Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1ß.

Interleukin 1 (IL-1) is an important mediator of innate immunity but can also promote inflammatory tissue damage. During chronic infections such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo, we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-γ (IFN-γ). This effect was mediated by nitric oxide (NO), which we found specifically inhibited assembly of the NLRP3 inflammasome via thiol nitrosylation. Our data indicate that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses elicited during persistent infections.

Intrathymic programming of effector fates in three molecularly distinct γδ T cell subtypes.

Innate γδ T cells function in the early phase of immune responses. Although innate γδ T cells have often been studied as one homogenous population, they can be functionally classified into effector subsets on the basis of the production of signature cytokines, analogous to adaptive helper T cell subsets. However, unlike the function of adaptive T cells, γδ effector T cell function correlates with genomically encoded T cell antigen receptor (TCR) chains, which suggests that clonal TCR selection is not the main determinant of the differentiation of γδ effector cells. A high-resolution transcriptome analysis of all emergent γδ thymocyte subsets segregated on the basis of use of the TCR γ-chain or δ-chain indicated the existence of three separate subtypes of γδ effector cells in the thymus. The immature γδ subsets were distinguished by unique transcription-factor modules that program effector function.

Late graft failure of pig-to-rhesus renal xenografts has features of glomerulopathy and recipients have anti-swine leukocyte antigen class I and class II antibodies.

Prolonged survival in preclinical renal xenotransplantation demonstrates that early antibody mediated rejection (AMR) can be overcome. It is now critical to evaluate and understand the pathobiology of late graft failure and devise new means to improve post xenograft outcomes. In renal allotransplantation the most common cause of late renal graft failure is transplant glomerulopathy-largely due to anti-donor MHC antibodies, particularly anti-HLA DQ antibodies. We evaluated the pig renal xenograft pathology of four long-surviving (>300 days) rhesus monkeys. We also evaluated the terminal serum for the presence of anti-SLA class I and specifically anti-SLA DQ antibodies. All four recipients had transplant glomerulopathy and expressed anti-SLA DQ antibodies. In one recipient tested for anti-SLA I antibodies, the recipient had antibodies specifically reacting with two of three SLA I alleles tested. These results suggest that similar to allotransplantation, anti-MHC antibodies, particularly anti-SLA DQ, may be a barrier to improved long-term xenograft outcomes.

Examining the Biosynthesis and Xenoantigenicity of Class II Swine Leukocyte Antigen Proteins.

Genetically engineered pig organs could provide transplants to all patients with end-stage organ failure, but Ab-mediated rejection remains an issue. This study examines the class II swine leukocyte Ag (SLA) as a target of epitope-restricted Ab binding. Transfection of individual α- and ß-chains into human embryonic kidney cells resulted in both traditional and hybrid class II SLA molecules. Sera from individuals on the solid organ transplant waiting list were tested for Ab binding and cytotoxicity to this panel of class II SLA single-Ag cells. A series of elution studies from an SLA-DQ cell line were performed. Our results indicate that human sera contain Abs specific for and cytotoxic against class II SLA. Our elution studies revealed that sera bind the SLA-DQ molecule in an epitope-restricted pattern. Site-specific mutation of one of these epitopes resulted in statistically decreased Ab binding. Humans possess preformed, specific, and cytotoxic Abs to class II SLA that bind in an epitope-restricted fashion. Site-specific epitope mutagenesis may decrease the Ab binding of highly sensitized individuals to pig cells.

Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion.

The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti-pig antibodies significantly improved survival in a pig-to-rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan-T cell depletion and an anti-CD154-based maintenance regimen. Selective depletion of CD4+ T cells but not CD8+ T cells resulted in long-term survival (median survival time >400 days vs 6 days). These studies suggested that CD4+ T cells may have a more prominent role in xenograft rejection compared with CD8+ T cells. Although animals that received selective depletion of CD8+ T cells showed signs of early cellular rejection (marked CD4+ infiltrates), animals receiving selective CD4+ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4+ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti-pig antibody and CD4 depletion resulted in consistent, long-term xenograft survival.

Examining epitope mutagenesis as a strategy to reduce and eliminate human antibody binding to class II swine leukocyte antigens.

Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.

Xenoantigen Deletion and Chemical Immunosuppression Can Prolong Renal Xenograft Survival.

OBJECTIVE: Xenotransplantation using pig organs could end the donor organ shortage for transplantation, but humans have xenoreactive antibodies that cause early graft rejection. Genome editing can eliminate xenoantigens in donor pigs to minimize the impact of these xenoantibodies. Here we determine whether an improved cross-match and chemical immunosuppression could result in prolonged kidney xenograft survival in a pig-to-rhesus preclinical model. METHODS: Double xenoantigen (Gal and Sda) knockout (DKO) pigs were created using CRISPR/Cas. Serum from rhesus monkeys (n = 43) was cross-matched with cells from the DKO pigs. Kidneys from the DKO pigs were transplanted into rhesus monkeys (n = 6) that had the least reactive cross-matches. The rhesus recipients were immunosuppressed with anti-CD4 and anti-CD8 T-cell depletion, anti-CD154, mycophenolic acid, and steroids. RESULTS: Rhesus antibody binding to DKO cells is reduced, but all still have positive CDC and flow cross-match. Three grafts were rejected early at 5, 6, and 6 days. Longer survival was achieved in recipients with survival to 35, 100, and 435 days. Each of the 3 early graft losses was secondary to IgM antibody-mediated rejection. The 435-day graft loss occurred secondary to IgG antibody-mediated rejection. CONCLUSIONS: Reducing xenoantigens in donor pigs and chemical immunosuppression can be used to achieve prolonged renal xenograft survival in a preclinical model, suggesting that if a negative cross-match can be obtained for humans then prolonged survival could be achieved.

Efficient generation of targeted and controlled mutational events in porcine cells using nuclease-directed homologous recombination.

BACKGROUND: Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. METHODS: Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by Sanger sequence, restriction enzyme digest and flow cytometry. RESULTS: Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events. Single-strand DNA was twice as efficient as double-strand DNA. Furthermore, nuclease-mediated insertion by homology-directed repair successfully drove locus-specific transgene expression in the porcine genome. CONCLUSIONS: A nonintegrating selection strategy based on fluorescence expression can increase the mutational efficiency of the CRISPR/Cas9 system. The precision of this system can be increased by the addition of a very short homologous template sequence and can serve as a method for locus-specific transgene delivery. Together these strategies may be used to efficiently control mutational events. This system may be used to better use the potential of nuclease-mediated genomic editing.

Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles: an alternative to direct complement regulation.

UNLABELLED: The future of solid organ transplantation is challenged by an increasing shortage of available allografts. Xenotransplantation of genetically modified porcine organs offers an answer to this problem. Strategies of genetic modification have 'humanized' the porcine model towards clinical relevance. Most notably, these approaches have aimed at either antigen reduction or human transgene expression. The object of this study was to evaluate the relative effects of both antigen reduction and direct complement regulation on the human-anti-porcine complement dependent cytotoxicity response. Genetically modified animals were created through CRISPR/Cas9-directed mutation and human transgene delivery. Pigs doubly deficient in GGTA1 and CMAH genes were compared to pigs of the same background that expressed a human complement regulatory protein (hCRP). A third animal was made deficient in GGTA1, CMAH and B4GalNT2 gene expression. Cells from these animals were subjected to measures of human antibody binding and antibody-mediated complement-dependent cytotoxicity by flow cytometry. Human IgG and IgM antibody binding was unchanged between the double knockout and the transgenic hCRP double knockout pig. IgG and IgM binding was reduced by 49.1 and 43.2 % respectively by silencing the B4GalNT2 gene. Compared to the double knockout, human anti-porcine cytotoxicity was reduced by 8 % with the addition of a hCRP (p = .032); It was reduced by 21 % with silencing the B4GalNT2 gene (p = .012). CONCLUSIONS: Silencing the GGTA1, CMAH and B4GalNT2 genes in pigs achieved a significant antigen reduction. Changing the porcine carbohydrate profile effectively mediates human antibody-mediated complement dependent cytoxicity.

Silencing the porcine iGb3s gene does not affect Galα3Gal levels or measures of anticipated pig-to-human and pig-to-primate acute rejection.

BACKGROUND: The Galα(1,3)Gal epitope (α-GAL), created by α-1,3-glycosyltransferase-1 (GGTA1), is a major xenoantigen causing hyperacute rejection in pig-to-primate and pig-to-human xenotransplantation. In response, GGTA1 gene-deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α-Gal epitope expressed in GGTA1(-/-) pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo-series glycosphingolipids with an α-GAL-terminal disaccharide (iGb3), creating the possibility that iGb3s may be a source of α-GAL epitopes in GGTA1(-/-) animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig-to-primate and pig-to-human immune cross-reactivity by creating and comparing GGTA1(-/-) pigs to GGTA1(-/-) - and A3GalT2(-/-) -double-knockout pigs. METHODS: We used the CRISPR/Cas 9 system to target the GGTA1 and A3GalT2 genes in pigs. Both GGTA1 and A3GalT2 genes are functionally inactive in humans and baboons. CRISPR-treated cells used directly for somatic cell nuclear transfer produced single- and double-gene-knockout piglets in a single pregnancy. Once grown to maturity, the glycosphingolipid profile (including iGb3) was assayed in renal tissue by normal-phase liquid chromatography. In addition, peripheral blood mononuclear cells (PBMCs) were subjected to (i) comparative cross-match cytotoxicity analysis against human and baboon serum and (ii) IB4 staining for α-GAL/iGb3. RESULTS: Silencing of the iGb3s gene significantly modulated the renal glycosphingolipid profile and iGb3 was not detected. Moreover, the human and baboon serum PBMC cytotoxicity and α-GAL/iGb3 staining were unchanged by iGb3s silencing. CONCLUSIONS: Our data suggest that iGb3s is not a contributor to antibody-mediated rejection in pig-to-primate or pig-to-human xenotransplantation. Although iGb3s gene silencing significantly changed the renal glycosphingolipid profile, the effect on Galα3Gal levels, antibody binding, and cytotoxic profiles of baboon and human sera on porcine PBMCs was neutral.

The fate of human platelets exposed to porcine renal endothelium: a single-pass model of platelet uptake in domestic and genetically modified porcine organs.

BACKGROUND: Thrombocytopenia may represent a significant challenge to the clinical application of solid-organ xenotransplantation. When studied in a pig-to-primate model, consumptive coagulopathy has challenged renal xenografts. New strategies of genetic manipulation have altered porcine carbohydrate profiles to significantly reduce human antibody binding to pig cells. As this process continues to eliminate immunologic barriers to clinical xenotransplantation, the relationship between human platelets and pig organs must be considered. METHODS: Genetically modified pigs that were created by the CRISPR/Cas9 system with α-1,3-galactosyltransferase (GGTA1)(-/-) or GGTA1(-/-) cytidine monophosphate-N-acetylneuraminic acid hydroxylase(-/-) phenotype, as well as domestic pigs, were used in this study. Autologous porcine platelets were isolated from donor animal blood collection, and human platelets were obtained from a blood bank. Platelets were fluorescently labeled and in a single-pass model, human, or autologous platelets were perfused through porcine organs at a constant concentration and controlled temperature. Platelet uptake was measured by sampling venous output and measuring sample florescence against input florescence. In vitro study of the interaction between human platelets and porcine endothelial cells was accomplished by immunohistochemical stain and confocal microscopy. RESULTS: Differences between human and autologous platelet loss through the porcine kidney were not significant in any genetic background tested (WT P = 0.15, GGTA1(-/-)P = 0.12, GGTA1(-/-) cytidine monophosphate-N-acetylneuraminic acid hydroxylase(-/-)P = 0.25). The unmodified porcine liver consumed human platelets in a single-pass model of platelet perfusion in fewer than 10 min. WT suprahepatic inferior vena cava fluoresce reached a maximum of 76% of input fluoresce within the human platelet cohort and was significantly lower than the autologous platelet control cohort (P = 0.001). Confocal microscopic analysis did not demonstrate a significant association between human platelets and porcine renal endothelial cells compared with porcine liver endothelial positive controls. CONCLUSIONS: Our results suggest that in the absence of immunologic injury, human platelets respond in a variable fashion to organ-specific porcine endothelial surfaces. Human platelets are not removed from circulation by exposure to porcine renal endothelium but are removed by unmodified porcine hepatic endothelium. Kidneys possessing genetic modifications currently relevant to clinical xenotransplantation failed to consume human platelets in an isolated single-pass model. Human platelets did not exhibit significant binding to renal endothelial cells by in vitro assay.

Chromatin decondensation and T cell hyperresponsiveness in diabetes-associated hyperglycemia.

Diabetes is linked to increased inflammation and susceptibility to certain infectious diseases including tuberculosis (TB). We previously reported that aerosol TB in mice with chronic (≥ 12 wk) hyperglycemia features increased bacterial load, overproduction of several cytokines, and increased immune pathology compared with normoglycemic controls. A similar phenotype exists in human patients with diabetes with TB. The mechanisms of increased T cell activation in diabetes are unknown. In the current study, we tested the hypothesis that hyperglycemia modifies the intrinsic responsiveness of naive T cells to TCR stimulation. Purified T cells from chronically hyperglycemic (HG) mice produced higher levels of Th1, Th2, and Th17 cytokines and proliferated more than T cells from normoglycemic controls after anti-CD3e or Ag stimulation. In this way, naive T cells from HG mice resembled Ag-experienced cells, although CD44 expression was not increased. Chromatin decondensation, another characteristic of Ag-experienced T cells, was increased in naive T cells from HG mice. That phenotype depended on expression of the receptor for advanced glycation end products and could be reversed by inhibiting p38 MAPK. Chromatin decondensation and hyperresponsiveness to TCR stimulation persisted following transfer of T cells from HG mice into normoglycemic mice. We propose that chronic hyperglycemia causes receptor for advanced glycation end products-mediated epigenetic modification of naive T cells leading to p38 MAPK-dependent chromatin decondensation. This preactivation state facilitates transcription factor access to DNA, increasing cytokine production and proliferation following TCR stimulation. This mechanism may contribute to pathological inflammation associated with diabetes and might offer a novel therapeutic target.

Robotic performance metrics model fellow proficiency in living donor nephrectomy.

We investigated the use of robotic objective performance metrics (OPM) to predict number of cases to proficiency and independence among abdominal transplant fellows performing robot-assisted donor nephrectomy (RDN). 101 RDNs were performed by 5 transplant fellows from September 2020 to October 2023. OPM included fellow percent active control time (%ACT) and handoff counts (HC). Proficiency was defined as ACT ≥ 80% and HC ≤ 2, and independence as ACT ≥ 99% and HC ≤ 1. Case number was significantly associated with increasing fellow %ACT, with proficiency estimated at 14 cases and independence at 32 cases (R2 = 0.56, p < 0.001). Similarly, case number was significantly associated with decreasing HC, with proficiency at 18 cases and independence at 33 cases (R2 = 0.29, p < 0.001). Case number was not associated with total active console time (p = 0.91). Patient demographics, operative characteristics, and outcomes were not associated with OPM, except for donor estimated blood loss (EBL), which positively correlated with HC. Abdominal transplant fellows demonstrated proficiency at 14-18 cases and independence at 32-33 cases. Total active console time remained unchanged, suggesting that increasing fellow autonomy does not impede operative efficiency. These findings may serve as a benchmark for training abdominal transplant surgery fellows independently and safely in RDN.

A novel assessment model for teaching robot-assisted living donor nephrectomy in abdominal transplant surgery fellowship.

BACKGROUND: An increasing number of transplant centers have adopted robot-assisted living donor nephrectomy. Thus, a transplant fellow assessment tool is needed for promoting operative independence in an objective and safe manner. METHODS: In this pilot study, data was prospectively collected on both fellow performance with focus on technique, efficiency, and communication ("overall RO-SCORE"), and operative steps ("operative steps RO-SCORE"). Robotic user performance metrics were analyzed from the da Vinci Xi system, including fellow percent active control time (ACT) and handoff counts. RESULTS: From July 2020 to February 2021, twenty-one robot-assisted donor nephrectomies were performed. In regression analysis, fellow performance (based on both RO-SCOREs and robot % ACT) was significantly associated with both time and case number, with time-to-independence modelled at 8.4-14.2 months, and case number-to-independence estimated at 15-22 cases. Robot user metrics provided valid objective measures alongside RO-SCOREs. CONCLUSIONS: This pilot study provides an effective assessment tool for promoting operative competency in robot-assisted donor nephrectomy among transplant fellows.

Diabetic mice display a delayed adaptive immune response to Mycobacterium tuberculosis.

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. We previously reported that streptozotocin-induced DM impaired TB defense in mice, resulting in higher pulmonary bacterial burden, more extensive inflammation, and higher expression of several proinflammatory cytokines known to play a protective role in TB. In the current study, we tested the hypothesis that DM leads to delayed priming of adaptive immunity in the lung-draining lymph nodes (LNs) following low dose aerosol challenge with virulent Mycobacterium tuberculosis. We show that M. tuberculosis-specific IFN-gamma-producing T cells arise later in the LNs of diabetic mice than controls, with a proportionate delay in recruitment of these cells to the lung and stimulation of IFN-gamma-dependent responses. Dissemination of M. tuberculosis from lung to LNs was also delayed in diabetic mice, although they showed no defect in dendritic cell trafficking from lung to LNs after LPS stimulation. Lung leukocyte aggregates at the initial sites of M. tuberculosis infection developed later in diabetic than in nondiabetic mice, possibly related to reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5 at early time points postinfection. We conclude that TB increased susceptibility in DM results from a delayed innate immune response to the presence of M. tuberculosis-infected alveolar macrophages. This in turn causes late delivery of Ag-bearing APC to the lung draining LNs and delayed priming of the adaptive immune response that is necessary to restrict M. tuberculosis replication.

Initiation of acquired immunity in the lungs of mice lacking lymph nodes after infection with aerosolized Mycobacterium tuberculosis.

Recent evidence points to lung draining lymph nodes as the site that initiates the immune response in mice infected with aerosolized Mycobacterium tuberculosis. Here we expanded these studies and showed that infection of mice that lack lymph nodes with aerosolized M. tuberculosis results in a massive mononuclear cell infiltrate in the lungs within 14 days postinfection. This infiltration clearly resembles an expansion of the bronchus-associated lymphoid tissue. As expected, no bronchus-associated lymphoid tissue was observed in M. tuberculosis-infected wild-type control mice. Importantly, acquired specific immune response to M. tuberculosis antigens could be detected in lung lymphocytes harvested from mice lacking lymph nodes as early as 14 days postinfection. In addition, the bacterial burden in these mice was indistinguishable from that observed in wild-type C57BL/6 control mice. These results indicate that in the absence of lymph nodes, priming of the immune response occurs in the lung tissues after infection of mice with aerosolized M. tuberculosis and clearly illustrate the enormous plasticity of the immune system to develop resistance to foreign pathogens.

CD4 T-cell-mediated heterologous immunity between mycobacteria and poxviruses.

The bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis is used in many parts of the world as a vaccine against Mycobacterium tuberculosis. Some epidemiological evidence has suggested that BCG immunization may have unpredicted effects on resistance to other pathogens. We show here in a mouse model that BCG immunization followed by antibiotic treatment to clear the host of the pathogen rendered three strains of mice partially resistant to infection with vaccinia virus (VV) but not to lymphocytic choriomeningitis virus (LCMV). VV-challenged BCG-immune mice developed a striking splenomegaly and elevated CD4 and CD8 T-cell responses by 6 days postinfection (p.i.). However, resistance to VV infection could be seen as early as 1 to 2 days p.i. and was lost after antibody depletion of CD4 T-cell populations. BCG- but not LCMV-immune memory phenotype CD4 T cells preferentially produced gamma interferon (IFN-gamma) in vivo after VV challenge. In contrast, LCMV-immune CD8 T cells preferentially produced IFN-gamma in vivo in response to VV infection. In BCG-immune mice the resistance to VV infection and VV-induced CD4 T-cell IFN-gamma production were ablated by cyclosporine A, which inhibits signaling through the T-cell receptor. This study therefore demonstrates CD4 T-cell-mediated heterologous immunity between a bacterium and virus. Further, it poses the question of whether BCG immunization of humans alters resistance to unrelated pathogens.

HLA Class I-sensitized Renal Transplant Patients Have Antibody Binding to SLA Class I Epitopes.

BACKGROUND: Highly sensitized patients are difficult to match with suitable renal allograft donors and may benefit from xenotransplant trials. We evaluate antibody binding from sensitized patients to pig cells and engineered single allele cells to identify anti-human leukocyte antigen (HLA) antibody cross-species reactivity with swine leukocyte antigen (SLA). These novel testing strategies assess HLA/SLA epitopes and antibody-binding patterns and introduce genetic engineering of SLA epitopes. METHODS: Sensitized patient sera were grouped by calculated panel reactive antibody and luminex single antigen reactivity profile and were tested with cloned GGTA1/CMAH/B4GalNT2 glycan knockout porcine cells. Pig reactivity was assessed by direct flow cytometric crossmatch and studied following elution from pig cells. To study the antigenicity of individual class I HLA and SLA alleles in cells, irrelevant sera binding to lymphoblastoid cells were minimized by CRISPR/Cas9 elimination of endogenous class I and class II HLA, B-cell receptor, and Fc receptor genes. Native HLA, SLA, and mutants of these proteins after mutating 144K to Q were assessed for antibody binding. RESULTS: Those with predominately anti-HLA-B&C antibodies, including Bw6 and Bw4 sensitization, frequently have low pig reactivity. Conversely, antibodies eluted from porcine cells are more commonly anti-HLA-A. Single HLA/SLA expressing engineered cells shows variable antigenicity and mutation of 144K to Q reduces antibody binding for some sensitized patients. CONCLUSIONS: Anti-HLA antibodies cross-react with SLA class I in predictable patterns, which can be identified with histocompatibility strategies, and SLA class I is a possible target of genetic engineering.

Hypercholesterolemia impairs immunity to tuberculosis.

We demonstrate that apolipoprotein E -deficient (ApoE(-/-)) mice are highly susceptible to tuberculosis and that their susceptibility depends on the severity of hypercholesterolemia. Wild-type (WT) mice and ApoE(-/-) mice fed a low-cholesterol (LC) or high-cholesterol (HC) diet were infected with approximately 50 CFU Mycobacterium tuberculosis Erdman by aerosol. ApoE(-/-) LC mice were modestly more susceptible to tuberculosis than WT LC mice. In contrast, ApoE(-/-) HC mice were extremely susceptible, as evidenced by 100% mortality after 4 weeks with tuberculosis. The lung pathology of ApoE(-/-) HC mice was remarkable for giant abscess-like lesions, massive infiltration by granulocytes, elevated inflammatory cytokine production, and a mean bacterial load approximately 2 log units higher than that of WT HC mice. Compared to WT HC mice, the gamma interferon response of splenocytes restimulated ex vivo with M. tuberculosis culture filtrate protein was delayed in ApoE(-/-) HC mice, and they failed to control M. tuberculosis growth in the lung. OT-II cells adoptively transferred into uninfected ApoE(-/-) HC mice had a weak proliferative response to their antigen, indicating impaired priming of the adaptive immune response. Our studies show that ApoE(-/-) deficiency is associated with delayed expression of adaptive immunity to tuberculosis caused by defective priming of the adaptive immune response and that elevated serum cholesterol is responsible for this effect.