Defining the Alloreactive T Cell Repertoire Using High-Throughput Sequencing of Mixed Lymphocyte Reaction Culture

Leventhal JR (2014) Defining the Alloreactive T Cell Repertoire Using High-Throughput Sequencing of Mixed Lymphocyte Reaction Culture. PLoS ONE 9(11): e111943. doi:10.1371/journal.pone.0111943 Defining the Alloreactive T Cell Repertoire Using High-Throughput Sequencing of Mixed Lymphocyte Reaction Culture Ryan O. Emerson 0 James M. Mathew 0 Iwona M. Konieczna 0 Harlan S. Robins 0 Joseph R. Leventhal 0 Jay Reddy, University of Nebraska-Lincoln, United States of America 0 1 Adaptive Biotechnologies Corporation , Seattle , Washington, United States of America, 2 Department of Surgery, Comprehensive Transplant center, Northwestern University , Chicago , Illinois, United States of America, 3 Department of Microbiology-Immunology, Northwestern University , Chicago , Illinois, United States of America, 4 Public Health Sciences Division, Fred Hutchinson Cancer Research Center , Seattle, Washington , United States of America The cellular immune response is the most important mediator of allograft rejection and is a major barrier to transplant tolerance. Delineation of the depth and breadth of the alloreactive T cell repertoire and subsequent application of the technology to the clinic may improve patient outcomes. As a first step toward this, we have used MLR and high-throughput sequencing to characterize the alloreactive T cell repertoire in healthy adults at baseline and 3 months later. Our results demonstrate that thousands of T cell clones proliferate in MLR, and that the alloreactive repertoire is dominated by relatively high-abundance T cell clones. This clonal make up is consistently reproducible across replicates and across a span of three months. These results indicate that our technology is sensitive and that the alloreactive TCR repertoire is broad and stable over time. We anticipate that application of this approach to track donor-reactive clones may positively impact clinical management of transplant patients. - . These authors contributed equally to this work. Cellular immune response is the most important mediator of transplant rejection and a major barrier to transplant tolerance [1 3]. It is largely mediated by memory T cell populations specific for allo-peptides presented either on allo-MHC (direct antigen presentation) or on self-MHC (indirect antigen presentation) [3 5]. Positive selection in the thymus requiring immature T cells to have some binding affinity for self-HLA means that a significant proportion of mature T cells also have off-target specificity for alloHLA alleles. Negative selection removes T cells specific for selfpeptides presented on self-HLA, buts leaves T cells specific for selfpeptides presented on allo-HLA [612]. The production of the alloreactive T cell repertoire is further complicated by molecular mimicry. Thus, in one well-studied example a public T cell response specific to EBV in the context of HLA-B*08:01 has been shown to exhibit cross-reactivity with a self-peptide presented by HLA-B*44:02 [1316]. These cross-reactive T cells have been observed in HLA-B*08:01/HLA-B*44:02 mismatched lung allografts, suggesting direct clinical relevance for this mode of T cell alloreactivity [17]. Even in individuals with no history of allo-HLA sensitization, viral exposure or vaccine administration can create HLA cross-reactive memory T cells [1822]. Many studies have identified public and private alloreactive T cell clones that can be primed by a variety of immunogenic events. However, while public T cell clones may play an important role in specific exposures they represent a very small proportion of the entire T cell repertoire; investigating private T cell specificities allows for a much broader view of the alloreactive T cell repertoire but private T cell responses must be measured anew in each subject. It is our hypothesis that the alloreactive T cell repertoire can be studied by performing mixed lymphocyte reaction cultures [23,24], followed by molecular analysis of clonotypes thus generated. The availability of high-throughput sequencing of rearranged T cell receptor genes, which act as unique molecular tags for each clonal population, now allows for unprecedented depth and accuracy in the characterization of T cell repertoires. Here, we employ this high-throughput TCR sequencing to test our hypothesis by thoroughly interrogating the alloreactive T cell repertoire between three pairs of healthy adult subjects as well as the persistence of alloreactive T cell clones across biological replicates and across time. Figure 1. Experimental design. We assayed three pairs of healthy adult subjects using mixed lymphocyte reaction cultures. For each pair, lymphocytes from a responder subject were mixed with inactivated lymphocytes from a stimulator subject and cultured in duplicate. Uncultured freshly isolated PBMC from the responder as well as proliferating T cell populations from the duplicate cultures were subjected to high-throughput sequencing: we sequenced nine samples in total across the three pairs of subjects. Three months later, the experiments were repeated to generate nine more samples for high-throughput TCRb sequencing. doi:10.1371/journal.pone.0111943.g001 Subjects Human peripheral blood samples were obtained from laboratory volunteers under a protocol following written informed consent approved and supervised by a Northwestern University Institutional Review Board. These healthy volunteers were HLAtyped by the Northwestern HLA laboratory using molecular methods (reverse sequence specific oligonucleotide probe hybridization). Mixed Lymphocyte Reaction (MLR) Culture and Alloreactive Responding Cell Isolation Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque. The responder cells were labeled with CFSE and the stimulator cells labeled with PKH26 as described previously [25,26]. The responders and stimulators were matched for 1 HLADR antigen to mimic the minimum requirement for some clinical transplants [27]. The PKH26 labeled stimulator cells were also irradiated at 3000 rads. The responder and stimulator cells were cultured in bulk in 15% normal AB serum containing RPMI 1640 culture medium (NAB-CM) at 16106/ml each. After 7 days these were harvested and the proliferating responders were then sorted on FACSAria (BD, San Jose, CA) by gating on the CFSE dim or negative cells after gating out both CFSE high non-proliferating and the very few PKH26+ stimulator cells that still survived. In parallel, flow cytometric analysis of the above MLR cultures was performed to determine which subsets of responder cells proliferated in response to allostimulation, using fluorochrome conjugated monoclonal antibodies. The data were acquired on an FC500 flow cytometer (Beckman-Coulter) and analyzed for cell subsets by gating on the CFSE dim or negative cells after gating out both CFSE high non-proliferating and the very few PKH26+ stimulator cells [25,26]. Additionally, standard 7-day 3H-thymidine incorporation ass (...truncated)