Skin Rejection Research Articles

Donor DNA can be detected in recipient tissues during rejection of allograft

Abstract The main source of donor DNA in recipients of allograft are “passenger” cells. It is claimed that they are responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have observed that besides cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of the allograft. In this study, we provide evidence for the presence in the recipient of both DNA in “passenger cells” and free DNA in tissues at the terminal stage of rejection. Male BN (RT1 n) rat heart or skin was transplanted to female LEW (RT1 1) rats followed by a vascularized bone marrow in a hind-limb transplant. In another group, heart and skin were transplanted followed by immediate i. v. infusion of donor-type bone marrow cells. CsA was given in a dose of 17 mg/kg body weight for 30 days, then the rats were followed up until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and day 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, as well as lymphoid organs, at the time of rejection of allograft, but not when the rats were maintained on CsA. Taken together, donor DNA was detected in recipient tissues at the time of heart or skin rejection. It appeared to be released from cells of rejecting grafts and not from “passenger” cells, representing only a minor cellular mass compared with the graft.

Durable donor-specific T and B cell tolerance in rhesus macaques induced with peritransplantation anti-CD3 immunotoxin and deoxyspergualin: absence of chronic allograft nephropathy.

Tolerance induction can prevent acute kidney allograft rejection without chronic immunosuppression. It is uncertain whether specific tolerance can prevent chronic allograft nephropathy (CAN), which involves both nonimmune and immune injury. This report provides evidence that immunologically tolerant macaques, induced with immunotoxin and deoxyspergualin, developed neither acute rejection nor CAN. Long survivors, bearing MHC-mismatched grafts without chronic immunosuppression for 0.8 to 3.4 years, exhibited general immune competence with donor-specific T and B cell tolerance and no functional or histological evidence of CAN. Stringent criteria for tolerance were satisfied by specific prolongation of donor skin grafts with rapid rejection of third-party skin, followed by indefinite acceptance of a second donor kidney graft and establishment of microchimerism. Primate tolerance with documented absence of CAN may give impetus to the clinical application of tolerance.

Donor DNA can be detected in recipient tissues during rejection of allograft

The main source of donor DNA in recipients of allograft are "passenger" cells. It is claimed that they are responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have observed that besides cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of the allograft. In this study, we provide evidence for the presence in the recipient of both DNA in "passenger cells" and free DNA in tissues at the terminal stage of rejection. Male BN (RT1 n) rat heart or skin was transplanted to female LEW (RT1 l) rats followed by a vascularized bone marrow in a hindlimb transplant. In another group, heart and skin were transplanted followed by immediate i.v. infusion of donor-type bone marrow cells. CsA was given in a dose of 17 mg/kg body weight for 30 days, then the rats were followed up until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and day 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, as well as lymphoid organs, at the time of rejection of allograft, but not when the rats were maintained on CsA. Taken together, donor DNAwas detected in recipient tissues at the time of heart or skin rejection. It appeared to be released from cells of rejecting grafts and not from "passenger" cells, representing only a minor cellular mass compared with the graft.

Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation.

Induction of tolerance to organ transplants will increase graft survival and decrease patient mortality and morbidity. Radiation-induced cytoreduction/ablation followed by donor hematopoietic cell reconstitution has been the most consistently successful approach to experimental tolerance induction. However, reluctance of clinicians to expose recipients to radiation has hampered its clinical application. In the studies described, administration of polyclonal antilymphocyte serum (ALS), donor-specific bone marrow (DSBM) (150x10(6) cells), and sirolimus (24 mg/kg) in a completely mismatched murine model (B10.A donor, C57B/10 recipient) produced 100% indefinite (>250 days) skin graft survival. The level and character of donor-specific chimerism was evaluated with flow cytometry. Specific tolerance was confirmed by continued acceptance of primary and secondary donor-specific skin allografts and rejection of third-party grafts. The level and duration of chimerism induced was directly related to the dose of DSBM administered. Mice given 150x10(6) DSBM cells showed levels of 8-10% donor peripheral blood mononuclear cell chimerism by 30 days, and these levels persisted indefinitely (>250 days) in association with permanent tolerance of donor grafts. Eighty percent of donor chimeric cells were B lymphocytes (MHC class I and II positive, Fc receptor positive, CD45/B220 positive but negative for CD4, CD8 and Thy 1.2) and 20% were sorted in the macrophage monocyte population. These studies demonstrate for the first time that cytoreduction/ablation with ALS combined with sirolimus and reconstitution with donor bone marrow induces tolerance and chimerism in a completely mismatched murine combination. The use of ALS and sirolimus, currently employed therapies in clinical transplantation, and the lack of requirement for radiation make this tolerance protocol attractive for clinical application.

Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model.

Bone marrow transplantation (BMT) has considerable potential for the treatment of malignancies, hemoglobinopathies, and autoimmune diseases, as well as the induction of transplantation allograft tolerance. Toxicities associated with standard preparative regimens for bone marrow transplantation, however, make this approach unacceptable for all but the most severe of these clinical situations. Here, we demonstrate that stable mixed hematopoietic cell chimerism and donor-specific tolerance can be established in miniature swine, using a relatively mild, non-myeloablative preparative regimen. We conditioned recipient swine with whole-body and thymic irradiation, and we depleted their T-cells by CD3 immunotoxin-treatment. Infusion of either bone marrow cells or cytokine-mobilized peripheral blood stem cells from leukocyte antigen-matched animals resulted in stable mixed chimerism, as detected by flow cytometry in the peripheral blood, thymus, and bone marrow, without any clinical evidence of graft-versus-host disease (GvHD). Long-term acceptance of donor skin and consistent rejection of third-party skin indicated that the recipients had developed donor-specific tolerance.

"The Kindergarten of New Consciousness": Gwendolyn Brooks and the Social Construction of Childhood

We watch strange moods fill our children, and our hearts swell with pain. The streets, with their noise and flaring lights, taverns, automobiles, and poolrooms claim them, and no voice of ours can call them back.... We cannot keep them in school; more than 1,000,000 of our black boys and girls of high school age are not in school.... It is not their eagerness fight that makes us afraid, but that they go death on city pavements faster than even disease and starvation can take them. As courts and morgues become crowded with our lost children, hearts of officials of city grow cold toward us. (Wright 136) I GIVE YOU MY GALLERY. So many boys. Boys. Lincoln West. Merle. Ulysses. Shabaka. Martin D. The Near- Johannesburg Boy. Diego. Kojo. Seven boys in a poolroom during schooltime. The Pool Players, Seven at The Golden Shovel- We real cool. We Left school. We Lurk late. We Strike straight. We Sing sin. We Thin gin. We Jazz June. We Die Die Today, many such boys--their girl friends, too--EXPECT die soon. In Chicago. In New York. In Springfield, in Philadelphia. In Whatalotago, Alabama. In Detroit. (In Washington D.C.?) They do not expect become twenty-one. They are designing their funerals. Their caskets will be lined with Kente cloth. They choose their music: they want rap, they want Queen Latifah. (Brooks, Rep art from Part Two 123-24) In Report from Part One, Gwendolyn Brooks gives an account of her conversion Black [1] militancy at 1967 Fisk Writers' Conference. Impressed by energy and anger in work of Amiri Baraka (then LeRoi Jones) and others, Brooks recognized that there is indeed a new black today. Acknowledging that for most of her life secretly [she] had felt that be black was good, she writes that she had 'gone gamut' from an almost angry rejection of my dark skin by some of my brainwashed brothers and sisters a surprised queenhood in new black sun. I...am qualified, Brooks proclaims, to enter at least of new now (84). Since In Mecca (1968), Brooks has published her work exclusively with Black presses such as Broadside, Third World Press, and her own David Company, work characterized by a turn toward free verse as well as increasingly direct political content. Although kindergarten of new consciousness fostered in Brooks a new Black identity an d a new sense of Black people as her primary audience, her poetry, as she insisted in an interview with Claudia Tate, has always been 'politically aware' (42). Part of her political project has been a clear-eyed, tough, and compassionate look at plight of children. From A Street in Bronzeville (1945) present, Brooks's work has used image and voice of child negotiate a complex poetic strategy that explores as a position from which critique prevailing constructs of class and race. For Brooks, subject of childhood represents a means through which she can interrogate and unmask dominant notions of domesticity and child-rearing as part of her own radical social and poetic agenda. Childhood as a subject would gain force in '40s and '50s for other American poets, including Robert Lowell, Elizabeth Bishop, and Randall Jarrell. But for most poets subject of childhood was steeped in nostalgia, indicative of growing trend toward introspection among White intellectuals occasioned by rise of a newly psychologized self. By contrast, Brooks chose write about the children of poor, borrow title of her sonnet sequence from Annie Allen. Critic Gary Smith argues that, if Brooks's poetry about adults is bleak, her poetry about children is even more so (130): Her children do not exist in a pastoral world apart from socioeconomic and psychological problems that beset her adult characters. …

Swine composite tissue allotransplant model for preclinical hand transplant studies.

Our laboratory previously developed and used an orthotopic radial forelimb osteomyocutaneous flap in the pig as a preclinical composite tissue allograft (CTA) model. To ensure that it mimicked the clinical situation as closely as possible we developed this model taking many immunologic and reconstructive considerations into account. While our original pig CTA model was ideal for studying the methods of preventing skin, muscle, bone, vessel and nerve rejection, and systemic toxicity, it did not include specialized tissues/structures of a joint and digit. Therefore, we were unable to evaluate rejection of these specialized tissues and their functional properties. Recognizing the importance of assessing joint rejection and function in hand transplantation research we developed a new swine forelimb CTA model that included the animal's medial digit. The present study describes the anatomy and the transplantation technique used in this new preclinical CTA model. We transplanted a radial osteomyocutaneous flap that included the medial digit between two size- (17-21 kg) and age- (6-8-week) matched farm pigs. We removed the digit from the recipient pig's forelimb in continuity with a section of the radial bone and replaced it with the same structure transplanted from a donor pig. After transplantation, a full-length cast was placed on the recipient pig's operated limb and changes in flap color, temperature and the presence of edema were monitored continuously for 6 h, and then regularly at predetermined intervals over 4 days. No weight bearing restrictions were placed on the animal's operated limb. After 4 days, the animal was euthanized. Direct visual monitoring of the allograft during 4 days revealed it was viable with no signs of graft failure due to technical complications associated with the transplant procedure. Upon waking from anesthesia, the animal stood and wandered freely about its cage with no apparent difficulty. Based on the animal's high level of activity at this time, we concluded that the procedure caused it minimal morbidity. At 4 days after the operation, early signs of rejection (skin erythema and edema) were observed. By incorporating a digit into our original CTA pig forelimb model we have made it a better model for performing preclinical hand transplant studies. The added advantage of being able to assess methods of preventing rejection in the specialized joint/digital tissues (articular cartilage, digital flexor and extensor systems, the nail complex) and assess long-term function of these structures is important. The fact that the procedure does not cause major morbidity to the animal makes it possible to conduct long-term graft survival and functional studies.

Characterization and manipulation of T cell immunity to skin grafts expressing a transgenic minor antigen.

Minor histocompatibility antigens play a significant role in allograft rejection when donor and recipient are matched at MHC loci. An improved understanding of T cell immunity directed toward a model minor antigen may provide new approaches for preventing graft rejection. C57BL/6 (B6) recipient mice were engrafted with skin from B6 beta-galactosidase transgenic (beta-gal tg) donors and the induced T cell immune responses were characterized by cytokine ELISA spot assay. beta-gal-specific immunity was manipulated prior to transplant through preinjection with beta-gal in complete Freund's adjuvant (CFA) or through preinjection with soluble beta-gal i.v. B6 mice rejected beta-gal tg skin by day 25. Rejection was associated with a low frequency of predominantly CD8+, interferon-gamma-producing T cells capable of directly recognizing both beta-gal tg cells and an immunodominant major histocompatibility complex I-restricted peptide derived from the beta-gal protein. Rejection of multiple minor antigen disparate skin and major histocompatibility complex-disparate skin occurred significantly faster, and was associated with a 10- to 30-fold higher frequency of alloreactive T cells, than rejection of beta-gal tg skin. Prepriming of recipients with beta-gal in complete Freund's adjuvant resulted in an increased frequency of beta-gal-specific T cells and accelerated rejection of beta-gal tg skin. Intravenous injection of soluble beta-gal-induced graft tolerance and a lack of detectable beta-gal-specific immunity. The findings reveal that transgenically expressed beta-gal behaves as a minor transplantation antigen and that manipulation of the beta-gal-specific T cell repertoire can dramatically affect rejection of beta-gal tg skin grafts. The work provides the foundation for mechanistic studies of tolerogenesis to minor antigenic determinants.

IL-5 Mediates Eosinophilic Rejection of MHC Class II-Disparate Skin Allografts in Mice

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts. C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins. Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.

Scoring of Skin Rejection in a Swine Composite Tissue Allograft Model

Background. For the first time, we define and correlate visual and histologic grading systems of composite tissue allograft (CTA) skin rejection in a large-animal model and determine the utility of these grading systems for early diagnosis and monitoring of rejection.Materials and methods. Sixteen pairs of outbred swine underwent transplant of a forelimb osteomyocutaneous free flap. Group I (n = 6) did not receive immunosuppressive therapy. Group II (n = 10) received oral cyclosporin A, mycophenolate mofetil, and prednisone. The flap was visually inspected and protocol skin biopsies were taken at frequent intervals over a 90-day period. Visual Grades 0 (no rejection) to 4 (severe rejection) were assigned based on skin color, bleeding from biopsy site, and blister formation. Histologic Grades 0 to 4 were assigned based on the degree of vasculitis, folliculitis, dermal inflammation, and epidermal degeneration present.Results. All Group I animals progressively rejected their graft by Day 7. Group II grafts survived from 19 and 90 days; 93% of 115 biopsy specimens were read to be within ±1 histologic score of their assigned flap visual grade. Visual assessment carried an 8% false positive and 39% false negative rate with regard to biopsy-proven rejection. However, 81% of missed rejection specimens were histologic Grade 1. Biopsy, when visually indicated, would detect all rejection episodes when histologically Grade 1 or 2 and still potentially reversible.Conclusions. Visual scoring of CTA skin serves as a useful tool for initially detecting rejection, but repeated histologic evaluation is necessary for monitoring the subsequent course of the graft.

EARLY SKIN BUT NOT WHOLE-BLOOD CYCLOSPORINE (CSA) LEVELS CORRELATE WITH REVERSIBILITY OF REJECTION IN A NOVEL SWINE COMPOSITE TISSUE ALLOGRAFT (CTA) MODEL

237 With an eye toward direct clinical application, we developed a porcine brachial artery-based radial forearm osteomyocutaneous flap allograft model to assess the antirejection efficacy of CSA/mycophenolate mofetil (MMF) treatment. Transplants were performed between size-matched, outbred donor-recipient pairs (n=5). A once-daily oral CSA/MMF/prednisone regimen was used: CSA dose was 40 mg/kg/d, adjusted to maintain 24 hr trough levels between 100-300 ng/ml by the EMIT assay; MMF dose was 1 g/d; and prednisone was tapered from 2.0 to 0.1 mg/kg/d over 30 days. Drug doses were not adjusted based on the clinical course. Graft skin biopsies were performed at 0, 2, 4, 7, 10, 14, 21, 30, 45, 60, and 90 days posttransplant. Histologic grading of skin rejection (I, mild-IV, severe) was based on the severity of vasculitis; dermal inflammation; folliculitis; and epidermal degeneration. CSA trough levels were determined daily for the first 3 weeks; then 3X/week for the next 3 weeks; and then weekly. Skin CSA levels were measured on days 2, 7, 14, 21, 30, 45, 60, and 90 using the EMIT assay. All 5 pigs developed grade I-II rejection by day 7. However, rejection reversed spontaneously in 3 pigs (#1-3), which remained rejection-free at 90 days, but progressed in 2 pigs (#4, 5), with graft loss from grade IV rejection on days 25 and 29. (Table)TableDespite lower mean blood [CSA] during the first week, pigs #1-3 were able to subsequently reverse established mild to moderate acute rejection occurring by day 7, presumably as a result of achieving higher local skin [CSA] within the allograft. Indeed, these pigs displayed higher Kp values on day 2, day 7, and overall during the first week (p=0.09, 0.09, and 0.01, respectively, by unpaired t-test vs #4,5), indicating that more CSA was being distributed to the skin relative to the blood in these animals in the early posttransplant period. There were no differences between the subgroups with regard to mean blood or skin [CSA] or Kp values during weeks 2 and 3. Skin [CSA] correlated significantly with blood [CSA] only in pig #3 (r = 0.97, p = 0.005 by Fisher's r to z test). Our preliminary data in a small number of animals suggests that (1) early attainment of adequate local [CSA] within CTA skin may be more important in inhibiting rejection than systemic blood levels and that this may be dependent upon the Kp; and (2) monitoring local tissue drug levels may be essential to proper immunosuppressive management of CTAs.

FROM BENCH TO BEDSIDE: PORCINE EXTREMITY COMPOSITE TISSUE ALLOGRAFT (CTA) TO HUMAN HAND TRANSPLANT

71 In order to proceed with clinical hand transplantation, it was necessary to demonstrate that rejection could be effectively suppressed in a large-animal CTA model with acceptable morbidity. We therefore developed a porcine brachial artery-based radial forearm osteomyocutaneous flap allograft model to assess the antirejection efficacy of combination cyclosporine (CSA)/mycophenolate mofetil (MMF) and tacrolimus (FK)/MMF treatment. Once-daily oral CSA/MMF/prednisone (pred) and FK/MMF/pred regimens were used. Initial CSA and FK506 doses were 40 and 1.5 mg/kg/d, adjusted to maintain 24 hr trough levels between 100-300 and 3-8 ng/ml, respectively; MMF dose was 0.5 g/d; and pred was tapered from 2.0 to 0.1 mg/kg/d over 30 days. Drug doses were not adjusted based on the clinical course. Graft skin biopsies were performed at 0, 2, 4, 7, 10, 14, 21, 30, 45, 60, and 90 days posttransplant. Visual and histologic scoring systems for grading of skin rejection were developed. In 5 control pigs, the flap was completely rejected on day 7. Of the 10 pigs receiving CSA, 1 died from pneumonia and an another from an anesthetic complication on days 19 and 30 without rejection. 2 flaps were lost on days 25 and 29 from severe rejection. 6 flaps survived 90 days, 5 with persistent mild to severe rejection. Of the 9 pigs receiving FK, 3 died from pneumonia on days 29, 30, and 83 without rejection and an another from gastric rupture on day 42 with mild rejection. The remaining 5 pigs were free of rejection at 90 days (p < 0.01 vs controls for both groups). Our results demonstrated for the first time that rejection can be significantly delayed in a large-animal CTA model including skin using only orally-administered agents dosed according to a clinically-relevant strategy without antibody induction and led to clinical trials. We assessed the risk/benefit ratio for a human hand transplant using risk analysis as an epidemiologic tool to measure quality of life values. The development of a clinical program was done as a transparent process, inviting public and medical commentary prior to the procedure. Ethics committee and institutional review board presentations led to final approval. Patient selection involved medical, surgical, psychiatric, and social services input. The protocol included HLA and blood group testing, as well as flow cytometric and CDC crossmatching. The heartbeating cadaver donor was matched by bone size (± 20%), gender, and color, and the donor hand was flushed with UW solution and cold-stored until blood flow was reestablished. The recipient received basilixamab 20 mg IV on days 0 and 4, FK (target trough level 15-20 ng/ml), MMF 1g bid, and pred (2 mg/kg/d tapered to 10 mg/d by 90 days). Protocol skin biopsies were performed on days 0, 5, 7, 10, 14, 21, and 28. Physical therapy and bracing started on day 3 and continued daily. No visual signs of skin rejection were apparent during the first month, with mild perivascular infiltrates seen on biopsy. Function is equivalent to that of the replanted autologous hand. No drug-specific toxicity or infectious complications were observed. We conclude that early success in clinical hand transplantation is achievable using currently available immunosuppressants.

Tolerance Induction in a Rat Osteomyocutaneous Allograft Model Across Major Immunologic Barrier

256 Whole limb allotransplantation presents a tremendous immunologic challenge due to high antigenicity of the combination of composite tissues. In our previous study, leflunomide (LEF) combined with cyclosporine (CsA) successfully prevented limb allograft rejection across a strong histocompatibility barrier. However, following cessation of the treatment on day 60, all recipients developed Graft-versus-Host Disease (GVHD) in 7 days. In this study osteomyocutaneous grafts composed of the whole lower thigh tissues were transplanted from Brown-Norway (BN) to Lewis (LEW) rats. Recipients were administered with LEF (10 mg/kg/24hr/PO) and CsA (5mg/kg/24hr/PO), LEF (10 mg/kg/24hr/PO) and FK506 (0.1 mg or 0.5 mg/kg/24hr/SC), or placebos. All medications were started 24 hours prior to transplantation and continued for 30 days or until graft rejection. As a result, all treated allografts survived the 30-day period of immunosuppression. Following the cessation of treatment, all rats received LEF and CsA developed rejection in 10 to 25 days with no or minimal signs of GVHD. One Recipient in the group received LEF and 0.1 mg/kg FK506 (n=6) rejected the graft skin in 25 days, while the other 5 rats tolerated the grafts for more than 100 days. And all recipients (n=6) received LEF and 0.5 mg/kg FK506 maintained the grafts for more than 100 days, with no signs of GVHD or rejection. Rejections were confirmed pathologically with or without a second BN skin grafting. Tolerance was confirmed by the rejection of the 3rd party skin. Microchimerism in tissues and blood of the recipients were detected by immunochemical method and flow cytometry. The results indicated that 30-day immunosuppresion was better than the 60-day program in preventing GVHD and inducing tolerance. And the combination of LEF and FK506 is superior to LEF and CsA in inducing composite tissues tolerance.

AN UNEXPECTED ROLE FOR CD28 AND LACK OF A ROLE FOR CD40L SIGNALING IN PERIPHERAL DELETION OF DONOR-REACTIVE CD4+ T CELLS AND ESTABLISHMENT OF MIXED CHIMERISM THROUGH COSTIMULATORY BLOCKADE

453 Purpose: Bone marrow transplantation plus costimulatory blockade allows the establishment of long-term multilineage chimerism and tolerance across a full MHC barrier. Tolerance is associated with rapid peripheral deletion of recipient CD4+ T cells bearing donor-reactive Vβ. We evaluated the role of CD28 and CD40L in this regimen using CD28−/− and CD40L−/− mice. Methods: C57BL/6-CD28−/− and C57BL/6 wild-type (WT) mice (H-2b) received 3Gy WBI and 20×106 B10.A (H-2a) bone marrow cells on day 0, followed by single injections of anti-CD40L mAb (MR1; 0.5mg; day 0) with or without CTLA4Ig (0.5mg, day 0 or day +2). H-2b B6/129-CD40L−/− and B6/129 mice received 3Gy WBI followed by 15×106 B10.A BMC on day 0, and single injections of CTLA4Ig (0.5mg; day +2) with or without MR1 (0.5mg; day 0). Levels of donor chimerism in peripheral blood and frequency of TCR using certain Vβ were followed by FCM analysis. Tolerance was tested by grafting donor (B10.A) and third party (A.SW) tail skin. Results: In the CD28−/− experiment, five of six WT mice receiving MR1 and CTLA4Ig developed multilineage mixed chimerism, with peripheral deletion of donor-reactive CD4+ T cells at week 1 that progressed further by week 3. In contrast, CD28−/− mice failed to demonstrate measurable chimerism or deletion, regardless of CTLA4Ig administration. In the CD40L−/− experiment, two of five CD40L-deficient mice receiving only CTLA4Ig showed high levels of multilineage chimerism, similar to results in WT mice receiving both CTLA4Ig and MR1 (3 of 5 mice). Similar deletion of donor-reactive Vβ was evident among CD4+ T cells of chimeric CD40L−/− mice which received only CTLA4Ig and wild-type controls receiving MR1 and CTLA4Ig. Donor-specific skin graft tolerance (>100 days) was observed in all chimeric mice, with prompt rejection of third party skin. Conclusion: Surprisingly, signaling through CD28 on host T cells plays a role in peripheral donor-reactive T cell deletion and in permitting allogeneic marrow engraftment in mice receiving costimulatory blockade. In contrast, signaling through CD40L is not necessary in this regimen of tolerance induction. The deletion of donor-reactive CD4+ T cells observed in CD40L−/− mice which did not receive MR1 suggests that direct destruction of anti-CD40L-coated activated T cells is unlikely, and that the importance of anti-CD40L treatment in tolerance induction is due to its ability to prevent activation of APC's.

Prolonged skin allograft survival in mice treated with flt3-ligand-induced dendritic cells and anti-CD154 monoclonal antibody

The problem of allogeneic skin rejection is a major limitation to more widespread application of clinical composite tissue allotransplantation (CTA). Previous research examining skin rejection has mainly studied rejection of conventional skin grafts (CSG) using standard histological techniques. The aim of this study was to objectively assess if there were differences in the immune response to CSG and primarily vascularized skin in composite tissue allotransplants (SCTT) using in vivo techniques in order to gain new insights in to the immune response to skin allotransplants.CSG and SCTT were transplanted from standard Lewis (LEW) ad Wistar Furth (WF) to recipient transgenic green fluorescent Lewis rats (LEW–GFP). In vivo confocal microscopy was used to observe cell trafficking within skin of the transplants. In addition, immunohistochemical staining was performed on skin biopsies to reveal possible expression of class II major histocompatibility antigens.A difference was observed in the immune response to SCTT compared to CSG. SCTT had a greater density cellular infiltrate than CSG (p < 0.03) that was focused more at the center of the transplant (p < 0.05) than at the edges, likely due to the immediate vascularization of the skin. Recipient dendritic cells were only observed in rejecting SCTT, not CSG. Furthermore, dermal endothelial class II MHC expression was only observed in allogeneic SCTT. The immune response in both SCTT and CSG was focused on targets in the dermis, with infiltrating cells clustering around hair follicles (CSG and SCTT; p < 0.01) and blood vessels (SCTT; p < 0.01) in allogeneic transplants.This study suggests that there are significant differences between rejection of SCTT and CSG that may limit the relevance of much of the historical data on skin graft rejection when applied to composite tissue allotransplantation. Furthermore, the use of novel in vivo techniques identified characteristics of the immune response to allograft skin not previously described, which may be useful in directing future approaches to overcoming allograft skin rejection.

Chronic rejection of major histocompatibility complex class II-disparate skin grafts after anti-CD3 therapy: a model of antibody-independent transplant vasculopathy.

Chronic rejection remains a leading cause of allograft loss. Histologically, it is characterized by arterial intimal thickening and parenchymal fibrosis. The immune mechanisms triggering chronic rejection are still uncompletely understood. We performed major histocompatibility complex (MHC) class H-incompatible skin grafts from C-H2bm12 (bm12, H2bm12) into C57BL/6 (C57BL/6, H2b) recipients immunosuppressed with a short course of anti-CD3 monoclonal antibodies to prevent acute rejection. More than 80% of grafts survived for prolonged periods, but eventually all displayed macroscopic and microscopic evidence of chronic rejection. At histology, there was a progressive arterial intimal thickening as well as intense dermal fibrosis. This was accompanied by an inflammatory infiltrate consisting of lymphocytes and macrophages, but also of a considerable number of eosinophils. Mice with chronic rejection were unable to generate anti-donor MHC class II cytotoxic T lymphocyte activity at either 20 or 60 days after transplant. Furthermore, transplantation of bm12 skins on C57BL/6-congenic, Ig knock-out mice was associated with the development of a chronic rejection that was identical to that occurring in wild-type C57BL/6 animals, indicating that alloantibodies are not necessary in this model. (1) Skin grafts may undergo chronic rejection with the characteristic lesions of vasculopathy and fibrosis; (2) chronic rejection of MHC class II-disparate skins may occur in the absence of direct cytotoxic T lymphocyte activity or alloantibodies.

Rejection of H-Y Disparate Skin Grafts by Monospecific CD4+ Th1 and Th2 Cells: No Requirement for CD8+ T Cells or B Cells

We wished to determine whether CD4+ T cells could reject a skin graft that was discordant for a single minor transplantation Ag in the absence of CD8+ T cells or Ab. Transgenic A1(M) mice were constructed that express the rearranged V beta 8.2 and V alpha 10 TCR genes from a T cell clone that is specific for the male Ag (H-Y) in the context of H2-Ek. In addition, the RAG-1(-/-) background was bred onto these mice to eliminate any endogenous TCR rearrangements. As expected, clonal deletion was found to be complete in the thymus of male A1(M) x RAG-1(-/-) mice, while only CD4+ T cells were positively selected and found in the periphery of females. Female A1(M) x RAG-1(-/-) mice were able to rapidly reject (in <14 days) male (but not female) skin grafts in a CD4-dependent fashion. After multiple grafts, it was confirmed that no CD8+ T cells or surface Ig+ B cells were present. An immunofluorescent analysis of spleen cells after grafting showed that the majority of T cells expressed activation markers (CD44, CD25, and intracytoplasmic IL-2) and a significant proportion were making IFN-gamma and IL-4. Surprisingly, the transfer of either Th1 or Th2 CD4+ T cell lines from these mice into T cell-depleted recipients was sufficient to cause a specific rejection of male skin.

INTERFERON-GAMMA (IFNγ) IS REQUIRED FOR LONGTERM ALLOGRAFT SURVIVAL INDUCED BY COMBINED ADMINISTRATION OF DONOR-SPECIFIC SPLENOCYTE TRANSFUSION(DSST) AND CTLA4Ig.

433 Administration of donor splenocytes or bone marrow enhances the salutary effect of B7-CD28 costimulation blockade on allograft survival in rodents. Because IFNγ has important immunoregulatory functions, we addressed whether this cytokine plays a role in DSST+CTLA4Ig-mediated immunosuppression by comparing the survival of fully allogeneic cardiac or skin grafts in wildtype (IFNγ+/+) recipients to that in IFNγ gene-knockout(IFNγ-/-) mice. DSST (1 × 107 donor splenocytes) was administered i.v. at the time of transplantation. CTLA4Ig (200μg) was administered i.p. in a single dose two days after transplantation. Cardiac rejection was defined as cessation of palpable heart beat and was confirmed histologically. Skin rejection was defined as >90% necrosis of the graft(trunk skin). Results: DSST+CTLA4Ig-mediated prolongation of cardiac allograft survival (C3H/He → BALB/c) was significantly greater in IFNγ+/+ than IFNγ-/- recipients:TableThese data are consistent with our previous findings that endogenous IFNγ facilitates induction of longterm cardiac allograft survival in mice treated with CTLA4Ig alone. Similarly, DSST+CTLA4Ig-mediated prolongation of skin allograft survival(BALB/c → C57BL/6), although modest, was significantly greater in IFNγ+/+ than IFNγ-/- recipients:TableConclusions: (1) Endogenous IFNγ does not hinder but rather facilitates induction of longterm allograft survival in mice treated with donor-specific splenocyte transfusion and CTLA4Ig. (2) These preliminary data suggest that IFNγ plays an essential regulatory role in alloimmunity. Because cyclosporine inhibits IFNγ production, cyclosporine could abrogate the potential clinical utility of donor blood, bone marrow, or splenocyte administration to recipients of solid organ allografts.

IDENTIFICATION OF AN INDIRECTLY PRESENTED EPITOPE IN A MOUSE MODEL OF SKIN ALLOGRAFT REJECTION1

The indirect pathway of allorecognition involves the processing and presentation of donor molecules by recipient antigen-presenting cells to alloreactive CD4+ T cells. Our objective was to assess the occurrence and significance of the indirect presentation of allogeneic major histocompatibility complex molecules in the rejection of major histocompatibility complex class I-disparate skin. A mouse model of allograft rejection was developed in which tail skin from C57.BL/10 (H2b) donors was transplanted onto B10.A(5R) recipients resulting in an allogeneic mismatch at the D locus. T-cell depletion studies were used to characterize T-cell subset involvement in rejection. B10.A(5R) mice were immunized with pools of overlapping peptides spanning the polymorphic region of Db in order to identify Db-derived epitopes involved in rejection. The relevance of these epitopes was assessed through immunization of recipient mice with peptides before skin grafting to observe the effect of presensitization on the kinetics of rejection. Rejection of Db-disparate skin by B10.A(5R) was delayed by CD4 and CD8+ T-cell depletion, indicating the significance of both cell types in rejection. At least six immunogenic peptides were identified, all of which contained a cryptic T-cell epitope. One peptide, however, was able to accelerate the rejection of Db-disparate skin. Presensitization of B10.A(5R) mice with this peptide also resulted in an increase in alloantibody, indicating the presence of a physiological as well as a cryptic epitope. Presensitization of mice with a peptide containing a distinct cryptic epitope, however, failed to influence rejection. These findings demonstrate a significant role for the indirect pathway of antigen presentation in allograft rejection.

Immunosuppressive applications of PHA and other plant mitogens.

Phytohemagglutinin was prominent in the evaluation of the immunosuppressive effects of mitogenic lectins that started in 1965 and continued for two decades. Basic studies elucidated the inhibitory actions of PHA on humoral and cellular immune responses in mice, rats, and guinea pigs. Some suppressive effects of this and other mitogens including lentil lectin and Con A were demonstrated on renal, skin, pancreas, and heart allograft rejections in mice, rats, and dogs. In addition to their inherent suppressive activities, these substances have been shown to potently augment the suppression generated by conventional agents. More relevant to tolerance-inducing modulations, the Rigas group showed that in vitro incubation of the parental spleen cells with PHA in the F1 hybrid model before giving them to the F1 mouse virtually abolished the GvH responses. Their explanation was that the donor cells were rendered unresponsive by their reversion to an immature state in which they lost the essential surface receptors. Similar spleen cell suppression was generated by systemic administration of PHA to the parental donor prior to their administration. A method is proposed here for establishing tolerance to either newly introduced or firmly established antigens applying the L4 isolectin of PHA to make non-reactive all T lymphocytes that remain functional in conjunction with full suppression by conventional agents. During the vulnerable period of drug-induced suppression, the host would be protected from infection and bleeding by full nonspecific proliferative activation of the lymphoid and myeloid systems. The establishment of allograft tolerance by preemptive inhibition of responses to newly introduced transplant antigens would be easier to achieve than the reversal of firmly established responses to antigens involved in GvH reactions and autoimmune diseases. The studies of von Boehmer and Kisielow in a transgenic mouse model confirmed that clonal deletion does occur in the thymus whereby corresponding specific thymocytes are destroyed when they encounter self-antigens. Their work suggested that tolerance to self-antigens might be established if immune responses against putative antigen peptides were blocked sufficiently that they would be released to reach the central or peripheral residence sites of their immunologically reactive clones for deletion to occur. This approach to suppressive therapy would be useful for managing the problems of allograft rejection, GvH reactions, and autoimmune disorders even if they fell short of generating complete tolerance. Suppressive treatment with mitogens would also be indicated for the immune-related group of aplastic anemias, but a discussion of these disorders will be deferred to a separate article because of certain aberrations they present.