Cytomegalovirus (CMV) infection is associated with allograft rejection but the mechanisms behind are poorly defined yet. Although cross-reactivity of T cells to alloantigen and CMV has been hypothesized, direct evidence in patients is lacking. In this observational cohort study, we tested the pre-transplant effector/memory T cell response to CMV peptide pools and alloantigen in 78 living donor/recipient pairs using the interferon-gamma Enzyme-Linked ImmunoSpot (ELISPOT) assay. To prove the hypothesis of cross-reactivity, we analyzed by applying next-generation sequencing the T cell receptor ß (TCR- ß) repertoire of CMV- and alloantigen-reactive T cells enriched from peripheral pre-transplant blood of 11 CMV-seropositive and HLA class I mismatched patients. Moreover, the TCR-repertoire was also analyzed in the allograft biopsies of those patients. There was a significant association between the presence of pre-transplant CMV immediate-early protein 1 (IE-1)-specific effector/memory T cells and acute renal allograft rejection and function (p = 0.01). Most importantly, we revealed shared TCR-ß sequences between CMV-IE1 and donor alloantigen-reactive T cells in all pre-transplant peripheral blood samples analyzed in CMV-seropositive patients who received HLA class I mismatched grafts. Identical TCR sequences were also found in particular in post-transplant allograft biopsies of patients with concomitant CMV infection and rejection. Our data show the presence of functional, cross-reactive T cells and their clonotypes in peripheral blood and in kidney allograft tissue. It is therefore likely that CMV-donor cross-reactivity as well as CMV specific T cell elicited inflammation is involved in the processes that affect allograft outcomes.

Table S1. Patient characteristics of the validation set AB0i (n = 18) HLAi (n = 9) P Type of donor, n (deceased, living) 0/18 9/0 < 0.001 Retransplantation, n (1 st /2 nd / 3 rd) 18/0/0 2/5/2/0 < 0.001 Recipient age, years 47 [24;68] 45 [21;78] 0.817 Recipient sex, male, n 11 8 GO terms upregulated in controls compared to the AB0i group Count Benjamini p value GO:0006952 defense response 33 1.44E-06 GO:0009611 response to wounding 28 3.15E-05 GO:0006954 inflammatory response 21 6.98E-05 GO:0006955 immune response 29 2.94E-04 GO:0050778 positive regulation of the immune response 13 7.49E-04 GO:0002684 positive regulation of the immune system process 16 1.13E-03 GO:0002253 activation of the immune response 10 2.94E-03 GO:0002526 acute inflammatory response 10 3.07E-03 GO:0006959 humoral immune response 9 4.50E-03 GO:0006956 complement activation 7 5.26E-03 GO terms upregulated in controls compared to the HLAi group Count Benjamini p value GO:0005578 proteinaceous extracellular matrix 46 7.95E-14

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Recipient sensitization is a major risk factor of antibody-mediated rejection (ABMR) and inferior graft survival. The predictive effect of solid-phase human leukocyte antigen antibody testing and flow cytometry crossmatch (FCXM) in the era of peritransplant desensitization remains poorly understood. This observational retrospective single-center study with 108 donor-specific antibody (DSA)-positive deceased donor kidney allograft recipients who had undergone peritransplant desensitization aimed to analyze variables affecting graft outcome. ABMR rates were highest among patients with positive pretransplant FCXM vs. FCXM-negative (76 vs. 18.7%, p &lt; 0.001) and with donor-specific antibody mean fluorescence intensity (DSA MFI) &gt; 5,000 vs. &lt;5,000 (54.5 vs. 28%, p = 0.01) despite desensitization. In univariable Cox regression, FCXM positivity, retransplantation, recipient gender, immunodominant DSA MFI, DSA number, and peak panel reactive antibodies were found to be associated with ABMR occurrence. In multivariable Cox regression adjusted for desensitization treatment (AUC = 0.810), only FCXM positivity (HR = 4.6, p = 0.001) and DSA number (HR = 1.47, p = 0.039) remained significant. In conclusion, our data suggest that pretransplant FCXM and DSA number, but not DSA MFI, are independent predictors of ABMR in patients who received peritransplant desensitization.