Disruption of murine cardiac allograft acceptance by latent cytomegalovirus.

Cytomegalovirus (CMV) reactivation is a well-described complication of solid organ transplantation. These studies were performed to (1) determine if cardiac allograft transplantation of latently infected recipients results in reactivation of CMV and (2) determine what impact CMV might have on development of graft acceptance/tolerance. BALB/c cardiac allografts were transplanted into C57BL/6 mice with/without latent murine CMV (MCMV). Recipients were treated with gallium nitrate induction and monitored for graft survival, viral immunity and donor reactive DTH responses. Latently infected allograft recipients had approximately 80% graft loss by 100 days after transplant, compared with approximately 8% graft loss in naïve recipients. PCR evaluation demonstrated that MCMV was transmitted to cardiac grafts in all latently infected recipients, and 4/8 allografts had active viral transcription compared to 0/6 isografts. Latently infected allograft recipients showed intragraft IFN-alpha expression consistent with MCMV reactivation, but MCMV did not appear to negatively influence regulatory gene expression. Infected allograft recipients had disruption of splenocyte DTH regulation, but recipient splenocytes remained unresponsive to donor antigen even after allograft losses. These data suggest that transplantation in an environment of latent CMV infection may reactivate virus, and that intragraft responses disrupt development of allograft acceptance.

Human allograft acceptance is associated with immune regulation.

The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-beta or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

Chronic remodeling pathology in grafts.

The theoretical basis of chronic allograft rejection remains poorly defined, but is clearly associated with the development of a relatively unique vasculopathy, transplant vascular sclerosis (TVS). The hypothesis that TVS is the cause of chronic rejection has yet to be proven, although it is widely accepted. Accumulating data suggest that, at best, the hypothesis is incomplete. The challenge for transplant investigators is to review the currently available data and to develop a testable, revised version of the hypothesis that explains both the antigen-dependent and antigen-independent contributions to graft vascular remodeling, and that encompasses all reasonable alternative mechanisms of graft failure.

Phenotype and functional activity of tumor-infiltrating lymphocytes isolated from immunogenic and nonimmunogenic rat brain tumors.

The purpose of the present study was to define the immunogenicity of two transplantable rat gliomas, designated F98 and D74, and to relate this to the phenotype and functional activity of tumor-infiltrating lymphocytes (TIL). Fischer rats, immunized with irradiated F98 tumor cells and challenged with intracerebral implants of ten F98 cells, had a median survival time of 49 days compared to 36 days for nonimmunized controls. In contrast, no statistically significant increases in survival times were noted in animals similarly immunized and challenged with the D74 tumors. No in vivo protection could be demonstrated in animals immunized and cross-challenged with either F98 or D74 glioma cells. Lymph node lymphocytes and TIL, isolated from animals immunized and challenged with F98 cells, were more cytolytically active than effector cells obtained from D74-immunized animals. Phenotypes of TIL isolated from intracerebral F98 gliomas of immunized rats were 52% OX-8+ and 21% W3/25+ compared to 31% OX-8+ and 19% W3/25+ for D74-immunized animals. Cytolytic activity against glioma targets was mediated by OX-8+ TIL, as determined by cell depletion experiments. Limiting dilution analysis showed that cytolytic T-lymphocyte precursors were present in TIL of F98 gliomas of immunized rats at a frequency of 1/3547 and were specific for F98 targets, while natural killer cell-like activity was low. Our data indicate that the F98 glioma was more immunogenic than the D74 glioma, as evidenced by increased numbers and activity of cytolytic effector cells and their precursors among TIL. This may explain in part the longer survival times observed in immunized animals challenged intracerebrally with the F98 gliomas compared to D74-immunized and -challenged hosts.

Limiting dilution analysis of human, tetanus-reactive helper T lymphocytes. A rapid method for the enumeration of helper T lymphocytes with specificity for soluble antigens.

The number of helper T lymphocytes (HTL) in human peripheral blood with specificity for the soluble protein, tetanus toxoid, was estimated by limiting dilution analysis (LDA). HTL were detected via antigen-induced interleukin-2 (IL-2) production, as measured by incorporation of tritiated thymidine by an IL-2-dependent indicator cell line, CTLL-20. Culture conditions optimizing assay sensitivity are described, and the ability to detect antigen-specific HTL using this LDA technique are demonstrated. Observed HTL frequencies in healthy human donors tested for tetanus-reactive helper T cells ranged from less than 1 HTL/268,749 peripheral blood mononuclear cells (PBMC) (undetectable) to 1 HTL/1486 PBMC. The LDA technique was also used to detect frequency shifts in human peripheral blood HTL following challenge with antigen. This assay offers distinct advantages over proliferative LDA techniques in that it is rapid (requiring only 2 days), and defines an antigen-reactive T cell subset with defined function (IL-2 secretion). Furthermore, the LDA technique can be adapted for the detection of other soluble protein antigens, such as PPD and Candida albicans. In general, this LDA technique provides a reliable, quantitative index of human HTL reactivity to any of a variety of soluble protein antigens, and has clinical as well as experimental applicability.

Quantitation of glioma-reactive cytolytic T lymphocyte precursors by means of limiting dilution analysis.

The purpose of the present study was to define the usefulness of limiting dilution analysis (LDA) to enumerate glioma-reactive cytolytic T lymphocytes (CTL) as a constituent of tumor infiltrating lymphocytes (TIL) isolated from rat gliomas. Optimum LDA microculture conditions were defined by co-cultivating graded numbers of responder TIL together with 10(5) irradiated syngeneic rat splenocytes, 10(3) irradiated glioma cells, and 10 U/well of recombinant interleukin-2 incubated for 8 days. Antigenic specificity of the anti-tumor response was demonstrated by high levels of [3H]thymidine incorporation by TIL derived from F98 gliomas following stimulation with irradiated F98 glioma cells compared to low levels following stimulation with the antigenically distinct D74 glioma cells. Limiting dilution analysis showed that cytolytic T lymphocyte-precursors were present in TIL of F98 gliomas of immunized rats at an approximate frequency of 300 CTL/10(6) TIL, indicating that less than 1% of the TIL were tumor-reactive CTL. As determined by cell depletion experiments using various MoAbs and complement, the majority of the cytolytic activity detected against glioma targets was mediated by OX-8+ TIL. Split culture experiments revealed that high levels of glioma-reactive CTL activity and low levels of NK activity, which are simultaneously detected among TIL, were mediated by separate cell populations. Our data demonstrate that LDA microcultures can be used as a powerful tool to differentiate tumor-reactive CTL from other effector cell populations.

Limiting dilution analysis for alloreactive, TCGF-secretory T cells. Two related LDA methods that discriminate between unstimulated precursor T cells and in vivo-alloactivated T cells.

We have developed a rapid limiting dilution analysis technique for quantitating alloantigen-specific TCGF-secretory T lymphocytes (operationally defined as helper T lymphocytes or HTL) in murine lymphoid populations. A simple permutation of this technique allows the distinction between HTL that have encountered antigen in vivo and the large population of naive precursor HTL (pHTL) with the same alloantigen specificity. We present evidence to validate these LDA techniques, and show that lymphoid tissue from mice bearing sponge matrix allografts, but not isografts, contain HTL that have been activated in vivo. In addition, we demonstrate a requirement for restimulation with alloantigen for secretion of lymphokine by the in vivo-activated cells. We suggest that this differential LDA technique could serve as a valuable tool in evaluating in vivo immunity and/or the in vivo efficacy of immunosuppressive, as well as immunopotentiating, therapies.

Suppression of in vitro cell-mediated lympholysis generation by alloactivated lymphocytes. Examination of radioresistant suppressive activity.

We investigated the radioresistant (1000 rads) suppression of CML generation mediated by alloactivated murine splenocytes. Suppressive cells were generated in MLCs by stimulation of (A X 6R)F1 splenocytes with irradiated C57BL/10 splenocytes. Suppressive cells could lyse targets bearing H-2b alloantigens, but would not lyse parental B10.T(6R) or B10.A targets. Suppressive activity was detected by including the alloactivated (A X 6R)F1 cells in B10.T(6R) anti-B10.A(1R) MLCs. Relative to the suppressive (A X 6R)F1 cells, the B10.A(1R) lymphocytes display both parental and suppressor-inducing alloantigens. In the absence of a suppressive population, B10.A(1R) stimulators cause B10.T(6R) splenocytes to generate cytolytic activity specific for both H-2Db (suppressor-inducing) and H-2Kk (suppressor-borne) target determinants. The irradiated, alloactivated (A X 6R)F1 cells decrease the H-2Db-specific CML generated in this system, thus mediating apparent antigen-specific suppression. However, cytolytic activity concomitantly generated in the same culture against the unrelated H-2Kk target determinants is similarly reduced by the (A X 6R)F1 cells. Thus, radioresistant suppression by alloactivated splenocytes is not necessarily antigen-specific. The irradiated (A X 6R)F1 cells would not suppress the generation of H-2Kk-specific CTL in B10.T(6R) anti-B10.A MLCs. Hence, the irradiated (A X 6R)F1 cells can impede CML generation against third-party alloantigens if, and only if, those alloantigens are coexpressed with suppressor-inducing alloantigens on the stimulator cells in suppressed MLCs. Similar results were also obtained using a different histoincompatible lymphocyte combination. Since the pattern of suppressor specificity and the pattern of CTL specificity were identical and concomitant under these experimental conditions, these data are consistent with the hypothesis that radioresistant suppression by alloactivated lymphocytes can reflect coincidental in vitro cytolytic T cell function in vitro.

Phorbol myristate acetate and in vitro T lymphocyte function. III. Selective impairment by PMA of lethal hit delivery by cloned CTL.

Phorbol myristate acetate (PMA) has little immediate effect on the lysis of antigenic tumor targets by the representative cytotoxic T lymphocyte (CTL) clones B6D/2-7c, 5MB6-5, and 5MB10-31. However, prolonged contact (24-48 hr) with PMA (10(-6)M) can profoundly depress the lytic activity of these and other cloned T lymphocytes. This concentration of PMA is neither toxic nor mitogenic for cloned T lymphocytes. B6D/2-7c cells that are treated with PMA lose some ability to bind tumor targets; however, the primary defect in PMA-treated B6D/2-7c cells appears to be at the level of lethal hit delivery, because cells remain essentially ineffective at tumor cell lysis in the presence of agglutinating lectin. Nonetheless, PMA-treated 5MB10-31 and B6D/2-7c cells continue to respond to the proliferative stimuli associated with alloantigens, especially in the presence of exogenous lymphokines. B6D/2-7c cells treated with PMA neither acquire the ability to suppress the cytolytic activity of untreated B6D/2-7c cells, nor undergo any significant alteration of Lyt-2 expression. PMA-induced loss of lytic activity is reversible, and cytolysis is reexpressed by PMA-treated B6D/2-7c cells if they are incubated with 2 degrees MLC SN, but not WEHI-3 SN. The reexpression of cytolysis occurs in the presence of cytostatic concentrations of cytosine arabinoside (ARA-C), indicating that cell proliferation is not required for this process. These data show that cloned CTL are capable of reversible cytotoxic function, and they establish the utility of PMA to probe requirements for expression of CTL function.

Detection of donor-reactive alloantibody in the early posttransplant period. Elimination of therapeutic ALG as a complicating factor.

We have developed an immunoabsorbent reagent that can differentially remove ALG from human serum samples in vitro--i.e., goat antihorse IgG covalently linked to Sepharose beads. When used according to protocol, this immunoabsorbent can effectively remove up to 0.78 mg/ml of ALG from human serum mixtures. To demonstrate that immunoabsorption is selective, an HLA-B7-specific alloserum was mixed with a known amount of ALG and absorbed with antibody-conjugated Sepharose beads. The addition of ALG to the serum sample caused high degrees of nonspecific lympholysis in standard microcytotoxicity assays. When this serum/ALG mixture was immunoabsorbed detectable ALG activity was lost but the original HLA alloantibody titer (1:2) against specific target lymphocytes was retained.

In vivo mechanisms of alloreactivity. I. Frequency of donor-reactive cytotoxic T lymphocytes in sponge matrix allografts.

To study the development of alloreactive cytolytic T cells in vivo, C57BL/6 mice were implanted s.c. with polyurethane sponges bearing allogeneic (DBA/2) splenocytes. On various days thereafter, cells that had accumulated in these sponge grafts were tested for cytolytic activity against DBA/2 target cells in 51Cr-release assays, and for frequency of DBA/2-reactive CTL as determined by limiting dilution analysis (LDA). During these studies we found that LDA was consistently more efficient at detecting alloreactive CTL than the traditional 51Cr-release assays. As determined by LDA, sponge grafts initially infused with DBA/2 splenocytes acquired high levels of DBA/2-reactive CTL, while sponge grafts infused only with saline acquired few DBA/2-reactive CTL. DBA/2-reactive CTL first became detectable in sponge allografts approximately four days after implantation, and reached a maximal frequency by the 10th day after implantation. This frequency was maintained for at least the next seven days. In contrast, the ability of cellular infiltrates from sponge allografts to lyse DBA/2 target cells in 51Cr-release assays was not detectable until the 7th day after implantation, was optimal by the tenth day, but declined thereafter to lower levels, as observed on the 13th and 17th day after implantation. Since the frequency of CTL remains stable through the 17th day after implantation, this decline in cytolytic activity may indicate that donor-reactive CTL remain in sponge allografts, but they continue to differentiate to a noncytolytic status. We further observed that previous allosensitization with skin grafts markedly accelerates the accumulation of alloreactive CTL in sponge allografts. The mechanism that promotes more rapid accumulation of CTL in allosensitized sponge graft recipients remains to be established. Throughout these studies, we observed that even at peak development, donor reactive CTL are, at most, 0.2% of the cells recovered from sponge allografts. This raises some questions regarding not only the fundamental role of the CTL in allograft rejection, but also the role of the remaining 99.8% of the allograft-infiltrating cells.

Frequency of human alloantigen-reactive T lymphocytes. II. Method for limiting dilution analysis of alloantigen-reactive helper T cells in human peripheral blood.

Quantitative immunologic techniques for analysis of human alloreactivity are currently lacking in transplantation immunology. We report a rapid, sensitive, and quantitative limiting dilution analysis technique that provides a minimal estimate of the number of peripheral blood mononuclear cells (PBMC) capable of secreting interleukin-2 (operationally defined as helpter T lymphocytes) when cultured in vitro with allogeneic PBMC bearing serologically identified MHC disparities. Using this LDA technique, we have estimated that approximately 1/500 to 1/2000 (0.2% to 0.05%) of the PBMC from various individuals can secrete IL-2 after in vitro contact with completely major-histocompatibility-complex-disparate PBMC. Under normal conditions the HTL frequency in human peripheral blood appears quite stable, based on serial analysis of HTL frequency in a healthy human donor. This LDA technique is more rapid and informative than the MLR, and may be useful for pretransplant evaluation and posttransplant monitoring of donor reactivity in transplant recipients.

In vitro stimulation of human endothelial cells by sera from a subpopulation of high-percentage panel-reactive antibody patients.

We have studied a serum activity that enhances in vitro ICAM-1 expression by human endothelial cells (EC) and report that this activity can be found in approximately 8% of pretransplant serum samples from individuals with a history of high %PRA. Hence, most high %PRA sera lack this activity, and, furthermore, mixing these negative sera does not result in an active serum pool. In patients with active serum, the ICAM-1 enhancing activity is found only sporadically, despite the continuous detection of endothelial-reactive antibodies. Absorption of Ig from a high %PRA serum reduced ICAM-1 enhancing activity, as well as endothelial-reactive antibodies. However, enhancing activity can sometimes be observed in sera that lack detectable endothelial-reactive antibodies, and none of several patient sera with defined MHC class I-specific alloantibodies displayed ICAM-1 enhancing activity. Together, these data suggest that ICAM-1 enhancing activity may not necessarily be mediated by anti-MHC alloantibodies. In addition to influencing this expression, ICAM-1 active patient sera also influence EC expression of VCAM-1 and MHC class I, but not MHC class II molecules, a pattern that is similar to that stimulated by TNF alpha. However, coincubation of EC with active serum plus soluble TNF receptor did not block the endothelial phenotypic changes, despite the ability of the soluble receptor to completely abrogate endothelial changes induced by TNF alpha. IFN gamma also increases endothelial ICAM-1 expression, but has response kinetics different from that of active serum. Interestingly, brief treatment of endothelial cells with IFN gamma greatly increased the amount of IgG bound from the active sera by EC. We conclude that some pretransplant patients occasionally express an activity in their serum that influences EC expression of several adhesion molecules, including ICAM-1, VCAM-1, and MHC class I. This activity may be associated with alloantibodies, but is independent of MHC class I-reactive antibodies, circulating TNF alpha, or IFN gamma. The relevance of a serum-borne component capable of activating EC is discussed.

The effect of injection of donor bone marrow on the frequency of donor-reactive CTL in antilymphocyte serum-treated, grafted mice.

The survival of C3H/He skin grafts can be prolonged on B6AF1 mice immunosuppressed with ALS by the injection of C3H/He marrow 1 week postgrafting. The precursor frequencies of donor-reactive CTL in the spleen and lymph nodes of ALS-treated, grafted mice given donor marrow were compared with CTL frequencies observed in ALS-treated, grafted controls. Spleens and nodes were removed from experimental and control mice on days +8, +14, +21, +58, and 1 year postgrafting, and used as effectors in the LDA. Donor-reactive CTL in the marrow-injected group remained suppressed as long as the recipients maintained their grafts. The frequency of CTL to third-party antigens was normal in mice bearing long-term C3H/He grafts. When marrow-injected mice rejected their grafts, the total donor-reactive CTL frequency returned to normal. In contrast, in ALS-treated controls that did not receive marrow, the total number of donor-reactive CTL returned to normal levels with recovery from the immunosuppressive effects of ALS. These results suggest that donor marrow suppresses the regeneration of donor-reactive CTL in the lymphoid tissues of ALS-treated mice, possibly by veto cell activity.

Frequency of human alloantigen-reactive T lymphocytes. III. Evidence that cyclosporine has an inhibitory effect on human CTL and CTL precursors, independent of CsA-mediated helper T cell dysfunction.

We have used limiting dilution analysis to study the behavior of alloantigen-reactive cytolytic T lymphocytes derived from human peripheral blood. During these studies, we found that the presence of cyclosporine in limiting dilution microcultures significantly impairs the subsequent development of alloantigen-reactive cytolytic T cell activity. As a result, CsA reduces the estimate of CTL precursor frequency by limiting dilution analysis. CTL frequency estimates are reduced by CsA in a dose-dependent manner, and concentrations of CsA that are readily achieved in human peripheral blood (100-1000 ng/ml) are capable of reducing estimates of CTL frequency by 90% to 100%. Further studies revealed that (1) human CTL derived either from fresh peripheral blood or from primary mixed lymphocyte cultures are sensitive to the suppressive effects of cyclosporine in limiting dilution microcultures, indicating that CsA influences both alloantigen-primed CTL and CTL precursors; (2) CsA impairs an immunologic event or events, that occurs for at least the first four days of limiting dilution microculture incubation; (3) CsA-mediated suppression is eliminated by separation of CTL from cyclosporine; (4) CsA blocks development of CTL generation, but not cell proliferation in limiting dilution microcultures; and (5) the CsA-mediated suppression is not reversed by supraoptimal concentrations of IL-2, high concentrations of gamma-IFN, or supplementation with the multiple lymphokines present in MLC supernatants. These data suggest that CsA may have a direct inhibitory influence on the differentiation of human CTL precursors that is independent of helper T cell dysfunction.

Development and evaluation of a limiting dilution analysis technique that can discriminate in vivo alloactivated cytotoxic T lymphocytes from their naive CTL precursors.

We describe a permutation of the conventional limiting dilution analysis (LDA) technique that allows, for the 1st time, the differential enumeration of alloantigen-specific CTL that have been activated by alloantigens in vivo. This technique does not detect nonactivated CTL precursors, even those with similar alloantigen specificity. Data are presented to validate this limiting dilution analysis technique. Using this LDA technique, we demonstrate that large numbers of the donor-reactive CTL arrive in sponge matrix allografts (f = 1/3,599 cells), most or all of which are in an activated state (f = 1/4,385 cells). In contrast, alloactivated CTL constitute only a small fraction (f = 1/57,208 cells) of the donor-reactive CTL in the regional lymph node (f = 1/1,873 cells) of the same sponge allograft recipients. As expected, regional lymph nodes from sponge isograft recipients contain DBA/2-reactive CTL precursors (f = 1/1,873 cells), but no activated DBA/2-reactive CTL (f less than 1/385,529 cells). This LDA technique should be useful in studies regarding activation and redistribution of alloreactive CTL caused by allograft implantation.

Alloreactive delayed-type hypersensitivity in graft recipients: complexity of responses and divergence from acute rejection.

BACKGROUND: Immunocompetent allograft recipients typically exhibit evidence of sensitization to graft antigens through alloantibody production and allograft rejection, as well as delayed-type hypersensitivity (DTH) reactivity to donor antigens. Most previous studies have relied on whole donor splenocytes, which primarily elicit allorestricted allogeneic responses, to test specific DTH responses and overlook the independent element of self-restricted responses in host-allograft interactions. METHODS: We tested expression of self-MHC-restricted versus allo-MHC-restricted allogeneic DTH responses in both nonimmunosuppressed and tolerized C57BL/6 mice. Mice were sensitized for allogeneic DTH either by rejection of skin or cardiac allografts, or by subcutaneous injection of intact allogeneic splenocytes. Patterns of alloreactive DTH were compared in allosensitized, tolerant, and naive hosts. RESULTS: All three methods of allosensitization resulted in equivalent self-restricted and allorestricted allogeneic DTH responses in nonimmunosuppressed mice. Gallium nitrate blocked acute rejection of cardiac allografts, and also blocked allosensitization of both self-restricted and allorestricted DTH responses, but did not influence the expression of DTH responses in presensitized mice. Gallium nitrate treatment could not block acute rejection of skin allografts, but interfered with sensitization for self-restricted, but not allorestricted, DTH responses in these recipients. This divergence of self- versus allo-MHC-restricted allosensitization for DTH was observed in two additional situations: the rates of allosensitization for self-restricted versus allorestricted DTH, and the acquisition of allorestricted, but not self-restricted, alloreactive DTH responses in cardiac allograft tolerant mice subsequently challenged with a skin allograft. CONCLUSIONS: These studies demonstrate that acute rejection correlates generally with allogeneic DTH, whereas tolerance is associated with a lack of alloreactive DTH. However, self-restricted and allorestricted allosensitization can operate independently in allograft recipients. Thus, the relationships between alloreactive DTH and graft-induced allosensitization, acute rejection, or tolerance are more complicated than previously appreciated.

Transforming growth factor-beta and interleukin-10 subvert alloreactive delayed type hypersensitivity in cardiac allograft acceptor mice.

We have previously reported that temporary treatment of cardiac allograft recipients with gallium nitrate (GN) results in indefinite graft survival, and the inability to mount donor-reactive delayed type hypersensitivity (DTH) responses. We report that antibodies to either transforming growth factor-beta (TGFbeta) or interleukin-10 (IL10) can uncover DTH responses to donor alloantigens in cardiac allograft acceptor mice. The DTH responses uncovered with TGFbeta-reactive antibodies can be blocked by exogenous IL10, and those uncovered with IL10-reactive antibodies can be blocked by exogenous TGFbeta. These data demonstrate that allograft acceptor mice are fully allosensitized, and poised to make donor-reactive cell-mediated immune responses. However, such responses are subverted by a donor alloantigen-dependent mechanism that involves TGFbeta and IL10, which in turn interfere with local cell-mediated immune responses.

Costimulation pathways in host immune responses to allogeneic hepatocytes.

BACKGROUND: This study investigated the role of the CD28/B7 (blocked by CTLA4Ig) and CD40/CD40L (blocked by MR1) costimulation pathways on in vivo host T-cell- mediated immune responses to allogeneic hepatocytes. METHODS: Survival of allogeneic hepatocytes (H-2q) in C57BL/6 (H-2b) mice untreated or treated with MR1, CTLA4Ig, L6 (control fusion protein), or a combination of MR1 and CTLA4Ig fusion protein was determined. RESULTS: Median survival time for hepatocellular allografts was 10, 84, 10, 10, and 84 days in untreated (n= 10), MR1-treated (n=7) (P<.0001), CTLA4Ig-treated (n=7) (P=0.02), L6-treated (n=3) (P, not significant), and the combination of MR1- and CTLA4Ig-treated (n=6) (P=0.0003) groups, respectively. CONCLUSIONS: Host treatment with MR1, but not CTLA4Ig, prolonged hepatocellular allograft survival. These data suggest that CD28/B7 interactions appear relatively unimportant, whereas CD40/CD40L interactions provide critical costimulator signals for T-cell-dependent immune responses to allogeneic hepatocytes.

Assessment of immunocompetence by limiting dilution analysis in long-term T cell depletion chimeras transplanted across the MHC barrier.

Chimeras were generated in a system in which donor C57BL/6 bone marrow plus spleen cells were T-cell-depleted prior to transplantation into lethally irradiated DBA/2 recipients. This protocol permits donor lymphohematopoietic engraftment and protects transplanted mice from development of lethal GVHD. The frequencies of alloantigen-specific cytotoxic T cells (CTL) and/or CTL precursors (CTL-P) in the chimera spleens were determined by limiting dilution analysis. This identified a small population of host-reactive CTL-P. The presence of host-reactive CTL-P in the absence of detectable anti-host immune response raises questions concerning the maintenance of the tolerant state in chimeras. Using mixtures of chimera and normal C57BL/6 splenocytes we found no evidence by limiting-dilution analysis for regulatory cells capable of dampening antihost immune reactivity in chimera spleens. We next measured the frequency of third-party-reactive CTL-P in chimeras. Chimeras displayed low CTL-P frequency by the 30th day posttransplant, which increased 15-21-fold over a five-month interval. Interestingly, both chimeric and irradiated syngeneic reconstituted control mice recovered anti-third-party CTL-P at a similar rate, but CTL-P levels never reached those measured in normal unirradiated control mice, suggesting that the radiation regimen has a long-lasting influence on host immunocompetence. In concomitant experiments we measured third-party CTL generation in MLC. Our findings suggest that measurement of CTL generation in MLC may be a less sensitive assessment of immunocompetence than LDA analysis. Our data also suggest that irradiated T-cell-depleted chimeras may suffer prolonged immunologic deficiencies based on reduced frequencies of alloreactive CTL-P.