Increased expression of class I major histocompatibility complex antigens on hepatocytes in rejecting human liver allografts

We studied hepatocellular expression of major histocompatibility (MHC) antigens in 43 serial liver transplant biopsies from 22 patients (42 percutaneous, 1 autopsy specimen), 4 normal liver biopsies, and 8 percutaneous biopsies of diseased livers from non-liver-transplant patients. Frozen tissue sections were stained by an indirect immunofluorescence technique using monoclonal antibodies (MCAb) that recognize nonpolymorphic human class I or class II MHC determinants. Ethidium bromide was used to stain nuclei and rhodamine-conjugated anti-basement-membrane antibodies to delineate epithelial and vascular structures. HLA-DR antigens recognized by MCAb OKIa1 and I2 were not detected on hepatocytes but were detected on the bile duct epithelium in 7 of 27 transplant biopsies, including 5 with acute rejection and 1 with chronic liver disease that later progressed to chronic rejection. HLA-A, B, C antigens recognized by MCAb 34/28 intensely stained cells lining the liver sinusoids but were negative on hepatocytes in 4 normal liver biopsies and 7 of 8 non-transplant biopsies. Expression of class I MHC antigens on hepatocyte membranes was increased in 17 of 21 (81%) biopsies from patients with acute rejection, in 4 of 4 with chronic transplant liver disease, but in only 3 of 18 (17%) biopsies from patients with no rejection (chi square = 8.62, P less than 0.01). Our observations demonstrate increased expression of MHC class I antigens in association with acute rejection in human orthotopic liver transplantation. Histologic resolution of the rejection episode is generally followed by a decrease in hepatocyte class I antigen expression. Further analysis of this response may have value in assessing the severity of the rejection and effectiveness of treatment.     

The frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting renal allografts

Abstract. We have estimated the frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting histoincompatible renal allografts. In a major plus minor antigen-incompatible DA-to-WF combination on day 4 post-transplantation, reverse protein A plaque assay demonstrated that in the graft the frequency of lymphoid cells secreting Ig was 1:850. A major locus-incompatible and minor locus-compatible, congeneic LBN-to-Lewis strain combination was then applied to estimate the specificity of the secreted antibody. The lymphoid inflammatory cells were fused with mouse myeloma cells, cultured under limiting dilution conditions, and assayed by ELISA to donor and irrelevant strain spleen cells. Among cells infiltrating the graft, the fusion frequency was 1:172 10³ the frequency of Ig-producing hybrids 1:400 10³ (i. e., this assay was approximately three log orders less sensitive than the reverse pA assay). The frequency of hybridomas secreting specific antibodies against donor MHC antigens was 1:720 10³ (i. e., every second hybridoma deriving from inflammatory population produced specific Ig). In addition, there was at least one obviously polyspecific population of hybridomas, detectable only in the spleen and reactive with all rat strains tested with a frequency of 1:700 10³. The inflammatory cells were also cultured directly under limiting dilution conditions, and the frequency of Ig-secreting cells was determined by ELISA. The frequency of inflammatory lymphocytes secreting detectable amounts of immunoglobulin in the supernatant was 1:14 10³ in the graft (i. e., this assay was approximately one log order less sensitive than the reverse protein A plaque assay). Donor-strain reactive cells were not detected in the spleen or blood of nontransplanted Lewis (recipient) rats, nor were they detected in the blood of the transplant recipient. Our results show that B cells secreting antibodies against graft donor MHC antigens are present in the graft-infiltrating cells' inflammatory population. In the inflammatory population only approximately 1:850 of the inflammatory cells are engaged in Ig secretion, and at most 1:2 of these produce specific antibody to graft donor MHC antigens. This finding suggests that a nonspecific (polyclonal) Ig-producing population is also present in situ. These results are also concordant with the reported low frequency of specific T cells of both cytotoxic and helper type at the site of inflammation and emphasize the importance of nonspecific inflammatory (“delayed hypersensitivity”) mechanisms in acute allograft rejection.     

The frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting renal allografts

We have estimated the frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting histoincompatible renal allografts. In a major plus minor antigen-incompatible DA-to-WF combination on day 4 post-transplantation, reverse protein A plaque assay demonstrated that in the graft the frequency of lymphoid cells secreting Ig was 1: 850. A major locus-incompatible and minor locus-compatible, congeneic LBN-to-Lewis strain combination was then applied to estimate the specificity of the secreted antibody. The lymphoid inflammatory cells were fused with mouse myeloma cells, cultured under limiting dilution conditions, and assayed by ELISA to donor and irrelevant strain spleen cells. Among cells infiltrating the graft, the fusion frequency was 1:172x10³ and the frequency of Ig-producing hybrids 1:400x10³ (i.e., this assay was approximately three log orders less sensitive than the reverse pA assay). The frequency of hybridomas secreting specific antibodies against donor MHC antigens was 1:720x10³ (i.e., every second hybridoma deriving from inflammatory population produced specific Ig). In addition, there was at least one obviously polyspecific population of hybridomas, detectable only in the spleen and reactive with all rat strains tested with a frequency of 1:700x10³. The inflammatory cells were also cultured directly under limiting dilution conditions, and the frequency of Ig-secreting cells was determined by ELISA. The frequency of inflammatory lymphocytes secreting detectable amounts of immunoglobulin in the supernatant was 1:14x10³ in the graft (i.e., this assay was approximately one log order less sensitive than the reverse protein A plaque assay). Donor-strain reactive cells were not detected in the spleen or blood of nontransplanted Lewis (recipient) rats, nor were they detected in the blood of the transplant recipient. Our results show that B cells secreting antibodies against graft donor MHC antigens are present in the graft-infiltrating cells' inflammatory population. In the inflammatory population only approximately 1:850 of the inflammatory cells are engaged in Ig secretion, and at most 1:2 of these produce specific antibody to graft donor MHC antigens. This finding suggests that a nonspecific (polyclonal) Ig-producing population is also present in situ. These results are also concordant with the reported low frequency of specific T cells of both cytotoxic and helper type at the site of inflammation and emphasize the importance of nonspecific inflammatory (delayed hypersensitivity) mechanisms in acute allograft rejection.     

Aberrant colonic expression of MHC class II antigens in Hirschsprung's disease.

The major histocompatibility complex (MHC) Class I and II cell surface antigens responsible for the recognition of self vs non-self were studied in patients with documented Hirschsprung's disease. Monoclonal antibodies reactive with monomorphic determinants of human lymphocyte antigen (HLA)-A,B,C (Class I) and HLA-DR (Class II) were used to demonstrate immunohistochemically the expression of MHC antigens in 27 biopsy specimens from a variety of colorectal disorders. The rectal specimens examined from patients with Hirschsprung's disease showed an unexpected, marked elevation of Class II antigens with abnormal localization in the mucosa and lamina propria. This ectopic expression was not seen in any portion of the small or large bowel of patients who did not have Hirschsprung's disease. Furthermore, proximal normal colon of children with Hirschsprung's disease failed to show increased expression of Class II antigen. In an attempt to better define the effector arm at a cellular level, the distribution of helper T cells (CD4+), cytotoxic/suppressor T cells (CD8+) and natural killer cells (NK; CD16+) was examined in 5 cases. In Hirschsprung's disease, rectal infiltration of CD8+ and CD16+ cells was found, but not CD4+ cells. Ectopic expression of Class II antigen with increased numbers of rectal T cells and NK cells suggested that an early immunologic event may be causal in Hirschsprung's disease.     

The role of class I and class II MHC antigens in the rejection of vascularized heart allografts in mice

We have examined the role of entire major histocompatibility complex (MHC) disparity, individual class II or class I alloantigens in the rejection of vascularized heart allografts. Our results demonstrate that entire MHC, as well as both class II and class I disparities, may induce acute heart graft rejection or severe and irreversible heart muscle destruction. However, in 1 of 2 combinations differing at class II and 1 of 5 differing at class I, hearts have shown a good function greater than 100 days postgrafting. Furthermore, each donor-recipient combination has demonstrated a unique pattern of heart allograft function as well as a degree of heart muscle damage. In conclusion, these data suggest that the rejection process depends upon multiple factors such as the immune-response-gene-regulated immunoresponsiveness of the recipient as well as the expression of alloantigens on heart grafts during the induction and effector phases of the immune response.     

Does high MHC class II gene expression in normal lungs account for the strong immunogenicity of lung allografts?

Clinical experience has demonstrated that lung allografts are considerably more immunogenic than liver allografts although both organs contain equivalent amounts of lymphoreticular tissue. Northern blot analysis of MHC class II gene expression in various murine organs with I-AB and I-EB gene-specific oligonucleotide probes revealed that MHC class II expression in lungs is semiquantitatively higher than in the liver and other organs with the exception of the spleen. We conclude that this high MHC class II expression strongly suggests that the lymphoreticular tissue in the lungs is in a state of activation. This may be an important reason for the strong immunogenicity of lung allografts.     

Expression of class II major histocompatibility complex antigens by bronchial epithelium in rat lung allografts

Variations in expression of class II major histocompatibility complex antigens on bronchial epithelial cells and vascular endothelium were investigated in normal rat lungs and allografted lungs during acute rejection and after cyclosporine (CsA) treatment. BN (RT1n) left lungs were transplanted into LEW (RT1l) recipients. Lungs were excised during acute rejection in untreated rats on postoperative days 1 through 5, and after CsA treatment (25 mg/kg on days 2 and 3) on days 5 and 100. Cryostat sections were examined for class II antigen expression with an immunoperoxidase technique, using various monoclonal antibodies. In the normal lung, class II antigens were not expressed by epithelial or endothelial cells. In the allografts, induction of class II antigens closely correlated with the rejection process: on day 2, the ciliated bronchial epithelium was locally positive; it became uniformly positive with increasing cellular peribronchial infiltration on days 3 and 4. CsA treatment prevented class II antigen expression to a certain extent, leaving the bronchial epithelium weakly positive at 100 days. Endothelial cells were invariably negative for class II antigens in all allografted lungs. The class II antigens expressed on the bronchial epithelial cells were of graft origin, except for recipient-type class II molecules found on the ciliated surface in CsA-treated animals. We conclude that expression of class II antigens by bronchial epithelium is the result of a bronchus-directed rejection process, and hypothesize that such a rejection process may have caused bronchiolitis obliterans in several of the patients with combined heart-lung transplants. Important is the observation that class II molecules can be present on the membranes of cells that do not themselves produce these antigens.     

The effects of cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in the rat

We have previously reported 5-30-fold increases in the expression of class I and class II major histocompatibility complex (MHC) antigens in rejecting heart and kidney allografts in the DA-to-PVG rat strain combination. We examine here the effects of immunosuppression with cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in this strain combination. Immunohistological studies and quantitative absorption analyses using monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens were used throughout. Heart allografts in cyclosporine-treated rats were examined on day 3,5,7,9,11, and 14 after transplantation, and kidney allografts in cyclosporine-treated rats were examined at day 7. In addition, untreated heart and kidney isografts were studied at days 1,3,5, and 7 after grafting. Immunohistological studies on frozen sections showed that cyclosporine-treated heart and kidney allografts showed no induction of class II MHC antigens, in contrast to untreated heart and kidney allografts. Class I MHC antigen induction did occur in spite of cyclosporine-therapy, but at levels lower than those seen in untreated allografts. Moreover, the pattern and degree of class I induction in the cyclosporine-treated allografts resembled very closely those seen in isografts, and so this induction was, in all probability, a consequence of the transplantation procedure rather than of specific immune responses. We also noted, in the cyclosporine-treated heart allografts, that all donor interstitial dendritic cells had disappeared and been replaced by recipient interstitial dendritic cells by the end of the second week after grafting. In addition, there was no reduction in the class II antigen content of kidney allografts treated for 7 days with cyclosporine. The absence of class II antigen induction in allografts where rejection is effectively suppressed with cyclosporine might be of clinical value in the differential diagnosis between rejection and cyclosporine toxicity in renal transplantation, and between active and inactive cellular infiltrates in heart transplantation.     

B cell-induced tolerance to class II MHC antigens in the chicken

Transplantation of cells from the bursa of Fabricius reconstitutes the B cell system of chemically bursectomized chickens. Even allogeneic bursa cells can restore the recipient's B cell system and induce tolerance to donor major histocompatibility complex antigens, but the chimeras cannot mount a T-dependent antibody response. In order to study the mechanisms of tolerance to class II MHC (B-L) antigens, we transplanted class II-incompatible bursa cells from 4-day-old donors into cyclophosphamide-treated recipients of the same age. Donor and host cells carried different allelic products of a genetically polymorphic B cell alloantigen (Bu-1), allowing us to detect cellular chimerism using monoclonal antibodies and immunofluorescence. The B cell-chimeric chickens were tested for tolerance by skin grafting, graft-versus-host splenomegaly assay, and mixed lymphocyte reaction. Specific unresponsiveness to donor MHC antigens was observed in all three tests. When spleen cells from chickens tolerant of donor class II antigens were transferred into irradiated secondary recipients of the same strain, several of the secondary recipients accepted primary donor-type skin grafts. Most secondary recipients were, however, reactive in the GVH assay and MLR. Depletion of chimeric B cells before spleen cell transfer impaired the transferability of tolerance to class II disparity. Altogether, our results indicate that tolerance to class II antigens can be induced with B cells. They suggest that at least two different mechanisms maintain the unresponsiveness in B cell-chimeric chickens.     

Cold preservation of nerve grafts decreases expression of ICAM-1 and class II MHC antigens.

Cold preservation has previously been shown to decrease the antigenicity of nerve allografts, while Schwann cells remain viable. The expression of intercellular adhesion molecule-1 (ICAM-1) and class II MHC antigens, both of which have been shown to play a major role in initiating graft rejection, was studied in fresh rat nerve, and after 2 and 7 weeks of cold preservation. Ten sciatic nerves harvested from Lewis rats were cut into three segments. One segment was processed immediately, while the other ones were preserved at 5 degrees C for 2 and 7 weeks, respectively, before processing. Immunostains using specific monoclonal antibodies and alkaline phosphatase development were performed on each sample. The relative level of expression of these antigens was compared using computer-assisted densitometry. Expression of ICAM-1 was significantly decreased at 7 weeks, as compared to fresh and 2-week groups, with no statistically significant difference between fresh and 2-week nerves. Expression of class II MHC was significantly decreased at 2 and 7 weeks, compared to fresh nerves, with no statistically significant difference between the preserved groups. The decrease in antigenicity of cold-preserved nerve allografts appears to be linked to a down-regulation of ICAM-1 and MHC class II expression.