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The microvascular endothelia of rejecting DBA/2----C57B1/6 murine cardiac allografts develop reactivity with the mAb M/K-2, which recognizes the murine homologue of the human leukocyte adhesion molecule VCAM-1. This reactivity does not develop in DBA/2----DBA/2 cardiac isografts or normal DBA/2 cardiac tissues. To determine whether endothelial VCAM-1 plays a role in allograft inflammation, cardiac allograft recipients were treated with M/K-2 antibody and monitored for leukocytic graft infiltration and graft survival. Treatment of the graft recipients with 200 micrograms/day M/K-2 Ig prolonged graft survival by 5 to 6 days (statistically significant); whereas treatment with 100 micrograms M/K-2 Ig every other day after transplant did not influence graft survival. Neither treatment interfered with leukocytic infiltration, as detected histologically or by limiting dilution analysis. The high dose treatment, but not the low dose treatment, resulted in high circulating levels of M/K-2, as detected by serum ELISA, and prominent antibody deposition on the graft vascular endothelia, as demonstrated by immunohistologic analysis. These data demonstrate that VCAM-1 is uniquely expressed on the vascular endothelia of rejecting murine cardiac allografts, and that a mAb to VCAM-1 can interfere with the allograft rejection process. Although this antibody can interfere with lymphocyte-endothelial adhesion in vitro, it has little effect on leukocytic infiltration in rejecting allografts suggesting that this process does not depend exclusively on VCAM-1 expression.

To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.

We have developed modified limiting dilution analysis (LDA) techniques that distinguish in vivo Ag-stimulated murine helper T lymphocytes (HTL) and CTL from unstimulated precursor T cells, even those with the same Ag specificity. We refer to these cells that are detectable in the modified LDA as "Ag-conditioned" T cells (cHTL and cCTL). We have used the modified LDA techniques in conjunction with conventional LDA techniques (which enumerate all Ag-specific T cells) to evaluate the in vivo distribution of Ag-conditioned cHTL and cCTL following in vivo sensitization to alloantigens via sponge matrix or skin allografts. In general, we observed the following regarding the distribution of cHTL and cCTL: 1) Ag-conditioned HTL and CTL were detectable only after in vivo sensitization with alloantigen: 2) not all Ag-reactive T cells became conditioned T cells after in vivo Ag deposition; 3) the percentage of Ag-reactive T cells that converted to conditioned T cells after Ag deposition varied among different lymphoid compartments; 4) a high percentage of cHTL, but a low percentage of cCTL, accumulated in regional lymph nodes and spleen; 5) cHTL accumulated in peripheral blood, whereas cCTL did not; 6) Ag-conditioned cHTL were detectable in various lymphoid tissues for greater than 60 days following Ag deposition, whereas cCTL were detectable for only 14 to 20 days; and 7) unlike the other lymphoid sites, the site of Ag deposition accumulated a high percentage of both Ag-stimulated cHTL and cCTL. Furthermore, cHTL and cCTL appeared to reside in phenotypically distinct T cell subsets in that in vivo treatment with anti-L3T4 mAb abrogated the accumulation of HTL, but not CTL, at the site of Ag deposition. These data demonstrate differential compartmentalization of Ag-conditioned cHTL and cCTL subsequent to in vivo Ag deposition. The implications of these findings regarding the monitoring of in vivo immune responses are discussed.

Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective in the prevention/control of acute and chronic rejection in animal models and currently in phase I clinical trials in human transplant recipients. This agent is also effective in the control of graft-versus-host disease, autoimmune reactions, and the growth of certain tumors. The importance of the endothelium in these disease processes led us to hypothesize that leflunomide might act directly upon the endothelial cell (EC).Assay of human EC colony formation demonstrated dose-dependent, leflunomide-mediated inhibition of EC proliferation. In addition, the organization of EC into capillary-like networks, which occurs during 18 hr of incubation on Matrigel, was progressively disrupted with increasing concentrations of leflunomide. Finally, fibrin-embedded transverse sections of murine aorta, which sprout numerous microvessels during an 11-day incubation, were inhibited from doing so in the presence of this agent. All drug concentrations used in these experiments were nontoxic and pharmacologically relevant, and none of these effects were reversible by exogenous uridine, implying that inhibition of these processes was not due to intracellular pyrimidine depletion. Furthermore, neither cyclosporine nor tacrolimus exerted inhibitory activity in any of the experiments described above.Data generated by these studies distinguish leflunomide among immunosuppressants as uniquely capable of inhibiting angiogenesis-related endothelial functions and suggest additional mechanisms by which this agent might intervene in the diverse array of disease processes against which it has shown therapeutic potential.

The purpose of the current study was to investigate the capacity of CD4+, CD8+, or non-T cells to independently initiate acute rejection of allogeneic hepatocytes using reconstituted SCID, CD4 or CD8 knockout (KO) recipient mice.Allogeneic hepatocytes (FVB/N, H-2q) were transplanted into C57BL/6.SCID (H-2b), CD4 KO (H-2b), CD8 KO (H-2b), or beige/beige (H-2b) mice. SCID mice with functioning hepatocellular allografts subsequently received purified non-T cells (NTC), CD4+, or CD8+ splenocytes. Some mice were treated with anti-CD4, anti-CD8, and/or anti-nkl.1 mAb. Recipient mice were also assessed for donor-reactive delayed-type hypersensitivity (DTH) responses and donor-reactive alloantibody production.Median hepatocellular allograft survival time (MST) was 28 days in CD4+ reconstituted SCID mice and 14 days in CD8+ reconstituted SCID mice. SCID hosts reconstituted with NTC demonstrated indefinite hepatocellular allograft survival (120 days). MST was 10 days in untreated beige/beige (NK cell deficient) mice. MST was 14 days in untreated, 35 days in anti-CD4 mAb treated, and 10 days in anti-nkl.1 mAb treated CD8 KO mice. MST was 10 days in untreated, 35 days in anti-CD8 mAb treated, and 7 days in anti-nk1.1 mAb treated CD4 KO mice. Donor-reactive DTH responses were not detected in reconstituted SCID mice, were minimal in CD4 KO mice, and were prominent in CD8 KO mice after rejection of allogeneic hepatocytes. Similarly, donor-reactive alloantibody, was not detected in CD4 KO hosts, but was readily detected in CD8 KO hosts.These studies show that both CD4+ and CD8+ T cells (but not host NTC) can independently initiate the rejection of allogeneic hepatocytes. While hepatocyte rejection by isolated CD4+ T cells is not surprising, rejection by CD8+ T cells (in the absence of CD4+ T cells) was unusual, and may explain the failure of "standard" immunosuppressive regimens to suppress acute rejection of allogeneic hepatocytes, as noted in prior studies. Furthermore, NK cells do not appear to be required for either CD4+ T cell or CD8+ T cell initiated hepatocyte rejection.

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.

This is the first in a series of reports that characterizes immune responses evoked by allogeneic hepatocytes using a functional model of hepatocyte transplantation in mice."Donor" hepatocytes expressing the transgene human alpha-1-antitrypsin (hA1AT-FVB/N, H2q) were transplanted into C57BL/6 (H2b) or MHC II knockout (H2b) hosts treated with anti-CD4, anti-CD8, or a combination of anti-CD4 and anti-CD8 monoclonal antibodies (mAbs). Hepatocyte rejection was determined as a loss of circulating ELISA-detectable transgene product (hA1AT). In addition, some C57BL/6 mice underwent transplantation with FVB/N heterotopic cardiac allografts and were treated with anti-CD4 mAb. Cardiac allograft rejection was determined by palpation. Graft recipients were tested for donor-reactive alloantibodies and donor-reactive delayed-type hypersensitivity (DTH) responses.The median survival time (MST) of allogeneic hepatocytes in normal C57BL/6 mice was 10 days (no treatment), 10 days (anti-CD4 mAb), 14 days (anti-CD8 mAb), and 35 days (anti-CD4 and anti-CD8 mAbs). The MST of hepatocytes in B6 MHC class II knockout mice was 10 days (no treatment) and 21 days (anti-CD8 mAb). The MST of cardiac allografts was 11 days (no treatment) and100 days (anti-CD4 mAb). Donor-reactive DTH responses were readily detected in both untreated and mAb-treated recipients. Donor-reactive alloantibody was barely detectable in untreated hosts.These studies demonstrate that allogeneic hepatocytes are highly immunogenic and stimulate strong cell-mediated immune responses by both CD4+ and CD8+ T cells, even when treated with agents that can cause acceptance of cardiac allografts. Indeed, CD4+ or CD8+ T cells seem to independently cause hepatocellular allograft rejection. Allogeneic hepatocytes evoked strong donor-reactive DTH responses but were poor stimuli for donor-reactive antibody production. This is an unusual pattern of immune reactivity in allograft recipients.

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.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.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.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.

In addition to transplant-associated vascular sclerosis, cardiac allografts also may be vulnerable to a previously unrecognized aspect of remodeling involving reactivation of fetal structural genes in the adult heart.Vascular smooth muscle (VSM) alpha-actin is encoded by a gene that normally is repressed in the ventricle during late gestation. Immunohistochemical analysis of accepted mouse cardiac allografts was performed to determine whether this fetal actin was reexpressed after transplant.VSM alpha-actin was detected within 30 days after transplant throughout the allograft myocardium, where it frequently exhibited a distinct periodicity suggestive of protein localization in sarcomeres. By 90 days after transplant, VSM alpha-actin filaments specifically accumulated in the left ventricular endocardium. Donor hearts and isografts did not express myocardial VSM alpha-actin, indicating that fetal gene activation was linked to chronic rejection.The results indicate that chronic rejection is associated with fetal muscle gene activation, which may facilitate parenchymal cell remodeling and impair graft function.

The development of long-term allograft survival and understanding the mechanism(s) by which it is induced are major goals of experimental transplantation. Studies by several different investigators have provided conflicting evidence for the role of interleukin (IL)-4 in the process of allograft rejection or long-term allograft survival. These studies examine the role of IL-4 in experimental cardiac allograft rejection and in inducing long-term allograft survival. A possible mechanism for long-term allograft maintenance via alternative allosensitization is discussed.Adult IL-4-intact or IL-4-deficient (knockout, KO) C57BL/6 (B6) mice were transplanted with heterotopic DBA/2 cardiac allografts and immunosuppressed either with gallium nitrate (GN), or the anti-CD4 monoclonal antibody, GK1.5. Cellular allosensitization was assessed by testing the allograft recipients for donor-reactive delayed-type hypersensitivity (DTH) responses. The presence of antigen-driven suppressive mechanisms was assessed using a linked unresponsiveness (bystander suppression) DTH assay.In general, the results were the same with either GN or GK1.5. We observed that (1) IL-4 is not required for acute allograft rejection or allogeneic DTH responses in nonsuppressed mice, (2) IL-4 is required for long-term allograft survival in immunosuppressed mice, (3) immunosuppression creates a requirement for IL-4 in major histocompatibility complex self-restricted, but not allorestricted, DTH responses, and (4) the development of alloantigen-dependent linked DTH unresponsiveness (bystander suppression) in allograft recipients requires IL-4.In summary, these studies demonstrate a common requirement for IL-4 during the development of long-term allograft survival and the concurrent development of alloantigen-dependent DTH down-regulation in cardiac allograft recipients after immunosuppression.

The immunologic characteristics of experimental allograft acceptance remain ill-defined. This study evaluates humoral and cell-mediated immunity in transiently immunosuppressed mice that have accepted cardiac allografts.DBA/2--C57BL/6 heterotopic cardiac allograft recipients were immunosuppressed with either GK1.5 monoclonal antibody or gallium nitrate and monitored for donor-reactive delayed-type hypersensitivity (DTH) assessed by ear challenge and for alloantibody production detected by flow cytometry.Cardiac allograft function continued for90 days in approximately 50% of GK1.5-treated and 97% of gallium nitrate-treated transplant recipients. All nonsuppressed recipients lost graft function within 7 to 10 days. Among mice that accepted allografts, donor-reactive IgG was produced by about 50% of GK1.5 monoclonal antibody-treated mice and 80% of gallium nitrate-treated mice. None of the these mice exhibited donor-reactive DTH responses, and all could down-regulate third-party DTH responses in a donor alloantigen-dependent manner. This down-regulation is not found in nonsuppressed allograft recipients or in naive mice. Importantly, transfer into SCID mice of splenocytes from mice that accepted allografts, but not naive splenocytes, provided them with a similar ability to accept cardiac allografts, even if the grafts co-expressed third-party alloantigens.IgG alloantibody production by murine cardiac allograft recipients is not a precise indicator of allosensitization leading to either cardiac allograft rejection or acceptance. However, expression of alloreactive DTH is a reliable indicator of allosensitization leading to acute rejection, and the absence of DTH in association with active DTH down-regulatory mechanisms is a reliable indicator of allograft acceptance in this experimental model. Thus, DTH analysis may hold more promise than alloantibody detection for clinical assessment of posttransplant immune status.

It has been reported previously that liver grafts and liver cells seem to be tolerogenic, based on the high frequency of spontaneous tolerance after orthotopic liver transplantation in rodents and on the phenomenon of portal venous tolerance in other models. The purpose of the current study was to characterize in vivo immune responses to allogeneic hepatocytes transplanted into the portal circulation.In this functional model of hepatocyte transplantation, "donor" hepatocytes from mice transgenic for human alpha1-antitrypsin (hA1AT) were transplanted by intrasplenic injection into host mice and the secreted hA1AT protein measured in host serum to determine hepatocellular graft survival. Host immune responses were assessed by measurement of donor-specific alloantibodies and delayed-type hypersensitivity responses. In some experiments, liver nonparenchymal cells (NPCs) were co-transplanted with the allogeneic hepatocyte transplant.Allogeneic hepatocyte transplant into immunocompetent hosts resulted in loss of host serum hA1AT by days 7-10 after transplant, whereas syngeneic hosts maintained long-term hepatocellular graft survival as reflected by persistence of serum hA1AT for20 weeks. Allogeneic hepatocyte transplantation resulted in the development of donor-specific alloantibody and delayed-type hypersensitivity responses, as well as a "second set" response of accelerated hepatocellular graft rejection after a second transplant. Pretransplantation or co-transplantation of donor-matched liver NPCs at the time of allogeneic hepatocyte transplantation did not prolong hepatocellular allograft survival.Allogeneic hepatocytes introduced into the portal circulation via intrasplenic injection are immunogenic not tolerogenic and stimulate a weak humoral and strong cell mediated host immune response in vivo. Co-transplantation or pretransplantation of allogeneic liver NPCs did not protect allogeneic hepatocytes from immunologic rejection.

Previous studies to determine the significant donor and host factors which contribute to immune responses to allogeneic hepatocytes have been pursued with in vitro and in vivo studies. However, in order to further characterize in vivo host immune responses to transplanted hepatocytes in conjunction with their consequences upon allograft survival, a functional model of hepatocyte transplantation in which the function and survival of transplanted hepatocytes could be serially assessed was pursued.A transgenic mouse line expressing the human alpha1-antitrypsin (hA1AT) gene was developed in an FVB/N (H2q) mouse (hA1AT-FVB/N). Hepatocytes from these "donor" transgenic mice were transplanted into host mice by intrasplenic injection, and subsequent survival of the transgenic hepatocytes was determined by assay for the secreted hA1AT protein in host serum by enzyme-linked immunosorbent assay.Transplantation of transgenic "donor" hepatocytes (hA1AT-FVB/N) into (a) syngeneic FVB/N (H2q), (b) allogeneic immunoincompetent C57BL/6.SCID (H2b), or (c) allogeneic T cell-deficient (outbred) hosts resulted in long-term survival (16 weeks) of hepatocytes. Transplantation of allogeneic hepatocytes (hA1AT-FVB/N, H2q) into C57BL/6 (H2b), C3H (H2k), or BALB/c (H2d) hosts resulted in loss of hA1AT in host serum by day 10 after transplant. Likewise, transplantation of allogeneic hepatocytes into hosts deficient in B cells, CD8+ T cells, or CD4+ T cells resulted in loss of hA1AT by day 10 after transplant.This functional model of hepatocyte transplantation is validated for the study of host immune responses to hepatocellular grafts and to assess efficacy of strategies designed to alter these in vivo immune responses. The immunologic rejection of allogeneic hepatocytes appears to be T cell-mediated.

The practice of clinical and experimental transplantation continues to evolve at a rapid pace. To appreciate the current transplant practices, it is first necessary to review transplant immunology in its proper context, ie, as a component of the complex series of events that promote the repair of damaged tissues. These processes are generally categorized as inflammation, immunity, and tissue repair/reinforcement. In general, there are 3 forms of graft rejection: hyperacute, acute, and chronic rejection. All 3 forms of graft rejection represent pathologic consequences of one or more of these repair-related processes. The various graft rejection responses also illustrate several complex immunologic principles that need to be considered. These include the definition of an alloantigen, the structure and function of major histocompatibility complex molecules, and the behavior of antigen-presenting cells and alloreactive T cells. This review combines these concepts and principles into a discussion of the 3 forms of graft rejection, each of which is addressed at the level of histopathology, pathobiology, incidence, and clinical strategies.

This study examined the relationship between transplant vascular sclerosis (TVS) and tissue fibrosis, features of chronic rejection that can develop rapidly in accepted heterotopic murine cardiac allografts.The rate of development of interstitial fibrosis or TVS development was determined by computerized analysis of tissue sections from DBA/2--C57BL/6 heterotopic cardiac allografts after immunosuppression with gallium nitrate.In accepted cardiac allografts, neointimal fibrosis developed by 30 days after transplant, whereas TVS was minimal by day 30, and maximal by day 60. Variable levels of fibrosis were found throughout the allografts. DBA/2--DBA/2 cardiac isografts never displayed TVS in this time period, but displayed allograft-like fibrosis within 60 days of transplantation.Interstitial fibrosis can be dissociated from the TVS development in this experimental model of chronic cardiac allograft rejection. Apparently, it is caused, at least in part, by alloantigen-independent factors other than TVS-related tissue ischemia.

We treated C57BL/6 mouse recipients of DBA/2 cardiac allografts with anti-CD4 monoclonal antibodies (mAb) or anti-vascular cell adhesion molecule 1 mAb to promote long-term allograft survival and subjected both the recipient animals and the long-surviving allografts to a battery of histologic and immunologic tests. The results were similar regardless of the mAb used for antirejection therapy. At all tested times after transplantation, the allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcription polymerase chain reaction analyses revealed continuous intragraft expression of messenger RNA for interleukin 1, interleukin 2, interleukin 4, interleukin 6, tumor necrosis factor, interferon gamma, and transforming growth factor beta. All grafts had histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and vascular neointimal hyperplasia. The graft recipients retained limiting dilution analysis--detectable, donor-reactive cytolytic T lymphocyte, and helper T lymphocyte in their spleens and produced high liters of donor-reactive alloantibodies. Variable amounts of allogeneic microchimerism were detectable in some, but not all of the long-surviving graft recipients. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 mAb or anti-vascular cell adhesion molecule-1 mAb was used for antirejection therapy, in spite of the major differences in lineage and distribution of cells targeted by these two mAbs, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes very similar to those observed during acute allograft rejection, and (3) in spite of these processes the allografts continue to function, although they invariably develop a chronic rejection-like histopathologic condition that may ultimately limit graft function. In this regard, the recipients of long-surviving allografts do not seem to be tolerant of their graft alloantigens.

Transplant vascular sclerosis (TVS) is manifested in transplanted human and murine hearts as a concentric, intimal lesion. The purpose of this study was to characterize the rate, location, and intensity of developing TVS lesions in murine cardiac allografts using quantitative morphometric analysis.Murine cardiac allografts, treated with the immunosuppressant gallium nitrate, were explanted at 30, 60, and 90 days after transplant. The grafts were histologically stained and evaluated for intimal thickening by deriving a neointimal index (NI) using a computerized image-analysis system.In cardiac allografts, mild vascular lesions of varying NI were detectable by day 30 and lesion severity increased significantly by day 60. Thereafter, average lesion severity stabilized, although the percentage of affected vessels continued to increase from day 30 to day 90. In contrast, day-90 cardiac isografts showed little to no TVS development. Vascular lesions developed randomly without regard for vessel location or size. TVS developed more regularly in vessels of the interventricular septum than in the right or left ventricular walls. The degree of TVS development fluctuated along the length of individual vessels, even as late as 90 days after transplant. The smaller vessels (85 microm in diameter) appeared to occlude more quickly than the larger vessels.TVS developed reproducibly in a random pattern throughout cardiac allografts over a 1-month to 3-month period after transplant. This development can be quantitatively monitored by computerized morphometric analysis. In general, under these experimental conditions, 30-day cardiac allografts seem to provide a useful experimental model for studying early aspects of TVS, whereas 60-day allografts may be better suited for analysis of advanced TVS.

Chemokines are cytokines with chemoattractant properties for leukocytes. They may play a critical role in directing leukocytes to graft sites and in amplifying intragraft inflammation during rejection. Previous studies have tested the intragraft expression of chemokine genes during the rejection of allogeneic skin grafts in mice. In the current study, we used a heterotopic heart transplant model in mice to test the intragraft expression of these genes in nonrejecting cardiac isografts, rejecting cardiac allografts, and cardiac allografts that were accepted due to immunosuppression with gallium nitrate. With the exception of low levels of interleukin-1beta and JE, intragraft expression of the the proinflammatory cytokine genes was not observed in either isografts or native heart. Two distinct patterns of chemokine mRNA were observed in the rejecting cardiac allografts. Intra-allograft expression of interleukin-1beta, interferon-gamma-inducible protein, JE, and KC was prominent by day 3 after transplantation. The expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated upon activation, normal T cell expressed and secreted (RANTES) was at low or undetectable levels at day 3 after transplantation but at high levels by day 8 after transplantation. Sixty days after transplantation, intra-allograft expression of chemokines in hearts from gallium nitrate-treated recipients indicated low levels of MIP-1alpha, MIP-1beta, and KC but high levels of interferon-gamma-inducible protein and RANTES.

We retrospectively compared the clinical and financial impact of a final cross-match by T cell flow cytometry (FXM) versus conventional complement-dependent cytotoxicity (CXM) in consecutive primary cadaveric kidney (K) and primary simultaneous cadaveric pancreas-kidney (SPK) transplant recipients. Mean follow-up was 14 months for both the K (range, 5-22 months) and SPK (range, 5-22 months) recipients. There were no instances of a positive CXM result if the FXM result was negative. However, 18 of the 102 (18%) K recipients and 11 of the 66 (17%) SPK recipients were FXM positive, CXM negative, but no grafts lost to hyperacute rejection in this group. In addition, patient survival, graft survival, incidence of acute rejection, and kidney and pancreas function (immediate and late) were not different in the FXM-positive versus the FXM-negative groups. Charges for the CXM and FXM methods were compared over a 6-month period. During that period, the FXM charges averaged $583 less per recipient than the CXM charges (58% reduction in charges), and the time required to perform the FXM method was 50% of that required for the CXM method. These results demonstrate that a clinical pathway for primary transplantation that utilizes the FXM rather than the CXM final cross-match is clinically safe, with no adverse effect on posttransplant outcome, reduces organ preservation time by shortening the waiting period for the final cross-match results, and significantly reduces the tissue typing charges. However, about 9% of all primary K and SPK recipients will be FXM positive, CXM negative on final cross-match and will be unnecessarily denied a transplant. In this study, we describe a method to identify these patients so that they can be tested by traditional CXM to avoid being denied access to donor organs.

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.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.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.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.

Transplant vascular sclerosis is expressed in transplanted human and murine hearts as a concentric intimal thickening. The purpose of this study was to characterize the location, distribution, and intensity of transplant vascular sclerosis in murine cardiac allografts using computerized morphometric analysis.Murine cardiac allograft recipients were treated with the immunosuppressant gallium nitrate to promote graft survival. The grafts were removed at 60 days after transplantation and histologically stained. The coronary arteries were analyzed for intimal thickening using a neointimal index (NI) derived with a computer imaging system.A cross-section taken from the middle of a cardiac allograft showed four major coronary arteries, each with widely different NI values (65, 0, 92, and 0). The same four vessels in two other grafts also showed highly variable NI values, but different patterns of vessel involvement. Next, NI values were determined along the length of a single vessel from aorta to apex. This revealed variable, fluctuating intimal thickening along the length of the vessel. In general, arteries from the aortic versus apical regions of the grafted hearts expressed similar amounts of intimal thickening (analysis of variance, P=0.4826). Finally, a method was devised to quantitate intimal thickening from a sampling of three tissue cross-sections taken from the middle of each cardiac allograft. This value was statistically indistinguishable from values obtained by analysis of intimal thickening in multiple sections covering the entire heart (P=0.6734, 0.9021, and 0.1474).Intimal thickening in the coronary arteries of murine cardiac allografts appears to be variable in terms of location, distribution, and intensity. This is true for different regions of the same vessel, different vessels in the same heart region, and the same vessels in different cardiac allografts.

Cytolytic T cells were generated in vitro by culturing purified Balb/c CD4+ T cells with irradiated C57Bl/6 (B6) splenocytes plus anti-IL-4 mAb. Matched, noncytotoxic T cells were similarly generated by culturing purified Balb/c CD4+ T cells with irradiated B6 splenocytes plus recombinant murine IL-4. The latter T cells displayed to cytolytic activity, even in lectin-mediated lysis assays, but produced characteristic cytokines upon contact with specific alloantigens. Transfusion of cytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts resulted in acute allograft rejection within 5 to 10 days. Transfusion of noncytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts also resulted in acute allograft rejection within 7 to 10 days. Limiting dilution analysis (LDA) of infiltrating cells recovered from rejected allografts after collagenase digestion demonstrated that the CD4+ T cells retained their cytolytic or noncytolytic functional phenotypes in vivo throughout the rejection process. These data demonstrate that isolated CD4+ T cell populations can promote rapid acute cardiac allograft rejection, and that cytolytic activity is not necessary for this acute rejection response.

It has been hypothesized that TH1 cells mediate the archetypical cell-mediated immune response of acute allograft rejection, whereas TH2 cells promote allograft acceptance. To test this, we transfused SCID cardiac allograft recipients with polarized TH1-like or TH2-like graft-reactive T cells, and monitored graft function and graft-reactive immune responses in the graft recipients. Polarized THl-like cells, which were generated in vitro by stimulating syngeneic splenocytes with donor alloantigens in the presence of anti-IL-4 mAb, produced IFNg but not IL-4 when restimulated with donor alloantigen. Polarized TH2-like populations, which are generated in vitro by stimulating syngeneic splenocytes with donor alloantigens in the presence of IL-4, produced IL-4 but not IFNg when restimulated with donor alloantigen. Interestingly, bioassays of culture SN from restimulated TH1 but not TH2 cells revealed IL-2 production, although LDA analyses revealed that the TH1 and TH2 cells had identical frequencies of IL-2-producing cells. Transfusion of THl-like cells into SCID cardiac allograft recipients resulted in acute rejection within 7-10 days that was characterized by cellular infiltration, myocyte necrosis, and edema. Graft-infiltrating cells (GIC) recovered from TH1-transfused animals contained large numbers of graft-reactive IL-2-producing cells (68-269/10(6) GIC), but no LDA-detectable IL-4-producing cells. These data support the hypothesis that donor-reactive TH1 cells can promote acute allograft rejection. In contrast to the hypothesis, transfusion of the polarized TH2-like population into SCID cardiac allograft recipients also resulted in histologically similar acute rejection within 7-10 days. Infiltrating cells recovered from TH1-transfused allografts contained large numbers of graft-reactive (109-1458/10(6) GIC), LDA-detectable, IL-4-producing cells--indicating that the TH2 cells had arrived at the graft-but promoted acute allograft rejection rather than allograft acceptance.

Gallium nitrate (GN) was evaluated for its ability to interfere with a cute rejection of DBA/2--C57BL/6 heterotopic cardiac allografts, in comparison with the depleting anti-CD4 mAb, GK1.5. The administration of GN for 30 days (s.c. 30 mg/kg elemental gallium on days 0 and 3, 10 mg/kg every third day) resulted in60-day graft survival in 78% (25 of 32) of the graft recipients, whereas 2 perioperative injections of anti-CD4 monoclonal antibody (mAb) resulted in60-day graft survival in 58% (24 of 41) of the graft recipients. Serum gallium levels peaked at about 2000 ng/ml after 2-3 weeks of treatment and decreased to about 300 ng/ml by day 60, a level that was maintained for at least 30 more days. During the early posttransplant period, 25% of GN-treated grafts, but not anti-CD4 mAb-treated grafts, exhibited an unusual, transient reduction in graft impulse strength, suggesting a transient rejection response. Macroscopically, the long-surviving (60 days) grafts from either treatment group exhibited none of the features of rejecting allografts. Histologically, they exhibited minor edema and rare epicardial inflammation but no tissue necrosis. However, there were vascular changes in allografts from GN-treated mice, including altered endothelial morphology, associated with moderate intimal hyperplasia and mild perivascular leukocytic infiltration. Allografts from anti-CD4 mAb-treated mice exhibited prominent neointimal hyperplasia associated with endothelial morphologic changes and prominent vascular and perivascular leukocytic infiltration. In general, both GN and anti-CD4 mAb promoted long-term allograft survival, but these allografts displayed the histopathologic signs of ongoing inflammation and chronic allograft rejection.

We have observed that most C57BL/6 (H-2b) recipients of DBA/2 (H-2d) heterotopic cardiac allografts retain their grafts for more than 60 days following treatment with anti-CD4 MAb (GK1.5), anti-VCAM-1 MAb (M/K-2), or gallium nitrate (GN). Nevertheless, many ongoing alloimmune responses are detectable more than 60 d post-transplant in these mice: (i) continuous inflammatory endothelial activation and low-grade cellular infiltration in the graft, (ii) modest frequencies of donor-reactive T cells in the graft and spleen, (iii) mRNA for various cytokines (IL-1, IL-2, IL-4, IL-6, IFN gamma, TNF alpha, TGF beta) in the graft, (iv) high levels of donor-reactive alloantibody in the circulation, (v) variable development of allogeneic chimerism in the recipient, and (vi) development of interstitial fibrosis and neointimal hyperplasia in the graft. Despite the widely differing targets of GK1.5, M/K-2, and GN, their therapeutic outcomes appear to be identical. Taken together, these observations suggest that long-term allograft survival in these experimental systems is not the result of immune acceptance, i.e. allogeneic tolerance. Although these therapies interrupt the destructive progression of acute rejection, they permit many related immune responses in the graft recipient in association with the development of chronic rejection-like histopathology.

This report provides evidence to support the hypothesis that tumor necrosis factor-alpha (TNF-alpha) and IL-4 promote the expression of new endothelial surface molecules in rejecting murine heterotopic cardiac allografts. The microvascular endothelia of these cardiac allografts all develop strong reactivity with the monoclonal antibodies (mAbs) YN1.1/74 (anti-ICAM-1), M/K-2 (anti-VCAM-1) and MECA-32 (undefined molecule) within 3 to 5 days of graft implantation. Daily treatment of the allograft recipients with pentoxifylline (PTX), soluble TNF receptor (TNFR:Fc), anti-interleukin-4 (IL-4) mAb (11B11), or soluble IL-4 receptor, each abrogate the expression of endothelial VCAM-1 and reduce the endothelial reactivity with the mAbs YN1.1/74 and MECA-32 to levels found in cardiac isografts. This is accompanied by a reduction, but not an elimination, of interstitial leukocytic infiltration. Despite this, cardiac allograft recipients treated with PTX or the mAb 11B11 rejected allografts at the same rate as untreated allograft recipients, ie, within 10 to 12 days after graft implantation. These rejected grafts contained mRNAs for TNF-alpha and IL-4, as well as for all other cytokines that have been associated with rejecting allografts. This suggests that endothelial VCAM-1 expression, which is characteristic of rejection, is not an essential element of the rejection process. Interestingly, the grafts from the PTX-treated recipients continued to display rare, isolated VCAM-1 positive cells in the interstitium, which may be dendritic cells. In general, these studies demonstrate a role for IL-4 and TNF-alpha in the alterations of vascular endothelial phenotype observed in rejecting cardiac allografts. They also demonstrate that endothelial VCAM-1 expression is not essential for the allograft rejection process, and that the role of VCAM-1 in this process may be more subtle than was initially suspected.

The immunosuppressive agents used clinically to prevent allograft rejection exert their effects by interfering with antigen-dependent T cell activation, endothelial cell function, or both. Gallium nitrate (GN) is immunosuppressive both in vitro and in vivo, and has potential for clinical use in transplant recipients. Therefore, we analyzed the influence of GN on gonadal vein endothelial cell (GVEC) and T cell activation. GVEC were stimulated with IFN gamma or TNF alpha in the presence or absence of GN, and tested for changes in levels of MHC class I, MHC class II, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression. GN did not interfere with the baseline or cytokine-enhanced expression of these molecules. Rather, it increased the expression of intercellular adhesion molecule-1 on GVEC, and this effect was further augmented in the presence of IFN gamma. In contrast, GN inhibited T cell proliferation stimulated by allogeneic GVEC or allogeneic monocytes in a dose-dependent manner. In transwell experiments, GN blocked the induction of MHC class II expression on isolated GVEC caused by alloactivated T cells, but not by recombinant IFN gamma. This suggests that GN can interfere indirectly with inflammatory responses of endothelial cells by interfering with local T cell activation and lymphokine production. Once lymphokines are produced, GN does not interfere with their effects on endothelial cells. GN is thought to act through transferrin receptors, but GVEC, unlike T cells, do not increase their expression of transferrin receptors, after stimulation with cytokines. This may explain their relative lack of sensitivity to GN. In general, GN appears to stimulate endothelial cells but suppress T cells. This paradoxic effect suggests that therapy with GN may enhance T cell-independent inflammatory responses, such as cellular infiltration and repair of tissue damage, while suppressing T cell-dependent responses, such as T cell-mediated tissue destruction and allograft rejection.

We have investigated the contribution of monocytes (Mo) to the activation of purified human T cells by allogeneic human vascular endothelial cells (HUVEC). We have previously demonstrated that allogeneic HUVEC stimulate IL-2 production by CD8+ helper T lymphocytes (HTL), but not CD4+ HTL, in the absence of accessory Mo. We now show that addition of responder-autologous Mo to such cultures stimulates a high frequency of CD4+ HTL (1/6500), but no additional CD8+ HTL (1/35,000), as detected by limiting dilution analysis (LDA). The CD4+ HTL production of IL-2 increased with increasing numbers of Mo. Monoclonal antibodies to MHC class II interfered with HTL responses to allogeneic HUVEC in the presence, but not in the absence of autologous Mo. In contrast, IFN-gamma-treated HUVEC stimulated a high frequency of CD4+ HTL in the absence of autologous Mo. However, deletion experiments revealed that the population of HTL responsive to IFN-gamma-treated HUVEC is distinct from the population that responds to HUVEC in the presence of autologous Mo. These data suggest that Mo promote IL-2 production by presenting HUVEC-derived alloantigens via MHC class II molecules to CD4+ HTL, rather than by providing cytokines that promote more efficient IL-2 production by CD8+ HTL, or by inducing MHC class II expression on the HUVEC. In general, these data demonstrate that autologous Mo can play a significant role in the response of T cells to allogeneic HUVEC. Further, they demonstrate that the activation of human HTL by allogeneic HUVEC is complex, and can occur by at least three pathways: (1) direct stimulation of a small number of CD8+ HTL, but no CD4+ HTL, by quiescent HUVEC, (2) direct stimulation of a large number of CD4+ HTL by IFN-gamma-treated HUVEC, and (3) indirect stimulation of a different subset of CD4+ HTL by HUVEC in the presence of autologous monocytes. These three pathways of alloactivation are not unique to allogeneic HUVEC, but this experimental system provides a convenient and relevant model with which each pathway can be easily and independently investigated.

We describe the recipient of a marrow graft from an HLA-serologically identical unrelated donor from whom highly potent host-reactive CTL of donor origin were isolated in association with acute GVHD. Extensive sequence and biochemical analysis of the HLA complex of this donor and recipient revealed several disparities in class I and class II HLA with the potential to be recognized by T cells from the donor or the host. The donor-derived CTL exclusively recognized a class I HLA difference associated with HLA-B44. Nucleotide sequencing of donor and recipient cells revealed that the patient possessed the HLA-B*4402 allele recognized by IEF as B44.2 while the donor possessed HLA-B*4403 (IEF variant B44.1). These alleles differ at one amino acid residue located at position 156 in the alpha 2 domain. The donor-derived CTL were shown to be specific for B44.2 by blocking studies and by the lysis of five different B44.2+ unrelated cell lines, two of which were confirmed by sequencing to be homozygous for B*4402. A host-specific difference involving a HLA-DRB1 allele was not recognized by the CTL, neither did HLA differences unique to the donor HLA-B*4403 and HLA-DQ8 elicit a host response. These data show that certain HLA disparities may be tolerated at the same time that other disparities elicit a potent immunologic response. The chemical nature of the difference, its structural impact, as well as the conditions of transplant appear to influence the type of response which occurs.

Most previous studies to evaluate endothelial cell-T lymphocyte interactions have used human peripheral blood as a source of T lymphocytes and human umbilical vein endothelial cells as a source of endothelia. Implicit in this experimental system are allogeneic lymphocyte-endothelial interactions, which are largely ignored. To overcome this problem, we isolated gonadal vein endothelial cells (GVEC) along with matched splenic macrophages and T lymphocytes from cadaveric donors, thus providing a completely autologous series of cells for experimentation. First, GVEC were analyzed for morphology, surface phenotype, and cytokine mRNA expression, and found to be indistinguishable from human umbilical vein endothelial cells. Using this system, we observed that irradiated GVEC were able to promote a 2- to 3-fold increase in the proliferation of matched autologous splenic T cells after PHA stimulation. This indicates that the costimulator activity of endothelial cells reported by others is an intrinsic property of endothelial cells, and is not a consequence of endothelial alloantigens. We also used this system to assess the relative abilities of GVEC and macrophages obtained from the same donor to stimulate the proliferation of purified allogeneic CD3+ PBL. We found the following hierarchy of alloantigenicity in this experimental system: splenic macrophagesIFN-gamma-treated GVECuntreated GVEC = TNF alpha-treated GVEC. These studies demonstrate that allogeneic macrophages are intrinsically more antigenic than endothelial cells derived from the same donor. Furthermore, they illustrate the utility of this experimental system to obtain data regarding lymphocyte-endothelial interactions that are otherwise unobtainable.

CMV has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system. Although endothelial HLA class II expression is a hallmark of vascular rejection, CMV does not directly induce these antigens on infected endothelial cells (EC) and, indeed, renders them refractory to HLA DR induction by IFN-gamma. Our earlier studies have demonstrated, however, that CMV-infected EC are capable of eliciting vigorous activation responses by allogeneic, CMV-seropositive donor-derived CD4+ T cells. We now show that T cells thus activated can induce HLA DR expression on noninfected EC. Human umbilical vein endothelial cells (HUVEC) were inoculated at low titer with CMV strain VHL/E, cocultured with allogeneic, CMV-seropositive or CMV-seronegative donor-derived CD4+ T cells, then dual immunohistochemically stained for CMV antigen and HLA DR, or assayed for HLA DR expression by fluorescence flow cytometry. Results demonstrated that HLA DR was induced in 20-70% of HUVEC in monolayers containing less than 0.5% CMV-infected EC after coculture with CMV-seropositive donor-derived T cells. No such induction was observed in experiments employing T cells isolated from CMV-seronegative individuals. Expression of this antigen was strictly limited to noninfected cells. Rare HLA DR induction was observed in virus-free cocultures. To determine whether endothelial HLA DR was induced by a soluble factor(s) elaborated by activated T cells, noninfected HUVEC monolayers were treated for 72 hr with cell-free supernatant from CMV-infected or noninfected HUVEC/T cell cocultures and assayed as above. Again, HLA DR expression was induced by supernatant from CMV-positive cocultures (only when cocultured T cells were isolated from CMV-seropositive donors), but not by supernatant from CMV-negative cocultures or from T cells cultured alone. The soluble factor was identified as IFN-gamma by the complete attenuation of HLA DR induction by anti-IFN-gamma mAb. These findings suggest that allograft endothelium harboring low level CMV may be capable of eliciting host CD4+ T cell activation, and that consequent release of IFN-gamma is capable of inducing endothelial HLA class II expression, as observed in vascular rejection and transplantation-associated arteriosclerosis. Results of these studies thus provide insight into mechanisms that may help elucidate the association between CMV and rejection-related immune events in the allograft.

We have employed a murine model of cardiac transplantation and two monoclonal antibodies, M/K-2 and MECA-32, to study the responses of graft endothelia during allograft rejection. Using immunohistologic techniques, we demonstrate that the monoclonal antibody M/K-2, which binds to the murine cellular adhesion molecule VCAM-1, reacts with an inducible endothelial epitope found in rejecting cardiac allografts, but not in cardiac isografts, normal cardiac tissues, or extracardiac vasculature from allografted mice. Similar, but focal, M/K-2 reactivity is also found in nontransplanted hearts undergoing virally induced myocarditis. M/K-2 reactivity does not develop in the nonrejecting cardiac allografts from nu/nu mice, and M/K-2 reactivity is found only in grafts that develop CD25+ graft-infiltrating cells--i.e., allografts but not isografts. PCR analyses of grafts during development of VCAM-1 expression indicate that allografts, but not isografts, contain mRNA for the cytokines IL-2 and IFN-gamma, and either of these cytokines may be associated with the expression of M/K-2 reactivity in rejecting allografts. Unlike M/K-2, MECA-32 identifies an inducible epitope that is observed on myocardial endothelia of both isografts and allografts, but not normal cardiac tissues. Further, expression of the MECA-32 epitope can occur in grafts that do not develop CD25+ infiltrating lymphocytes, since it is observed in isografts and the native hearts of transplanted or sham-operated mice. Indeed, MECA-32 reactivity may be T cell independent, since it is also found in nonrejecting allografts of nu/nu mice. PCR analyses of grafts during development of MECA-32 reactivity indicate that cardiac isografts contain mRNA for IL-1, IL-6, TNF, and lymphotoxin. One or more of these might be associated with induction of MECA-32 reactivity.

The microvascular endothelia of rejecting DBA/2----C57B1/6 murine cardiac allografts develop reactivity with the mAb M/K-2, which recognizes the murine homologue of the human leukocyte adhesion molecule VCAM-1. This reactivity does not develop in DBA/2----DBA/2 cardiac isografts or normal DBA/2 cardiac tissues. To determine whether endothelial VCAM-1 plays a role in allograft inflammation, cardiac allograft recipients were treated with M/K-2 antibody and monitored for leukocytic graft infiltration and graft survival. Treatment of the graft recipients with 200 micrograms/day M/K-2 Ig prolonged graft survival by 5 to 6 days (statistically significant); whereas treatment with 100 micrograms M/K-2 Ig every other day after transplant did not influence graft survival. Neither treatment interfered with leukocytic infiltration, as detected histologically or by limiting dilution analysis. The high dose treatment, but not the low dose treatment, resulted in high circulating levels of M/K-2, as detected by serum ELISA, and prominent antibody deposition on the graft vascular endothelia, as demonstrated by immunohistologic analysis. These data demonstrate that VCAM-1 is uniquely expressed on the vascular endothelia of rejecting murine cardiac allografts, and that a mAb to VCAM-1 can interfere with the allograft rejection process. Although this antibody can interfere with lymphocyte-endothelial adhesion in vitro, it has little effect on leukocytic infiltration in rejecting allografts suggesting that this process does not depend exclusively on VCAM-1 expression.

To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.

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.

In a one-stage, interleukin-2 (IL-2), limiting-dilution analysis, peripheral blood mononuclear cells from patients with uveitis and normal control subjects were assayed for S-antigen specific, tetanus-specific, and in vivo activated helper T cells. Controls subjects consistently demonstrated tetanus-specific responses, but neither in vivo activation nor S-antigen specific helper T cell responses were seen. Patients with active forms of diffuse, posterior, and anterior uveitis were found to have significant frequencies of both in vivo activated and S-antigen specific helper T cells in their peripheral blood. These data show that patients with certain forms of uveitis have a measurable frequency of lymphocytes in the peripheral immunologic compartment capable of secreting IL-2 in response to autologous presentation of ocular autoantigen (S-antigen). Limiting-dilution analysis techniques, generating minimal responder cell frequency estimates and distinct IL secretion patterns, may provide an index of disease activity and critical information about the mechanism(s) of ocular inflammation.

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.

The interface between an allograft and the recipient's immune system is the endothelium of the allograft vasculature. In this boundary position, endothelial cells may play important roles in the afferent and efferent phases of allograft rejection, in the response of the allograft to pretransplant perfusion and to drug therapy, and in the response to viral infection of the host. The expression by endothelial cells of granule membrane protein-140 (GMP-140) and endothelial leukocyte adhesion molecule-1 (ELAM-1), increased tissue factor activity, increased secretion of plasminogen activator inhibitor, and decreased thrombomodulin may all contribute to hyperacute rejection. Similarly, endothelial cells may actively participate in acute cellular rejection and in the development of transplant-associated arteriopathy as a result of induction of antigen-presenting function (ie, HLA class II expression), upregulation of adhesion molecules for lymphocytes and monocytes, and release of platelet-derived growth factors. Endothelial cell functions, which are important for normal inflammatory responses and vessel behavior, may be pathogenic in the allograft.

We have developed modified limiting dilution analysis (LDA) techniques that distinguish in vivo Ag-stimulated murine helper T lymphocytes (HTL) and CTL from unstimulated precursor T cells, even those with the same Ag specificity. We refer to these cells that are detectable in the modified LDA as "Ag-conditioned" T cells (cHTL and cCTL). We have used the modified LDA techniques in conjunction with conventional LDA techniques (which enumerate all Ag-specific T cells) to evaluate the in vivo distribution of Ag-conditioned cHTL and cCTL following in vivo sensitization to alloantigens via sponge matrix or skin allografts. In general, we observed the following regarding the distribution of cHTL and cCTL: 1) Ag-conditioned HTL and CTL were detectable only after in vivo sensitization with alloantigen: 2) not all Ag-reactive T cells became conditioned T cells after in vivo Ag deposition; 3) the percentage of Ag-reactive T cells that converted to conditioned T cells after Ag deposition varied among different lymphoid compartments; 4) a high percentage of cHTL, but a low percentage of cCTL, accumulated in regional lymph nodes and spleen; 5) cHTL accumulated in peripheral blood, whereas cCTL did not; 6) Ag-conditioned cHTL were detectable in various lymphoid tissues for greater than 60 days following Ag deposition, whereas cCTL were detectable for only 14 to 20 days; and 7) unlike the other lymphoid sites, the site of Ag deposition accumulated a high percentage of both Ag-stimulated cHTL and cCTL. Furthermore, cHTL and cCTL appeared to reside in phenotypically distinct T cell subsets in that in vivo treatment with anti-L3T4 mAb abrogated the accumulation of HTL, but not CTL, at the site of Ag deposition. These data demonstrate differential compartmentalization of Ag-conditioned cHTL and cCTL subsequent to in vivo Ag deposition. The implications of these findings regarding the monitoring of in vivo immune responses are discussed.