An ongoing question regarding the primary allograft response is the relative contribution of the 'direct' (donor MHC-restricted) versus 'indirect' (host MHC-restricted) pathways of T cell recognition in vivo. While the frequency of direct donor-reactive T cells is quite high (1-10%), the frequency of indirect T cells is estimated to be 2-3 orders of magnitude less. In unprimed T cells, the dominance of the direct pathway is illustrated by the vigorous primary in vitro response while the indirect is nearly undetectable. In vivo, however, donor APCs are limited in number and can be rapidly eliminated. Therefore, the magnitude of the less frequent indirect response during the primary alloresponse in vivo remains unclear. In this study, we tracked the relative primary response of T cell receptor transgenic C57Bl/6 (B6; H-2b) CD4 T cells of direct versus indirect reactivity to allogeneic BALB/c (H-2d) cells in vivo.

To model relative indirect and direct alloreactive frequencies, 100 fold fewer CD45.1 congenic indirect TCR75 (1e4) versus direct 4C (1e6) TCR transgenic CD4 T cells were transferred to responding CD45.2 B6 mice. 4C CD4 T cells engrafted to .4-.6% of total CD4 T cells while TCR75 CD4 T cells were essentially undetectable without allogeneic challenge. Mice were challenged with 1e6 BALB/c splenocytes in each footpad and draining popliteal lymph nodes and spleens were harvested from separate mice on days 3, 4, and 5. Proliferating cells (Ki67+) and TCR Vβ staining was used to distinguish direct 4C (Vβ13+) from indirect TCR75 (Vβ8.3+) cells. The frequency of CD45.1+ cells among total CD4+Ki67+ cells increased dramatically between day 3 and 5 (1.8 % to 27%). Importantly, the percentage of direct 4C cells within CD4+Ki67+CD45.1+ cells decreased from 94% on day 3 to 50% on day 5. Meanwhile, the percentage of indirect TCR75 cells in increased from 0 on day 3 to 50% on day 5. Despite having nearly undetectable indirect T cells prior to stimulation, this response rapidly increases and approximates the absolute magnitude of the direct response during the primary response. Thus, while the biologic significance of the indirect pathway of donor recognition has been increasingly appreciated, these results indicate that the indirect pathway may actually comprise a dramatic proportion of the primary alloresponse in vivo.

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