Gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 prolongs cardiac allograft survival and inhibits donor-specific immunity.

Introducing immunomodulatory molecules into allografts by gene transfer may avoid the side-effects of systemic immunosuppression. vMIP-II and MC148 are two recently identified chemokine homologues encoded by human herpes virus 8 and Molluscum contagiosum, respectively, that have antagonistic activities against multiple different CC and CXC chemokine receptors. We hypothesized that introduction of these molecules into cardiac allografts may block leukocyte infiltration into the grafts and prolong survival. Vascularized and nonvascularized cardiac allografts in mice were performed and plasmid DNA encoding vMIP-II, MC148 and/or vIL-10 was transferred into the allograft at the time of transplantation. Gene transfer of either vMIP-II or MC148 into cardiac allografts markedly prolonged graft survival. Combining gene transfer of either one of these chemokine antagonists with vIL-10 gene transfer, which has a mechanistically different immunosuppressive action, further enhanced graft survival. vMIP-II and MC148 gene transfer both resulted in a marked decrease of donor-specific cytotoxic T lymphocytes (CTL) infiltrating the grafts and inhibited alloantibody production. These results demonstrate that plasmid-mediated gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 can block donor-specific lymphocyte immunity within cardiac allografts and prolong graft survival. This is a new mechanistic approach to analyze, treat, and prevent graft rejection. Delivery of these or related molecules by gene transfer or conventional pharmacologic means may represent a novel therapeutic modality for alloactivation.

Gene transfer of immunomodulatory peptides correlates with heme oxygenase-1 induction and enhanced allograft survival.

BACKGROUND: Decapeptides derived from human HLA class I sequences have been shown to prolong allograft survival. The mechanism of action of these peptides has been uncertain, because they act in an MHC unrestricted manner. Recently, it was found that these peptides bind heme oxygenase 1 (HO-1). In the present study, we sought to determine whether local delivery of these peptides through gene transfer could extend allograft survival, and to explore the underlying mechanisms. METHODS: C57BL/6 neonatal hearts were transplanted to CBA/J recipients and the peptide, or plasmid DNA encoding the peptide, was injected directly into the allograft at the time of the transplant. RESULTS: Direct injection of 1 microg of the B2702 peptide into the allograft did not prolong survival (13.3+/-0.8 vs. 13.4+/-0.8 days for untreated controls), but injection of 400 microg of peptide did extend survival (22.0+/-0.6). Injection of plasmid DNA encoding the B2702 peptide was superior to peptide delivery, extending graft survival to 30.8+/-1.5 days. Similar results were obtained using another plasmid encoding the rationally designed peptide BC1 (28.5+/-1.7), whereas no significant prolongation was observed using a plasmid encoding the control peptide B2705 (16.5+/-1.0). To explore the hypothesis that these peptides exert their immunosuppressive effect by altering HO-1 activity, animals were treated with iron protoporphyrin, an inducer of HO-1 activity, or tin protoporphyrin, an inhibitor of HO-1. Treatment with iron protoporphyrin alone extended graft survival (24.5+/-1.6) and did not alter the benefit in survival seen with BC1 gene transfer (28.0+/-0.8). In contrast, treatment with tin protoporphyrin abolished the benefit of BC1 gene transfer (17.0+/-0.6). CONCLUSIONS: These results demonstrate that plasmid mediated gene transfer is an effective means for delivering immunosuppressive peptides to extend allograft survival. The experiments suggest that these peptides may act by increasing HO-1 activity and support a role for HO-1 in immune regulation and allograft survival.

Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses.

The gene encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10) was introduced into BALB/c (H-2d) vascularized cardiac allografts by perfusing the graft vasculature with DNA-liposome complexes, utilizing the experimental cationic lipid gamma AP DLRIE/DOPE and a plasmid encoding vIL-10 under the control of the HCMVie promoter. The DNA to lipid ratio and DNA dose were critical factors in obtaining optimal biologic effects. Gene transfer of vIL-10 with a 3:1 DNA to lipid weight ratio using 375 micrograms DNA significantly prolonged allograft survival in MHC-mismatched C57BL/6 (H-2b) recipients (16.00 days) compared with both unmodified allografts (8.14 days) and vIL-10 antisense controls (8.28 days). Enhanced graft survival was specific to vIL-10 expression since treatment with antisense plasmid or anti-vIL-10 monoclonal antibody (mAb) abrogated the effect. Prolonged survival was associated with a novel histology characterized by a moderate mononuclear infiltrate, edema, and diffuse fibrillar/collagen deposition in the interstitium. Despite these morphologic changes, myocytes remained viable and vessels were patent. Limiting dilution analysis revealed transient infiltration of IL-2 secreting, donor-reactive, helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) in vIL-10 expressing grafts on day 7, that decreased significantly by day 14. Similarly, vIL-10 gene transfer inhibited the accumulation of donor-specific HTL and CTL in the spleen, compared with antisense controls. Prolonged survival was also associated with a marked decrease in IgM and IgG alloantibody production, with little to no IgG isotype switching. These results show that viral IL-10 gene transfer inhibits graft rejection in a clinically relevant model by inhibiting donor-specific cellular and humoral immune responses.

Direct transfer of a foreign MHC gene into human melanoma alters T cell receptor V beta usage by tumor-infiltrating lymphocytes.

The direct introduction of foreign genes into tumors shows promise as a therapeutic modality to enhance tumor immunogenicity. Hence, melanoma nodules were directly injected with a vector encoding an allogeneic MHC class I molecule, HLA-B7. Tumor-infiltrating lymphocytes (TIL) were isolated from cutaneous melanoma biopsies before and after HLA-B7 gene transfer. TIL were expanded in interleukin-2 (IL-2) by standard techniques for approximately 4 weeks, then analyzed for T cell receptor V beta usage by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Prior to gene transfer. TIL V beta usage was found to be highly restricted, the only one to four V beta families being expressed and one or two of these families representing more than 90% of the repertoire. As anticipated, TIL V beta usage varied among patients expressing different HLA types. However, V beta 13 was over-represented in that six of eight patients utilized V beta 13 as a dominant family, regardless of HLA type. Following HLA-B7 gene transfer, TIL V beta usage was markedly altered: (1) V beta families that dominated following gene transfer differed from the V beta families utilized by TIL prior to treatment, and (2) introduction of the HLA-B7 gene resulted in a more diverse repertoire with an increase in the number of V beta families represented. In two patients, TIL were evaluated before treatment and from multiple, distinct melanoma nodules following gene transfer. In these two patients, a comparison was made between TIL V beta profiles obtained after treatment from nodules that had been injected with the HLA-B7 gene or left untreated. Interestingly, the V beta repertoires of TIL from uninjected nodules following gene transfer were similar to that of TIL from injected nodules, rather than pretreatment TIL. These data demonstrate a direct immunological effect of foreign MHC gene transfer into human melanoma, and suggest that local expression of an allogeneic MHC molecule generates systemic alterations in the antitumor immune response.

Restricted V beta usage by T cells infiltrating rejecting human lung allografts.

TCR expression was evaluated in lung transplant patients to determine whether T cells infiltrating rejecting lung allografts employed restricted V beta elements. Serial bronchoalveolar lavage (BAL) specimens were obtained from six lung transplant recipients at approximately 3 wk, 6 wk, and 3 mo post-transplant. T cell lines were established by culturing lavage cells with irradiated donor splenocytes in the presence of low dose IL-2 for 3 wk, and TCR V beta usage was determined by quantitative reverse transcriptase-PCR. Patients were grouped into three categories based on TCR V beta profiles and the clinical status of the allograft. 1) In one patient, BAL-derived T cells expressed heterogeneous V beta repertoires at all time points evaluated. This patient did not experience graft rejection during the 16-mo period of observation, though respiratory infections were diagnosed. 2) In three patients, V beta usage by BAL-derived T cells was restricted during allograft rejection episodes, but was heterogeneous in the absence of rejection and during respiratory infections. In one of these patients, similar V beta repertoires were employed by BAL cells during multiple rejection episodes. 3) In two patients, restricted V beta usage by BAL-derived T cells was observed before and during rejection episodes. Collectively, these data illustrate that human lung allograft rejection, but not pulmonary infection, is associated with T cells expressing a limited number of V beta families. Restricted V beta usage by graft-reactive T cells may allow for the selective elimination of these cells using TCR-specific reagents, thereby promoting allograft-specific tolerance.

T cell receptor (TCR) repertoire in alloimmune responses.

A large number of alloantigenic determinants could be generated by both the direct and indirect alloantigen presentation pathways. Hence, a heterogeneous population of T cells expressing a wide variety of receptors would be expected to respond to this diverse array of alloantigenic determinants. However, T cells expressing highly restricted T cell receptor (TCR) variable genes have been reported in a variety of alloimmune responses. A similar phenomenon has been observed in a wide variety of other immune responses, from those induced by superantigens, to very specific responses induced by a single peptide presented by a single MHC molecule. Given this scenario, the limited number of T cell clones which dominate an allograft rejection response, or for that matter an autoimmune response or a tumor specific response, could be therapeutically targeted by virtue of the selected TCR expression.

Regulation of cardiac allograft rejection by T lymphocytes.

Cardiac allograft rejection is diagnosed by routine, surveillance endomyocardial biopsy (EMB). EMB is an invasive and expensive procedure that detects ongoing, rather than impending, rejection, and may be influenced by sampling error due to nonuniform distribution of myocardial lesions. T cells are the principal regulators of allograft rejection; therefore, identifying T cell functions that are associated with cardiac allograft rejection should provide insight for the development of noninvasive monitoring techniques that may augment and reduce the frequency of EMB. Experiments that define the protective and deleterious activities of Th1 and Th2 T cell subsets are clearly warranted, and will direct the potential use of cytokine manipulating therapies aimed at inducing tolerogenic T cell responses. (Trends Cardiovasc Med 1996;6:168-173).

Dissociation of mouse cardiac transplant rejection and donor alloantigen-specific T cell responsiveness.

Mouse hearts transplanted into MHC disparate donors are usually rejected 1 week after placement. It is widely accepted that alloantigen-reactive helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) are the key mediators of acute allograft rejection. This report demonstrates that the presence or absence of 'traditional' graft-reactive HTL and CTL is not necessarily related to allograft survival. In these studies, donor/recipient combinations disparate only at MHC or only at minor histocompatibility (mH) loci were employed. Allograft survival was monitored, donor-reactive IL-2 (interleukin-2) producing HTL and CTL were quantified by modified limiting dilution analysis, and serum levels of cytolytic alloantibody were determined. C57BL/10 hearts (H-2b) transplanted into B10.BR (H-2k) recipients (full MHC disparity) enjoyed prolonged survival despite massive infiltration of the allograft by donor-reactive HTL and CTL. IgM, but not IgG, donor-reactive alloantibody was present in the sera of these mice. Hence, traditional IL-2 producing HTL and CTL were not capable of mediating acute graft rejection, nor of providing help for alloantibody isotype switching in this MHC disparate combination. In contrast, C3H/HeN (H-2k) hearts transplanted into B10.BR (H-2k) recipients (mH disparity only) were acutely rejected. Donor-reactive HTL and CTL were rare or not detectable in these recipients, and cytolytic alloantibody was not detectable. Similar observations were made when B10.BR hearts were transplanted into C3H/HeN recipients. Interestingly, treatment of recipients with anti-CD4 monoclonal antibody prevented rejection of mH disparate allografts. Therefore, CD4+ T cells were required for rejection of mH disparate allografts, but this process was independent of detectable IL-2 production or CTL function. Hence, the significance of monitoring 'traditional' T cell functions should be questioned in certain donor/recipient combinations.

In vivo depletion of CD8+ T cells results in Th2 cytokine production and alternate mechanisms of allograft rejection.

A current hypothesis states that Th1 cytokines promote allograft rejection and that Th2 cytokines promote graft acceptance. We present data that question the tolerogenic activity of Th2 cytokines, and we suggest that Th2 cytokines may evoke allograft rejection by recruitment of alternate effector mechanisms. Unmodified rejection of mouse heterotopic cardiac allografts is associated with the accumulation of large numbers of donor-reactive CD8+ CTL within the allograft, which is indicative of a Th1-driven cellular response. However, when recipients are depleted of CD8+ CTL, rejection still occurs and is associated with an aggressive cellular infiltrate rich in eosinophils, large mononuclear cells, and fibroblast-like cells. Eosinophils, which are responsive to the Th2 cytokines IL-4 and IL-5, are not present in unmodified rejecting allografts. Differential production of Th1 versus Th2 cytokines was further suggested by altered levels of IgG2a (promoted by IFN gamma) and IgG1 (promoted by IL-4) alloantibody in the sera of these mice; IgG2a dominated the alloantibody response in unmodified allograft recipients, whereas IgG1 levels increased in recipients depleted of CD8+ CTL. Altered intragraft cytokine gene expression was verified by RT-PCR; Th1 (IL-2, IFN gamma), but not Th2 (IL-4, IL-5, IL-10), cytokine mRNAs were readily detectable in the allografts of unmodified recipients. In contrast, both Th1 and Th2 cytokine genes were expressed in the allografts of mice depleted of CD8+ CTL. These data suggest that donor-reactive CD8+ CTL inhibit intragraft production of Th2 cytokines, thereby promoting a Th1 dominated-rejection response. Elimination of CD8+ cells allows Th2 cytokine production, which may have deleterious, rather than protective, effects.

Evidence that human cardiac allograft acceptance is associated with a decrease in donor-reactive helper T lymphocytes.

We have reported that acute cardiac allograft rejection is associated with increased numbers of donor-reactive helper T lymphocytes (HTL) in the peripheral blood of patients. Further, increased frequencies of circulating donor-reactive HTL may predict allograft rejection episodes diagnosed by endomyocardial biopsy. The present study evaluates the relationship between donor-reactive HTL and allograft "acceptance" in cardiac transplant recipients bearing long-term allografts (> 1 year). Patients were categorized as either long-term acceptors or persistent rejecters based on the number of rejection episodes and the ability to withdraw steroid therapy. Limiting dilution analysis for IL-2-producing HTL was utilized, with cadaver donor splenocytes as a source of donor alloantigens. Donor-reactive HTL frequencies were determined from peripheral blood samples obtained before transplant, and at 1 month and 1 year after transplant. Individuals who accommodated their allografts and were withdrawn from steroid therapy had reduced numbers of donor-reactive HTL at 1 year after transplant as compared with earlier time points. Further, PBMC obtained from these individuals at 1 year after transplant responded weakly to donor alloantigens in a mixed lymphocyte response (MLR). This relationship between donor-reactive HTL and allograft accommodation was exemplified in a cardiac/liver transplant patient who was diagnosed with progressive multifocal leukoencephalopathy and removed from all immunosuppression. No subsequent rejection episodes were diagnosed. Donor-reactive HTL were not detectable and this individual failed to mount an MLR to donor alloantigens. However, a vigorous donor-reactive response was observed when MLR cultures were supplemented with exogenous IL-2. Therefore, nonresponsiveness to the allograft appeared to be due to a deficit in IL-2 production. In contrast, patients who experienced persistent rejection episodes and required continued steroid therapy maintained large numbers of donor-reactive HTL at 1 year after transplant. PBMC from these individuals responded vigorously to donor alloantigens in an MLR. Hence, monitoring donor-reactive HTL may identify individuals who have accommodated their graft and may tolerate a reduction in immunosuppression.

Increased frequency of alloantigen-reactive helper T lymphocytes is associated with human cardiac allograft rejection.

Endomyocardial biopsy (EMB) is the standard method of monitoring heart transplant recipients for the development of allograft rejection. To date, noninvasive methods to detect cardiac allograft rejection have lacked adequate sensitivity and specificity for wide clinical application. In this study, limiting dilution analysis (LDA) was used to quantitate the number of donor alloantigen-reactive helper T lymphocytes (HTLs) in the peripheral blood of cardiac transplant recipients. Cadaveric donor splenocytes were cryopreserved, providing a source of donor alloantigenic stimulation for these assays. Peripheral blood mononuclear cells were harvested from cardiac transplant recipients before transplantation and at the time of EMB. LDA of donor-reactive HTLs was conducted simultaneously on all time points to minimize experimental variation, and these data were related to EMB scores. Frequencies of donor-reactive HTLs in pretransplant samples were highly variable, ranging from 1/1381 to < 1/200,000, and correlated poorly with the degree of HLA disparity. During episodes of moderate rejection, donor-specific HTL frequencies increased an average of 6 times their post-transplant baseline frequency. Additionally, 10-fold increases in HTL frequencies were seen preceding EMB-diagnosed rejection in several individuals. These data indicate that episodes of allograft rejection are associated with increases in the number of circulating donor-reactive HTL which are frequently detected before the development of histologically defined rejection. Thus, monitoring HTL frequencies may serve as a non-invasive method for detecting and predicting cardiac allograft rejection. Furthermore, this assay may provide a valuable means of assessing the in vivo efficacy of various immunosuppressive therapies.