The cytokine transcription profiles of developing T helper 1 and T helper 2 cells are imprinted and induced appropriately following stimulation of differentiated cells. Epigenetic regulation combines several mechanisms to ensure the inheritance of transcriptional programs. We found that the expression of the polycomb group proteins, whose role in maintaining gene silencing is well documented, was induced during development in both T helper lineages. Nevertheless, the polycomb proteins, YY1, Mel-18, Ring1A, Ezh2, and Eed, bound to the Il4 and Ifng loci in a differential pattern. In contrast to the prevailing dogma, the binding activity of the polycomb proteins in differentiated T helper cells was associated with cytokine transcription. The polycomb proteins bound to the cytokine genes under resting conditions, and their binding was induced dynamically following stimulation. The recruitment of the polycomb proteins Mel-18 and Ezh2 to the cytokine promoters was inhibited in the presence of cyclosporine A, suggesting the involvement of NFAT. Considering their binding pattern at the cytokine genes and their known function in higher order folding of regulatory elements, we propose a model whereby the polycomb proteins, in some contexts, positively regulate gene expression by mediating long-distance chromosomal interactions.

| INTRODUC TI ONAdvances in HLA typing and HLA antibody testing over the past two decades transformed our ability to assess donor/recipient compatibility in the context of organ transplantation. Beginning from serologic donor/recipient HLA matching through emphasis on avoidance of preformed donor-specific HLA antibodies (DSAs), current approaches delve into amino acid sequences of HLA alleles, assessing the so-called molecular mismatch between donor and recipient, using one or more of the currently available tools: HLAMatchmaker, [1][2][3][4] Amino-Acid comparison, Electrostatic Mismatch Score (EMS 5-7 ), or PIRCHE-Predicted Indirectly ReCognizable HLA Epitopes presented by recipient HLA-Class II antigens. 8-10 A short description of each method is provided in the supplemental material.It is notable that only minor variations were found when comparing the ability of these different approaches to predict poor graft outcome (correlation ranging between R 2 of .85-.96). 8,11 In addition, when different patient populations were studied (eg, pediatric vs adult; kidney vs lung), different cutoff values were reported, 12-19 indicating the need Funding information Paul I Terasaki Research FoundationMolecular mismatch load analysis was recently introduced as a means for performing risk stratification following organ transplantation. However, although good correlation was demonstrated between molecular mismatch load and generation of de novo donor-specific HLA antibody (DSA), quite a few exceptions exist, and the underlying factors that define HLA immunogenicity remain unclear. Herein, we present a new paradigm to interrogate differences between molecular mismatches that lead to the generation of de novo DSA and those that do not (the 2MM1DSA cohort).Specifically, patients transplanted across 2 HLA-DQ mismatches, who formed de novo DSA only to one mismatch (foe) but not the other (friend), provide a unique environment in which patient-specific factors that affect the immune response other than immunogenicity, such as infection and immunosuppression, can be controlled for. It further permits focusing on mismatches uniquely exhibited by the de novo DSA allele, rather than mismatches shared by both DSA and non-DSA alleles. This concept paper illustrates several examples, highlights the need for center-specific or population-specific cutoff values for posttransplant risk stratification, and mostly argues that if there is no direct correlation between molecular mismatch load and immunogenicity, then molecular mismatch load must not be adopted as an approach for equitable organ allocation. K E Y W O R D Salloantibody, antigen presentation/recognition, clinical research/practice, histocompatibility, organ allocation

BackgroundFollowing antigen recognition, naive T helper (Th; CD4+) cells can differentiate toward one of several effector lineages such as Th1 and Th2; each expressing distinctive transcriptional profiles of cytokine genes. These cytokines eventually instruct the strategy of the immune response. In our search for factors that propagate the transcriptional programs of differentiated Th cells, we previously found that Polycomb group (PcG) proteins, which are known as epigenetic regulators that maintain repressive chromatin states, bind differentially the signature cytokine genes. Unexpectedly, their binding to the Ifng (Interferon-g) in Th1 cells and Il4 (Interleukin-4) in Th2 cells, was correlated with transcriptional activation. Therefore, in this study we aimed to determine the functional role of PcG proteins in the regulation of the expression of the signature cytokine genes.MethodsPcG proteins were knocked down in primary and established murine Th cells using transduction of lentiviruses encoding short hairpin RNAs (shRNAs) directed to Mel-18, Ezh2, Eed and Ring1A, representative of two different PcG complexes. The chromatin structure and the binding activity of PcG proteins and transcription factors at the Ifng promoter were assessed by chromatin immunoprecipitation (ChIP) assays.ResultsDownregulation of PcG proteins was consistent with their function as positive regulators of the signature cytokine genes in primary and established Th1 and Th2 cells. Moreover, the PcG protein Mel-18 was necessary to recruit the Th1-lineage specifying transcription factor T-bet, and the T cell receptor (TCR)-inducible transcription factor NFAT1 to the Ifng promoter in Th1 cells. Nevertheless, our results suggest that PcG proteins can function also as conventional transcriptional repressors in Th cells of their known target the Hoxa7 gene.ConclusionsOur data support a model whereby the non-differentially expressed PcG proteins are recruited in a Th-lineage specific manner to their target genes to enforce the maintenance of specific transcriptional programs as transcriptional repressors or activators. Although our results suggest a direct effect of PcG proteins in the regulation of cytokine gene expression, indirect functions cannot be excluded.

We have previously shown that in differentiated T-helper (Th)1 and Th2 cells, polycomb group (PcG) proteins are associated differentially with the promoters of the signature cytokine genes. The correlation of the binding activity of PcG proteins with gene expression is unusual, since they are well known as epigenetic regulators that maintain transcriptional silencing. Here we show that in Th17 cells, the more phenotypically flexible Th lineage, the PcG proteins Mel-18 and less strikingly Ezh2 are associated differentially with the Il17a promoter. Using the RNAi approach, we found that Mel-18 and Ezh2 positively regulate the expression of Il17a and Il17f. The inducible binding of Mel-18 and Ezh2 at the Il17a promoter was dependent on signaling pathways downstream of the TCR. However, a continuous presence of TGF-b, the cytokine that is necessary to maintain Il17a expression, was required to preserve the binding activity of Mel-18, but not of Ezh2, following restimulation. The binding of Mel-18 at the Il17a promoter was correlated with the recruitment of the lineage-specifying transcription factor RORct. Altogether, our results suggest that in Th17 cells the TCR and polarizing cytokines synergize to modulate the binding activity of Mel-18 at the Il17a promoter, and consequently to facilitate Il17a expression.

Background: Reduction of immunosuppression (IS) upon detection of Polyomavirus (BK) viremia is widely used to prevent BK virus nephropathy. This retrospective casecontrol study assesses the frequency of de novo donor-specific antibodies (dnDSA) in renal transplant recipients with IS modulation due to BK viremia and the associated risk of antibody mediated rejection. Methods: Our cohort included recipients of kidney transplantation between 2007 and 2017 with clinical, HLA antibody, and biopsy data. BK positivity was defined as viremia >10 000 c/ml or biopsy proven BK nephropathy. A total of 190 BK cases matched our inclusion criteria, each case was matched with two controls based on gender, donor type, and transplant within 1 year (N = 396).Results: Despite lower number of HLA antigen mismatches (mean = 3.5 vs. 4.4, p < .001), dnDSA rates were higher in BK cases than in control group (22.1% vs. 13.9%, p = .02), with the majority detected following IS reduction for BK infection, and arising earlier posttransplant compared with no BK infection (294d vs. 434d, p < .001). Antibody mediated rejection rates were similar between cases and controls (8.9% and 8.3%, respectively), but rejection was more likely to occur earlier posttransplant in the BK cases (354d vs. 602d, p = .03). Conclusion:Our data suggest a link between IS reduction and the generation of dnDSA and/or rejection, supporting close monitoring for DSA in patients with reduced IS due to BK infection given their increased risk to develop dnDSA.