Cell subsets responding to purified hepatocytes and evidence of indirect recognition of hepatocyte major histocompatibility complex class I antigen. II. In vitro-generated "memory" cells to class I+ class II- hepatocytes.

Purified hepatocytes stimulate the development of L3T4-, Ly2+ allospecific cytolytic T cells from naive splenocytes after 5 days in primary mixed lymphocyte-hepatocyte culture (MLHC). Previous studies indicate that the immunogenicity of purified hepatocytes relates to the expression of MHC class I antigen. The purpose of the following experiments was to identify the cell subsets that specifically recognize hepatocyte MHC class I antigen. We employed primed lymphocyte testing (PLT) in order to test for a "second set" response. Cells from primary MLHC reverted to a functionally quiescent state when they were grown in culture for an additional 7-9 days. The cells were then tested for cytotoxicity or rechallenged with allogeneic, syngeneic, or "third party" hepatocytes and tested for proliferation. Allocytotoxicity was low on day 12 in MLHC, but the sensitized cell population demonstrated peak proliferation in response to allogeneic hepatocytes 48 hr after restimulation. When bulk PLT cells were immunodepleted, both L3T4+, Ly2- and L3T4-, Ly2+ T cell subsets demonstrated a "second set" response to allogeneic hepatocytes consistent with specific recognition of and retention of "memory" for hepatocyte MHC class I alloantigen.     

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.     

Pretransplant sensitization with major histocompatibility complex class I+ class II- hepatocytes leads to accelerated skin graft rejection.

The immunogenicity of major histocompatibility complex (MHC) class I+ class II- hepatocytes is controversial. We studied the effect of pretransplant donor-specific sensitization with either purified hepatocytes (HC) or splenocytes (Spl) on subsequent skin allograft survival. Five million Percoll-purified DBA HC or 10 x 10(6) DBA Spl were injected into C57BL/6 recipients either intraperitoneally (ip) or into a sponge matrix allograft. Twelve days later, sensitized mice received a DBA skin graft. On the same day, allogeneic (DBA) and syngeneic (BL/6) skin grafts were placed on naive BL/6 mice. In naive BL/6 mice, allogeneic skin graft survival was 7.8 +/- 0.5 days (n = 4), and syngeneic survival was indefinite (n = 5). Skin graft survival (mean +/- SD in days) in recipients sensitized with hepatocytes ip was 6.0 +/- 1.2 days (n = 5) compared with 5.6 +/- 0.5 days in recipients sensitized with splenocytes ip. Similarly, graft survival in recipients that received hepatocytes into a sponge matrix allograft was 5.67 +/- 1 days (n = 6) compared with 5.2 +/- 1.1 days (n = 8) in those that received splenocytes into the sponge. There was no difference in graft survival between mice sensitized with HC vs Spl, nor between mice injected ip vs with the sponge. All sensitized mice experienced accelerated graft rejection compared with naive controls (P less than 0.000). These results demonstrate that purified MHC class I+, class II- murine HCs are immunogenic in vivo. Sensitization with donor-specific HCs led to accelerated rejection of subsequent skin grafts, similar to the accelerated rejection seen after sensitization with MHC class I+ and class II+ splenocytes.     

The role of class I major histocompatibility complex antigens in prolonging the survival of hepatic allografts in the rat

In an attempt to study the role of class I major histocompatibility complex antigens in inducing immunological unresponsiveness, the survival rates of hepatic allografts were compared in rats pretreated with blood taken from various rat strains. A single intravenous injection of 1 ml fresh heparinized whole blood seven days before transplantation significantly prolonged the survival of subsequent donor-specific hepatic allografts in the fully allogeneic ACI(RT1a)-to-LEW(RT1l) rat combination. However, pretreatment with blood taken from the third-party strain BN(RT1n) did not produce suppression of rejection, attesting to the specificity of the pretransplant transfusion effect. Interestingly, pretransplant transfusion of PVG.r1 blood, sharing only the RT1.A MHC region with ACI, significantly prolonged the survival of ACI-to-LEW hepatic allografts. In addition, no lymphocytotoxic antibodies could be detected at 30 or 100 days after transplantation in animals with long-surviving hepatic allografts pretreated with either PVG.r1 or ACI whole blood. On the other hand, pretreatment with PVG(RT1c) blood increased the survival of ACI-to-LEW hepatic allografts only moderately compared with controls. This finding may be consistent with a partial effect of some third-party blood transfusion. The experimental data suggest that the class I MHC antigens can be immunosuppressive in rat hepatic allografts. Adoptive transfer of 5 x 10(7) splenocytes taken from long-term-surviving hepatic allografts pretreated with donor ACI whole blood or PVG.r1 blood into irradiated (750 rads) LEW rats prolonged the survival of donor-type skin grafts, whereas third-party strain (BN) grafts were rejected. This finding suggests the presence of donor-specific suppressor cells.     

Cytomegalovirus-induced regulation of major histocompatibility complex class I antigen expression in human aortic smooth muscle cells.

Cardiac allograft vasculopathy (accelerated transplant atherosclerosis) is considered by most to involve a chronic allogeneic immune response to one or more constituents in the coronary vascular wall. Recent evidence suggests that there is an association between cytomegalovirus infection and the development of cardiac allograft vasculopathy (CAV). To determine whether CMV directly infects and/or potentially influences immunogenicity of vascular tissue, human umbilical vein (HU-VECs) or human aortic (HAECs) endothelial cells and human aortic smooth muscle cells (HASMCs) were isolated, cultured, and infected with CMV strain AD 169. Infection was detected using an immunoperoxidase-labeled monoclonal antibody to CMV immediate-early antigen (L-14). The presence and relative quantity of MHC class I and II antigens were determined flow cytometrically using monoclonal antibodies to monomorphic class I and class II HLA determinants. Gamma interferon was used as a positive control stimulant for the upregulation of MHC determinants. Both pooled HUVECs as well as 2 cell lines of HAECs served as targets for CMV infection though less than 10% of the cells were infected despite inocula of 10 pfu/cell. Infection of the pooled HUVECs resulted in no significant changes in the cell surface density of either MHC class I or II determinants. In contrast, HASMCs were excellent targets for CMV infection with virtually 100% of cells infected. CMV infection of 2 distinct HASMC cultures resulted in an increase of 254 +/- 158 relative fluorescence units (RFUs) in MHC class I antigen expression, as assessed by fluorescence intensity, in a variable portion of the HASMCs. A second population of cells exhibited a decrease of 73 +/- 16 RFUs in MHC class I antigen expression. No significant change in MHC class II antigen expression was noted. These results demonstrate that while HUVECs and HAECs are targets of CMV infection, human aortic smooth muscle cells can more readily be infected by CMV. Furthermore, CMV can regulate smooth muscle cell MHC class I expression, hence potentially altering immunogenicity. A pathophysiologic link between cardiac allograft vasculopathy and CMV disease can therefore be hypothesized.     

Transplantation effects of a unique major histocompatibility complex class I mutation.

This study describes a novel MHC class I mouse mutant that was discovered because of loss of reactivity of its cells to monoclonal antibodies. The mutation occurred in the H-2Ks molecule and is the first in vivo mutation described that has a single altered amino acid residue (amino acid 107) distant from the regions considered to be peptide or TCR contacts. Nevertheless, skin grafts from the mutant to the parent are rejected by CD8+ T-cells. In the reciprocal direction, the mutant shows partial tolerance to parental skin grafts, suggesting that the mutant is inefficient in selecting alloreactive T-cells specific for the wild-type Ks molecule.     

Human cytomegalovirus blocks interferon-gamma stimulated up-regulation of major histocompatibility complex class I expression and the class I antigen processing machinery

Interferon-gamma stimulates major histocompatibility complex (MHC) class I antigen processing and presentation by inducing the expression of major histocompatibility complex class I heavy chains, beta2-microglobulin, the transporter associated with antigen processing, and components of the proteasome complex. We demonstrate that this effect of interferon-gamma on the major histocompatibility complex class I pathway is inhibited in human cytomegalovirus-infected fibroblasts and endothelial cells. This is the result of a direct human cytomegalovirus/cell interaction leading to a block in interferon-gamma signal transduction beginning at early times after infection and peaking at 72 hr after infection. These observations suggest a novel level of herpesvirus interference with antigen processing: protection of infected cells from the immunoregulatory effects of interferon-gamma. Thus protected, human cytomegalovirus persists and may exacerbate graft rejection or lead to fulminant infection in the immunocompromised transplant recipient.     

Cross-reactivity of cytomegalovirus-specific CD8+ T cells to allo-major histocompatibility complex class I molecules

BACKGROUND: In transplantation settings, cytomegalovirus (CMV) infection is a common complication. CMV infection is associated with a higher incidence of graft rejection in solid organ transplantation and graft-versus-host disease in bone marrow transplantation. The underlying mechanism of this association could be the generation of CMV-specific CD8 T cells capable of cross-reacting with alloantigens present on graft and host, respectively. METHODS: Whereas as to date, no direct ex vivo analysis can be performed of the CD8 T-cell repertoire directed at allo-major histocompatibility complex (MHC) class I molecules, virus-specific cells can be readily enumerated by use of MHC-peptide tetrameric complexes. In this study, the authors used this technique to analyze potential overlapping CD8 T-cell repertoires between self-MHC-viral peptide and allo-MHC complexes by stimulating CMV-specific CD8 T cells with alloantigens. RESULTS.: The authors found that CMV-specific CD8 T cells are activated and proliferate on stimulation with alloantigens. CONCLUSIONS: Although these cells are cytotoxic against CMV-peptide pulsed target cells, no cytotoxicity of CMV-specific cells to alloantigens could be detected, inferring that there are other mechanisms of graft damage by alloantigen-stimulated virus-specific CTL.     

Interferon-gamma is necessary for initiating the acute rejection of major histocompatibility complex class II-disparate skin allografts.

BACKGROUND: Although interferon (IFN)gamma has immunostimulatory functions, it is not essential for the acute rejection of fully allogeneic grafts in mice. It is not known whether IFNgamma plays a critical role in the acute rejection of MHC class I- or MHC class II-disparate allografts. METHODS: We studied the survival of skin allografts transplanted from fully allogeneic (BALB/c), MHC class I-disparate (bml), or MHC class II-disparate (bm12) donors to C57BL/6 wild-type (IFNgamma+/+) and IFNgamma gene-knockout (IFNgamma-/-) recipients. We also investigated the in vitro responses of IFNgamma+/+ and IFNgamma-/- T cells to MHC class II-disparate splenocytes. RESULTS: We found that IFNgamma-/- recipients reject BALB/c and bml skin grafts at the same rate as IFNgamma+/+ mice but are not capable of rejecting bm12 skin. Despite the inability of IFNgamma-/- mice to reject bm12 skin grafts, IFNgamma-/- T cells displayed vigorous proliferation and cytotoxic responses when stimulated with bm12 splenocytes in vitro. Furthermore, priming IFNgamma-/- recipients with bm12 splenocytes enabled these mice to reject bm12 skin grafts at a normal rate and to mount a cutaneous delayed-type hypersensitivity response to the bm12 antigen. CONCLUSION: The data demonstrate that IFNgamma is not necessary for generating effector mechanisms associated with acute transplant rejection but that it is required for initiating alloimmune responses to MHC class II-disparate skin grafts.     

Immunomodulation of glioma cells after gene therapy: induction of major histocompatibility complex class I but not class II antigen in vitro

OBJECTIVE: Acquired immunity has been demonstrated in Fischer rats bearing syngeneic 9L tumors after herpes simplex virus (HSV) thymidine kinase (TK) gene transfection and ganciclovir treatment. The nature of this immunity in rats and its relevance to the HSV TK/ganciclovir protocol for human subjects remain to be determined. In this study, levels of major histocompatibility complex (MHC) Class I and II antigen expression were measured before and after HSV TK transfection, in an effort to document immunomodulatory changes caused by gene therapy. METHODS: Tumor cells from the 9L gliosarcoma cell line, three primary human glioma cultures, and the human glioma cell line U87 MG were transduced with HSV TK vector-containing supernatant from fibroblast-producing cells (titer of 5 x 10(6) colony-forming units/ml) and selected in G418 medium for neomycin resistance. Clones were pooled or individually selected for cell-killing assays with ganciclovir, to confirm TK expression (10(3) cells/well in a 96-well dish). Northern analyses using MHC Class I and Class II complementary deoxyribonucleic acid probes were performed on blots containing total ribonucleic acid from wild-type tumor cells and HSV TK transfectants. A beta-actin complementary deoxyribonucleic acid probe served as an internal control. Cell surface expression was confirmed with flow cytometry. The induction of MHC Class I was tested for cycloheximide and genistein sensitivity. RESULTS: All cell cultures exhibited increases in MHC Class I but not MHC Class II expression, as determined by Northern analysis densitometry and flow cytometry. Cycloheximide treatment did not diminish the up-regulation of MHC Class I after retroviral transfection, implicating a signal transduction pathway that does not require ongoing protein synthesis. Genistein pretreatment of cell cultures did diminish the up-regulation of MHC Class I, implicating a tyrosine kinase in the signaling cascade. CONCLUSION: Induction of MHC Class I in rat and human glioma cells after HSV TK retroviral gene therapy is a primary effect that is dependent on tyrosine kinase activity. Specific immune responses generated after transfection may represent an important general side effect of gene therapy protocols. Elucidation of the mechanism of immunomodulation after gene therapy will likely yield safer and more effective clinical protocols.     

Suppression of anchorage-independent growth of human glioblastoma cell by major histocompatibility complex class I gene-transfection.

The host's immune system discriminates tumor cells from normal cells by recognizing the major histocompatibility complex (MHC) class I antigen expressed on the tumor cell membrane. However, the role of MHC class I antigen in tumor cells has not yet been clarified. In this study, the influence of MHC class I antigen expression on the tumorigenicity of a human glioblastoma cell line (KMG4) is examined. Barely detectable levels of MHC class I messenger ribonucleic acid were found to express in KMG4 cells by Northern blot analysis using mouse MHC class I (H-2Ld) and human leukocyte antigen (HLA)-B7 genes as probes. The H-2Ld gene connected at the downstream end of murine mammary tumor virus (MMTV)-promoter was cotransfected with the neomycine-resistant gene pSV2-neo into KMG4 cells, and the drug-resistant cells were selected. The KMG4 cells (KMG4-MMTV-Ld), which acquired the MHC class I gene were detected by Northern blot analysis with H-2Ld as the probe, and by immunohistochemistry using the H-2Ld-specific monoclonal antibody. Tumorigenicity, as determined by colony-forming ability in soft agar, was then compared between MHC class I-expressing KMG4-MMTV-Ld and nonexpressing control cells. The MHC class I-expressing cells were found to be deprived of colony-forming ability, indicating that MHC class I antigen could negatively influence the anchorage-independent cell growth of the human glioblastoma cell line KMG4.     

Prolonged survival of cardiac allografts in rats following the administration of heat-treated donor lymphocytes. A possible immunosuppressive role for class I major histocompatibility complex antigens

Pretransplant transfusions of spleen and lymph node cells heated to 45 degrees C or 50 degrees C for 1 hr prolong the survival of subsequent donor-specific heart grafts in the fully allogeneic donor-host combination DA (RT1a)----AS (RT1l). The results are comparable to survival times recorded following pretransplant transfusions of purified donor specific red blood cells (RBC) in the same strain combination. Both class I and class II major histocompatibility complex (MHC) antigens are serologically detectable on heat-treated cells; by contrast only class I antigens are expressed on red blood cells. Although heat-treated cells stimulate alloantibody formation, they fail to provoke a proliferative response in an in vivo host-versus-graft assay. Both red blood cells and heat-treated inocula persist in the host for long periods, possibly an important consideration in relation to their capacity to prolong the survival of subsequent donor strain allografts. The experimental data support the contention that class I MHC antigens can be immunosuppressive in the context of allografting. The present results recall the experiments carried out early in the century, which used heat-treated tumor cells to prolong the survival of subsequent viable tumor allografts, and which are sometimes cited as the first example of active enhancement.     

CD4+ T cell recognition of a single discordant HLA-A2-transgenic molecule through the indirect antigen presentation pathway induces acute rejection of murine cardiac allografts.

To further define the role of indirect allorecognition, cardiac allografts from HLA-A2-transgenic (HLA-A2+) C57BL/6 mice were heterotopically transplanted into normal C57BL/6, CD4 T cell-knockout (KO) C57BL/6 mice, CD8 T cell-KO C57BL/6 mice, fully MHC-discordant BALB/c mice (allogeneic control), and HLA-A2+ C57BL/6 mice (syngeneic control). HLA-A2+ grafts were acutely rejected when transplanted into BALB/c mice (mean survival time: 10+/-0.8 days), normal C57BL/6 mice (mean survival time: 16.5+/-2.1 days) as well as CD8-KO mice (mean survival time: 12.8+/-1.3 days). Histopathological analysis revealed classical acute cellular rejection with moderate to severe diffuse interstitial CD4+ and CD8+ cellular infiltrates and significant intra-graft deposition of IgG and complement. In contrast, HLA-A2+ grafts were not rejected when transplanted into CD4-KO mice or HLA-A2+ mice. CD8-KO recipients treated with an anti-CD4 monoclonal antibody, but not with an anti-NK monoclonal antibody, failed to reject their allografts with prolonged administration of antibody (30 days). Spleen cells from mice rejecting HLA-A2+ allografts failed to lyse HLA-A2+ target cells indicating a lack of involvement of CD8+ T cells in the rejection process. In contrast, spleen cells from rejecting animals proliferated significantly to both HLA-A2+ cells and to a peptide derived from the HLA-A2 molecule. Development of anti-HLA-A2 antibodies was observed in all animals rejecting HLA-A2+ allografts. These results suggest that indirect allorecognition of donor MHC class I molecules leads to rejection of cardiac allografts and development of alloantibodies in this unique transplant model in which there is a single MHC discordance between donor and recipient.     

Indirect allorecognition of donor class I and II major histocompatibility complex peptides promotes the development of transplant vasculopathy.

Recent clinical and experimental evidence suggests that indirect allorecognition may promote the development of chronic rejection, but definitive experimental studies are lacking. To study the contribution of indirect allorecognition to chronic rejection, na?ve Lewis (RT1(1)) rats were immunized with synthetic Wistar Furth (WF) class II-RT1(u).D (HLA-DR-like) or -RT1(u).B (HLA-DQ-like) or class I-RT1(u).A (HLA-A-like) peptides emulsified in complete Freund's adjuvant 7 d before transplantation (n = 5 to 7/group). Experimental and control animals then acted as recipients of fully mismatched WF vascularized cardiac allografts. Recipients received immunosuppression in the form of cyclosporine at a tapering dose that allows for long-term allograft survival. Animals were sacrificed at either 3 or 6 mo, with allograft arterial luminal occlusion scored on elastin stains by a blinded observer. At 3 mo, mean vessel scores were significantly higher in the RT1(u).A-immunized versus class II-immunized and control groups (P < 0.05). By 6 mo, there was progression of chronic allograft vasculopathy and a significantly higher mean vessel score in the RT1(u).A- and RT1(u).D-immunized versus RT1(u).B and control groups (P < 0.05). In vitro studies show evidence of shifting MHC allopeptide immunogenicity. It was concluded that T cells primed by specific donor class I and II MHC allopeptides promote the development of chronic vascularized allograft rejection. These novel observations provide definitive evidence of a link between indirect allorecognition and the development and progression of chronic rejection.     

Regulatory role of host CD8+ T lymphocytes in experimental graft-versus-host disease across a single major histocompatibility complex class II incompatibility

BACKGROUND: CD8+ T cells are known to regulate type 2 helper T cell (Th2) alloreactive immune responses but their mode of activation is unclear. We investigated the role of host CD8+ T cells in experimental Th2-type graft-versus-host disease (GVHD) where donor/recipient disparity is restricted to a single major histocompatibility complex (MHC) class II antigen. METHODS: Immunoglobulin (Ig) E serum levels, eosinophilia and lymphoid tissue hyperplasia were compared after injection of bm12 CD4+ T cells in either wild-type or CD8+ T cell-deficient (CD8-/-) C57BL/6 mice. In vitro, we explored effects of the addition of CD8+ T cells from wild-type or IFN-gamma-/- mice in mixed leukocyte cultures prepared with beta2 microglobulin-deficient (beta2m-/-) CD4+ T cells as responders or beta2m dendritic cells as stimulators. RESULTS: HyperIgE resolved after 3 weeks in wild-type hosts whereas it persisted for 6 weeks in CD8-/- hosts. Eosinophil infiltrates in lymph nodes were significantly enhanced in CD8-/- hosts. Increased serum levels of IL-5 and IL-13 in CD8-/- hosts confirmed the enhancement of Th2-type responses in the context of recipient CD8+ T cell deficiency. Hyperplasia of lymph nodes and spleen were similar in both groups, as well as in vivo proliferation of donor CD4+ T cells. In vitro, CD8+ T cell regulation of the alloreactive Th2 response depended on their production of IFN-gamma and did not require expression of beta2m on CD4+ T cells or antigen-presenting cells. CONCLUSIONS: Host CD8+ T cells regulate alloreactive Th2 responses during graft-versus-host disease through an IFN-gamma dependent pathway, independently of the recognition of beta2m-associated MHC class I molecules.     

Detailed analysis and demonstration of differences in the kinetics of induction of class I and class II major histocompatibility complex antigens in rejecting cardiac and kidney allografts in the rat

In this paper, we analyze in detail donor class I and class II major histocompatibility complex (MHC) antigen induction in heart and kidney allografts in the DA-to-PVG rat strain combination. The immunohistological techniques and quantitative absorption analyses utilize monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens, to enable precise interpretation of the results in terms of the MHC antigens of the graft. Quantitative absorption analyses were performed on homogenates comprising 4-6 allografts pooled at each interval examined (days 1-5 for kidneys, days 3-7 for hearts). In the heart allografts, donor class I antigen induction begins at day 3 after transplantation and proceeds rapidly on the 4th and 5th postoperative days. The maximum level (a 10-fold increase in comparison with normal heart) occurs at day 6, and thereafter the level declines. Donor class II antigen induction in the heart allografts follows a similar pattern. In kidney allografts, it was of particular interest that donor class I induction occurred much more rapidly, being already evident on the first postoperative day, and reaching levels 20-fold greater than normal kidney by day 3. Maximum levels (approximately 30-fold that of normal kidney) of donor class I antigens were reached on days 4 and 5. Donor class II induction, by contrast, developed in kidney grafts with kinetics similar to that seen for class II induction in heart grafts (beginning at day 3 and reaching a maximum of 7-fold over normal kidney at day 5). Immunohistological studies were performed at days 1, 3, 5, and 7 after transplantation. These confirmed the early induction of donor class I antigen in the kidney allografts. In kidney, by the fifth postoperative day, all tubules in the cortex and medulla, and the arteriolar vascular endothelium, were strongly positive for class II antigens. However, the glomerulus, including the glomerular capillary endothelium, remained donor-class-II-negative, except for induction of class II antigens on Bowman's capsule. The endothelium of interstitial capillaries also probably remained class-II-negative. These results have potentially important implications for understanding the development of the rejection response.     

Correlation between major histocompatibility complex class I molecules and CD8+ T lymphocytes in prostate,and quantification of CD8 and interferon-gamma mRNA in prostate tissue specimens

OBJECTIVE: To investigate the immunology of host-tumour interaction, critical for the development of immunotherapy against cancers, by assessing the major histocompatibility complex (MHC) class I expression in both benign and malignant prostate disease, and the relationship between their expression and degree of tumour-infiltrating lymphocytes. MATERIALS AND METHODS: Direct serial analysis of gene expression in tumours is an extremely sensitive and powerful tool for monitoring immunological changes in the immunotherapy of solid tumours. Most previous monitoring protocols rely mainly on the analysis of patient's peripheral blood but in the present study the direct molecular analysis of small tissue samples was used, and its accuracy compared with that of conventional immunohistochemical analysis. Twenty-four formalin-fixed, paraffin-embedded prostate samples (11 benign and 13 carcinoma) were used for the immunohistochemical analysis of CD8+ T lymphocytes and MHC class I expression. CD8+ T lymphocytes were counted using an ocular grid and MHC class I measured using digital image-analysis software. Twenty-seven frozen prostate tissue samples (12 benign and 15 carcinoma) were used for direct gene measurements of CD8 and interferon-gamma using a quantitative real-time polymerase chain reaction. RESULTS: There were significantly fewer CD8+ T lymphocytes in prostate carcinoma nests than in benign prostate. There was a significant correlation between the number of CD8+ T lymphocytes and MHC class I expression in the prostate. There was a strong correlation between the immunohistochemical estimates of CD8+ T lymphocytes and CD8 gene by polymerase chain reaction, but no significant difference between benign prostate and prostate carcinoma tissue in gene measurements. CONCLUSION: Down-regulation of MHC class I expression by prostate cancer cells is associated with fewer CD8+ T lymphocytes and hence might be important in cancer growth. In addition, the measurement of gene expression in small tissue samples might be useful for monitoring the efficacy of treatment throughout cancer therapy.     

Autoimmune mechanisms in thromboangiitis obliterans (Buerger's disease): the role of tobacco antigen and the major histocompatibility complex.

This study is a continuation of our previous work that showed that patients with thromboangiitis obliterans (TAO; Buerger's disease) demonstrate a cell-mediated immune response to human artery type-specific collagens. To investigate the role of cigarette smoking in patients with TAO, cellular and humoral sensitivity was tested to a tobacco glycoprotein (TGP) antigen in 13 patients with Buerger's disease, 16 healthy smokers, and 12 nonsmoking healthy young male subjects. In this study, patients with Buerger's disease and healthy smokers had the same rate of cellular response to TGP, whereas nonsmokers did not respond. All three groups had a 30% to 40% measurable antibody response to TGP. If TGP has an immunologic role in the pathogenesis of TAO, an additional factor (or factors) may be operative. A specific genetic makeup may be one such factor, although at this stage other pathogenic mechanisms cannot be ruled out. Eleven patients with Buerger's disease and two control groups of 10 young healthy smoking male subjects and 12 young nonsmokers underwent histocompatibility leukocyte antigen (HLA) typing. Patients with Buerger's disease had a statistically significantly higher frequency of HLA-DR4 and a significantly lower frequency of the HLA-DRW6 antigen than had both control groups. Because similar findings have been reported in other autoimmune diseases, this observation may serve as further evidence that an autoimmune mechanism is involved in Buerger's disease.