Direct Allorecognition Research Articles

TCR Sequencing in Mouse Models of Allorecognition Unveils the Features of Directly and Indirectly Activated Clonotypes.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

Taking the A(llorecognition) train: connecting passenger T cells to DSA

Refers to: Charmetant X, Chen C-C, Hamada S, et al. Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation. Sci Transl Med. 2022;14:eabg1046.

Membrane attack complexes, endothelial cell activation, and direct allorecognition.

Endothelial cells (ECs) form a critical immune interface regulating both the activation and trafficking of alloreactive T cells. In the setting of solid organ transplantation, donor-derived ECs represent sites where alloreactive T cells encounter major and minor tissue-derived alloantigens. During this initial encounter, ECs may formatively modulate effector responses of these T cells through expression of inflammatory mediators. Direct allorecognition is a process whereby recipient T cells recognize alloantigen in the context of donor EC-derived HLA molecules. Direct alloresponses are strongly modulated by human ECs and are galvanized by EC-derived inflammatory mediators.Complement are immune proteins that mark damaged or foreign surfaces for immune cell activation. Following labeling by natural IgM during ischemia reperfusion injury (IRI) or IgG during antibody-mediated rejection (ABMR), the complement cascade is terminally activated in the vicinity of donor-derived ECs to locally generate the solid-phase inflammatory mediator, the membrane attack complex (MAC). Via upregulation of leukocyte adhesion molecules, costimulatory molecules, and cytokine trans-presentation, MAC strengthen EC:T cell direct alloresponses and qualitatively shape the alloimmune T cell response. These processes together promote T cell-mediated inflammation during solid organ transplant rejection.In this review we describe molecular pathways downstream of IgM- and IgG-mediated MAC assembly on ECs in the setting of IRI and ABMR of tissue allografts, respectively. We describe work demonstrating that MAC deposition on ECs generates ‘signaling endosomes’ that sequester and post-translationally enhance the stability of inflammatory signaling molecules to promote EC activation, a process potentiating EC-mediated direct allorecognition. Additionally, with consideration to first-in-human xenotransplantation procedures, we describe clinical therapeutics based on inhibition of the complement pathway. The complement cascade critically mediates EC activation and improved understanding of relevant effector pathways will uncover druggable targets to obviate dysregulated alloimmune T cell infiltration into tissue allografts.

Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation.

The generation of antibodies against donor-specific major histocompatibility complex (MHC) antigens, a type of donor-specific antibodies (DSAs), after transplantation requires that recipient's allospecific B cells receive help from T cells. The current dogma holds that this help is exclusively provided by the recipient's CD4+ T cells that recognize complexes of recipient's MHC II molecules and peptides derived from donor-specific MHC alloantigens, a process called indirect allorecognition. Here, we demonstrated that, after allogeneic heart transplantation, CD3ε knockout recipient mice lacking T cells generate a rapid, transient wave of switched alloantibodies, predominantly directed against MHC I molecules. This is due to the presence of donor CD4+ T cells within the graft that recognize intact recipient's MHC II molecules expressed by B cell receptor-activated allospecific B cells. Indirect evidence suggests that this inverted direct pathway is also operant in patients after transplantation. Resident memory donor CD4+ T cells were observed in perfusion liquids of human renal and lung grafts and acquired B cell helper functions upon in vitro stimulation. Furthermore, T follicular helper cells, specialized in helping B cells, were abundant in mucosa-associated lymphoid tissue of lung and intestinal grafts. In the latter, more graft-derived passenger T cells correlated with the detection of donor T cells in recipient's circulation; this, in turn, was associated with an early transient anti-MHC I DSA response and worse transplantation outcomes. We conclude that this inverted direct allorecognition is a possible explanation for the early transient anti-MHC DSA responses frequently observed after lung or intestinal transplantations.

334.8: Extracellular Vesicles as Mediators of Infectious Tolerance in Human Alloreactive Regulatory Cells

Introduction: We have developed a method for ex vivo expansion of donor antigen specific T regulatory cell enriched lines (ASTRLs) from stable human kidney transplant recipients (KTRs). Rat derived ASTRLs induced prolonged graft survival and donor specific tolerance in a rat kidney transplant model. Human ASTRLs are suppressive in their phenotype and function. We examined the various mechanisms of ASTRL mediated suppression including extracellular vesicle (EV)-mediated suppression. Method: ASTRLs were expanded ex vivo from PBMCs of stable KTRs by stimulation with donor allopeptides in presence of IL-2. ASTRLs were characterized for conventional Treg markers by flow cytometry. Functional characterization of ASTRLs were performed using standard suppression assays. Extracellular ATP hydrolysis was determined by malachite green assay. EVs were isolated from the ASTRL expansion media via differential ultracentrifugation and characterized by liquid chromatography-mass spectrometry. Results: The suppressive mechanism of ASTRLs was alloantigen-specific and dependent on cell-cell contact as shown in Figure 1 (top panel). ASTRLs upregulated the expression CD39 and CD73, ectonucleotidases associated with the adenosinergic pathway, and ASTRLs demonstrated increased extracellular ATP hydrolysis (eATP) that was inhibited by POM1, a CD39 specific inhibitor (Figure 1 left bottom panel) which also abrogated the suppressive effect of ASTRLs. In KTRs ASTRLs specific for one donor alloantigen were able to suppress an effector response against other donor alloantigens demonstrating bystander/ linked suppression and inhibit effector T cell proliferation in response to both direct and indirect allorecognition (Figure 1 bottom right panel). EVs isolated from ASTRLs enhanced the suppressive ability of ASTRLs and hydrolyze eATP independently, which was inhibited by POM1 (Fig. 2) and increase production of IL-4, IL-13, and IL-1RA by alloantigen stimulated T cells, indicating a biasing towards a regulatory response (Fig. 2). Global proteomics identified 1,709 unique proteins in the large EV fraction and 1,356 unique proteins in the small EV fraction. Pathway analysis revealed that both EV fractions under stimulation were enriched for proteins involved in T cell receptor (TCR) signaling and Treg differentiation (Fig. 2) with 145 differentially enriched proteins (DEPs) in the large and 162 DEPs in the small EVs (q<0.05). Pathway analysis of DEPs will be presented. Conclusion: ASTRLs expanded from stable KTRs suppress donor antigen specific effector responses predominantly via the adenosinergic pathway and demonstrate bystander/linked immunosuppression. ASTRL-EVs enhance the suppressive capacity of ASTRLs and exhibit an increase in CD39 function. These findings indicate that human ASTRLs may be useful as autologous regulatory cell therapy in transplant recipients and that ASTRL related EVs may be useful adjuncts to ASTRL cell therapy. Saxena Transplantation Fund.

Higher risk for chronic graft-versus-host disease (GvHD) in HLA-G mismatched transplants following allogeneic hematopoietic stem cell transplantation: A retrospective study.

Graft-versus-host disease (GvHD) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is highly influenced by the degree of HLA matching between recipient and donor. The HLA-class Ib molecule HLA-G has been shown to promote tolerogenicity through its interaction with inhibitory receptors found on several immunocompetent cells. We hypothesized that in an allo-HSCT setting, HLA-G mismatches may negatively impact the HLA-G-mediated tolerogenicity either due to inefficient interaction with the inhibitory receptors of the transplanted immune cells or due to direct allorecognition of mismatched HLA-G on host cells by the immune cells of the donor. In order to explore this hypothesis, we investigated the impact of HLA-G mismatching in 2.083 10/10 matched high resolution HLA-typed allo-HSCT transplants. We found that the risk of chronic GvHD was significantly higher in HLA-G-mismatched transplant cases as compared with the HLA-G-matched control group (HR: 1.46, 95%CI=1.11-1.91, p= 0.006). Sub-analysis of the mismatch vector revealed that this effect was only detectable in the GvH (HR: 1.89, 95%CI 1.39-2.57, p < 0.001) but not the HvG direction (HR: 1.01, 95%CI=0.63-1.63, p= 0.967). In addition, the negative impact of HLA-G mismatching on chronic GvHD was only significant in younger patients (<30y HR: 3.02, 95%CI=1.25-7.28, p=0.014; >29y HR: 1.28, 95%CI=0.94-1.72, p= 0.113). Our results indicate that HLA-G mismatches may contribute to the onset of chronic GvHD, especially in younger patients and should therefore be avoided when possible.

The role of self-peptides in direct T cell allorecognition.

Direct allorecognition, the ability of host T cells to recognize intact allogeneic MHC molecules on transplanted tissues, is often assumed to be less dependent on the peptide bound to the MHC molecule than are other antigen recognition pathways. In this issue of the JCI, Son et al. provide unequivocal, in vivo evidence that direct allorecognition depends on the self-peptides bound to the non-self MHC molecule. The authors demonstrate that the induction of allospecific tolerance required the presentation of self-peptides by the non-self MHC molecule, and that only a handful of these peptides accounted for a sizeable proportion of the immunogenicity of the MHC antigen. These are important findings for transplant immunologists because they provide molecular insights into the biology of direct allorecognition, the prime driver of the alloimmune response to MHC-mismatched grafts, and much-needed tools, peptide-MHC multimers, to track and study polyclonal alloreactive T cells.

The self-peptide repertoire plays a critical role in transplant tolerance induction.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

A Role for Human Renal Tubular Epithelial Cells in Direct Allo-Recognition by CD4+ T-Cells and the Effect of Ischemia-Reperfusion.

Direct allorecognition is the earliest and most potent immune response against a kidney allograft. Currently, it is thought that passenger donor professional antigen-presenting cells (APCs) are responsible. Further, many studies support that graft ischemia-reperfusion injury increases the probability of acute rejection. We evaluated the possible role of primary human proximal renal tubular epithelial cells (RPTECs) in direct allorecognition by CD4+ T-cells and the effect of anoxia-reoxygenation. In cell culture, we detected that RPTECs express all the required molecules for CD4+ T-cell activation (HLA-DR, CD80, and ICAM-1). Anoxia-reoxygenation decreased HLA-DR and CD80 but increased ICAM-1. Following this, RPTECs were co-cultured with alloreactive CD4+ T-cells. In T-cells, zeta chain phosphorylation and c-Myc increased, indicating activation of T-cell receptor and co-stimulation signal transduction pathways, respectively. T-cell proliferation assessed with bromodeoxyuridine assay and with the marker Ki-67 increased. Previous culture of RPTECs under anoxia raised all the above parameters in T-cells. FOXP3 remained unaffected in all cases, signifying that proliferating T-cells were not differentiated towards a regulatory phenotype. Our results support that direct allorecognition may be mediated by RPTECs even in the absence of donor-derived professional APCs. Also, ischemia-reperfusion injury of the graft may enhance the above capacity of RPTECs, increasing the possibility of acute rejection.

CD8+ T-Cell Repertoire in Human Leukocyte Antigen Class I-Mismatched Alloreactive Immune Response.

In transplantation, direct allorecognition is a complex interplay between T-cell receptors (TCR) and HLA molecules and their bound peptides expressed on antigen-presenting cells. In analogy to HLA mismatched hematopoietic stem cell transplantation (HSCT), the TCR CDR3β repertoires of alloreactive cytotoxic CD8+ responder T cells, defined by the cell surface expression of CD137 and triggered in vitro by HLA mismatched stimulating cells, were analyzed in different HLA class I mismatched combinations. The same HLA mismatched stimulatory cells induced very different repertoires in distinct but HLA identical responders. Likewise, stimulator cells derived from HLA identical donors activated CD8+ cells expressing very different repertoires in the same mismatched responder. To mimic in vivo inflammation, expression of HLA class l antigens was upregulated in vitro on stimulating cells by the inflammatory cytokines TNFα and IFNβ. The repertoires differed whether the same responder cells were stimulated with cells treated or not with both cytokines. In conclusion, the selection and expansion of alloreactive cytotoxic T-cell clonotypes expressing a very diverse repertoire is observed repeatedly despite controlling for HLA disparities and is significantly influenced by the inflammatory status. This makes prediction of alloreactive T-cell repertoires a major challenge in HLA mismatched HSCT.

Suppression of liver transplant rejection by anti-donor MHC antibodies via depletion of donor immunogenic dendritic cells.

BackgroundWe previously found two distinct passenger dendritic cell (DC) subsets in the rat liver that played a central role in the liver transplant rejection. In addition, a tolerance-inducing protocol, donor-specific transfusion (DST), triggered systemic polytopical production of depleting alloantibodies to donor class I MHC (MHCI) antigen (DST-antibodies).MethodsWe examined the role of DST-antibodies in the trafficking of graft DC subsets and the alloresponses in a rat model. We also examined an anti-donor class II MHC (MHCII) antibody that recognizes donor DCs more selectively.ResultsPreoperative transfer of DST-antibodies or DST pretreatment eliminated all passenger leukocytes, including both DC subsets and depleted the sessile DCs in the graft to ~20% of control. The CD172a+CD11b/c+ immunogenic subset was almost abolished. The intrahost direct or semi-direct allorecognition pathway was successfully blocked, leading to a significant suppression of the CD8+ T-cell response in the recipient lymphoid organs and the graft with delayed graft rejection. Anti-donor MHCII antibody had similar effects without temporary graft damage. Although DST pretreatment had a priming effect on the proliferative response of recipient regulatory T cells, DST-primed sera and the anti-donor MHCII antibody did not.ConclusionDST-antibodies and anti-donor MHCII antibodies could suppress the CD8+ T-cell-mediated liver transplant rejection by depleting donor immunogenic DCs, blocking the direct or semi-direct pathways of allorecognition. Donor MHCII-specific antibodies may be applicable as a selective suppressant of anti-donor immunity for clinical liver transplantation without the cellular damage of donor MHCII– graft cells and recipient cells.

Transplant Immunology: Basic Immunology and Clinical Practice

Engraftment of a transplanted organ into an allogeneic host triggers a cascade of immunologic responses in the host that are designed to facilitate graft rejection. Modern donor-to-host matching techniques and immunosuppression protocols have successfully tempered this natural immune response so that graft survival has dramatically improved. However, optimizing graft survival by precisely downregulating the host response to graft rejection while preserving host immune defenses against pathologic and infectious agents remains poorly understood and elusive in current clinical practice. This review discusses transplant immunology with respect to host versus graft and the basis of allorecognition, as well as clinical management of the transplanted allograft. Figures show human leukocyte antigen (HLA), direct allorecognition, T cell receptor and CD3, T cell–associated second messenger signaling pathway, CD8 molecules directly ligating class I HLAs and CD4 molecules directly binding HLA class II, detection of alloantibodies by enzyme-linked immunosorbent assay or flow cytometry, recipient-donor crossmatch, histopathology of kidney allograft with antibody-mediated rejection, and an algorithm for assessment and management of renal allograft rejection. This review contains 9 figures, 6 tables and 61 references. Keywords: Transplantation, immunology, human leukocyte antigen, crossmatch, donor, acute rejection, chronic rejection

Transplant Immunology: Basic Immunology and Clinical Practice

Engraftment of a transplanted organ into an allogeneic host triggers a cascade of immunologic responses in the host that are designed to facilitate graft rejection. Modern donor-to-host matching techniques and immunosuppression protocols have successfully tempered this natural immune response so that graft survival has dramatically improved. However, optimizing graft survival by precisely downregulating the host response to graft rejection while preserving host immune defenses against pathologic and infectious agents remains poorly understood and elusive in current clinical practice. This review discusses transplant immunology with respect to host versus graft and the basis of allorecognition, as well as clinical management of the transplanted allograft. Figures show human leukocyte antigen (HLA), direct allorecognition, T cell receptor and CD3, T cell–associated second messenger signaling pathway, CD8 molecules directly ligating class I HLAs and CD4 molecules directly binding HLA class II, detection of alloantibodies by enzyme-linked immunosorbent assay or flow cytometry, recipient-donor crossmatch, histopathology of kidney allograft with antibody-mediated rejection, and an algorithm for assessment and management of renal allograft rejection. This review contains 9 figures, 6 tables and 61 references. Keywords: Transplantation, immunology, human leukocyte antigen, crossmatch, donor, acute rejection, chronic rejection

Transplant Immunology: Basic Immunology and Clinical Practice

Engraftment of a transplanted organ into an allogeneic host triggers a cascade of immunologic responses in the host that are designed to facilitate graft rejection. Modern donor-to-host matching techniques and immunosuppression protocols have successfully tempered this natural immune response so that graft survival has dramatically improved. However, optimizing graft survival by precisely downregulating the host response to graft rejection while preserving host immune defenses against pathologic and infectious agents remains poorly understood and elusive in current clinical practice. This review discusses transplant immunology with respect to host versus graft and the basis of allorecognition, as well as clinical management of the transplanted allograft. Figures show human leukocyte antigen (HLA), direct allorecognition, T cell receptor and CD3, T cell–associated second messenger signaling pathway, CD8 molecules directly ligating class I HLAs and CD4 molecules directly binding HLA class II, detection of alloantibodies by enzyme-linked immunosorbent assay or flow cytometry, recipient-donor crossmatch, histopathology of kidney allograft with antibody-mediated rejection, and an algorithm for assessment and management of renal allograft rejection. This review contains 9 figures, 6 tables and 61 references. Keywords: Transplantation, immunology, human leukocyte antigen, crossmatch, donor, acute rejection, chronic rejection

Galectin-1-induced tolerogenic dendritic cells combined with apoptotic lymphocytes prolong liver allograft survival

Donor-derived tolerogenic dendritic cells (DCs) and apoptotic lymphocytes (ALs) are practical tools for controlling rejection after transplantation by targeting direct and indirect allorecognition pathways, respectively. To date, few studies have investigated the combination of donor-derived tolerogenic DCs and ALs infusion in organ transplantation protection. In the present study, we generated galectin-1-induced tolerogenic DCs (DCgal-1s) and ultraviolet irradiation-induced ALs with stable immune characteristics in vitro and potential immune regulatory activity in vivo. A rat model of acute liver transplant rejection was established, and the intrinsic tolerogenic profiles associated with the short-term alleviation of rejection and the long-term maintenance of tolerance in the absence of immunosuppressive drugs were evaluated. The DCgal-1-AL treatment prolonged allograft survival more significantly than a transfusion of DCgal-1s or ALs alone. This benefit was associated with CD4+ Treg cell expansion and decreased interferon (IFN)-γ+ T cell levels. Moreover, DCgal-1-AL treatment led to different cytokine/chemokine changes in the allograft and peripheral blood, that indicated an alleviation of local and systemic inflammation on day 7 post-transplantation. TGF-β1 and TGF-β2 were significantly increased in the long-term surviving allografts after DCgal-1-AL treatment. Our results indicate that the combination of DCgal-1s with ALs effectively prolongs liver allograft survival and represents a novel therapeutic strategy for liver transplant rejection.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction.

Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

TIGIT+ iTregs\xa0elicited by human regulatory macrophages control T cell immunity

Human regulatory macrophages (Mreg) have shown early clinical promise as a cell-based adjunct immunosuppressive therapy in solid organ transplantation. It is hypothesised that recipient CD4+ T cell responses are actively regulated through direct allorecognition of donor-derived Mregs. Here we show that human Mregs convert allogeneic CD4+ T cells to IL-10-producing, TIGIT+ FoxP3+-induced regulatory T cells that non-specifically suppress bystander T cells and inhibit dendritic cell maturation. Differentiation of Mreg-induced Tregs relies on multiple non-redundant mechanisms that are not exclusive to interaction of Mregs and T cells, including signals mediated by indoleamine 2,3-dioxygenase, TGF-β, retinoic acid, Notch and progestagen-associated endometrial protein. Preoperative administration of donor-derived Mregs to living-donor kidney transplant recipients results in an acute increase in circulating TIGIT+ Tregs. These results suggest a feed-forward mechanism by which Mreg treatment promotes allograft acceptance through rapid induction of direct-pathway Tregs.

OR2 Recipient NK cell receptor repertoire comprising activating KIR2DS5 and licensed KIR3DL1 is associated with improved chronic lung allograft dysfunction (CLAD) free survival when donor misses HLA-BW4 ligand

Aim Alloimmune responses manifesting as chronic lung allograft dysfunction (CLAD) limit long-term survival of lung transplant. Natural killer (NK) cells expressing an inhibitory KIR (iKIR) recognizing self-HLA can be activated when confronted with allograft lacking a ligand for that iKIR. Here we investigated if allograft missing ligand for recipient iKIR that had a cognate HLA ligand would enhance CLAD-free survival. We also analyzed the role of recipient’s activating KIRs (aKIR) on CLAD-free survival. Methods Using luminex rSSO methods, KIR and HLA genes were typed for 263 lung transplant recipients and HLA genes were typed for all donors. CLAD was determined by date of FEV1 or FVC drop to KIR-HLA genotypes. Results Bx KIR genotype recipients (i.e., carrying more a KIR s) had higher median CLAD-free survival than those carrying AA genotypes (Fig. A). Recipients with all 5 B haplotype-associated a KIR s ( 2DS1, S2, S3, S5, 3DS1 ) had enhanced CLAD-free survival relative to those missing one or more of these a KIR s. Particularly, recipients carrying 2DS5 showed enhanced CLAD-free survivals than those missing 2DS5 (Fig. B). Further, CLAD-free survival was higher for recipients carrying KIR3DL1 + Bw4 when donors missing Bw4 ligand (Fig. C). Allografts missing relevant ligands for recipients carrying 2DL1 + C2, 2DL2/L3 + C1, and/or 3DL2 + A3/A11 pairs were not associated with differential survival. Recipients with 2DS5, 3DL1, and Bw4 receiving donors missing Bw4 ligand showed the longest CLAD-free survival (p = 0.01, 9.2 median survival years; Fig. D). Conclusions Recipient NK cells expressing aKIRs and/or licensed KIR3DL1 may respond and deplete donor-derived antigen presenting cells that do not express Bw4 ligand, and thus impair direct allorecognition pathways which in turn delay CLAD. Functional studies will be required to understand the mechanism of KIR-HLA interactions in protecting from CLAD.

Recognition of Intact Donor Class I by Recipient CD8+ T Cells is Required for Tolerance Induction Following Liver-Directed Transgene Expression

Introduction: AAV-mediated expression of donor MHC I (H-2Kb or H-2Kd) in recipient liver induces donor-specific tolerance in a mouse skin transplant model where Kb or Kd are mismatched between donor and host [1]. Recognition of both intact and processed allogeneic class I could contribute to tolerance induction. Aims and Methods: To determine the relative importance of intact and processed class I in tolerance induction, we generated rAAV-Kb-D227K and Kd-D227K vectors where a point mutation in the class I alpha 3 domain abrogates CD8 binding [2] and direct allorecognition but does not interfere with recognition of processed peptides. Expression of WT or mutant class I on hepatocytes was assessed by FACS and IHC. TCR-transgenic T cells recognizing intact (Des-RAG) or processed (TCR75) allogeneic class I were used as reporter cells in adoptive transfer experiments. Skin bearing the mismatched MHC I was grafted onto uninjected recipients or mice inoculated with rAAV encoding WT or D227K mutant Kb or Kd. In some recipients, Tregs were depleted using anti-FR4 or Diphtheria toxin (DT). Interferon gamma production by responding T cells was determined using ELISpot. Results: WT and D227K class I were expressed on hepatocytes at comparable levels. Adoptive transfer assays confirmed that the D227K mutation abrogated recognition of intact Kb by CD8+ Des-RAG T cells, whilst recognition of processed Kd peptides by TCR75-RAG cells was unimpaired. Uninjected B10.BR mice rejected Kb-mismatched 178.3 skin (MST = 16 d), while grafts onto rAAV-Kb-injected mice survived indefinitely (p < 0.001). rAAV-Kb-D227K only slightly prolonged graft survival (MST = 27d, p < 0.05). These results were recapitulated in the B6.Kd to C57BL/6 model. The modest survival prolongation of skin grafts in mice inoculated with Kb-D227K was abrogated when the recipients were treated with anti-FR4 (clone TH6) to deplete Tregs. Similarly, the survival prolongation of Kd-mismatched B6.Kd grafts onto DEREG recipients treated with rAAV-Kd-D227K (MST = 28d compared with 20d in mice receiving DT alone) was blocked when mice receiving Kd-D227K were also treated with DT (MST 18 days, p = 0.0046). Treg depletion did not shorten survival of skin grafts in mice inoculated with WT MHC class I vectors. Interferon-gamma production by unfractionated or CD8-enriched splenocytes from primed C57BL/6 mice in response to B6.Kd stimulators was over fourfold lower in mice inoculated with WT Kd than in those receiving Kd-D227K (p = 0.038). Conclusions: Recognition of intact MHC class I by alloreactive CD8+ T cells is required for tolerance induction via liver-directed expression of donor MHC class I. Indirect recognition of class I allopeptides and generation of Tregs can produce a modest prolongation of class I-mismatched skin graft survival but does not induce tolerance.FigureFigure