Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium.

BACKGROUND: We investigated whether differentiation of embryonic stem cells (ESCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection. METHODS AND RESULTS: In one series, 129/SvJ-derived mouse ESCs (ES-D3 line) were transplanted by direct myocardial injection (1 x 10(6) cells) into murine hearts of both allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients after left anterior artery ligation. Hearts were procured at 1, 2, 4, and 8 weeks after ESC transplantation and analyzed by immunohistochemistry to assess immune cell infiltration (CD3, CD4, CD8, B220, CD11c, Mac-1, and Gr-1) and ESC differentiation (hematoxylin and eosin). In a second series (allogeneic n=5, sham n=3), ESC transplantation was performed similarly; however after 2 weeks, left anterior descending artery-ligated and ESC-injected hearts were heterotopically transplanted into naive BALB/c recipients. After an additional 2 weeks, donor hearts were procured and analyzed by immunohistochemistry. In the first series, the size of all ESC grafts remained stable and there was no evidence of ESC differentiation 2 weeks after transplantation; however, after 4 weeks, both allogeneic and syngeneic ESC grafts showed the presence of teratoma. By 8 weeks, surviving ESCs could be detected in the syngeneic but not in the allogeneic group. Mild inflammatory cellular infiltrates were found in allogeneic recipients at 1 and 2 weeks after transplantation, progressing into vigorous infiltration at 4 and 8 weeks. The second series demonstrated similar vigorous infiltration of immune cells as early as 2 weeks after heterotopic transplantation. CONCLUSIONS: In vivo differentiated ESCs elicit an accelerated immune response as compared with undifferentiated ESCs. These data imply that clinical transplantation of allogeneic ESCs or ESC derivatives for treatment of cardiac failure might require immunosuppressive therapy.

Graft coronary artery disease in murine cardiac allografts: proposal to meet the need for standardized assessment.

BACKGROUND: Inconsistency exists in assessing the severity of graft coronary artery disease (GCAD) in studies that use mouse models. The central issue associated with this inconsistency is the lack of a standardized approach for assessing mouse GCAD. METHODS: We propose a new histologic definition of GCAD based on 3 successive stages (endotheliitis, premature lesion, and mature lesion) that mark the progression of this condition. In addition to these qualitative measures of GCAD, we propose including 2 additional morphometric parameters (percentage of luminal narrowing and intima-to-media ratio) and a measure of distribution (percentage of affected vessels) in the routine quantification of GCAD. RESULTS: We introduce 2 new mouse models of GCAD as examples that satisfy these criteria. CONCLUSION: The proposed assessment criteria may simplify data collection and interpretation of results in various models of GCAD.

T-cell response to cardiac myosin persists in the absence of an alloimmune response in recipients with chronic cardiac allograft rejection.

BACKGROUND: Immune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection. METHODS: The frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-gamma-producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts. RESULTS AND CONCLUSIONS: MR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to naïve, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.

The relative contribution of direct and indirect antigen recognition pathways to the alloresponse and graft rejection depends upon the nature of the transplant.

In this study, we measured direct and indirect T-cell alloresponses mediated by CD4(+) and CD8(+) T cells in three mouse transplantation models: skin, cornea, and retina. We show that the contribution of direct and indirect antigen recognition pathways to the alloresponse to fully allogeneic grafts varies depending upon the nature of the tissue/organ transplanted. The implications of this finding for understanding the cellular mechanisms by which rejection is mediated in different transplant models are discussed.

Progression of alloresponse and tissue-specific immunity during graft coronary artery disease.

Chronic rejection remains the major obstacle for long-term transplant survival. Both indirect alloresponse and tissue-specific autoimmunity have been implicated in its pathogenesis. The interrelationship between these two types of host anti-graft response remains poorly understood. We have developed an immunosuppression-free mouse model of graft coronary artery disease (GCAD), in which all FVB (H-2(q)) cardiac allografts placed into minor Ag (mHC)-mismatched DBA/1 (H-2(q)) hosts survived more than 112 days, and developed GCAD. We then examined the kinetics of both anti-mHC alloresponse and host autoimmunity against heart-specific antigen, cardiac myosin (CM). At 8 days post-transplantation, recipient mice showed minimal intragraft inflammation and apoptosis, and limited expansion of allo-specific T cells. In addition, we observed early production of anti-myosin IgG1 autoantibodies, which occurred in the absence of activated CM-specific T lymphocytes. By day 56, GCAD indices, the numbers of mHC- and CM-reactive T cells, and the levels of circulating allo- and CM-specific antibodies were all significantly increased. While host alloresponse was exhausted at 112 days post-transplant, T-cell reactivity against CM persisted. Our data suggest that both allo- and tissue-specific immunity might contribute to the induction of GCAD. They indicate that continual autoimmune response against graft tissue antigens may provide for GCAD sustenance.

Enzyme-linked immunosorbent spot assay analysis of peripheral blood lymphocyte reactivity to donor HLA-DR peptides: potential novel assay for prediction of outcomes for renal transplant recipients.

Chronic allograft dysfunction, which is the most common cause of late allograft failure, is in part caused by an ongoing immune response orchestrated by T lymphocytes primed by the indirect pathway of allorecognition. The low frequencies of such T cells have made it difficult to study indirect alloreactivity by using currently available assays. The development of a sensitive, clinically useful method of measuring indirect alloreactivity among human renal transplant recipients was thus attempted. Furthermore, in a pilot immunologic study, the contribution of the indirect pathway was studied in two groups of renal transplant recipients, i.e., patients with no prior acute rejection episodes and stable renal function ("stable" patients) and patients with at least one previous episode of biopsy-proven acute rejection, who were thus at risk for the development of chronic rejection ("high-risk" patients). The frequencies of type 1 T helper (interferon-gamma-producing) and type 2 T helper (interleukin-5- and -10-producing) peripheral blood lymphocytes reactive with a panel of synthetic peptides (corresponding to sequences from donor HLA-DR molecules) were determined for renal transplant recipients and normal control subjects by using an enzyme-linked immunosorbent spot assay (ELISPOT). Among recipients of DR-mismatched allografts, a cut-off value of 60 interferon-gamma spots/10(6) cells significantly (P = 0.02) separated stable patients (creatinine concentration, 1.1 +/- 0.3 mg/dl) from high-risk patients (creatinine concentration, 2.3 +/- 1.7 mg/dl). This is the first demonstration that the enzyme-linked immunosorbent spot assay can be used to monitor indirect alloreactivity to donor HLA-DR peptides among renal transplant recipients. These data provide the rationale for the prospective study of indirect alloreactivity among transplant recipients, to allow predictions of which patients would be at risk for the development of chronic rejection and thus allow appropriate planning of future interventions.

Modulation of tissue-specific immune response to cardiac myosin can prolong survival of allogeneic heart transplants.

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4(+) Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-gamma-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient's CD4(+) T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.