean±SD of 8.7±1.1×10⁴ vs 2.4±0.3×10⁴; p<0.05), suggesting that OTII cell activation by antigen reduced the “resources” available to other GVH-reactive CD4 T cells. Additionally OVA-B6D2F1 FI mice showed more rapid mortality compared to the wild-type recipients (median survival time: 7 days vs 32 days; p<0.05), suggesting a significant effect of OVA mHA recognition, even in the presence of GVH response across a full haplotype barrier. We then compared the fate of directly class II MHC alloreactive TCR transgenic CD4 T cells and irrelevant cells in a similar mouse model. Wild-type B6D2F1 mice underwent lethal TBI and received B6 BMC and a lymphocyte inoculum of 10⁶ OTII CD4 T cells (not recognizing any antigen in the recipient), 10⁶ 4C TCR transgenic CD4 T cells (directly alloreactive for recipient IA^d class-II antigen) and 10⁷ polyclonal B6 spleen cells. On day 6, 4C CD4 T cells showed marked accumulation, whereas OTII CD4 T cells failed to accumulate in the spleen (mean±SD of 1.6±0.4×10⁶ vs 0.02±0.01 × 10⁶; p<0.05). Moreover, 4C T cells acquired the activated/memory phenotype, with increased expression of CD44, CD25 and CD69 and downregulation of CD62L and CD45RB. Evaluation of the above markers on the OTII population was not possible due to the low numbers of cells recovered. Our data suggest that activation and proliferation of irrelevant non-GVH-reactive T cells does not occur in the setting of GVHD, despite the presence of an inflammatory and lymphopenic environment. Activation and proliferation of GVH-reactive CD4 T cells appears to be predominantly antigen-driven. Moreover, competition for “resources” among GVH-reactive clones seems to occur in this environment.