Quantifying the Size and Diversity of the Human Alloresponse Via High-Throughput T Cell Receptor Sequencing

Alloreactive T lymphocytes are the primary mediators of the immune response in transplantation, both in the graft-versus-host and host-versus graft directions; however, the large size and diversity of the alloreactive T cell population has presented a great challenge for quantitative analysis of this important response. We have therefore developed and validated a method for defining the biologically-relevant alloreactive T cell repertoire for any HLA-mismatched human responder-stimulator pair by combining the in vitro mixed lymphocyte reaction with high-throughput T cell receptor (TCR) sequencing. Here we use this method to characterize the normal human alloresponse (n = 9) at an unprecedented level of resolution. We provide the first quantification of alloreactive T cell repertoire diversity comparing CD4 and CD8 populations. In addition to standard techniques for comparing population diversity such as entropy, clonality, and R20 (fraction of clones accounting for the top 20 percent of reads), we have established a new approach for quantitative population diversity comparisons measuring power-law slopes (S) of circulating and alloreactive T cell populations, enabling the comparison of key repertoire information not captured in other analyses. Using S, we observe a significant increase in alloreactive repertoire diversity with increased HLA mismatches between responder and stimulator. In addition, we are able to quantitatively measure repertoire divergence, and have discovered that the alloreactive repertoire is highly allo-specific: the alloreactive repertoires generated by the same responder to two different stimulators are markedly distinct. However, when the stimulators share Class I HLA, we identify shared CD8 alloreactive clones. Perhaps most strikingly, we have found that although alloreactive clones are abundant, they circulate at such low frequencies that the majority are undetectable with deep sequencing. Cumulative CD4 and CD8 alloreactive clones by frequency detected in circulation account for 0.83% ± 0.73% and 0.76% ± 0.75%, respectively (mean ± SD). We have also developed a statistical model to estimate the frequency of undetected alloreactive clones, which added a total frequency of 1.5% ± 0.69% for CD4s and 0.53% ± 0.31% for CD8s. Lastly, we sought to identify clones known to be public viral-reactive clones to EBV, CMV, HSV, and Influenza A within our alloreactive T cell populations. Contrary to a common hypothesis that a large fraction of alloreactive clones are cross-reactive to viral antigens, very few public viral clones were found in our alloreactive repertoires in the presence of relevant responder HLA alleles. Taken together, high-throughput TCR sequencing yielded new insight into the alloimmune response, the key immune response in GVHD. Our approach has the potential to provide novel mechanistic information about the fate and frequency of alloreactive clones in hematopoietic stem cell transplantation, particularly in the haploidentical setting. Furthermore, our new analytic approaches including a model to extrapolate the circulating frequency of an population of "unseen" clones has a broad range of applications for TCR sequencing analysis in any setting in which only a sub-group of clones is identified within a circulating T cell population.