The contributions of Dr. Hilliard Seigler to the founding of the Duke kidney transplantation program were considerable in both surgery and immunology. Some of these highlights are summarized based upon interviews with Dr. Seigler by the authors.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

Optimizing natural killer (NK) cell alloreactivity could further improve outcome after allogeneic hematopoietic cell transplantation (alloHCT). The donor\'s Killer-cell Immunoglobulin-like Receptor (KIR) genotype may provide important information in this regard. In the past decade, different models have been proposed aiming at maximizing NK cell activation by activating KIR-ligand interactions or minimizing inhibitory KIR-ligand interactions. Alternative classifications intended predicting outcome after alloHCT by donor KIR-haplotypes. In the present study, we aimed at validating proposed models and exploring more classification approaches. To this end, we analyzed samples stored at the Collaborative Biobank from HLA-compatible unrelated stem cell donors who had donated for patients with acute myeloid leukemia (AML) or myelodysplastic neoplasm (MDS) and whose outcome data had been reported to EBMT or CIBMTR. The donor KIR genotype was determined by high resolution amplicon-based next generation sequencing. We analyzed data from 5,017 transplants. The median patient age at alloHCT was 56 years. Patients were transplanted for AML between 2013 and 2018. Donor-recipient pairs were matched for HLA-A, -B, -C, -DRB1, and -DQB1 (79%) or had single HLA mismatches. Myeloablative conditioning was given to 56% of patients. Fifty-two percent of patients received anti-thymocyte-globulin-based graft-versus-host disease prophylaxis, 32% calcineurin-inhibitor-based prophylaxis, and 7% post-transplant cyclophosphamide-based prophylaxis. We tested several previously reported classifications in multivariable regression analyses but could not confirm outcome associations. Exploratory analyses in 1,939 patients (39%) who were transplanted from donors with homozygous centromeric (cen) or telomeric (tel) A or B motifs, showed that the donor cen B/B-tel A/A diplotype was associated with a trend to better event-free survival (HR 0.84, p=.08) and reduced risk of non-relapse mortality (NRM) (HR 0.65, p=.01). When we further dissected the contribution of B subtypes, we found that only the cen B01/B01-telA/A diplotype was associated with a reduced risk of relapse (HR 0.40, p=.04) while all subtype combinations contributed to a reduced risk of NRM. This exploratory finding has to be validated in an independent data set. In summary, the existing body of evidence is not (yet) consistent enough to recommend use of donor KIR genotype information for donor selection in routine clinical practice.

Prolonging the lifespan of transplanted organs is critical to combat the shortage of this life-saving resource. Chronic rejection, with irreversible demise of the allograft, is often caused by the development of donor-specific HLA antibodies. Currently, enumerating molecular (amino acid) mismatches between recipient and donor is promoted to identify patients at higher risk of developing HLA antibodies, for use in organ allocation, and immunosuppression-minimization strategies. We have counseled against the incorporation of such approaches into clinical use and hypothesized that not all molecular mismatches equally contribute to generation of donor-specific immune responses. Herein, we document statistical shortcomings in previous study design: for example, use of individuals who lack the ability to generate donor-specific-antibodies (HLA identical) as part of the negative cohort. We provide experimental evidence, using CRISPR-Cas9-edited cells, to rebut the claim that the HLAMatchmaker eplets represent \"functional epitopes.\" We further used unique sub-cohorts of patients, those receiving an allograft with two HLA-DQ mismatches yet developing antibodies only to one mismatch (2MM1DSA), to interrogate differential immunogenicity. Our results demonstrate that mismatches of DQα05-heterodimers exhibit the highest immunogenicity. Additionally, we demonstrate that the DQα chain critically contributes to the overall qualities of DQ molecules. Lastly, our data proposes that an augmented risk to develop donor-specific HLA-DQ antibodies is dependent on qualitative (evolutionary and functional) divergence between recipient and donor, rather than the mere number of molecular mismatches. Overall, we propose an immunological mechanistic rationale to explain differential HLA-DQ immunogenicity, with potential ramifications for other pathological processes such as autoimmunity and infections.

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The innate and adaptive immune responses elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection are known to be poor. This study investigates the impact of PRRSV-induced transforming growth factor beta 1 (TGFβ1) on the expressions of type I and II interferons (IFNs), transcription factors, major histocompatibility complexes (MHC), anti-inflammatory and pro-inflammatory cytokines in PRRSV-infected co-cultures of monocytes and peripheral blood lymphocytes (PBL). Phosphorothioate-modified antisense oligodeoxynucleotide (AS ODN) specific to the AUG region of porcine TGFβ1 mRNA was synthesized and successfully knocked down TGFβ1 mRNA expression and protein translation. Monocytes transfected with TGFβAS1 ODN, then simultaneously co-cultured with PBL and inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) showed a significant reduction in TGFβ1 mRNA expression and a significant increase in the mRNA expressions of IFNα, IFNγ, MHC-I, MHC-II, signal transducer and activator of transcription 1 (STAT1), and STAT2. Additionally, transfection of TGFβAS1 ODN in the monocyte and PBL co-culture inoculated with cPRRSV-2 significantly increased the mRNA expression of interleukin-12p40 (IL-12p40). PRRSV-2 RNA copy numbers were significantly reduced in monocytes and PBL co-culture transfected with TGFβAS1 ODN compared to the untransfected control. The yields of PRRSV-2 RNA copy numbers in PRRSV-2-inoculated monocytes and PBL co-culture were sustained and reduced by porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα), respectively. These findings highlight the strategy employed by PRRSV to suppress the innate immune response through the induction of TGFβ expression. The inclusion of TGFβ as a parameter for future PRRSV vaccine and vaccine adjuvant candidates is recommended.

BACKGROUND: Pediatric heart transplant (HT) candidates experience high waitlist mortality due to a limited donor pool that is constrained in part by anti-HLA sensitization. We evaluated the impact of CDC and Flow donor-specific crossmatch (XM) results on pediatric HT outcomes.
METHODS: All pediatric HTs between 1999 and 2019 in the OPTN database were included. Donor-specific XM results were sub-categorized based on CDC and Flow results. Primary outcomes were treated rejection in the first year and time to death or allograft loss. Propensity scores were utilized to adjust for differences in baseline characteristics.
RESULTS: A total of 4,695 pediatric HT patients with T-cell XM data were included. After propensity score adjustment, a positive T-cell CDC-XM was associated with 2 times higher odds of treated rejection (OR 2.29 (1.56, 3.37)) and shorter time to death/allograft loss (HR 1.50 (1.19, 1.88)) compared to a negative Flow-XM. HT recipients who were Flow-XM positive with negative/unknown CDC-XM did not have higher odds of rejection or shorter time to death/allograft loss. An isolated positive B-cell XM was also not associated with worse outcomes. Over the study period XM testing shifted from CDC- to Flow-based assays.
CONCLUSIONS: A positive donor-specific T-cell CDC-XM was associated with rejection and death/allograft loss following pediatric HT. This association was not observed with a positive T-cell Flow-XM or B-cell XM result alone. The shift away from performing the CDC-XM may result in loss of important prognostic information unless the clinical relevance of quantitative Flow-XM results on heart transplant outcomes is systematically studied.

The international high-resolution external proficiency testing (EPT) started in 2004 with high-resolution typing of human leucocyte antigen (HLA) class I (HLA-A,B,C) and HLA class II (HLA-DRB1, DRB345, DQB1, and DPB1) alleles, since possibilities for such an EPT within Europe were limited and all existing EPTs at that time made use of the comparison of HLA typing results without a reference. This EPT was set up as a collaboration between the HLA laboratory of Leiden, providing DNA samples to the participants, and the laboratory of Maastricht, performing the high-resolution typing as the reference result and evaluating the results of all participants according to the prevailing European Federation for Immunogenetics (EFI) standards. Once a year, 12 samples were sent to the participating laboratories, and evaluation and certificates were provided at the end of that same year. During the years, the EPT was extended to low-resolution HLA class I and II typing, high-resolution typing including DQA1 and DPA1, and allelic resolution typing for HLA class I, the latter one being unique in this field. Evaluation of the high-resolution typing results of the last 19 years showed a clear increase in the number of loci tested by the participating laboratories and a clear change of method from Sanger sequencing with additional other techniques (SSO/SSP) to the nowadays widely used next-generation sequencing method. By strictly using the EFI rules for high-resolution HLA typing, the participants were made aware of the ambiguities within exons 2 and 3 for class I and exon 2 for class II and the presence of null alleles even in a two-field HLA typing. There was an impressive learning curve, resulting in >98% correctly typed samples since 2017 and a 100% fulfillment of EFI rules for all laboratories for all loci submitted in the last 2 years. Overall, this EPT meets the need of an EPT for high-resolution typing for EFI accreditation.

Allogeneic transplantation is a multi-step process involving many clinicians and laboratory personnel working together to achieve a common goal-to maximize the recipients\' chance of survival and to improve their quality of life. One of the key elements of the process is to ensure high quality, accuracy, and reliability of histocompatibility testing. This manuscript presents: the development and organizational principles of the national system of supervision and control of histocompatibility laboratories in Poland, problems faced by these laboratories, availabe proficiency testing schemes, as well as suggestions and prospects for the future raised by members of the Polish histocompatibility community.

BACKGROUND: Pediatric (age < 18 years) kidney transplant (KT) candidates face increasingly complex choices. The 2014 kidney allocation system nearly doubled wait times for pediatric recipients. Given longer wait times and new ways to optimize compatibility, more pediatric candidates may consider kidney-paired donation (KPD). Motivated by this shift and the potential impact of innovations in KPD practice, we studied pediatric KPD procedures in the US from 2008 to 2021.
METHODS: We describe the characteristics and outcomes of pediatric KPD recipients with comparison to pediatric non-KPD living donor kidney transplants (LDKT), pediatric LDKT recipients, and pediatric deceased donor (DDKT) recipients.
RESULTS: Our study cohort includes 4987 pediatric DDKTs, 3447 pediatric non-KPD LDKTs, and 258 pediatric KPD transplants. Fewer centers conducted at least one pediatric KPD procedure compared to those that conducted at least one pediatric LDKT or DDKT procedure (67, 136, and 155 centers, respectively). Five centers performed 31% of the pediatric KPD transplants. After adjustment, there were no differences in graft failure or mortality comparing KPD recipients to non-KPD LDKT, LDKT, or DDKT recipients.
CONCLUSIONS: We did not observe differences in transplant outcomes comparing pediatric KPD recipients to controls. Considering these results, KPD may be underutilized for pediatric recipients. Pediatric KT centers should consider including KPD in KT candidate education. Further research will be necessary to develop tools that could aid clinicians and families considering the time horizon for future KT procedures, candidate disease and histocompatibility characteristics, and other factors including logistics and donor protections.