Direct Allorecognition Research Articles

Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.

Preexisting, donor-specific antibodies (DSAs) are culprits of hyperacute rejection. Donor-specific antibodies are also formed de novo, and their role in acute and chronic rejection is increasingly appreciated. However, it is difficult to assess damage inflicted exclusively by DSAs when alloreactive T cell and B cell responses coincide. We reasoned that allosensitization with "costimulation-deficient" cells should induce DSA synthesis but not naive cytotoxic T lymphocyte (CTL) precursors' priming via direct allorecognition. Accordingly, we have developed a novel model to quantify DSA-mediated cytotoxicity in vivo. C57BL/6 (H-2b) mice were sensitized with H-2 kidney epithelial cells, and a cytofluorimetric killing assay was tailored to the measurement of allocytotoxicity. We took cell/complement depletion, costimulation blockade, and serum transfer approaches to reveal the mediators of cytotoxicity. "Third-party" controls and a skin allotransplantation model were used to confirm DSAs' specificity for allo-major histocompatibility complex. We validated our experimental approach in other mouse strains primed with different allogeneic cell types, including endothelial cells. To demonstrate the usefulness of our model/method for drug efficacy testing, we examined the effect of CTLA4-Ig and rapamycin on DSA-mediated cytolysis. Allosensitization of MHC-disparate mouse strains with costimulation-deficient cells led to robust cytotoxicity mediated by complement-fixing DSAs and phagocytic cells. This response was independent of CTLs, natural killer or natural killer T cells. It required CD4 T cell help, CD40 signaling and CD28-based costimulation during allosensitization and could be reversed by sustained rapamycin treatment. The unique model described herein should enable mechanistic studies on sensitization and effector phases of humoral alloreactivity as well as efficacy testing of future immunotherapies to prevent DSA-induced pathology.

Altered Immunogenicity of Donor Lungs via Removal of Passenger Leukocytes Using Ex Vivo Lung Perfusion

Passenger leukocyte transfer from the donor lung to the recipient is intrinsically involved in acute rejection. Direct presentation of alloantigen expressed on donor leukocytes is recognized by recipient T cells, promoting acute cellular rejection. We utilized ex vivo lung perfusion (EVLP) to study passenger leukocyte migration from donor lungs into the recipient and to evaluate the effects of donor leukocyte depletion prior to transplantation. For this purpose, female pigs received male left lungs either following 3 h of EVLP or retrieved using standard protocols. Recipients were monitored for 24 h and sequential samples were collected. EVLP-reduced donor leukocyte transfer into the recipient and migration to recipient lymph nodes was markedly reduced. Recipient T cell infiltration of the donor lung was significantly diminished via EVLP. Donor leukocyte removal during EVLP reduces direct allorecognition and T cell priming, diminishing recipient T cell infiltration, the hallmark of acute rejection.

The road to transplant tolerance is paved with good dendritic cells

After transplantation, recipient T cells can recognize donor antigens either by interacting with MHC class II on donor bone marrow-derived cells (direct allorecognition), or by recognizing allogeneic peptides bound to self-MHC class II molecules on recipient antigen presenting cells (indirect allorecognition). The activation of pro-inflammatory T cells via either of these pathways leads to allograft rejection, so the suppression of both of these pathways is needed to achieve transplantation tolerance. A study in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43: 734-746] shows that allogeneic dendritic cells (DCs) modified to either lack expression of CD80/86 or over-express indoleamine 2,3-dioxygenase (IDO) are able to inhibit direct and/or indirect alloresponses in vitro and in vivo in mice. Notably, both allorecognition pathways were suppressed by the coexpression of self- and allo-MHC molecules on semi-allogeneic DCs. This Commentary discusses the challenges and potential of using genetically-modified DCs to suppress alloreactivity in the context of transplant tolerance.

Assessment of Longevity of the Direct and Indirect CD4 T Cell Allorecognition Response

Introduction: Recipient CD4 T cells can recognise alloantigen ‘directly’, intact on donor antigen presenting cells (APCs), and ‘indirectly’, as self-restricted processed allopeptide. The relative duration of each pathway remains unclear, and further complicated by our recent description that the longevity of indirect responses differed according to target alloantigen. This work however examined different CD4 T cell responses in different murine models, and to control for differences in the immunogenicity of each, we developed a new model of chronic allograft vasculopathy that would allow definitive examination of all CD4 T cell allorecognition pathways operating in each recipient. Methods: Several mouse strains were intercrossed to generate a novel strain, bm12.Kd.IE, which differs from wild-type B6 by possessing additional MHC class I (H-2Kd) and MHC class II (IAbm12 and IE) molecules, but lacks the IAb MHC class II antigen. Heterotopic cardiac allografts were performed using bm12.Kd.IE donors into B6 recipients, and the model characterised by measuring rejection kinetics, degree of allograft vasculopathy, and production of alloantibody and autoantibody. The relative duration of direct and indirect allorecognition of different alloantigens was assessed by comparing division of monoclonal populations of alloreactive, CFSE-labelled TCR-transgenic CD4 T cells that were adoptively transferred either on day 0 or 28 post transplantation. Indirect allorecognition of MHC I was assessed using TCR75 CD4 T cells (I-Ab-restricted, H2-Kd peptide-specific); class II using TEa CD4 T cells (I-Ab-restricted, I-E peptide-specific) and minor antigen using Mar CD4 T cells (I-Ab-restricted, H-Y peptide-specific). Direct allorecognition of I-Abm12 MHC II was assessed using ABM CD4 T cells. Results: Bm12.Kd.IE hearts survived >50 days in B6 recipients, with histological evidence of chronic allograft vasculopathy and production of anti-Kd and anti-I-E alloantibody. Autoantibody, assessed by indirect immunfluosrescent binding to nuclear-antigen-expressing HEp-2 cells, also developed. Flow cytometric analysis of CFSE labelled transgenic CD4 T cells confirmed that direct pathway responses were robust immediately after transplant, with extensive division of the transferred ABM CD4 T cells, but were minimal four weeks later. Similarly, strong indirect pathway responses against MHC II alloantigen occurred immediately after transplant, but were surprisingly undetectable at later time points. In contrast, indirect alloresponses against MHC I alloantigen were equally strong at early and late times after transplant. Comparable results were obtained when indirect alloresponses against minor H-Y antigen were studied, although division at five weeks was less marked than immediately after transplant. Conclusions: Our results provide experimental evidence to support the paradigm that direct pathway CD4 T cells responses are short-lived, but also highlight that the duration of indirect pathway responses varies according to target alloantigen. The transient nature of anti-class II responses presumably reflects rapid loss of donor APC after transplantation.

Two immunogenic passenger dendritic cell subsets in the rat liver have distinct trafficking patterns and radiosensitivities

The aim of this study was to investigate the trafficking patterns, radiation sensitivities, and functions of conventional dendritic cell (DC) subsets in the rat liver in an allotransplantation setting. We examined DCs in the liver, hepatic lymph, and graft tissues and recipient secondary lymphoid organs after liver transplantation from rats treated or untreated by sublethal irradiation. We identified two distinct immunogenic DC subsets. One was a previously reported population that underwent blood-borne migration to the recipient's secondary lymphoid organs, inducing systemic CD8(+) T-cell responses; these DCs are a radiosensitive class II major histocompatibility complex (MHCII)(+) CD103(+) CD172a(+) CD11b(-) CD86(+) subset. Another was a relatively radioresistant MHCII(+) CD103(+) CD172a(+) CD11b(+) CD86(+) subset that steadily appeared in the hepatic lymph. After transplantation, the second subset migrated to the parathymic lymph nodes (LNs), regional peritoneal cavity nodes, or persisted in the graft. Irradiation completely eliminated the migration and immunogenicity of the first subset, but only partly suppressed the migration of the second subset and the CD8(+) T-cell response in the parathymic LNs. The grafts were acutely rejected, and intragraft CD8(+) T-cell and FoxP3(+) regulatory T-cell responses were unchanged. The radioresistant second subset up-regulated CD25 and had high allostimulating activity in the mixed leukocyte reaction, suggesting that this subset induced CD8(+) T-cell responses in the parathymic LNs and in the graft by the direct allorecognition pathway, leading to the rejection. Conventional rat liver DCs contain at least two distinct immunogenic passenger subsets: a radiosensitive blood-borne migrant and a relatively radioresistant lymph-borne migrant. LNs draining the peritoneal cavity should be recognized as a major site of the intrahost T-cell response by the lymph-borne migrant. This study provides key insights into liver graft rejection and highlights the clinical implications of immunogenic DC subsets.

Immunomodulatory Effect of Nuclear Factor-κB Inhibition by Dehydroxymethylepoxyquinomicin in Combination With Donor-Specific Blood Transfusion

Nuclear factor-κB (NF-κB) is a key molecule in alloimmune responses, however, its role in tolerance induction is not clear. We have previously reported that dehydroxymethylepoxyquinomycin (DHMEQ), a novel NF-κB inhibitor, prolongs cardiac allograft survival. In this study, we evaluated the immunomodulatory effects of DHMEQ when combined with a donor-specific blood transfusion (DST), and assessed whether the treatment induces tolerance in a mouse heart transplantation model. DST (20×10 splenocytes) was given intravenously at day -7. DHMEQ (30 mg/kg/day) was administered intraperitoneally for 14 days after DST. Graft survival and histology were evaluated. The underlying mechanisms of immunomodulation by DST and DHMEQ treatments were investigated by assessing alloimmune responses after transplantation. In fully mismatched H2-to-H2 heart transplants, DST alone prolonged allograft median survival time to 15 days, whereas when DST was combined with DHMEQ treatment, the graft median survival time was prolonged to 39.5 days. When the donor-recipient strain combination was reversed, that is, H2-to-H2, heart transplants were accepted (>150 days survival) in more than 60% of recipients treated with a DST and DHMEQ, whereas control allografts were all rejected within 8 days. The combined therapy markedly inhibited immune responses by both the direct and indirect allorecognition pathways mainly attributed to promotion of activation-induced cell death and Treg generation. Our results demonstrate the distinctive ability of NF-κB inhibition in combination with donor alloantigen to promote transplantation tolerance through multiple cellular mechanisms.

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

BackgroundDirect allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.MethodsUsing transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2d) prior to transplanting into C57BL/6 mice (H-2b), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).ResultsDepleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.ConclusionCollectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

Inhibition of T-Cell Expansion Caused by Inducible Costimulator/B7h Costimulation Blockade in Direct Allorecognition Pathway

Objective Inducible costimulator (ICOS)/B7h costimulation plays a crucial role in acute and chronic allograft rejection. To test the role of the ICOS signal in T-cell activation and expansion, we used ICOS-Fc–targeted B cells as donor antigen presenting cells to challenge the allogeneic response in vitro. Methods In vitro, the binding of ICOS-Fc with B7h on splenic B cells was confirmed by flow cytometry analysis. To evaluate the capacity of ICOS-Fc–targeted B cells to elicit an allogeneic response in vitro, we performed mixed lymphocyte reactions. Results The binding of B7h on splenic B cells by ICOS-Fc was confirmed at a saturating concentration of 100 μg/mL. Blockade of ICOS/B7h in direct allorecognition depressed proliferation of alloreactive T cells in vitro. Conclusions ICOS/B7h signal plays an important role in direct allorecognition, eliciting allogeneic responses in vitro.

Contributions of Direct and Indirect Alloresponses to Chronic Rejection of Kidney Allografts in Nonhuman Primates

The relative contribution of direct and indirect allorecognition pathways to chronic rejection of allogeneic organ transplants in primates remains unclear. In this study, we evaluated T and B cell alloresponses in cynomolgus monkeys that had received combined kidney/bone marrow allografts and myeloablative immunosuppressive treatments. We measured donor-specific direct and indirect T cell responses and alloantibody production in monkeys (n = 5) that did not reject their transplant acutely but developed chronic humoral rejection (CHR) and in tolerant recipients (n = 4) that never displayed signs of CHR. All CHR recipients exhibited high levels of anti-donor Abs and mounted potent direct T cell alloresponses in vitro. Such direct alloreactivity could be detected for more than 1 y after transplantation. In contrast, only two of five monkeys with CHR had a detectable indirect alloresponse. No indirect alloresponse by T cells and no alloantibody responses were found in any of the tolerant monkeys. Only one of four tolerant monkeys displayed a direct T cell alloresponse. These observations indicate that direct T cell alloresponses can be sustained for prolonged periods posttransplantation and result in alloantibody production and chronic rejection of kidney transplants, even in the absence of detectable indirect alloreactivity.

Immune Recognition and Rejection of Allogeneic Skin Grafts

The transplantation of allogeneic skin grafts is associated with a potent inflammatory immune response leading to the destruction of donor cells and the rejection of the graft. Shortly after transplantation, skin dendritic cells (DCs) migrate out of the graft through lymphatic vessels and infiltrate the recipient's draining lymph nodes where they present donor antigens via two mechanisms: the direct pathway, in which T cells recognize intact donor MHC antigens on donor DCs; and the indirect pathway, involving T-cell recognition of donor peptides bound to self-MHC molecules on recipient DCs. Some recent studies have suggested that T cells can become activated via recognition of donor MHC molecules transferred on recipient antigen-presenting cells (semidirect pathway). Activation of T cells via direct or indirect allorecognition is sufficient to trigger acute rejection of allogeneic skin grafts. In addition, allospecific antibodies contribute to the rejection process either by killing allogeneic targets in a complement-dependent fashion or by opsonizing donor cells and forming immune complexes. Finally, several studies demonstrate that NK cells, activated due to missing self-MHC class I molecules on allogeneic cells, are involved in allogeneic skin graft rejection via direct killing of donor cells and through the production of proinflammatory cytokines including IFN-γ and TNF-α.

CCR2 Regulates Monocyte Recruitment As Well As CD4+ Th1 Allorecognition After Lung Transplantation

Graft rejection remains a formidable problem contributing to poor outcomes after lung transplantation. Blocking chemokine pathways have yielded promising results in some organ transplant systems. Previous clinical studies have demonstrated upregulation of CCR2 ligands following lung transplantation. Moreover, lung injury is attenuated in CCR2-deficient mice in several inflammatory models. In this study, we examined the role of CCR2 in monocyte recruitment and alloimmune responses in a mouse model of vascularized orthotopic lung transplantation. The CCR2 ligand MCP-1 is upregulated in serum and allografts following lung transplantation. CCR2 is critical for the mobilization of monocytes from the bone marrow into the bloodstream and for the accumulation of CD11c(+) cells within lung allografts. A portion of graft-infiltrating recipient CD11c(+) cells expresses both recipient and donor MHC molecules. Two-photon imaging demonstrates that recipient CD11c(+) cells are associated with recipient T cells within the graft. While recipient CCR2 deficiency does not prevent acute lung rejection and is associated with increased graft infiltration by T cells, it significantly reduces CD4(+) T(h)1 indirect and direct allorecognition. Thus, CCR2 may be a potential target to attenuate alloimmune responses after lung transplantation.

Transferable Cardiac Allograft Acceptance Induced by Transfusion of Donor B Cells With Impaired Inducible Costimulator/B7h Allorecognition

Objective Donor-specific transfusion (DST) of leukocytes with an impaired costimulatory signal has been proven to be an effective way to improve allograft survival. Inducible costimulator (ICOS) has been shown to play a crucial role in acute and chronic allograft rejection. To test the role of ICOS signaling during DST, we employed ICOS-Fc-targeted B cells as antigen of DST to challenge the allogeneic engraftment in vivo. Materials and Methods A murine cardiac allograft model was employed using BALB/c donors and C57BL/6 recipients, while various transfusions were performed according to treatment protocols. Results Allograft survival was prolonged by infusion of ICOS-Fc-targeted B cells; however, allograft acceptance could not be achieved unless additional systemic injections of ICOS-Fc were given. Adoptive transfer of splenic CD4 + but not CD4 +CD25 − subsets from long-term allograft survival (LTAS) mice to lightly irradiated naive recipients resulted in subsequent BALB/c allograft acceptance without additional immunosuppression. Conclusions ICOS/B7h signaling during direct allorecognition played an important role in prolonging allograft survival, and an allograft acceptance can be established by DST with complete blockade of ICOS/B7h in both direct and indirect allorecognition. Interestingly, this allograft acceptance was transferable and maintained at least partly by the immune regulation of CD4 +CD25 + T cells. These findings may help to design a potential therapeutic treatment to prevent allograft rejection by DST in combination with ICOS/B7h blockade.

End-stage renal failure and regulatory activities of CD4+CD25bright+FoxP3+ T-cells

The defensive immune system in patients with end-stage renal failure is impaired at multiple levels. This state of immune incompetence is associated with continuous activation of the immune system. An additional explanation for this state of activation may be the disturbed function of CD4(+)CD25(bright+)FoxP3(+) regulatory T-cells. The phenotype and function of peripheral regulatory T-cells from patients with end-stage renal failure (N = 80) and healthy controls (N = 17) was studied by flow cytometry, RT-PCR and mixed lymphocyte reaction. Patients were on haemodialysis (N = 40), peritoneal dialysis (N = 26) or not treated with dialysis yet (N = 14). The latter group had a glomerular filtration rate of <20 ml/min/ 1.73 m(2). The basal IL-2 mRNA level was high in patient-PBMC (P = 0.0002 versus healthy controls). The absolute number of CD4(+)CD25(bright+) T-cells was low in patients (P < 0.05 versus healthy controls). Furthermore, proliferation of patient-PBMC upon allogeneic stimulation was impaired (P < 0.0001 versus healthy controls). The regulatory function of CD4(+)CD25(bright+) T-cells was determined in the setting of direct allorecognition. First, the effect of depletion of CD25(bright+) cells from patient-PBMC on proliferation was low. Second, co-culture of CD25(bright+) cells with CD25(neg/dim) cells (1:10 ratio) showed impaired regulatory function (P < 0.001 versus healthy controls), which was especially pronounced in patients on dialysis. The FOXP3 mRNA level was also low upon stimulation (P = 0.0002 versus healthy controls). In line with previous studies, we observed an overactivated but functionally compromised immune system in patients with end-stage renal failure. It now appears that in this setting, regulation by CD4(+)CD25(bright+)FoxP3(+) T-cells is also impaired.

Differential Roles of Direct and Indirect Allorecognition Pathways in the Rejection of Skin and Corneal Transplants

It is generally accepted that all transplants are not rejected in the same fashion. However, the extrinsic and intrinsic factors that control the recognition and rejection of a particular allograft by the host are not well characterized. We compared the mechanisms underlying the response with donor antigens by T cells activated after transplantation of fully allogeneic skin and corneal grafts in mice. In corneal-transplanted mice, the CD4+ T-cell response was exclusively mediated by T cells recognizing minor antigens in an indirect fashion and producing low levels of interleukin-2. In contrast, skin grafts elicited both direct and indirect CD4+ T-cell responses primarily directed to major histocompatibility complex antigens and characterized by high interleukin-2 levels. Although CD8+ T-cells producing gamma interferon were activated directly in both skin- and corneal-grafted mice, only CD8+ T cells from skin-transplanted mice mounted a cytotoxic response. Next, we investigated whether failure of corneal transplants to induce a CD4+ direct alloresponse is due to their poor immunogenicity or due to the site of placement (eye). We observed that corneas transplanted under the skin and splenocytes transplanted in the eye were both capable of inducing direct CD4+ T-cell alloreactivity. This shows that failure of orthotopic corneal allotransplants to elicit a CD4+ T-cell direct alloresponse is associated with the combination of two factors, their low immunogenicity and the immune-privileged properties of the eye.

Ascidian Sperm Glycosylphosphatidylinositol-anchored CRISP-like Protein as a Binding Partner for an Allorecognizable Sperm Receptor on the Vitelline Coat

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

Circulating Alloreactive T Cells Correlate with Graft Function in Longstanding Renal Transplant Recipients

Monitoring for alloreactive memory T cells after organ transplantation may allow individualization of immunosuppression. Two pathways of T cell allorecognition have been implicated in chronic graft dysfunction: Direct (recipient T cells respond to donor peptides presented by donor antigen-presenting cells) and indirect (donor peptides are processed and presented by recipient antigen-presenting cells). Previous studies have assessed these alloresponses only during the first 2 yr after kidney transplantation,so this study correlated the presence of circulating donor-reactive memory/effector T cells, primed by both pathways, in 34 longstanding living-donor renal transplant recipients using the highly sensitive IFN-gamma Elispot assay. Remarkably, 59% of patients had directly primed donor-reactive T cells, and their presence correlated directly with serum creatinine (P = 0.001) and inversely with estimated GFR (P = 0.042). Multivariate analysis revealed that hyporesponsiveness of direct, donor-specific T cells was the only variable that significantly correlated with graft function and that antidonor indirect alloreactivity was the only variable that significantly correlated with proteinuria. Interestingly, when both allorecognition pathways were considered together, patients with undetectable direct alloreactivity had better longterm graft function, independent of allosensitization by the indirect pathway. In conclusion, circulating donor-specific alloreactive T cells primed by both pathways are detectable long after transplantation and are associated with graft injury. Assessment of alloreactive memory/effector T cells might be helpful to tailor individual immunosuppression regimens for transplant recipients in the future.

Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation.

The interaction of donor passenger leukocytes and host leukocytes in recipient secondary lymphoid tissues during the early posttransplantation period is crucial in directing host immune reactions toward allograft rejection or acceptance. Responsible T cell clones could be activated through the direct and indirect pathways of allorecognition. We examined the role of the indirect pathway in liver transplantation (LT) tolerance by depleting host antigen-presenting cells (APC) with phagocytic activity [e.g., cluster domain (CD)68+/CD163+ macrophages, CD11c+ dendritic cells (DC)] using liposome-encapsulating clodronate (LP-CL). After Lewis rat cell or liver graft transplantation, Brown Norway (BN) rat recipients pretreated with LP-CL showed a significantly reduced type 1 helper T cell cytokine up-regulation than control-LP-treated recipients. In the LT model, LP-CL treatment and host APC depletion abrogated hepatic tolerance; Lewis liver grafts in LP-CL-treated-BN recipients developed mild allograft rejection, failed to maintain donor major histocompatibility complex (MHC) class II+ leukocytes, and developed chronic rejection in challenged donor heart allografts, while control-LP-treated BN recipients maintained tolerance status and donor MHC class II+ hepatic leukocytes. Furthermore, in the BN to Lewis LT model, LP-CL recipient treatment abrogated spontaneous hepatic allograft acceptance, and graft survival rate was reduced to 43% from 100% in the control-LP group. In conclusion, the study suggests that host cells with phagocytic activity could play significant roles in developing LT tolerance.

Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation

Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation

Monitoring of indirect allorecognition: wishful thinking or solid data?

Monitoring of T cells involved in the alloimmune response after transplantation requires the availability of reliable in vitro assays for the detection of T cells with both direct and indirect allospecificity. While generally accepted assays exist to measure helper and cytotoxic T cells involved in direct allorecognition, consensus about an assay for monitoring indirect T-cell allorecognition in clinical transplantation is lacking. Many studies claim a relationship between the reactivity of T cells with indirect allospecificity and graft rejection, but different protocols are used and essential controls are often lacking. In this review, the disadvantages and pitfalls of the current approaches are discussed, in some cases supported by the results of our own in vitro experiments. We conclude that an international workshop is necessary to establish and validate a uniform, robust and reliable assay for the monitoring of transplant recipients and to study the actual role of indirect allorecognition in acute and chronic rejection.

Influence of direct and indirect allorecognition pathways on CD4+CD25+regulatory T-cell function in transplantation

While both direct and indirect allorecognition are involved in allograft rejection, evidence to date suggests that tolerance is primarily dependent on indirect pathway-triggered CD4+CD25+ T cell-mediated immunoregulation. However, the precise influence of these two pathways on CD4+CD25+ T-cell function has not been addressed. In the current study, we have utilized an adoptive transfer model to assess selectively how the absence of either direct or indirect allorecognition affects CD4+CD25+ T-cell function. The effects of the loss of the direct pathway were assessed by transplanting skin grafts from minor histocompatibility mismatched B10.D2 (H-2d) donors onto Balb/c (H-2d) recipients, or by placing bone marrow chimeric DBA/2 (H-2d/H-2b) allografts onto C57BL/6 (H-2b) hosts. The requirement for indirect allorecognition was tested by grafting DBA/2 skin allografts onto either C57BL/6- or MHC-II-deficient C57BL/6 recipients. We report here that although CD4+CD25+ regulatory T cells can suppress both directly and indirectly generated alloresponses, immunoregulation is favored when indirect presentation is the sole mechanism of allorecognition. Hence, in the absence of indirect presentation, net CD4+CD25+ T cell-dependent immunoregulation is weak, and high ratios of CD4+CD25+ to CD4+CD25 T cells are required to ensure graft survival.