Indirect Recognition Pathway Research Articles

312.6: Immunological challenges to control indirect recognition pathway for long-term outcome in xenotransplantation

312.6: Immunological challenges to control indirect recognition pathway for long-term outcome in xenotransplantation

Recipient dendritic cells modified by RNA interference targeting CD80 and CD86 elicit T cell hyporesponsiveness via enhanced T cell apoptosis.

Despite extensive research, the mechanism of immature dendritic cells (DCs) induced immune hyporesponsiveness remains incompletely understood. Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin (IL)-2, IL-4, IL-10, and interferon (INF)-γ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining. There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups (P < 0.05), indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups (P < 0.05). There was no significant difference in IL-4 levels between the groups (P > 0.05). We also showed that CD80/86 low DCs loaded with alloantigen (1) stimulated low T cell proliferative responses via the indirect recognition pathway and (2) enhanced apoptotic activity (P < 0.05) in co-cultured T cells. Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinically applicable.

Cross‐species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin/26S proteasome‐mediated proteolysis

The recognition of ubiquitylated substrates is an essential element of ubiquitin/26S proteasome-mediated proteolysis (UPP), which is mediated directly by the proteasome subunit RPN10 and/or RPN13, or indirectly by ubiquitin receptors containing ubiquitin-like and ubiquitin-associated domains. By pull-down and mutagenesis assays, we detected cross-species divergence of the major recognition pathways. RPN10 plays a major role in direct recognition in Arabidopsis and yeast based on the strong affinity for the long and K48-linked ubiquitin chains. In contrast, both the RPN10 and RPN13 homologs play major roles in humans. For indirect recognition, the RAD23 and DSK2 homologs (except for the human DSK2 homolog) are major receptors. The human RAD23 homolog is targeted to the 26S proteasome by the RPN10 and RPN13 homologs. In comparison, Arabidopsis uses UIM1 and UIM3 of RPN10 to bind DSK2 and RAD23, respectively. Yeast uses UIM in RPN10 and LRR in RPN1. Overall, multiple proteasome subunits are responsible for the direct and/or indirect recognition of ubiquitylated substrates in yeast and humans. In contrast, a single proteasome subunit, RPN10, is critical for both the direct and indirect recognition pathways in Arabidopsis. In agreement with these results, the accumulation of ubiquitylated substrates and severe pleiotropic phenotypes of vegetative and reproductive growth are associated with the loss of RPN10 function in an Arabidopsis T-DNA insertion mutant. This implies that the targeting and proteolysis of the critical regulators involved are affected. These results support a cross-species mechanistic and functional divergence of the major recognition pathways for ubiquitylated substrates of UPP.

Endoplasmic Reticulum Stress-Mediated Apoptosis Involved in Indirect Recognition Pathway Blockade Induces Long-Term Heart Allograft Survival

Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

A Novel Flow Assay for the Detection of Cytokine Secreting Alloreactive T Cells: Application to Immune Monitoring

The direct and indirect allorecognition pathways play an important role in graft rejection. We hypothesized that the presence of alloreactive memory T cells in the recipient’s circulation increases the risk of rejection after transplantation. The objective of this study was to develop a noninvasive, immune monitoring tool that simultaneously measures donor-specific responses via both the direct and indirect recognition pathways. Our laboratory developed a whole blood flow cytometric cytokine secretion assay to identify interferon (IFN)-γ secreting memory T cells in whole blood of renal transplant patients. The assay readily detected IFN-γ producing CD3+ T cells in response to recall antigens tetanus toxoid, purified protein derivative, and alloantigens in whole blood from healthy controls. Analysis of sequential posttransplant blood samples from 19 renal allograft recipients showed that alloimmune responses were higher in transplant recipients who had undergone acute rejection than in those without acute rejection episodes. In addition, patients showing increased creatinine levels 3 months after transplantation were more likely to exhibit alloimmune responses than recipients with stable graft function. The flow cytokine secretion assay provides a reliable and simple method for identification of patients at risk of acute rejection and early graft dysfunction.

Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4(+) T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8(+) T cells and transplanted with class II transactivator (CIITA)-deficient cardiac allografts, which cannot directly present class II alloantigens to CD4(+) T cells. In this manner, the rejection response by CD4(+) cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8(+) cells, both BALB/c and C57BL/6 mice rejected CIITA-/- allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA-/- allografts were depleted of CD8(+) T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8-depleted C57BL/6 recipients of CIITA-/- allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4(+) T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2-dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.

Prolonged survival of donor-specific rat intestinal allograft by administration of bone-marrow-derived immature dendritic cells

It has been reported that intraportal administration of donor antigens induced donor-specific hyporesponsiveness. We studied here the effects of transplantation of BM-derived immature dendritic cells (imDCs) and mature DCs (mDCs) via portal vein on rat small intestinal allograft survival. This study comprised four treatment groups: 1) untreated controls; 2) FK506 alone; 3) intraportal donor-specific BM-derived imDCs transplantation+FK506; 4) mDCs/Tx+FK506. Allograft survival was minimal in control group (5.2±0.8 days) and maximal in imDC+FK506 group (28.4±3.0 days). The rats in mDC+FK506 group showed systemic inflammatory reaction due to GVHR, and died within 10 days after transplantation. The in vitro MLR reaction using imDCs was also strongly inhibited both in direct and indirect recognition pathways. The impact of imDCs for the specific induction of transplant tolerance may suggest that immunization with donor-specific imDCs has therapeutic potential in organ transplantation.

Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is associated with indirect recognition by CD4 + T cells

T-cell mediated immune responses play a critical role in chronic allograft dysfunction. The complex nature of allograft rejection, particularly with respect to the vast repertoire of alloantigens and their mode of recognition by T cells, presents a major challenge for the design of well-controlled studies into the immunobiology of chronic rejection. The purpose of this study was to develop a rat model with restricted antigenic specificity that develops chronic rejection without any immunologic manipulation to study the T-cell response. PVG.1U allogeneic hearts disparate for one single class I antigen, RT.1A u, were transplanted into PVG.R8 rat recipients. Grafts from PVG.R8 were used as syngeneic controls. Chronic rejection was studied by histological analysis of the grafted hearts at various time points posttransplantation (20–100 days). Donor specific alloreactive response was studied in a mixed lymphocyte reaction assay. All allografts survived more than 90 days and showed extensive evidence of chronic rejection, which was characterized by interstitial fibrosis, vasculitis, and occlusive myointimal thickening. Chronic rejection was evident by day 20 and most extensive by day 100 posttransplantation. In marked contrast, syngeneic grafts remained free of chronic lesions. Lymphocytes harvested from graft recipients showed a more vigorous proliferative response to allogeneic splenocytes as compared with that of lymphocytes from naı̈ve animals. The proliferative response was primarily mediated by CD4 + T cells recognizing the RT1.A a molecule via the indirect pathway. A single class I disparity in this model generates chronic rejection associated with potent CD4 + T-cell responses induced by the indirect recognition pathway. The use of this antigenically restricted model may facilitate the design of well-controlled studies for the characterization of immune mechanisms responsible for chronic rejection.

Comparison of Ab−/−β, H2-DM−, and CIITA−/− in Second-Set Skin Allograft Rejection

Background. Responses against donor MHC antigens are the major contributor to allograft rejection. Currently, it is unclear whether both direct and indirect recognition pathways are necessary and/or sufficient for allograft rejection. Previously, we found donor MHC class II and H2-DM to have dramatic effects on cardiac allograft survival.Methods. Here, we used H2-DM− mice, which express CLIP–MHC class II complexes, and CIITA−/− mice, which lack all class II proteins, to examine the role of direct and indirect recognition on skin allograft rejection. Recipients were primed with donor cultured keratinocytes and later tested for accelerated memory response by challenge with full-thickness tail skin grafts.Results. As previously reported, Ab−/−β grafts survived longer than wild-type grafts, while H2-DM− grafts were rejected as rapidly as wild-type grafts. Skin grafts deficient for both β2m and H2-DM survived longer than grafts lacking only H2-DM, but not as long as Ab−/−β grafts. Additionally, CIITA−/− grafts survived as long as Ab−/−β grafts.Conclusions. The delayed rejection of Ab−/−β compared to H2-DM− suggests that indirect recognition of surface-expressed donor MHC class II is sufficient to mediate rapid skin allograft rejection. The equivalent survival of CIITA−/− and Ab−/−β grafts suggests that indirect presentation of donor class II molecules (Aα or Eβ) present in Ab−/−β but not CIITA−/− mice does not contribute to graft rejection. These results reveal a modest role for surface-expressed donor class II in primed keratinocyte rejection, but also reveal a dramatic contrast to the cardiac allograft system and indicate tissue/organ-specific mechanisms of rejection.

CONRIBUTION OF THE INDIRECT RECOGNITION PATHWAY TO CARDIAC GRAFT REJECTION IN THE RAT-to-MOUSE XENOGENIC MODEL

49 The T cell response to transplanted xenogenic organs/tissues is mediated via direct (donor MHC plus peptides) and indirect (recipient MHC plus donor-derived peptides) xenorecognition pathways. The role of the indirect pathway in xenoantigen recognition and xenograft rejection has not been fully investigated. In this study, we, therefore, used cardiac grafts in the rat-to-mouse (PVG.R8-to-C57BL/B10) species combination as a model system to determine relative contribution of indirect recognition to the rejection of vascularized xenografts. The T cell response to xenoantigens was determined using lymph node cells or splenocytes harvested from naive or xenograft recipients at rejection in a standard mixed lymphocyte reaction assay. The responses to auto and allogenic antigens were used as negative and positive controls, respectively. We obtained a higher (30,000-35,000 c.p.m.) T cell response to xenoantigens when compared with that for alloantigens (18,000-21,000 c.p.m.) at all time points of cell proliferation examined. The proliferative response of immune T cells was more robust and vigorous than that of naive T cells (45,000-50,000 vs 30,000-35,000 c.p.m.). We used monoclonal antibodies to mouse CD80 and CD86 costimulatory molecules at various doses (1-25 μg/ml) to determine the contribution of indirect recognition to the observed T cell responses. There was a dose-dependent inhibition of the proliferative response of both naïve and immune T cells when these antibodies used either individually or in combination. The maximum inhibition (95-98%), however, was observed when both antibodies used in combination at a dose of 25 μg/ml. The suppression of cell proliferation by antibodies against mouse CD80 and CD86 costimulatory molecules provide clear evidence that the indirect recognition pathway plays a critical role in xenoreactive immunity in this model as these antibodies can only block antigen presentation by the recipient (mouse) antigen presenting cells (indirect recognition). The dominant role of indirect recognition in T cell responses to xenoantigens suggests that the modulation of this pathway will be critical for prevention of cellular immunity and long-term graft survival in a xenosetting.

Intensity and mechanisms of in vitro xenorecognition of adult pig pancreatic islet cells by CD4+ and CD8+ lymphocytes from type I diabetic or healthy subjects.

The intensity and mechanisms of cell-mediated rejection of pig islet cells were studied in 49 Type I diabetic and 34 healthy subjects. Human peripheral mononuclear cells proliferated strongly in response to pig islet cells (p<0.001), though with notable interindividual variations (stimulation index 2 to 215). The variance of stimulation index was higher in diabetic than healthy subjects (p<0.0001). The response to islet cells was stronger (p<0.01) than that to pig splenocytes. Proliferation in response to islet cells was strongly decreased (p<0.01) when CD4+ T cells were blocked with monoclonal antibodies, whereas the blocking of CD8+ cells or NK cells gave less pronounced effects. The response to islet cells was decreased (p<0.01), but not abolished, after antigen-presenting cells were removed. Purified CD4+ cells alone did not proliferate in response to islet cells but recovered their proliferative ability when mixed with antigen-presenting cells, whereas CD8+ cells alone proliferated in the presence of interleukin-2 in response to islet cells. Proliferation was blocked (p<0.01) by anti-DR monoclonal antibodies. During proliferation in response to islet cells, interleukin-10 increased 43-fold (p<0.01) but interferon-gamma increased only slightly. No statistical differences were detected between diabetic and control subjects with respect to lymphocyte subsets and the recognition mechanisms or to interferon-gamma/interleukin-10 production in response to islet cells. These results provide the first detailed information on human cell-mediated xenoreaction to pig islet cells. This situation involves a dominant CD4 class II-restricted Th2 response, with an indirect recognition pathway, as well as a CD8 T-cell response resulting from direct recognition. This strong reaction constitutes a serious obstacle which may vary in degree among subjects.

Specific immunosuppression of the indirect recognition pathway

Specific immunosuppression of the indirect recognition pathway

Specific immunosuppression of the indirect recognition pathway

Specific immunosuppression of the indirect recognition pathway

Role of the indirect recognition pathway in the development of chronic liver allograft rejection

Role of the indirect recognition pathway in the development of chronic liver allograft rejection

Mechanism of liver allograft rejection: The indirect recognition pathway

Transplant rejection is mediated by the direct and indirect pathways. To explore the role of the indirect recognition pathway in the rejection of liver allografts, T cells obtained from peripheral blood were expanded in medium containing IL-2 and tested in LDA for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial investigations of 17 recipients showed that T-cell reactivity to donor HLA-DR peptides was strongly associated with acute rejection episodes. In recipients carrying a graft that was mismatched by two HLA-DR alleles, a single donor antigen was targeted during primary rejection, although allopeptide reactivity against the second HLA-DR antigen was observed during subsequent episodes of acute rejection. The finding that allopeptide reactivity occurs early following transplantation and is predictive of rejection is consistent with the notion that processing of donor alloantigens by recipient APCs activates the indirect T-cell recognition pathway that plays a major role in initiating and amplifying allograft rejection.

T and B cell responsiveness to donor class I MHC molecules and peptides in long survivors with kidney allografts.

LEW rats with long-surviving (> 100 days) (DA x LEW)F1 kidney allografts were generated by treating the recipients with cyclosporine for 14 days after grafting. All rats were monitored after transplantation for the development of antibodies to intact donor class I MHC molecules. Cyclosporine completely suppressed the early antibody response to intact DA class I MHC molecules in all 19 LEW rats. However, 17 of the 19 rats developed antibodies between four and six weeks after grafting-i.e., between two and four weeks after the cessation of cyclosporine therapy, and maintained high levels of antibody to the donor class I molecules in spite of the long-term presence of the allograft. The 2 rats that did not produce antibodies to donor class I MHC molecules, along with one of the 17 that did produce antibodies, were immunized with a synthetic peptide corresponding to a region of the DA class I MHC molecule known to be recognized by LEW CD4+ T cells via the indirect recognition pathway. All 3 long survivors developed self APC-dependent CD4+ T cell proliferation to the immunizing donor peptides, and strong antibody responses to these peptides. However, none of these long survivors suffered rejection episodes as a consequence of the peptide immunization. In one of the two long-surviving rats without antibodies to intact donor class I MHC molecules at the time of peptide priming, the peptide priming resulted in the prompt development of strong antibodies to intact donor class I molecules. However, the other of these 2 rats did not produce such antibodies after peptide priming. Thus in this model of kidney allograft tolerance, with long-term exposure of the recipient's immune system to donor antigens without evidence of rejection, none of the animals develops tolerance for the indirect T cell recognition of donor class I MHC antigens. In occasional animals, B cells potentially reactive to intact donor class I molecules are present and are adequately exposed to antigen but are quiescent because of the absence of T cell help, perhaps as a consequence of reversible T cell suppression or anergy. In other occasional animals, B cell nonreactivity (anergy or tolerance) to intact donor class I molecules appears to develop.

Indirect Allorecognition of Platelets by T Helper Cells During Platelet Transfusions Correlates With Anti-Major Histocompatibility Complex Antibody and Cytotoxic T Lymphocyte Formation

To study the cellular immunology of platelet-induced alloimmunization, a murine transfusion model was developed. BALB/c (H-2d) recipient mice were transfused weekly with 2 x 10(8) platelets or 10(3) leukocytes from C57BL/6 (H-2b) donor mice. Recipient antidonor major histocompatibility complex (MHC) class I alloantibodies could be detected in flow cytometric assays by the fifth platelet transfusion. In contrast, when leukocytes only were transfused, alloantibodies were not detected. In vitro assays demonstrated that murine H-2b platelets were positive for MHC class I expression but lacked MHC class II molecules on their membranes and were unable to stimulate proliferation or cytokine production when incubated with naive H-2d spleen cells. In vivo, however, platelet transfusions induced two distinct patterns of cell-mediated reactivity. First, during the initial transfusions and before alloantibody formation, there was induction of T-cell anergy, characterized by the inability of recipient T cells to respond to Concanavalin A (ConA) or to proliferate in an antidonor mixed lymphocyte reaction (MLR), together with suppressed natural killer (NK) cell activity. This unresponsiveness was associated with a transient increase in nitric oxide (NO)-dependent cytotoxicity and interleukin-1 (IL-1) production. Second, once alloantibodies developed, significantly increased antidonor CD8+ cytotoxic T lymphocyte (CTL) and NK cell responses were observed. At this time, when recipient spleen cells were depleted of CD8+ T cells and incubated with only donor platelets in 7-day antigen-presenting cell (APC) assays, enhanced proliferation and IL-2 production occurred. These cellular responses were not seen when 10(3) allogeneic leukocytes were transfused. Thus, the results suggest that leukoreduced platelet transfusions induce antidonor MHC antibodies and CD8+ CTL responses in recipient mice. At the same time, the transfusions induced recipient CD4+ T-cell activation when incubated with donor platelets in the presence of syngeneic APCs, an indirect recognition pathway that correlates with the time of alloantibody production.

Contribution of direct and indirect recognition pathways to T cell alloreactivity.

T cells from an HLA-DR11/DR12 responder were stimulated in mixed lymphocyte culture with cells carrying the DR1 antigen. After priming, T cells proliferated in response to both DR1-positive-stimulating cells and a peptide derived from a polymorphic region of the HLA-DR beta 1*0101 chain presented by responder's antigen-presenting cells (APC). The dominant epitope recognized by the primed T cells corresponded to residue 21-42 and was presented by the responder's HLA-DR12 antigen. The DR1 peptide-reactive T cells express T cell receptor V beta 3. The results demonstrate that allopeptides derived from the processing and presentation of donor major histocompatibility complex molecules by host-derived APC trigger alloreactivity. The frequency of T cells engaged in the indirect pathway of allorecognition is about 100-fold lower than that of T cells participating in the direct recognition of native HLA-DR antigen. However, indirect allorecognition may play an important role in chronic allograft rejection, a phenomenon that is mediated by the activation of T helper cells and of alloantibody-producing B cells.

Contribution of direct and indirect recognition pathways to T cell alloreactivity: [formula omitted], Y.K. Sun, Y.P. Xi, A. Maffei, E. Reed, P. Harris, and N. Suciu-Foca. Department of Pathology, Columbia University, New York, NY

Contribution of direct and indirect recognition pathways to T cell alloreactivity: [formula omitted], Y.K. Sun, Y.P. Xi, A. Maffei, E. Reed, P. Harris, and N. Suciu-Foca. Department of Pathology, Columbia University, New York, NY