RESUMO
The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from 4 patients who experienced severe acute rejection leading to graft loss. Alloreactive T cell receptor (TCR) clones were identified in pretransplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of recipient-derived TRM cells expressing an alloreactive TCR in the 4 kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, these clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.
Assuntos
Rejeição de Enxerto , Transplante de Rim , Células T de Memória , Humanos , Células T de Memória/imunologia , Rejeição de Enxerto/imunologia , Masculino , Feminino , Memória Imunológica , Pessoa de Meia-Idade , Sobrevivência de Enxerto/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Adulto , Prognóstico , Seguimentos , Linfócitos T CD8-Positivos/imunologia , Falência Renal Crônica/cirurgia , Falência Renal Crônica/imunologia , Doadores de TecidosRESUMO
HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.
Assuntos
Receptor 4 Toll-Like , Doenças Vasculares , Humanos , Metaloproteinase 9 da Matriz , Selectina-P , Macrófagos , Endotélio , Antígenos HLA , Aloenxertos , Imunoglobulina GRESUMO
Antibody-mediated rejection (ABMR) is a significant obstacle to achieving optimal long-term outcomes after solid organ transplantation. The presence of donor-specific antibodies (DSA), particularly against HLA, increases the risk of allograft rejection and subsequent graft loss. No effective treatment of ABMR currently exists, warranting novel approaches to target the HLA-specific humoral alloimmune response. Cellular therapies may hold promise to this end. According to publicly available sources as of now, three independent laboratories have genetically engineered a chimeric HLA-antibody receptor (CHAR) and transduced it into human T cells, based on the demonstrated efficacy of chimeric antigen receptor T cell therapies in malignancies. These CHAR-T cells are designed to exclusively eliminate B cells that produce donor-specific HLA antibodies, which form the cornerstone of ABMR. CHAR technology generates potent and functional human cytotoxic T cells to target alloreactive HLA-specific B cells, sparing B cells with other specificities. Thus, CHAR technology may be used as a selective desensitization protocol and to treat antibody-mediated rejection after solid organ transplantation.
Assuntos
Autoanticorpos , Glomerulonefrite Membranosa , Glomerulonefrite Membranosa/imunologia , Glomerulonefrite Membranosa/terapia , Humanos , Autoanticorpos/sangue , Autoanticorpos/imunologia , Resultado do Tratamento , Receptores de Antígenos Quiméricos/imunologia , Masculino , Terapia Baseada em Transplante de Células e Tecidos/métodosRESUMO
Donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules are a major risk factor for rejection of transplanted organs (in antibody-mediated rejection [ABMR]), particularly in patients who have prior sensitization or receive insufficient immunosuppression through minimization or noncompliance. These DSA are measured routinely in the serum of patients prior to transplantation mainly using bead-based technologies or cell-based assays. However, the absence of detectable serum DSA does not always reflect the absence of sensitization or histologically defined ABMR, and so it has been proposed that the detection and measurement of memory B cells capable of secreting antibodies against donor HLA antigens could be carried out using B-cell ImmunoSpot, to better inform the degree of immune sensitization of transplant patients prior to as well as after transplantation. Such an assay is described here.
Assuntos
Isoanticorpos , Transplante de Rim , Humanos , Células B de Memória , Rejeição de Enxerto , Antígenos HLA , Antígenos de Histocompatibilidade Classe II , Imunoglobulina G , Doadores de Tecidos , Sobrevivência de EnxertoRESUMO
Background: Kidney transplantation is the preferred treatment option for patients with end-stage renal disease. However, long-term graft survival remains a challenge. The enzyme indoleamine 2,3 dioxygenase (IDO) has been reported to have immunomodulatory effects with IDO transcripts being elevated in both antibody-mediated rejection and T cell-mediated rejection. Methods: A metal-conjugated antibody panel for the staining of kidney biopsies was developed, allowing the visualization of 41 structural and immune markers on a single tissue slide to gain in-depth insight into the composition and localization of the immune cell compartment. Staining was applied to week 4 and 24 protocol biopsies of 49 patients as well as on 15 indication biopsies of the TRITON study and 4 additional transplantation biopsies with glomerulitis. Results: A highly distinctive and specific glomerular IDO expression was observed in biopsies from 3 of 49 patients in imaging mass cytometry. Immunohistochemistry confirmed IDO expression in glomeruli of 10 of 10 cases with glomerulitis. IDO was found to be expressed by CD31+ glomerular endothelial cells, accompanied by the presence of granzyme-B+Tbet+CD7+CD45RA+ natural killer cells and CD68+ macrophages. Furthermore, a proportion of both the immune cells and endothelial cells expressed Ki-67, indicative of cell proliferation, which was not observed in control glomeruli. Conclusions: Our results show glomerular IDO expression in transplanted kidneys with glomerulitis, which is accompanied by increased numbers of natural killer cells and macrophages and likely reflects local immune activation.
RESUMO
Background: Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). Methods: We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. Results: At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan-Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). Conclusion: This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.
Assuntos
Rejeição de Enxerto , Antígenos HLA , Isoanticorpos , Transplante de Rim , Humanos , Transplante de Rim/efeitos adversos , Estudos Retrospectivos , Masculino , Feminino , Pessoa de Meia-Idade , Rejeição de Enxerto/imunologia , Isoanticorpos/imunologia , Isoanticorpos/sangue , Adulto , Antígenos HLA/imunologia , Células B de Memória/imunologia , Doadores de Tecidos , Idoso , Transplantados , Sobrevivência de Enxerto/imunologiaRESUMO
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.
Assuntos
Isoanticorpos , Transplante de Órgãos , Humanos , Alelos , Teste de Histocompatibilidade , Antígenos HLA-DQ/genética , Rejeição de Enxerto/genéticaRESUMO
In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.
Assuntos
Anticorpos Monoclonais , Antígenos HLA-DR , Humanos , Epitopos , Alelos , Doadores de Tecidos , Antígenos HLA-B , Antígenos HLA , Teste de Histocompatibilidade , Rejeição de EnxertoRESUMO
HSCT with mismatched unrelated donors (MMUD) is associated with inferior outcome compared to matched unrelated donors. We aimed to identify permissible mismatches using HLA-EMMA, which determines permissibility by analyzing amino-acid (AA) sequences, in a single center cohort of 70 pediatric 9/10 MMUD HSCTs and 157 10/10 MUDs for comparison. AA matching was evaluated for the whole HLA protein, the α-helices, and the ß-sheets, in both Host versus Graft (HvG) and Graft vs Host (GvH) direction. Superior EFS was found in 13 patients permissibly mismatched in the HvG direction (totalHvG, 92% vs 58% at 1 year, p=0.009), and in 21 patients matched for AA on the α-helices (αHvG, 90% vs 53%, p=0.002), similar to EFS with 10/10 MUDs (90% vs 80%, p=0.60). EFS was not related to ß-sheet AA matching, nor to matching in the GvH direction. OS trended similarly as EFS for AA mismatches (totalHvG, 92% vs 74%, p=0.075 and αHvG90% vs 71%, p=0.072). These findings were reproduced in an EBMT inborn errors cohort of 271 pediatric 9/10 MMUD HSCTs and 929 10/10 MUD HSCTs, showing a significant effect of αHvG matching on both OS and EFS and similar OS and EFS between αHvG matched MMUDs and 10/10 MUDs. In summary, HvG-AA matching on the α-helices identifies 9/10 MMUD with permissible mismatches correlated with a favorable transplant outcome similar to matched donors.
RESUMO
Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in immunocompromised individuals such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in immunosuppressed individuals have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8+ T cells in immunocompromised patients and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8+ T cells in KTRs with non-controlled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8+ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8+ T cells could be targeted by inhibiting CD38 to reverse hypo-responsiveness in individuals who fail to control chronic viral infection.
RESUMO
In kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of those scores in relation to transplant outcome is unknown. In a large Dutch kidney transplant cohort, Ellipro scores of pretransplant donor antibodies that can be assigned to known eplets (donor epitope specific HLA antibodies [DESAs]) were compared between early graft failure and long surviving deceased donor transplants. We did not observe a significant Ellipro score difference between the two cohorts, nor significant differences in graft survival between transplants with DESAs having high versus low total Ellipro scores. We conclude that Ellipro scores cannot be used to identify DESAs associated with early versus late kidney graft loss in deceased donor transplants.
Assuntos
Nefropatias , Transplante de Rim , Humanos , Sobrevivência de Enxerto , Alelos , Anticorpos , Rim , Epitopos , Rejeição de Enxerto , Antígenos HLA , Doadores de TecidosRESUMO
In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope-specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan-Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre-transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations (p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA-DR, and 1 of HLA-DQ, and most antibodies were directed to HLA-B (47%). Fifty-three patients (69.7%) had DESA against one donor epitope (range 1-5). Long-term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84-3.25) in deceased donation, and 2.22 (1.25-3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA-based risk analysis on antigen level.