Dynamic Establishment Of Recipient Tissue Resident Memory T Cell’s TCR Repertoire In Human Intestinal Transplant Allografts

Purpose:Intestinal transplantation(ITx)is the only long-term treatment for end stage irreversible intestinal failure.The clinical outcome of ITx is limited by rejection and side-effects of immunosuppression such as increased infection rate and tumor occurrence rate.Among solid organ transplantations,liver transplantation has a median survival time of 8.5 years,heart transplantation has a median survival time of12 years,and kidney transplantation has a median survival time from 12 to 16 years while the median survival time of ITx is only 3.6 years.As an immunological barrier organ,intestine is highly enriched for tissue resident memory T cells(TRMs).TRMs are believed to persist in non-lymphoid tissue(NLT),performing significant roles in homeostasis maintain,infection control,and repair of tissues.However,the function of TRMs in organ transplantation is still not clear and the mechanism of TRMs’ taking part in rejection and post-Tx opportunistic infection is under investigated.As a result,studying the TRM repertoire reconstitution in intestinal transplant allografts is significant to understand the immune status and promote the clinical outcome of ITx patients.The research of human TRMs is limited by a lack of access to longitudinal human NLT samples.Fortunately,ITx provides a unique opportunity to study human TRM repertoire establishment,as serial surveillance biopsies are taken from ileal allografts post-Tx for clinical monitoring.Applying flow cytometry,researchers have found out that dynamic acquisition of TRM features,reflected by upregulation of CD69 and CD103 among graft repopulating recipient T cells after ITx and high levels of donor T cell chimerism in peripheral blood of ITx recipients(peak level ≥4% donor T cells,defined as “macrochimerism”)is associated with slower replacement of intestinal mucosal T cells by the recipient and reduced rejection rates.These phenomena are closely related to immune status of ITx patients.However,the dynamic establishment of recipient TRM repertoire post-ITx remains unclear yet.With specificity and diversity,the T cell receptor(TCR)has been used to identify and track unique T cells as the biometric fingerprint.We can reveal the mechanism of recipient TRM repertoire dynamic establishment in intestinal allograft mucosa by integrating high throughput TCR sequencing and flow cytometry.Methods:We collected pre-Tx samples from 16 ITx patients and gathered plenty of samples at multiple time points from different tissues through up to 5 years’ post-Tx follow-up.Donor and recipient T cells were divided by human leukocyte antigen specific monoclonal antibodies and TCR repertories of pre-and post-Tx samples were established by bulk TCR sequencing.Combining mixed lymphoid reaction and TCR sequencing,we distinguished the TCRs with donor antigen recognize potential.Combing flow cytometry data and TCR sequencing data,we investigated the velocity,cell resource,distribution,and antigen recognize ability of recipient TRM repertoires.Results:1.Newly detected TCRs continually reinforced the ileal mucosa TCR repertoire and finally contributed to a stable TCR repertoire.Both donor age ≥1 year and macrochimerism resulted in a delayed stable recipient TCR repertoire formation.2.Both TCRs that appeared in pre-Tx lymphoid tissue and TCRs that appeared in pre-Tx gut were detected in the post-Tx ileal mucosa TCR repertoires.The TCRs detected in both pre-Tx lymphoid tissue and gut were more likely to appear and persist in post-Tx ileal mucosa.3.Increased TCR repertoire crosstalk was found among paired blood samples and ileal samples post-ITx,compared to the ulcerative colitis and healthy control.4.Similar TCR repertoires were formatted in the native colon mucosa and allograft mucosa when vast majorities of donor T cells had been replaced by recipient T cells in ileal allograft.5.Not only donor antigen reactive TCRs but also microbe antigen reactive TCRs contributed to the post-Tx ileum mucosa TCR repertoire establishment.Conclusion:Recipient T cells derived from recipient lymphoid tissue and NLT with donor antigen recognition ability or microbe antigen recognition ability infiltrated into intestinal allograft mucosa from circulation and capture TRM features gradually,forming a relative stable recipient TRM repertoire in the entire gut post-ITx.This process was delayed in patients with macrochimerism and/or donor age ≥1 year.After the TRM repertoire established,some TRMs in that repertoire had re-circulating ability and T cells from circulation kept reinforcing the TRM repertoire,contributing to a dynamic stability.We revealed the dynamic establishment of recipient TRM repertoire in intestine allograft and related T cell origin,distribution,and antigen recognize function post-ITx on clonal level and promoted the understanding of human immune system in transplant settings.