Cell-mediated Lympholysis Assays Research Articles

Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine.

This study tested the hypothesis that vascularized composite allografts (VCA) could be accepted in a robust model of hematopoietic chimerism by injecting allogeneic bone marrow cells (BMC) into swine fetuses. Outbred Yorkshire sows and boars were screened to ensure the absence of the major histocompatibility (MHC) allele SLA(cc) of inbred MGH miniature swine and then mated. Bone marrow harvested from an SLA(cc) swine donor was T-cell depleted and injected intravenously into the fetuses between days 50-55 of gestation. After birth, the piglets were studied with flow cytometry to detect donor cells and mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays to assess their response to donor. Donor-matched VCAs from SLA(cc) donors were performed on four chimeric and two nonchimeric swine. The results showed donor cell engraftment and multilineage macrochimerism after the in utero transplantation of adult BMC, and chimeric animals were unresponsive to donor antigens in vitro. Both control VCAs were rejected by 21days and were alloreactive. Chimeric animals accepted the VCAs and never developed antidonor antibodies or alloreactivity to donor. These results confirm that the intravascular, in utero transplantation of adult BMC leads to donor cell chimerism and donor-specific tolerance of VCAs across a full MHC barrier in this animal model.

Carbon Monoxide Administered Only to the Donor Is Sufficient to Prolong Pulmonary Allograft Survival Across a Fully MHC-Mismatched Barrier in CLAWN Miniature Swine

Objective: To attempt carbon monoxide (CO) to apply for the clinical setting, we have extensively studied the role of CO using miniature swine as a translational research. We have previously reported that perioperative low-dose CO inhalation decreases IRI using in situ porcine lung IRI model and that perioperative CO inhalation to both donor and recipient prolongs lung graft survival in MHC-inbred CLAWN miniature swine. To understand the mechanisms involved in prevention of graft damage following CO inhalation, we examined whether donor or recipient treatment alone is sufficient to prolong graft survival. Methods: Eighteen CLAWN miniature swine received fully MHC mismatched lungs with 12 days of tacrolimus (days 0-11; 35-45 ng/ml). In the control group (n=6), recipients received FK506 alone. In D+R group (n=5), both donor and recipient additionally inhaled 200-250 ppm CO (180 min for donor; 390 min for recipient until 2-hr reperfusion). In D group (n=4), only donor was treated with CO for 180 min. In R group (n=3), CO was administered only to the recipient for 390 min. The concentration of inhaled CO was adjusted, such that carboxyhemoglobin levels did not exceed 15%. Graft function was monitored by chest radiography and serial open lung biopsy. Immunologic responses were assessed by mixed lymphocyte reaction (MLR), cell-mediated lympholysis (CML) assays and allo-antibody (Ab) development using flow cytometry. Results: All control recipients rejected the grafts by POD63 (47 ± 7 days) with development of cytotoxic antidonor antibodies. Four out of 5 recipients in D+R group accepted the grafts over 63 (82 ± 13) days. Surprisingly, CO therapy only with donor (D group) was effective in prolonging graft survival. Three out of 4 recipients accepted the grafts over 63 (97 ± 26) days with the longest survivor lasting 150 days. In contrast, all three recipients in R group rejected the grafts by POD63 (54 ± 6 days). Development of anti-donor Abs and MLR and CML responses was delayed in D+R and D group. Furthermore, fewer inflammatory cell infiltrates on POD2 biopsies, and decreased serum concentrations of IL-1β and IL-6 were mostly observed in D+R and D group. Conclusions: These data suggest that donor CO alone is sufficient to improve lung graft survival in a clinically relevant large animal model. CO pretreatment might have the effects through some analogue of ischemic preconditioning. Donor cytoprotective preconditioning with CO during organ harvest or preservation could be accomplished in a clinical setting, potentially improving graft survival with little risk of adverse effects on the recipient.

Tolerance and Long-Lasting Peripheral Chimerism After Allogeneic Intestinal Transplantation in MGH Miniature Swine

Clinical intestinal transplantation (Int-Tx) is limited by high rates of rejection, infection, and graft versus host disease. To improve clinical outcomes and eliminate the comorbidities associated with chronic immunosuppression, the induction of donor-specific tolerance to intestinal grafts is desirable, especially in the pediatric population. This study determined the ability of intestinal grafts to facilitate tolerance induction in major histocompatibility complex (MHC)-inbred miniature swine. Seven MGH-miniature swine received heterotopic intestinal grafts, two across MHC-matched, minor-antigen disparities, three across a class I MHC disparity with 12 days of cyclosporine A, and two across a class I MHC disparity without an immunosuppressant. Chimerism was assessed by FACS analysis and immunohistochemistry. Cell-mediated lympholysis assays were used to assess antidonor responses. Two animals receiving intestinal grafts without an immunosuppressant developed antidonor IgG in 14 days and rejected these completely. All other grafts were accepted with 12 days of cyclosporine A across both MHC-matched and MHC class I barriers. Cell-mediated lympholysis assays showed donor-specific unresponsiveness by day 30 across MHC class I barriers. Greater than 15% peripheral donor cell chimerism persisted for more than 60 days after MHC-matched Int-Tx. Although less than 1.5% peripheral donor cell chimerism was seen during the maintenance period after class I-mismatched Int-Tx, 5% to 10% myeloid chimerism was found in the peripheral blood 14 to 90 days after Int-Tx. FACS analysis demonstrated that 1% to 2% of lymphocytes in the graft mesenteric lymph nodes were CD4/CD25(HIGH+)/Foxp3(+) cells. To our knowledge, this is the first demonstration of tolerance induction and persistence of chimerism in a large animal intestinal transplant model.

Porcine CFSE mixed lymphocyte reaction and PKH-26 cell-mediated lympholysis assays

Mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) are widely used to assess T cell responses. A major limitation of the traditional MLR and CML assays is that they require radioisotope labeling with 3H for MLR and 51Cr for CML, thereby limiting their use to laboratories with the capabilities to deal safely with these materials. Recently, flow cytometry with CFSE labeling has been used to detect cell division in rodent and human assays, and flow cytometry with PKH-26 labeling has been used to study cytotoxicity in murine models. Partially inbred miniature swine provide a unique large animal preclinical model for experimental transplantation, helping to bridge the gap between rodent and clinical studies. In this study, we modified the reported CFSE and PKH-26 labeling procedures for use with porcine cells, and established that these radioactive-free MLR and CML assays are comparable to traditional radioactive CML and MLR assays for assessing immunologic responses in miniature swine. To our knowledge, this is the first report that has directly compared the traditional CML/MLR with radiation-free CML/MLR in MHC-defined swine models. Objective The aim of this study is to establish non-radiolabeled CSFE and PKH-26 labeling procedures for flow cytometry based CML/MLR assays that are comparable to radioactive CML/MLR assays in preclinical large animals.

Administration of Donor-Derived Mesenchymal Stem Cells Can Prolong the Survival of Rat Cardiac Allograft

Background Mesenchymal stem cells (MSCs) are multipotent adult elements that have recently been shown to have profound immunomodulatory effects both in vitro and in vivo. Herein we have examined the impact of intravenous infusion of donor MSCs on the survival of transplanted hearts in a rat allograft model. Methods Recipient Fisher344 rats were transplanted with hearts from inbred Wistar rats. Wistar rat MSCs were infused via the tail vein at designated intervals. In vitro mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays were performed to assess whether MSCs downregulated T-cell responses in vivo. Real-time polymerase chain reaction (PCR) was used to analyze the Th1/Th2 balance in MSC-treated and control groups. Results The MSCs cultured in vitro exhibited multipotential for differentiation. Survival of the allografts was markedly prolonged by administration of MSCs compared with the controls, namely mean survivals of 12.4 vs 6.4 days, respectively. Real-time PCR showed a shift in the Th1/Th2 balance toward Th2. By MLR and CML assays, untreated control rats showed greater alloreactivity than did MSC-treated rats. Conclusion Our results indicated that MSCs suppressed allogeneic T-cell responses both in vitro and in vivo. Intravenous administration of MSCs prolonged the survival of transplanted hearts, possibly by induction of allograft tolerance through changing the Th1/Th2 balance.

Long-Term Acceptance of Renal Allografts following Prenatal Inoculation with Adult Bone Marrow

The aim was to investigate if intravascular in utero injection of adult bone marrow into swine fetuses could lead to macrochimerism and tolerance to the donor. Outbred Yorkshire sows and boars screening negative for MHC allele SLA of MGH miniature swine were bred. A laparotomy was performed on the sows at 50 days gestation to expose the uterus. Bone marrow harvested from SLA miniature swine was T-cell depleted and injected intravascularly into seventeen fetuses. Flow cytometry was performed to detect donor cells (chimerism) in the peripheral blood after birth. Mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays were used to assess the response to donor MHC. Previously frozen skin grafts from the bone marrow donor were placed on the offspring from the first litter. Donor-matched renal transplant from SLA donors were performed on chimeric swine, with and without a short 12-day course of cyclosporine, and one nonchimeric littermate. Nine inoculated offspring demonstrated donor cell chimerism in the peripheral blood and lymphohematopoietic tissues. All animals with detectable chimerism within the first three weeks were consistently nonreactive to donor MHC in vitro. Animals challenged with donor skin grafts displayed prolonged graft survival without producing antidonor antibodies. All chimeric animals accepted donor-matched kidney allografts, even one without cyclosporine. The kidney in the nonchimeric littermate rejected by day 21. Transplantation of allogeneic adult bone marrow into immunocompetent fetal recipients resulted in chimerism. In utero inoculation led to operational tolerance to the donor's major histocompatibility antigens and long-term acceptance to organ allografts.

A CD8 DE loop peptide analog prevents graft-versus-host disease in a multiple minor histocompatibility antigen-mismatched bone marrow transplantation model

Donor CD8(+) T cells can be potent mediators of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation to either major histocompatibility complex (MHC) class I-or multiple minor histocompatibility antigen-mismatched recipients. To develop small molecular inhibitors of CD8(+) T-cell activity, theoretical structural analysis of the human CD8 alpha molecule was previously used to identify potential functional surface epitopes that interact with the MHC class I molecule. The DE loop (p71-78) was identified as such a target region, and a panel of synthetic cyclized peptide mimics of this region were tested for their inhibitory effects on cytotoxic T lymphocyte activity in human cell-mediated lympholysis assays. Peptide 1109 (CKRLGDTFVC) was most effective at inhibiting specific target cell lysis. Accordingly, studies were conducted to determine whether there was sufficient cross-species homology in the DE loop region and its nonpolymorphic interactive site on the beta(2)-microglobulin domain of the MHC class I molecule to allow similar inhibition of murine CD8(+) cytotoxic T lymphocyte activity. On the basis of strong in vitro inhibitory activity of 1109 in the murine system, the capacity of the peptide to inhibit in vivo CD8(+) T-cell effector functions in skin and hematopoietic stem cell transplantation models was examined. In the C57BL/6 anti-bm1 skin allograft rejection model, across an MHC class I barrier, a single injection of 1109 at the time of transplantation significantly prolonged graft survival. Moreover, 1109 administered at the time of transplantation in the multiple minor histocompatibility antigen-disparate B10.BR-->CBA GVHD model significantly prolonged the survival of lethally irradiated mice that underwent transplantation with donor bone marrow cells and CD8(+) T cells. Histopathologic analysis confirmed that mice treated with the synthetic peptide exhibited diminution of epithelial target cell injury. Specificity of the peptide effect was evidenced by draining lymph node cells from B10.BR mice that had been challenged with CBA lymphocytes and simultaneously treated with 1109. These cells could not generate secondary proliferative responses in vitro upon stimulation with CBA splenocytes but could respond to third-party C57BL/6 stimulation. Thus, the 1109 peptide has potential application in the prevention of CD8-mediated GVHD development.

Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine.

Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.

The Role of Regulatory Cells in Miniature Swine Rendered Tolerant to Cardiac Allografts by Donor Kidney Cotransplantation

To determine the mechanism by which cotransplantation of a kidney allograft induces tolerance to a donor heart in miniature swine, we examined the role of CD25+ cells in heart/kidney recipients. Tolerance was induced to class‐I MHC mismatched hearts by cotransplanting a donor‐specific kidney with a 12‐day course of cyclosporine. Peripheral blood leukocytes (PBL) were isolated from tolerant heart/kidney recipients and used in cell‐mediated lympholysis (CML) coculture assays as either unmodified PBL, PBL enriched for CD25+ cells or PBL depleted of CD25+ cells to assess their ability to suppress CML responses of naïve recipient‐matched leukocytes against mismatched target cells. Primed PBL from tolerant heart/kidney recipients completely suppressed lysis by naïve cells. Complete suppression of the response of naïve recipient‐matched leukocytes against donor‐matched target cells was lost following the depletion of CD25+ cells from tolerant heart/kidney animal PBL, but it was reestablished by incubation of naïve cells with small populations of CD25+ cells from tolerant heart/kidney animals. These data suggest that peripheral blood from tolerant heart/kidney recipients contains regulatory cells that, upon priming, can suppress the response of naïve‐matched PBL in coculture CML assays, and that suppression appears to be dependent on cells expressing CD25.

Propagation of activated T lymphocytes from endomyocardial biopsy samples of cardiac allografts: Influence of the addition of recombinant interleukin‐4 to the culture environment

In vivo, activated T cells can be propagated from endomyocardial biopsy (EMB) samples of cardiac allografts in cultures containing recombinant interleukin-2 (rIL-2). However, T cells are sometimes not propagated in such cultures, even when rejection is present, and at other times the yield of lymphocytes is too small to allow further studies of these graft-infiltrating cells. The current study investigated the effects of the addition of recombinant interleukin-4 (rIL-4) to the culture environment. Cultures were performed on 532 consecutive EMB samples from 120 adult and pediatric heart transplant recipients. Each sample was divided into multiple fragments. Half of the fragments were cultured in media containing 30 U/mL of rIL-2 and the remaining half were cultured under identical conditions but with the addition of 200 U/mL of rIL-4. After 14 days, cell counts were performed, the cell phenotypes were assessed by flow cytometry, and donor specificity and cytotoxicity were assessed using the primed lymphocyte test (PLT) and cell-mediated lympholysis (CML) assay, respectively. Lymphocyte growth occurred in 18% of grade 0-1a EMB in the presence of rIL-2 and in 29% of grade 0-1a EMB in the presence of rIL-2/rIL-4 (p = 0.02). For higher-grade EMB (equivalent to grade >or=1b), the proportion of positive cultures (approximately 39%) was similar in both conditions. For positive cultures, there was a 5-fold increase in the number of cells in the rIL-2/4 cultures compared to rIL-2 alone (1.6 x 10(6) versus 3.4 x 10(5)). Flow cytometry revealed an increase in the proportion of CD8+ cells in the rIL-2/4 cultures (42% versus 23%, p = 0.004). Proliferative responses to donor antigens (as assessed by using the PLT) were comparable between the two groups, but donor-specific cell-mediated cytotoxicity was enhanced on addition of rIL-4. Hence, addition of rIL-4 enhances the propagation of donor-specific T cells from heart biopsy samples, especially in the presence of minimal rejection. This will provide a greater quantity of material for further studies of graft-infiltrating cells.

Regulatory cells in the maintenance phase of tolerance in miniature swine: coculture cell mediated lympholysis assay as approach for a tolerance assay

that CM/IFA-injected animals grafted with MHC class II KO hearts experienced significant prolongation of heart survival as compared to untreated mice (11 1 d vs 24 2 d). We conclude that modulation of CM response can on its own achieve graft prolongation when CD4 alloresponse is oligoclonal, i.e. mediated via indirect allorecognition. Our findings have important implications for the design of future therapies in heart transplantation.

Analysis of the immune status in the recipients with long-term well-functioning kidneys allografts.

The immune status of thirteen living and related kidney transplant recipients with stable allografts were examined. The immunological assays consisted of a mixed lymphocyte reaction (MLR), cell-mediated lympholysis (CML) assay, interleukin-2 (IL-2) production in mixed lymphocytes culture (MLC) and IL-2 receptor (IL-2 R) expression on MLC cells. The suppression rates of the monoclonal antibodies (mAbs) against IL-2 R were tested on MLRs. The stimulation indices (SI) of the MLR against both donor and third-party cells increased compared with those of pretransplantation. The MLC responder cells stimulated by donor cells produced detectable amounts of IL-2, these amounts were lower than those by third-party cells. The MLC cells against donor cells expressed IL-2 R alpha and beta chains to the same degree as those against third-party cells. Anti-IL-2 R mAbs equally inhibited the MLRs between recipient and donor or third-party cells. Cytotoxic T lymphocytes (CTL) against donor cells were not generated, even with the addition of recombinant IL-2 in any of recipients except one, while anti-donor CTL had been detected prior to transplantation and the CTL against third-party cells were induced in posttranspalnt CML assays. These results indicate that the clonal anergy phenomenon might mediate the specific CTL unresponsiveness observed in kidney transplant recipients and the anergy phenomenon might serve in the long-term acceptance of allograft.

Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance.

Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them. C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells. Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance. One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.

An allelic non-histocompatibility antigen with wide tissue distribution as a marker for chimerism in pigs.

It is frequently useful in studies of transplantation to have available an antibody to a cell surface antigen, which is not itself responsible for transplant rejection. In this paper, we identify and describe such an antibody/antigen system in miniature swine. The monoclonal antibody, 1038H-10-9, was found to react to a pig allelic antigen (called PAA), found on a variety of pig cells and tissues, including peripheral blood mononuclear cells (PBMC), thymocytes, lymph node, bone marrow, and skin. Analysis for recipient sensitization against PAA was performed by in vitro cell-mediated lympholysis (CML) assay, mixed lymphocyte reaction (MLR) assay, antibody binding studies, and skin graft rejection patterns were examined. No evidence was found to indicate detection of PAA by any of these assays of alloreactivity. We therefore conclude that PAA is an allelic swine cell surface antigen, with wide tissue distribution, and that it is not a histocompatibility antigen. It should provide a powerful tool for studies of transplantation biology in miniature swine, such as identification and quantification of chimerism following organ transplantation.

Role of the thymus in transplantation tolerance in miniature swine. III. Surgical manipulation of the thymus interferes with stable induction of tolerance to class I-mismatched renal allografts.

Previous studies have demonstrated that long-term tolerance of class I mismatched renal allografts in miniature swine is induced by a short course of cyclosporine (CyA), and that a total thymectomy 21 days before transplantation abrogates the induction of stable tolerance. We have now examined the effects of surgical manipulation of the thymus, with or without a reduction in the thymic volume, on the induction of tolerance. Miniature swine receiving a transplant of a class I-mismatched renal allograft and 12 days of CyA underwent either (1) a partial thymectomy 21 days before kidney transplantation (day -21), (2) serial thymic biopsies (to evaluate the effect of surgical trauma and reduction in volume of the thymus) or serial incisions of the thymus thymus (to evaluate the effect of surgical trauma without changes in thymic volume), (3) a sham thymectomy on day -21, or serial sham thymic surgery on the same POD as the thymic biopsies and incisions (control animals). Control animals had a stable plasma creatinine, had donor-specific unresponsiveness in cell-mediated lympholysis (CML) assays, had absence of rejection in kidney biopsy specimens, and did not develop anti-donor class I immunoglobulin (Ig)G alloantibodies. Animals undergoing a partial thymectomy on day -21 or serial thymic biopsies showed severe renal dysfunction, histological evidence of rejection in kidney biopsy specimens and anti-donor reactivity in CML assays; all but one animal developed anti-donor class I IgG alloantibodies. Serial incisions of the thymus induced an increase in plasma creatinine and histological rejection in 1 of 3 animals and anti-donor cytotoxic T cells in vitro in all 3 animals. A partial thymectomy or serial thymic biopsies markedly interfere with the induction of tolerance to renal allografts. Serial thymic incisions also interfere with the induction of tolerance, but to a lesser degree. These studies may have implications for tolerance-inducing protocols that involve thymic manipulation.

Peripheral Tolerance to Class I Mismatched Renal Allografts in Miniature Swine: Donor Antigen-Activated Peripheral Blood Lymphocytes from Tolerant Swine Inhibit Antidonor CTL Reactivity

Studies utilizing partially inbred miniature swine have demonstrated that a short course of cyclosporin A leads to indefinite survival of two haplotype class I mismatched renal allografts. In the present study, we have examined peripheral regulatory mechanisms that may be involved in maintenance of tolerance by coculturing PBL from long-term tolerant animals with naive recipient-matched PBL in cell-mediated lympholysis assays. We show that PBL from tolerant animals, primed in vitro with donor Ag, suppress antidonor CTL reactivity by naive recipient-matched PBL. Suppression was not observed when PBL from naive animals, primed with donor-matched PBL, were cocultured with PBL from a second naive animal, nor did PBL from either tolerant or naive recipient-matched control animals, primed with third-party Ag, suppress the generation of anti-third-party CTL by a second naive animal. The suppression was cell dose-dependent, radiation-sensitive, required cell-to-cell contact not reversed by the provision of exogenous IL-2, and associated with lower levels of IL-2R expression on the suppressive effector group (particularly the CD8 single positive cells) when compared with the control effector group. These data indicate an association between the presence of peripheral regulatory cells demonstrable in vitro and the maintenance of tolerance to renal allografts.

Expression Screening of a Yeast Artificial Chromosome Contig Refines the Location of the MouseH3aMinor Histocompatibility Antigen Gene

AbstractThe H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include β2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3aa Ag by using them as targets in a cell-mediated lympholysis assay with H3aa-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.

Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen.

Mixed hematopoietic chimerism induced with a nonmyeloablative conditioning regimen leads to donor-specific transplantation tolerance. Analyses of specific Vbeta-bearing T-cell families that recognize endogenous superantigens demonstrated that donor-specific tolerance is due mainly to an intrathymic deletional mechanism in these mixed chimeras. However, superantigens are not known to behave as classical transplantation antigens. We therefore used T-cell receptor (TCR) transgenic (Tg) recipients expressing a clonotypic TCR specific for an allogeneic major histocompatibility complex antigen to further assess deletional tolerance. 2C TCR Tg mice (H2b), whose Tg TCR recognizes major histocompatibility complex class I Ld, were used as recipients of Ld+ bone marrow cells after conditioning with depleting anti-CD4 and CD8 monoclonal antibodies, 3 Gy whole-body irradiation, and 7 Gy thymic irradiation. Chimerism and deletion of CD8+ 2C recipient T cells was evaluated by flow cytometry and by immunohistochemical staining. Tolerance was tested with in vitro cell-mediated lympholysis assays and in vivo by grafting with donor skin. Intrathymic and peripheral deletion of 2C+ CD8-single-positive T cells was evident in mixed chimeras, and deletion correlated with the presence of donor-type cells with dendritic morphology in the thymus, and with chimerism in lymphohematopoietic tissues. Chimeras showed tolerance to the donor in cell-mediated lympholysis assays and specifically accepted donor skin grafts. Tolerance to transplantation antigens is achieved through intrathymic deletion of donor-reactive T cells in mixed chimeras prepared with a nonmyeloablative conditioning regimen and allogeneic bone marrow transplantation.

CYTOTOXICITY OF LAK CELLS AND EBV-SPECIFIC T CELLS AGAINST LYMPHOBLASTOID EBV-POSITIVE B CELLS: A MODEL FOR CELLULAR THERAPY OF PTLD

696 Posttransplant lymphoproliferative disorder (PTLD) is a significant cause of morbidity and mortality in transplant patients. The presence of the Epstein-Barr virus (EBV) in many of these lymphomas has prompted the use of cellular immunotherapy using either EBV-directed T cells or autologous lymphokine-activated killer (LAK) cells. EBV-specific T cells recognize viral epitopes in association with HLA Class I molecules, whereas LAK cell cytotoxicity is enhanced by their depletion. The purpose of this study was to examine the relative levels of in vitro cytotoxicity of these effector cells against either untreated or HLA Class I-depleted EBV-immortalized B lymphocytes. LAK cells, EBV-specific T cell lines and EBV-immortalized lymphoblastoid cell lines were prepared from healthy donors. The degree of cell-mediated lympholysis (CML) was determined by using a standard 4 hour 51Cr release and the results were expressed as lytic units (LU-number of effectors required for 20% lysis of 5×103 targets). In some cases, HLA Class I expression in the target cells was depleted by a brief exposure to pH 3.3 buffer. Cell viability and Class I depletion were confirmed by trypan blue exclusion and flow cytometry, respectively prior to execution of CML assays. LAK cell-mediated cytotoxicity against autologous EBV target B cells was 14.4 LU which increased to 69.6 LU when target cells were HLA-depleted(p<0.05, Wilcoxon test). On the contrary, EBV-specific T cell-mediated cytotoxicity against untreated B cell targets was 765 LU, and this decreased to 316 LU when HLA-Class I depleted cells were used as targets (p<0.05). Additionally, as compared to that of LAK cells, T cell-mediated cytotoxicity against untreated targets was significantly greater (Friedman's test p=0.03, Tukey multiple comparison p<0.05). We conclude that EBV-specific T cells show superior in vitro cytotoxicity against autologous EBV-positive B cell targets, regardless of the level of HLA Class I expression. The results may explain in part the uniform response of PTLD to T cell therapy, and the less frequent responses to that of LAK cell. The level of cytotoxicity for both effector cell types is influenced by the degree of expression of HLA Class I in this model system. This approach may be useful in examining other factors which may enhance or decrease the cytotoxic response against EBV-positive B cells.

Preservation of immunological and colony-forming capacities of long-term (15 years) cryopreserved cord blood cells.

Cryopreserved cord blood may be stored for decades before being used for allogeneic stem cell transplantation. Little is known about the effect of long-term cryopreservation in liquid nitrogen on the viability and function of cord blood cells. We examined the recovery, viability, clonogenic capacity, and T-cell reactivity to HLA alloantigens of cord blood samples cryopreserved up to 15 years. Progenitor cell recoveries were studied by (colony-forming unit-granulocyte-macrophage) clonogenic assays from 18 cord blood samples short-term frozen for 2-8 weeks and from 8 samples cryopreserved for 15 years. Proliferative and cytotoxic responses against HLA antigens of thawed cord blood mononuclear cells after short-term or long-term cryopreservation were tested in standard mixed lymphocyte cultures and cell-mediated lympholysis assays. After thawing, the mononuclear cell recovery from long-term frozen cord blood low-density fractions averaged 80% (range, 64% to 92%). The presented data show that long-term frozen cord blood cells keep their clonogenic potential. No damaging effect was seen on the proliferative and cytotoxic capacities of long-term frozen cord blood T cells. The results support the possibility of long-term storage of progenitor cells from umbilical cord blood for future bone marrow reconstitution.