RESUMEN
HIV infection persists during antiretroviral therapy (ART) due to a reservoir of latently infected cells that harbor replication-competent virus and evade immunity. Previous ex vivo studies suggested that CD8+ T cells from people with HIV may suppress HIV expression via non-cytolytic mechanisms, but the mechanisms responsible for this effect remain unclear. Here, we used a primary cell-based in vitro latency model and demonstrated that co-culture of autologous activated CD8+ T cells with HIV-infected memory CD4+ T cells promoted specific changes in metabolic and/or signaling pathways resulting in increased CD4+ T cell survival, quiescence, and stemness. Collectively, these pathways negatively regulated HIV expression and ultimately promoted the establishment of latency. As shown previously, we observed that macrophages, but not B cells, promoted latency in CD4+ T cells. The identification of CD8-specific mechanisms of pro-latency activity may favor the development of approaches to eliminate the viral reservoir in people with HIV.
Asunto(s)
Infecciones por VIH , Humanos , Linfocitos T CD8-positivos , Latencia del Virus , Linfocitos T CD4-Positivos , Replicación ViralRESUMEN
Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Menor , Células T Invariantes Asociadas a Mucosa , Especificidad de la Especie , Animales , Células T Invariantes Asociadas a Mucosa/inmunología , Células T Invariantes Asociadas a Mucosa/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ratones , Bovinos , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/química , Porcinos , Macaca , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
The cytokine interleukin-2 (IL-2) plays a critical role in controlling the immune homeostasis by regulating the proliferation and differentiation of immune cells, especially T cells. IL-2 signaling is mediated via the IL-2 receptor (IL-2R) complex, which consists of the IL-2Rα (CD25), the IL-2Rß, and the IL-2Rγ. While the latter are required for signal transduction, IL-2Rα controls the ligand-binding affinity of the receptor complex. A soluble form of the IL-2Rα (sIL-2Rα) is found constitutively in human serum, though its levels are increased under various pathophysiological conditions. The sIL-2Rα originates partly from activated T cells through proteolytic cleavage, but neither the responsible proteases nor stimuli that lead to IL-2Rα cleavage are known. Here, we show that the metalloproteases ADAM10 and ADAM17 can cleave the IL-2Rα and generate a soluble ectodomain, which functions as a decoy receptor that inhibits IL-2 signaling in T cells. We demonstrate that ADAM10 is mainly responsible for constitutive shedding of the IL-2Rα, while ADAM17 is involved in IL-2Rα cleavage upon T cell activation. In vivo, we found that mice with a CD4-specific deletion of ADAM10, but not ADAM17, show reduced steady-state sIL-2Rα serum levels. We propose that the identification of proteases involved in sIL-2Rα generation will allow for manipulation of IL-2Rα cleavage, especially as constitutive and induced cleavage of IL-2Rα are executed by different proteases, and thus offer a novel opportunity to alter IL-2 function.
Asunto(s)
Proteína ADAM10 , Subunidad alfa del Receptor de Interleucina-2 , Receptores de Interleucina-2 , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Animales , Eliminación de Gen , Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Ratones , Receptores de Interleucina-2/genéticaRESUMEN
Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell-bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.
Asunto(s)
Quimiocina CCL21/metabolismo , Células Dendríticas/inmunología , Plasminógeno/metabolismo , Linfocitos T/metabolismo , Antifibrinolíticos , Quimiocinas , Células Dendríticas/metabolismo , Fibrinolisina/metabolismo , Humanos , Ligandos , Activadores Plasminogénicos/metabolismo , alfa 2-AntiplasminaRESUMEN
The Sensitization in Transplantation: Assessment of Risk workgroup is a collaborative effort of the American Society of Transplantation and the American Society of Histocompatibility and Immunogenetics that aims at providing recommendations for clinical testing, highlights gaps in current knowledge, and proposes areas for further research to enhance histocompatibility testing in support of solid organ transplantation. This report provides updates on topics discussed by the previous Sensitization in Transplantation: Assessment of Risk working groups and introduces 2 areas of exploration: non-human leukocyte antigen antibodies and utilization of human leukocyte antigen antibody testing measurement to evaluate the efficacy of antibody-removal therapies.
Asunto(s)
Trasplante de Órganos , Trasplante de Órganos/efectos adversos , Factores de Riesgo , Histocompatibilidad , Prueba de Histocompatibilidad , Procesos de Grupo , Rechazo de Injerto/etiología , IsoanticuerposRESUMEN
Belatacept-based immunosuppression in kidney transplantation confers fewer off-target toxicities than calcineurin inhibitors but comes at a cost of increased incidence and severity of acute rejection, potentially due to its deleterious effect on both the number and function of Foxp3+ regulatory T cells (Tregs). TIGIT is a CD28 family coinhibitory receptor expressed on several subsets of immune cells including Tregs. We hypothesized that coinhibition through TIGIT signaling could function to ameliorate costimulation blockade-resistant rejection. The results demonstrate that treatment with an agonistic anti-TIGIT antibody, when combined with costimulation blockade by CTLA-4Ig, can prolong allograft survival in a murine skin graft model compared with CTLA-4Ig alone. Further, this prolongation of graft survival is accompanied by an increase in the frequency and number of graft-infiltrating Tregs and a concomitant reduction in the number of CD8+ T cells in the graft. Through the use of Treg-specific TIGIT conditional knockout animals, we demonstrated that the TIGIT-mediated reduction in the graft-infiltrating CD8+ T cell response is dependent on signaling of TIGIT on Foxp3+ Tregs. Our results highlight both the key functional role of TIGIT on Foxp3+ Tregs under conditions in which CTLA-4 is blocked and the therapeutic potential of TIGIT agonism to optimize costimulation blockade-based immunosuppression.
Asunto(s)
Trasplante de Riñón , Linfocitos T Reguladores , Animales , Ratones , Abatacept/farmacología , Linfocitos T CD8-positivos , Factores de Transcripción Forkhead , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/etiología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Trasplante de Riñón/efectos adversos , Trasplante HomólogoRESUMEN
T cells must be activated and become effectors first before executing allograft rejection, a process that is regulated by diverse signals and transcription factors. In this study, we studied the basic leucine zipper ATF-like transcription factor (BATF) family members in regulating T cell activities in a heart transplant model and found that mice deficient for both BATF and BATF3 (Batf-/- Batf3-/- mice) spontaneously accept the heart allografts long-term without tolerizing therapies. Similarly, adoptive transfer of wild type T cells into Rag1-/- hosts induced prompt rejection of heart and skin allografts, whereas the Batf-/- Batf3-/- T cells failed to do so. Analyses of graft-infiltrating cells showed that Batf-/- Batf3-/- T cells infiltrate the graft but fail to acquire an effector phenotype (CD44high KLRG1+ ). Co-transfer experiments in a T cell receptor transgenic TEa model revealed that the Batf-/- Batf3-/- T cells fail to expand in vivo, retain a quiescent phenotype (CD62L+ CD127+ ), and unable to produce effector cytokines to alloantigen stimulation, which contrasted sharply to that of wild type T cells. Together, our data demonstrate that the BATF and BATF3 are critical regulators of T effector functions, thus making them attractive targets for therapeutic interventions in transplant settings.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Linfocitos T , Aloinjertos/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Regulación de la Expresión Génica , Factores Reguladores del Interferón , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/metabolismoRESUMEN
Oral antigen exposure is a powerful, non-invasive route to induce immune tolerance to dietary antigens, and has been modestly successful at prolonging graft survival in rodent models of transplantation. To harness the mechanisms of oral tolerance for promoting long-term graft acceptance, we developed a mouse model where the antigen ovalbumin (OVA) was introduced orally prior to transplantation with skin grafts expressing OVA. Oral OVA treatment pre-transplantation promoted permanent graft acceptance and linked tolerance to skin grafts expressing OVA fused to the additional antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party allografts were spontaneously accepted. Oral OVA treatment promoted an anergic phenotype in OVA-reactive CD4+ and CD8+ conventional T cells (Tconvs) and expanded OVA-reactive Tregs pre-transplantation. However, skin graft acceptance following oral OVA resisted partial depletion of Tregs and blockade of PD-L1. Mechanistically, we revealed a role for the proximal gut draining lymph nodes (gdLNs) in mediating this effect, as an intestinal infection that drains to the proximal gdLNs prevented tolerance induction. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance.
Asunto(s)
Isoantígenos , Trasplante de Piel , Animales , Antígenos , Antígeno B7-H1 , Supervivencia de Injerto , Tolerancia Inmunológica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina , Tolerancia al TrasplanteRESUMEN
The potential of adoptive cell therapy with regulatory T cells (Tregs) to promote transplant tolerance is under active exploration. However, the impact of specific transplant settings and protocols on Treg manufacturing is not well-delineated. Here, we compared the use of peripheral blood mononuclear cells (PBMCs) from patients before or after liver transplantation to the use of healthy control PBMCs to determine their suitability for Treg manufacture using ex vivo costimulatory blockade with belatacept. Despite liver failure or immunosuppressive therapy, the capacity for Treg expansion during the manufacturing process was preserved. These experiments did not identify performance or quality issues that disqualified the use of posttransplant PBMCs-the currently favored protocol design. However, as Treg input correlated with output, significant CD4-lymphopenia in both pre- and posttransplant patients limited Treg yield. We therefore turned to leukapheresis posttransplant to improve absolute yield. To make deceased donor use feasible, we also developed protocols to substitute splenocytes for PBMCs as allostimulators. In addition to demonstrating that this Treg expansion strategy works in a liver transplant context, this preclinical study illustrates how characterizing cellular input populations and their performance can both inform and respond to clinical trial design and Treg manufacturing requirements.
Asunto(s)
Trasplante de Hígado , Linfocitos T Reguladores , Abatacept/farmacología , Humanos , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Leucocitos Mononucleares , Receptores de Trasplantes , Tolerancia al TrasplanteRESUMEN
Studies are urgently needed to characterize immunogenicity, efficacy, and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in kidney transplant (KT) recipients, excluded from major clinical trials. Complex ELISPOT and other cellular response techniques have been applied, but simpler tools are needed. An easy-to-use real-world monitoring of SARS-CoV-2 IgG antibodies against the Spike protein and QuantiFERON® SARS-CoV-2 IFNγ release assay (IGRA) were performed at baseline and 28 days after the second dose in KT recipients and controls (dialysis patients and healthy ones). All healthy controls and >95% dialysis controls became positive for anti-S IgG antibodies, while only 63.3% of KT patients seroconverted with a very low antibody level. A positive IGRA was documented in 96.9% of controls, 89.3% peritoneal dialysis, 77.6% hemodialysis, 61.3% of KT patients transplanted more than 1 year ago and only 36% of those transplanted within the previous 12 months. Overall, 100% of healthy controls, 95.4% of dialysis patients and 78.8% KT recipients developed any immune response (humoral and/or cellular) against SARS-CoV-2. KT patients showed low rates of immune responses to mRNA Coronavirus infectious disease 2019 vaccines, especially those with recent transplantations. Simple humoral and cellular monitoring is advisable, so that repeated doses may be scheduled according to the results.
Asunto(s)
COVID-19 , Trasplante de Riñón , Aloinjertos , Anticuerpos Antivirales , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Inmunidad , Ensayos de Liberación de Interferón gamma , Trasplante de Riñón/efectos adversos , ARN Mensajero/genética , Diálisis Renal , SARS-CoV-2RESUMEN
Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However, CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here, we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation, proliferation, and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen, we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99 days, whereas median survival with polyclonal Treg infusion was 35 days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors, A2-CAR Tregs slightly increased median graft survival from 11 to 14 days, which was further extended to >100 days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs, alone or in combination with immunosuppressive agents, toward protecting vascularized grafts in fully immunocompetent recipients.
Asunto(s)
Receptores Quiméricos de Antígenos , Aloinjertos , Animales , Rechazo de Injerto/etiología , Supervivencia de Injerto , Antígeno HLA-A2 , Isoantígenos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T ReguladoresRESUMEN
Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage-inducible C-type lectin (Mincle, Clec4e) expression is up-regulated in B cells from heart allograft recipients treated with murine anti-thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely, treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non-transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell-depleted, but not in Mincle-deficient, recipients indicating that CD40 signaling induces T cell proliferation via a Mincle-dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage-associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation.
Asunto(s)
Linfocitos B , Trasplante de Corazón , Aloinjertos , Animales , Linfocitos B/metabolismo , Citocinas/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos , Ratones , Ratones Endogámicos C57BLRESUMEN
CCR5 KO kidney transplant (KTx) recipients are extraordinarily high alloantibody producers and develop pathology that mimics human antibody-mediated rejection (AMR). C57BL/6 and CCR5 KO mice (H-2b ) were transplanted with A/J kidneys (H-2a ); select cohorts received adoptive cell therapy (ACT) with alloprimed CXCR5+ CD8+ T cells (or control cells) on day 5 after KTx. ACT efficacy was evaluated by measuring posttransplant alloantibody, pathology, and allograft survival. Recipients were assessed for the quantity of CXCR5+ CD8+ T cells and CD8-mediated cytotoxicity to alloprimed IgG+ B cells. Alloantibody titer in CCR5 KO recipients was four-fold higher than in C57BL/6 recipients. The proportion of alloprimed CXCR5+ CD8+ T cells 7 days after KTx in peripheral blood, lymph node, and spleen was substantially lower in CCR5 KO compared to C57BL/6 recipients. In vivo cytotoxicity towards alloprimed IgG+ B cells was also reduced six-fold in CCR5 KO recipients. ACT with alloprimed CXCR5+ CD8+ T cells (but not alloprimed CXCR5- CD8+ or third-party primed CXCR5+ CD8+ T cells) substantially reduced alloantibody titer, ameliorated AMR pathology, and prolonged allograft survival. These results indicate that a deficiency in quantity and function of alloprimed CXCR5+ CD8+ T cells contributes to high alloantibody and AMR in CCR5 KO recipient mice, which can be rescued with ACT.
Asunto(s)
Trasplante de Riñón , Animales , Linfocitos T CD8-positivos , Rechazo de Injerto/patología , Inmunoglobulina G , Isoanticuerpos , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Cytomegalovirus (CMV) persists as the most frequent opportunistic infection among solid organ transplant recipients. This multicenter trial aimed to test whether treatment with everolimus (EVR) could decrease the incidence of CMV DNAemia and disease. We randomized 186 CMV seropositive kidney transplant recipients in a 1:1 ratio to receive EVR or mycophenolic acid (MPA) in association with basiliximab, cyclosporin, and steroids and 87 in each group were analyzed. No universal prophylaxis was administered to either group. The composite primary endpoint was the presence of CMV DNAemia, CMV treatment, graft loss, death, and discontinuation of the study at 6 months posttransplant. In the modified intent-to-treat analysis, 42 (48.3%) and 70 (80.5%) patients in the EVR and MPA groups reached the primary endpoint (OR = 0.21, 95% CI: 0.11-0.43, p < .0001). Fewer patients of the EVR group received treatment for CMV (21.8% vs. 47.1%, p = .0007). EVR was discontinued in 31 (35.6%) patients. Among the 56 patients with ongoing EVR treatment, only 7.4% received treatment for CMV. In conclusion, EVR prevents CMV DNAemia requiring treatment in seropositive recipients as long as it is tolerated and maintained.
Asunto(s)
Infecciones por Citomegalovirus , Trasplante de Riñón , Antivirales/uso terapéutico , Citomegalovirus , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/epidemiología , Infecciones por Citomegalovirus/etiología , Everolimus/uso terapéutico , Humanos , Inmunosupresores/efectos adversos , Incidencia , Trasplante de Riñón/efectos adversos , Ácido Micofenólico/uso terapéutico , Receptores de TrasplantesRESUMEN
Unlike immunocompetent hosts, the duration of viral persistence after infection with severe acute respiratory syndrome coronavirus 2 can be prolonged in immunosuppressed patients. Here, we present a case of viral persistence for over 19 weeks in a patient with a history of solid organ transplant and explore the clinical, virologic, and immunologic course. Our patient still demonstrated viral persistence at 138 days with low polymerase chain reaction cycle threshold values and evidence of continuing viral sequence evolution indicative of ongoing virus replication. These findings have important implications for infection prevention and control recommendations in immunosuppressed patients. Immune response, including neutralizing antibody titers, T cell activity, and cytokine levels, peaked around days 44-72 after diagnosis. Anti-S trimer antibodies were low at all time points, and T cell response was attenuated by day 119. As immune response waned and viral load increased, increased genetic diversity emerged, suggesting a mechanism for the development of viral variants.
Asunto(s)
COVID-19 , Trasplante de Órganos , Anticuerpos Antivirales , Humanos , Trasplante de Órganos/efectos adversos , SARS-CoV-2 , Carga ViralRESUMEN
Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells. PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance to monitor real-time binding of recombinantly-expressed and -purified proteins, we found that W110PD-L2 acts as an "elbow" that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Furthermore, we identified a "latch" between the C and D ß-strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110PD-L2 "elbow" and a C-D region "latch." Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2-affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.
Asunto(s)
Antígeno B7-H1/química , Placenta/metabolismo , Proteína 2 Ligando de Muerte Celular Programada 1/química , Secuencia de Aminoácidos , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Femenino , Humanos , Ligandos , Activación de Linfocitos , Ratones , Mutagénesis Sitio-Dirigida , Filogenia , Embarazo , Proteína 2 Ligando de Muerte Celular Programada 1/clasificación , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Alineación de Secuencia , Electricidad EstáticaRESUMEN
The immune co-receptor CD8 molecule (CD8) has two subunits, CD8α and CD8ß, which can assemble into homo or heterodimers. Nonclassical (class-Ib) major histocompatibility complex (MHC) molecules (MHC-Ibs) have recently been identified as ligands for the CD8αα homodimer. This was demonstrated by the observation that histocompatibility 2, Q region locus 10 (H2-Q10) is a high-affinity ligand for CD8αα which also binds the MHC-Ib molecule H2-TL. This suggests that MHC-Ib proteins may be an extended source of CD8αα ligands. Expression of H2-T3/TL and H2-Q10 is restricted to the small intestine and liver, respectively, yet CD8αα-containing lymphocytes are present more broadly. Therefore, here we sought to determine whether murine CD8αα binds only to tissue-specific MHC-Ib molecules or also to ubiquitously expressed MHC-Ib molecules. Using recombinant proteins and surface plasmon resonance-based binding assays, we show that the MHC-Ib family furnishes multiple binding partners for murine CD8αα, including H2-T22 and the CD94/NKG2-A/B-activating NK receptor (NKG2) ligand Qa-1b We also demonstrate a hierarchy among MHC-Ib proteins with respect to CD8αα binding, in which Qa-1b > H2-Q10 > TL. Finally, we provide evidence that Qa-1b is a functional ligand for CD8αα, distinguishing it from its human homologue MHC class I antigen E (HLA-E). These findings provide additional clues as to how CD8αα-expressing cells are controlled in different tissues. They also highlight an unexpected immunological divergence of Qa-1b/HLA-E function, indicating the need for more robust studies of murine MHC-Ib proteins as models for human disease.
Asunto(s)
Antígenos CD8/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/química , Animales , Antígenos CD8/genética , Dimerización , Humanos , Interferón gamma/metabolismo , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Células Jurkat , Ligandos , Ratones , Ratones Endogámicos C57BL , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Resonancia por Plasmón de Superficie , Antígenos HLA-ERESUMEN
Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell-mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I-ß2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV-PA123-130 We determined the structure of the BF2*1501-PA123-130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123-130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123-130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV-PA123-130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123-130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.
Asunto(s)
Epítopos/química , Antígenos de Histocompatibilidad Clase I/química , Subtipo H5N1 del Virus de la Influenza A/inmunología , Animales , Pollos , Secuencia Conservada , Mapeo Epitopo , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Subtipo H5N1 del Virus de la Influenza A/química , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
T-cell activation is a critical part of the adaptive immune system, enabling responses to foreign cells and external stimulus. In this process, T-cell antigen receptor (TCR) activation stimulates translocation of the downstream kinase PKCθ to the membrane, leading to NF-κB activation and thus transcription of relevant genes. However, the details of how PKCθ is recruited to the membrane remain enigmatic. It is known that annexin A5 (ANXA5), a calcium-dependent membrane-binding protein, has been reported to mediate PKCδ activation by interaction with PKCδ, a homologue of PKCθ, which implicates a potential role of ANXA5 involved in PKCθ signaling. Here we demonstrate that ANXA5 does play a critical role in the recruitment of PKCθ to the membrane during T-cell activation. ANXA5 knockout in Jurkat T cells substantially inhibited the membrane translocation of PKCθ upon TCR engagement and blocked the recruitment of CARMA1-BCL10-MALT1 signalosome, which provides a platform for the catalytic activation of IKKs and subsequent activation of canonical NF-κB signaling in activated T cells. As a result, NF-κB activation was impaired in ANXA5-KO T cells. T-cell activation was also suppressed by ANAX5 knockdown in primary T cells. These results demonstrated a novel role of ANXA5 in PKC translocation and PKC signaling during T-cell activation.
Asunto(s)
Anexina A5/inmunología , Activación de Linfocitos , Proteína Quinasa C-theta/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Anexina A5/genética , Humanos , Células Jurkat , Ratones , Proteína Quinasa C-theta/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
The cytokine content in tissue microenvironments shapes the functional capacity of a T cell. This capacity depends on the integration of extracellular signaling through multiple receptors, including the T-cell receptor (TCR), co-receptors, and cytokine receptors. Transforming growth factor ß (TGF-ß) signals through its cognate receptor, TGFßR, to SMAD family member proteins and contributes to the generation of a transcriptional program that promotes regulatory T-cell differentiation. In addition to transcription, here we identified specific signaling networks that are regulated by TGFßR. Using an array of biochemical approaches, including immunoblotting, kinase assays, immunoprecipitation, and flow cytometry, we found that TGFßR signaling promotes the formation of a SMAD3/4-protein kinase A (PKA) complex that activates C-terminal Src kinase (CSK) and thereby down-regulates kinases involved in proximal TCR activation. Additionally, TGFßR signaling potentiated CSK phosphorylation of the P85 subunit in the P85-P110 phosphoinositide 3-kinase (PI3K) heterodimer, which reduced PI3K activity and down-regulated the activation of proteins that require phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) for their activation. Moreover, TGFßR-mediated disruption of the P85-P110 interaction enabled P85 binding to a lipid phosphatase, phosphatase and tensin homolog (PTEN), aiding in the maintenance of PTEN abundance and thereby promoting elevated PtdIns(4,5)P2 levels in response to TGFßR signaling. Taken together, these results highlight that TGF-ß influences the trajectory of early T-cell activation by altering PI3K activity and PtdIns levels.