Direct Allorecognition Research Articles

Modulation of alloreactivity to MHC-derived peptides and transplantation tolerance

The recognition by T cells of intact foreign MHC molecules at the surface of transplanted cells (direct pathway) was originally thought to represent the driving force behind acute rejection of allogeneic transplants. Over the past decade, however, a body of evidence has been provided demonstrating that T cell recognition of donor peptides presented by recipient APCs (indirect pathway) is sufficient on its own to ensure both acute and chronic rejection of allografts. While the direct allorecognition leads to an exceptionally vigorous inflammatory T cell response, it is thought to be short lived due to the rapid depletion of donor professional APCs and it can be controlled with a short course of immunosuppressive drugs including calcineurin inhibitors. In contrast, while the indirect alloresponse is oligoclonal and much weaker, it is long-lived and tends to spread to formerly cryptic determinants on donor and self-tissue specific antigens. This feature of indirect alloreactivity is presumably associated with the sustained presence of recipient professional APCs that can maintain a chronic inflammatory response similar to that observed in autoimmune diseases. Consequently, the indirect alloresponse may be more difficult to suppress than its direct counterpart. On the other hand, there is accumulating evidence showing that administration of alloantigen in a "tolerogenic fashion" mediates allograft acceptance via the activation of regulatory T cells recognizing donor antigens via the indirect allorecognition pathway. Apparently, these regulatory T cells can suppress both direct and indirect inflammatory T cell responses to donor antigens. This suggests that modulation of indirect alloreactivity may represent the best strategy to achieve long-term donor-specific tolerance to allotransplants.

Direct versus Indirect Allorecognition: Visualization of Dendritic Cell Distribution and Interactions During Rejection and Tolerization

Interactions of donor and recipient dendritic cells (DCs) with CD4+ T cells determine the alloantigenic response in organ transplantation, where recipient T cells respond either directly to donor MHC, or indirectly to processed donor MHC allopeptides in the context of recipient MHC molecules. The present study evaluates donor and recipient alloantigen-presenting DC trafficking and their interactions with CD4+ T cells in the lymph nodes (LNs) and the spleen under tolerogenic treatment with anti-CD2 plus anti-CD3 mAb compared with untreated rejecting conditions. CX3CR1(GFP) BALB/c (I-A(d)) donor hearts were transplanted into C57BL/6 (I-A(b)) mice and quantification of donor DC direct (GFP+ or I-A(d+)) and recipient DC indirect (YAe+) trafficking and interactions with host CD4+ T cells was performed by fluorescent microscopy. Our data indicate that although both direct and indirect interactions between CD4+ T cells and donor and recipient DCs occur shortly after engraftment, only indirect presentation persists in the LN, but not the spleen, of tolerized recipients. These data suggest that distinct anatomic lymphoid compartments play a critical role in peripheral tolerance induction and maintenance, and persistent indirect presentation to CD4+ T cells within the LNs is an important process during tolerization.

ILT3+ ILT4+ Tolerogenic Endothelial Cells in Transplantation

T cells can recognize allogeneic major histocompatibility complex (MHC) antigens by two distinct routes: either directly as intact molecules or indirectly as processed peptides presented by syngeneic antigen-presenting cells (APC). The graft endothelium plays an important role in rejection eliciting and serving as a target of T cells activated via the direct and/or indirect allorecognition pathway. Recent evidence demonstrates, however, that endothelial cells are also endowed with the capacity to downregulate alloreactivity inducing tolerogenic responses. Similar to professional APC (such as dendritic cells), endothelial cells express high levels of inhibitory receptors (ILT3 and ILT4 in humans and PIR-B in rodents) and low levels of costimulatory and adhesion molecules upon interaction with allospecific CD8 T suppressor cells or exposure to inhibitory cytokines. Because of the importance of endothelial cells in the activation and control of T cell reactivity, understanding of their biology is crucial for the development of new strategies for induction of transplantation tolerance and treatment of cancer, chronic infection, and autoimmunity.

Transplant-related Immunosuppression: A Review of Immunosuppression and Pulmonary Infections

Solid organ and hematopoietic stem cell transplantation are definitive therapies for a variety of end-stage diseases. Immunosuppression has improved graft survival but leaves the patient susceptible to infectious complications. Of these, pulmonary infections are the leading cause of morbidity and mortality in the transplant recipient. Allograft rejection is mediated primarily by T cells, with B cells playing a role via antibody production. Depending on the transplant type, rejection can be hyperacute, acute, or chronic. Hyperacute rejection occurs as an immediate response to preformed antibodies to donor human leukocyte antigens. Acute cellular rejection involves recipient T-cell recognition of human leukocyte antigen molecules expressed on donor-derived, antigen-presenting cells (direct allorecognition) or presentation of donor-derived peptides by recipient antigen-presenting cells to recipient T cells (indirect allorecognition). Once the alloantigens are recognized as foreign, the activation, proliferation, and production of cytokines by T lymphocytes and other immune cells lead to the amplification of the alloimmune response. This complex process involves the generation of effector T cells, antibody production by activated B cells, and macrophage activation. Alloimmunity is facilitated by the production of many cytokines, chemokines, and other effector molecules, such as complement. The immunosuppressants involve many classes of drugs, including antibody therapies that eliminate specific groups of cells or alter signaling pathways used by effector cells. The article reviews the agents and associated infections.

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.

Direct Allorecognition Promotes Activation of Bystander Dendritic Cells and Licenses Them for Th1 Priming: A Functional Link Between Direct and Indirect Allosensitization

T-cell sensitization to indirectly presented alloantigens (indirect pathway of allorecognition) plays a critical role in chronic rejection. The usual very efficient priming of such self-restricted, T helper type 1 (Th1)-deviated CD4+ T cells obviously conflicts with the fact that allogeneic MHC molecules are poorly immunogenic per se. The aim of the present study is to elucidate whether direct allosensitization induces production of inflammatory mediators that may affect recruitment and activation of immature bystander (host) dendritic cells (DC). These potential mechanisms were studied in vitro by conducting primary allogeneic mixed leucocyte reactions (MLR), mimicking the priming phase in secondary lymphoid organs, and secondary MLR, mimicking the effector phase within the graft. Primary, and particularly secondary, MLR supernatants were found to contain high levels of monocyte/immature DC-recruiting CC chemokines and pro-inflammatory cytokines. Exposure of immature DC to primary or secondary MLR supernatants was found to upregulate CD40 expression and further enhanced lipopolysaccharide-induced interleukin-12 (IL-12) p70 production. Secondary MLR supernatants additionally induced upregulation of CD86 and deviated allogeneic T-cell responses towards Th1 (enhanced interferon-gamma production without concomitant induction of detectable IL-4 or IL-10 production). These findings indicate that direct allorecognition may act as a Th1-deviating adjuvant for indirect allosensitization.

Dendritic Cell Adhesion Is Enhanced on Endothelial Cells Preexposed to Calcineurin Inhibitors

Chronic rejection remains a major complication in solid organ transplantation. Host alloreactive T cells (TC) can be activated by donor dendritic cells (DCs; direct allorecognition) or by recipient DCs (indirect allorecognition). A fundamental aspect of DC function is vascular invasion to present donor antigens to recipient naive TC in secondary lymphoid organs. We investigated the impact of calcineurin inhibitors on DC binding and transmigration to allogeneic human microvascular endothelial cells (ECs) with and without blocking of specific adhesion molecules. Recipient immature DCs were generated by culturing CD14 human peripheral blood monocytes with GM-CSF and IL-4. DC adhesion and transmigration were investigated on allogeneic ECs preincubated with increasing concentrations of cyclosporine and tacrolimus. Experiments were repeated in the presence of blocking antibodies against LFA-1, PECAM-1, VCAM-1, and ICAM-1. Endothelial stimulation with cyclosporine A (100 and 300 ng/mL) and tacrolimus (15 ng/mL) significantly enhanced DC-EC adhesion and transmigration (P<0.01). LFA-1 blockade on DCs significantly reduced cyclosporine- and tacrolimus-induced DC adhesion (P<0.001). VCAM-1 blockade on ECs partially reversed cyclosporine-induced DC adhesion (P<0.001), whereas DC adhesion under tacrolimus exposure was significantly decreased by ICAM-1 (P<0.01) and PECAM-1 (P<0.001) blockade. DC binding and transmigration on allogeneic ECs exposed to calcineurin inhibitors is concentration-dependently increased. Different adhesion molecule patterns on ECs are responsible for enhanced DC invasion under cyclosporine and tacrolimus exposure. We speculate that long-term immunosuppression mediates enhanced invasion of recipient DCs to the donor organ and therefore may aggravate chronic rejection.

Frontline: Peripheral priming of alloreactive T cells by the direct pathway of allorecognition.

Recent studies, though controversial, have suggested that secondary lymphoid organs may not constitute an essential site for the initiation of immune responses to transplant antigens. However, this issue has never been examined in the context of direct and indirect allorecognition. Here, we characterized immune responses arising in draining lymph nodes and skin allografts, in a murine model based on a single T cell clonotype where these two pathways can be independently studied. In this model, graft rejection by the direct or the indirect pathway occurred with similar kinetics, although initiation of the alloreactive responses was clearly different. During indirect responses, expansion and activation of alloreactive T cells were first observed in draining lymph nodes, at day 7 post-transplant, and graft-infiltrating T cells were observed later, at day 11. In striking contrast, directly activated alloreactive T cells were detected at an early stage inside the graft, and only later in the draining lymph nodes, after skin allograft rejection was almost completed. These results suggest that sensitization of naive T cells through the direct pathway could take place outside secondary lymphoid organs.

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival

Abstract Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct aliorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule Kb or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2k) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2b) cardiac allograft. Wildtype, as well as truncated Kb genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reversetranscriptase PCR showed expression of the Kb genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival.

Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct allorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule K(b) or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2(k)) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2(b)) cardiac allograft. Wildtype, as well as truncated K(b) genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reverse-transcriptase PCR showed expression of the K(b) genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

Vascular endothelium does not activate CD4+ direct allorecognition in graft rejection.

Expression of MHC class II by donor-derived APCs has been shown to be important for allograft rejection. It remains controversial, however, whether nonhemopoietic cells, such as vascular endothelium, possess Ag-presenting capacity to activate alloreactive CD4(+) T lymphocytes. This issue is important in transplantation, because, unlike hemopoietic APCs, allogeneic vascular endothelium remains present for the life of the organ. In this study we report that cytokine-activated vascular endothelial cells are poor APCs for allogeneic CD4(+) T lymphocytes in vitro and in vivo despite surface expression of MHC class II. Our in vitro observations were extended to an in vivo model of allograft rejection. We have separated the allostimulatory capacity of endothelium from that of hemopoietic APCs by using bone marrow chimeras. Hearts that express MHC class II on hemopoietic APCs are acutely rejected in a mean of 7 days regardless of the expression of MHC class II on graft endothelium. Alternatively, hearts that lack MHC class II on hemopoietic APCs are acutely rejected at a significantly delayed tempo regardless of the expression of MHC class II on graft endothelium. Our data suggest that vascular endothelium does not play an important role in CD4(+) direct allorecognition and thus does not contribute to the vigor of acute rejection.

TOLEROGENIC ENDOTHELIAL CELLS INHIBIT THE DIRECT RECOGNITION PATHWAY

P928 Aims: Allograft rejection is mediated by T cells with direct and indirect allorecognition capacity. The direct pathway involves T cells which interact directly with professional (dendritic cells, macrophages) or nonprofessional (endothelial cells) Antigen Presenting Cells (APC) of the donor. The indirect pathway involves T cells which recognize dominant allopeptides derived from the processing of necrotic or apoptotic cells of the graft by host APC. Both in the periphery and within the graft, the frequency of T cells with direct allorecognition capacity is 10,000 times higher than that of allopeptide reactive T cells at all times. We have explored the hypothesis that tolerogenic endothelial inhibit the direct recognition pathway. Methods: Human endothelial cells (EC) were conditioned by use of IFN-γ and used for T cell priming. Conditioned EC were incubated with allospecific CD4+CD25+CD45RO+ FOXP3+ T regulatory cells (TR) or CD8+CD28- T suppressor cells. The expression on EC of CD31, CD40, CD54, CD62E CD106 CD58, HLA class I, HLA class II, ILT3, and ILT4, prior and following exposure to TS or TR was measured using flow cytometry and RT-PCR. T suppressor cells were tested in proliferation assays for their capacity to inhibit TH reactivity. Results: The ILT3high, ILT4high phenotype is characteristic of tolerogenic DC which induce regulatory T cells inhibiting the direct allorecognition pathway. However, after the departure of donor APC from the graft, rejection can be triggered by activated endothelial cells (EC). We have explored the hypothesis that EC may be involved in the maintenance of quiescence. Our studies have shown that alloantigen specific CD8+CD28- T cells with suppressor activity are FOXP3 positive and interact with human dendritic cells as well as EC. The interaction of TS with EC results in the induction of the inhibitory receptor ILT4, down-regulation of costimulatory and adhesion molecules and tolerization of EC. In turn, tolerized ILT4high EC elicit the differentiation of CD8+CD28- FOXP3+ T suppressor cells. Similarly alloantigen specific TR elicited the transcriptional activation of ILT3 and ILT4 in EC. Alloantigen specific CD8+CD28- FOXP3+ T cells, which trigger the transcriptional activation of ILT4 in EC, were found in the circulation of 21 heart allograft recipients in quiescence. Patients with a history of early acute rejection episodes (N=7) and/or chronic rejection (N=3) showed no TS or TR. The assay which we developed facilitates the identification of patients who may benefit from partial or complete cessation of immunosuppression, a goal of obvious importance given the morbidity and mortality associated with chronic immunosuppression. Conclusions: Endothelial cells are an important target of antigen specific T effector cells not only in transplantation but also in numerous autoimmune diseases. Agents which inhibit EC activation and/or render them tolerogenic are likely to have a beneficial effect in treatment of rejection, autoimmunity and possibly atherosclerosis.

LONG TERM OBSERVATIONS IN RENAL ALLOGRAFT RECIPIENTS TREATED WITH A CHIMERISM- INDUCING NONMYELOABLATIVE REGIMEN WITH CD154 BLOCKADE

O111* Aims: Cynomolgus monkey renal allograft recipients treated with a chimerism-inducing regimen with perioperative CD154 blockade were studied long-term (>5 years) to establish reliable tolerance assays that are capable of predicting the long-term outcome. Methods: Cynomolgus monkeys in Group A were treated with a conditioning regimen consisting of low dose total body irradiation (TBI 1.5 Gy x2), thymic irradiation (TI 7 Gy), anti-thymocyte globulin (ATG), perioperative anti-CD154 monoclonal antibody (mAb) treatment, combined donor bone marrow (DBM) and kidney transplants on day 0 and one month course of cyclosporine (CyA). No immunosuppression was given after day 28. Control Groups include Group B: donor splenocyte infusion instead of DBM, Group C: no donor cell infusion, or Group D: no TI. Results: All eight recipients in Group A developed multilineage chimerism up to day 100 and 7/8 survived long-term after discontinuation of immunosuppression (>1850, >1323, 837, 755, 401, 373, 206, 58 days). Four of seven long-term survivors did not developed any evidence of rejection with the longest survival exceeding five years. Two of four long-term recipients accepted donor skin graft indefinitely while rejecting third-party skin graft within 10 days. However, 3/8 recipients in Group A eventually developed chronic rejection. All recipients in control groups B and C did not develop chimerism and lost their kidney allograft function either by acute or chronic rejection (CR) (292, 274, 250, 113, 93, 51 days). 2/3 recipients in Group D developed chimerism and one of them survived long-term without rejection (289, 71, 44). Anti-donor alloantibody (ADA), measured by flow cytometry and C4d staining of the graft biopsy were significantly relevant to CR. All six recipients that developed ADA eventually developed CR. Allograft biopsies of all six recipients with positive ADA later developed C4d in their renal allograft biopsies. In contrast, five recipients without ADA have never developed C4d or CR. In Group A, there was no significant difference found between tolerant recipients and rejecters in the level and duration of mixed chimerism or postoperative MLR and CML. In the ELISPOT assay, where posttransplant peripheral blood mononuclear cells (PBMC) were activated via either direct (donor APC) or indirect allorecognition pathway (sonicated donor cells), tolerant recipients showed specific inhibition of donor-reactive, gamma-interferon (gIFN) production in the indirect pathway (6.5 ±6 spots per 106 PBMC). In contrast, the recipients with CR showed vigorous gIFN production (132 ±17). In the direct pathway, however, there was no significant difference detected in gIFN production between tolerant recipients and rejecters (31.5 ±15 vs 82 ±25). Conclusions: Long-term stable renal allograft survival was achieved in 50% of recipients treated with a chimerism inducing nonmyeloablative regimen with perioperative CD154 blockade. Posttransplant ADA by flow cytometry, positive C4d in renal biopsies and positive gIFN production in the indirect pathway appear to correlate with the development of chronic rejection.

THE ROLE OF ALLOANTIGEN-INDUCED ACTIVATION OF T CELLS AND MACROPHAGES IN PARATHYROID ALLOGRAFT REJECTION

P895 Aims: Hypoparathyroidism is an endocrine dysfunction that is associated with reduced blood calcium levels due to insufficient levels of parathyroid hormone (PTH). The best known treatment is the transplantation of parathyroid glands. In order to prevent graft rejection without general immunosuppression it is necessary to get more knowledge about the immune response to allogeneic parathyroid glands. Methods: Male hypocalcaemic Lewis rats (LEW, RT1l) received parathyroid glands under the kidney capsule from Wistar-Furth (WF, RT1avl) or LEW rats (syngeneic control). Calcium serum levels were detected repeatedly after transplantation. Levels higher than 2.0 mmol/l proved function of the transplant, levels lower than 2.0 mmol/l indicated transplant rejection (Microsurgery, 2003). At various time points grafts were explanted and expression of inducible nitric oxide synthase (iNOS) were determined by RT-PCR. Grafts were also examined for infiltration of inflammatory immunocompetent host cells by immunohistochemistry. Some of the LEW rats were immunized prior to transplantation with P1, a synthetic peptide derived from the MHC class I molecules of WF. P1 induces a strong activation of alloreactive LEW T cells (Human Immunol, 2002). Some glands were depleted of passenger leukocytes prior to transplantation by in vitro cultivation. Results: LEW rats transplanted syngeneically returned to normal serum calcium levels. In the allogeneic transplanted animals serum calcium levels stayed normal until rejection on day 15±2. During this time period the allografts were continuously infiltrated by recipient’s leukocytes mainly T cells and macrophages. Activated MHC-class II positive, ED1-positive macrophages were observed on day 2 after transplantation within the allografts, on day 3 T cells were additionally detected. Few activated (CD25+) T cells appeared on day 4 and stayed within the infiltrate during the whole examination period. In activated macrophages the expression of iNOS was shown. LEW rats immunized with peptide P1 showed accelerated rejection at day 10±1. Depletion of passenger leukocytes had no effect on the onset of rejection. Conclusions: Direct allorecognition apparently is not important for the rejection of parathyroid allografts, since the depletion of passenger leukocytes did not prolong graft function. Alloreactive T cells activated by the indirect pathway of allorecognition after immunization with peptide P1 led to accelerated rejection. This indicates that T cells are involved in allograft destruction. In non-immunized animals only a low amount of activated T cells within the graft was detected. In contrast, the strong presence of activated MHC-class II positive macrophages in allografts of non-immunized LEW rats indicates their involvement in graft rejection. These macrophages produce the cytotoxic effector NO. Therefore therapeutic strategies should include the inhibition of macrophage infiltration and their allogeneic activation.

Transplant: immunology and treatment of rejection

Transplant: immunology and treatment of rejection

Role of hla molecules in the induction of alloimmune responses: clinical significance in the cyclosporine era

HLA molecules are peptide-presenting molecules for T cells. T-cell receptors (TCR) recognize antigen-derived peptides bound to HLA molecules in cell membranes, namely, peptide-HLA (pHLA) complexes. An allograft will be recognized as foreign by recipient T cells via two slightly different mechanisms—direct and indirect allorecognition. In direct allorecognition, recipient T cells directly recognize foreign pHLA complexes on donor cells, in particular on donor dendritic cells (DC). In indirect allorecognition, recipient T cells recognize foreign peptides from the graft presented by self-HLA molecules on recipient DC. Cyclosporine (CsA) inhibits some activation signals in T cells as the result of allorecognition. Notwithstanding the strong immunosuppressive effects of CsA, reducing the degree of allorecognition through HLA matching is still of clinical significance. Data from our own transplant center and results from others demonstrate that the introduction of CsA as a main immunosuppressive drug does not nullify the significant beneficial effects of HLA matching on the frequency and strength of rejection episodes as well as on graft survival after allotransplantation. In cadaveric donor transplantation we observe significant effects of matching just for a limited number of (broad) HLA-DR molecules. We conclude that one should take advantage both of the strong immunosuppressive effects of CsA and other modern immunosuppressants as well as the beneficial effects of optimal HLA matching in clinical transplantation.

MD-1 expression regulates direct and indirect allorecognition.

Expression of the molecule MD-1 was previously described to regulate allogeneic and xenogeneic skin graft survival, as documented by the decrease in rejection seen following functional blockade of MD-1 expression in vivo, using antisense oligodeoxynucleotides (ODNs) or anti-MD-1 antibodies. It was unclear from these data whether blockade of expression of MD-1 on donor or recipient cells was crucial. We have investigated the effect on allorecognition of treating skin graft donors, and/or recipients, of either fully major histocompatibility complex (MHC)-mismatched allogeneic skin grafts (C3H with C57BL/6 grafts and vice versa) or grafts differing at only multiple minor alloantigens (C3H with B10.BR grafts; C57BL/6 with C3H.SW), with antisense ODNs to MD-1, or in some cases, following transplantation of class II-deficient cells into class I-deficient mice. Graft-specific cytotoxic T lymphocytes (CTLs) were measured in spleen cells recovered at sacrifice of recipients and following donor-specific restimulation in vitro. In the latter case, we also measured cell proliferation and (by enzyme-linked immunosorbent assay) production of interleukin-2 (IL-2)/interferon-gamma (IFN-gamma) or IL-4/IL-10 in vitro (nominal type-1 vs type-2 cytokines). CTL responses to minor-incompatible grafts were diminished, only if graft recipients were treated with ODNs. However, treatment of graft donor and/or recipient of MHC-incompatible grafts produced inhibition of CTL production. Optimal inhibition came from treating both. Specific suppression of CTL production coincided with inhibition of proliferation and preferential production of IL-4 and IL-10 at the expense of IL-2 and IFN-gamma. Our data are consistent with the hypothesis that MD-1 expression regulates both the direct and indirect pathways of allorecognition and that regulation of MD-1 expression may thus help regulate clinical graft rejection.

Role of resident corneal leukocytes and draining cervical lymph nodes in corneal allograft rejection.

We review recently published experimental evidence on corneal transplant immunology involving cornea and draining cervical lymph nodes (CLNs) in the mouse. In the cornea, major histocompatibility complex (MHC) class II- dendritic cells (DCs) are present in the corneal epithelium. These DCs can express MHC class II antigen in vivo and in vitro. In the corneal stroma, there are many leukocytes of monocyte or macrophage lineage. Normal cornea has been reported to contain a significant number of bone marrow-derived resident cells, which may be able to act as antigen-presenting cells. Allograft rejection does not occur if draining CLNs are removed before corneal transplantation, indicative of an essential role of CLNs in promoting corneal allorejection. Moreover, donor cornea-derived DCs were detected in host draining CLNs in a mouse corneal transplantation model. These findings provide direct evidence that MHC class II- bone marrow-derived antigen-presenting leukocytes exist in the part of cornea used for transplantation and that direct allorecognition of antigen is, at least in part, relevant to the occurrence of corneal allograft rejection in which draining CLNs play a central role.

The role of graft-resident Kupffer cells and lymphocytes of donor type during the time course after liver transplantation?a clinico-pathological study

Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. The present study evaluated by non-isotopic in situ hybridization and immunohistochemistry if and during which time period after transplantation Kupffer cells (KC) and lymphocytes of the liver were replaced by recipient cells and if there is any correlation with the occurrence of rejection episodes and the clinical course. A successive re-population of the liver by recipient lymphocytes and KC was observed after transplantation but a smaller portion of lymphocytes and KC with donor genotype was detectable during the whole time course studied. There was no correlation between the portion of recipient-derived KC and donor-derived lymphocytes and histopathological alterations of the liver tissue. The biopsy content of KC with recipient origin has had no prognostic significance for the probability of survival, but patients with a low portion of donor lymphocytes in the liver biopsy obtained during the first week after transplantation have had a better prognosis for survival. The present results indicate that graft-resident KC and lymphocytes are potentially not the main cell types involved in tolerance induction after liver transplantation.

T-cell receptor beta-chain usage in directly activated alloreactive CD4+ T cells: unrestricted Vbeta gene usage with CDR3 size limitations.

Alloreactive immune responses may engage both direct and indirect antigen allorecognition. This study focuses on T-cell receptor (TCR) beta-chain usage by in vitro generated alloreactive CD4+ T cells involved in direct allorecognition pathway. We have established Lewis anti-Brown Norway rat CD4+ T-cell lines and confirmed their reactivities against cell-surface, but not soluble, alloantigens. TCR Vbeta-specific reverse-transcriptase polymerase chain reaction detected all 22 Vbeta genes in these cell lines at all stages, regardless of the lengths of in vitro stimulation. By using spectrotyping, we found that Vbeta complementarity determining region (CDR)3 length distribution pattern altered dramatically after repeated allostimulation. Such a skewed CDR3 distribution occurred in most Vbeta genes without any obvious preference, indicating that expansion occurred in all Vbeta expressing cells by allostimulation. Fluorescence-activated cell sorter analysis of Vbeta expression in alloreactive CD4+ T-cell lines with anti-Vbeta-specific monoclonal antibodies showed, quantitatively, similar percentages of individual Vbeta-expressing cells in the alloreactive pool after repeated allostimulation. To test whether preferential TCR Vbeta gene usage occurred in "high responder" cells, we sorted CD4+ T cells that underwent three or more divisions from primary mixed leukocyte reactions. Unlimited Vbeta usage with CDR3 alterations was observed, as in unsorted alloreactive T cells. TCR Vbeta gene usage in directly alloreactive CD4+ T-cell population is unrestricted. Clonal expansion occurs in all Vbeta expressing T cells by allostimulation.