Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

Antisera Recognising HLA-A, -B and DRW Antigens Raised in Rhesus Monkeys

Rhesus monkeys were immunized with partially purified HLA-A, -B, -C and DR antigens. The resulting sera were shown to have activity against species-specific determinants on both HLA-A, -B, -C chains and beta 2 microglobulin by the use of somatic cell hybrids. When this was removed by absorption, the sera showed activity against three of the four HLA-A and -B antigens in the immunogen when tested on a panel of peripheral blood lymphocytes and T cells. Antibodies recognizing HLA-DR antigens were detected by testing platelet absorbed sera on a panel of typed lymphoblastoid cell lines. After absorption to remove activity against species-specific determinants on the HLA-DR antigens, two cross reacting specificities were defined. One consisted of a determinant in common between HLA-DRw1, 2 and 6 and the other a putative determinant in common between HLA-DRw4, and 5. The nature and significance of these cross-reacting groups of HLA-DR antigens is discussed in the light of current HLA-DR serology and the nature of HLA antigens in general.     

HLA-D typing in Austria. A panel study using 26 newly defined homozygous typing cells

A panel of 120 HLA-A, -B, -C and -DR typed Austrians has been typed for HLA-D by the use of 26 Homozygous Typing Cells (HTC). The new Austrian HTC, partly defined by the 9th International Histocompatibility Workshop (9WS), partly by a checkerboard experiment with internationally well defined reference HTC, type for HLA-Dw1 to -Dw7 and an obviously new, so far unknown HLA-DR2 related HLA-D determinant. Associations of HLA-DR and HLA-D antigens in Austria and their frequencies are determined. Antigen frequencies in Austria are compared to frequencies in other Caucasoid populations.     

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.     

Nonimmune human cells can express MHC class II antigens in the absence of invariant chain--an immunohistological study on normal and chronically inflamed small intestine

The expression of HLA-DR, HLA-DP and HLA-DQ antigens and of the associated invariant chain (Ii) was studied immunohistologically in sessile cells of normal ileum and ileum affected by Crohn's disease which was taken as a model for chronic inflammation. Corresponding to the local inflammatory cell density, a considerable neo-expression of MHC class II antigens and Ii was observed in epithelial cells, vascular endothelial cells, and Schwann cells of the enteric nerve plexus. While HLA-DP and HLA-DQ antigens were undetectable in sessile cells of normal ileum, class II antigens expression in ileitis followed the order HLA-DR greater than or equal to HLA-DP greater than or equal to HLA-DQ. In various cell types a differential expression of Ii and class II antigens was noted. In normal crypt enterocytes and in arterial endothelial cells of inflamed ileum, Ii was found in the absence of class II antigens. On the other hand, most Schwann cells and internodal nerve strands of the submucous and myenteric plexus exhibited HLA-DR (-DP, -DQ) antigens in the absence of detectable Ii. Moreover, HLA-DR+ endothelial cells in normal tissue specimens were Ii-, and in inflamed intestine HLA-DR expression of venous/venular and capillary endothelium greatly exceeded Ii expression. The observation of both Ii+/HLA-DR- and Ii-/HLA-DR+ (HLA-DP+, HLA-DQ+) cells questions the previously assumed close association of Ii and class II antigen expression.     

Planned immunization of human volunteers with HLA-DR incompatible lymphocytes

Aiming at the production of anti HLA-DR test sera, eight healthy human volunteers were immunized by repeated intradermal injections of lymphocytes which were selected to be incompatible for one HLA-DR antigen, and matched as well as possible for HLA-A,-B,-C antigens. One out of 3 recipients immunized exclusively against HLA-DR produced lyrnphocytotoxic HLA-DR antibodies. The remaining 5 recipients were immunized against 1 or more HLA-A,-B,-C antigens in addition to one HLA-DR antigen. After 3 immunizations, 3 of these reacted with strong HLA-A or -B antibody production; however, only one showed a parallel anti HLA-DR antibody response detectable by complement dependent lymphocytotoxicity.
Testing of the recipient sera in the antibody dependent cell-mediated cytotoxicity (ADCC) assay revealed that 6 of the 8 recipients did react early to the immunizations with HLA specific antibody production. However, in spite of repeated booster injections it was not possible to obtain more than the above-mentioned 2 sera with HLA-DR antibodies strong enough to react in the lymphocytotoxicity microtechique.     

Evidence for Two Series of B-Cell Antigens in Man and Their Comparison with HLA-D

Population and family studies of five sera with antibodies against B-cell determinants show an excellent fit of some of them with the HLA-D determinants, suggesting that the HLA-D determinants themselves or closely linked structures can be recognized by serology. One serum, Ag, might be coded for by a locus different from HLA-D, which could be located between HLA-A and -B. A two-color fluorescence test to detect B-cell antigens was used and found to save time and reagents and to give very good results.     

HLA-DR genes and antigens in the Danish population.

Five hundred unrelated Danes, including 202 healthy individuals, 35 cadaveric kidney donors, and 263 patients in terminal uraemia were typed for the HLA-DR antigens DR1—w8.
The HLA-DR gene frequencies of healthy Danes are in good agreement with frequencies estimated during The Eighth International Histocompatibility Workshop of the Scandinavian population, but differ to some extent from other groups of European Caucasians. This indicates geographical variation in the distribution of DR genes within Europe.
Very close associations were found between the HLA-DR antigens and their corresponding HLA-D specificities, except for Dw4 and Dw6, which were included in DR4 and DRw6, respectively.
Linkage disequilibrium was calculated between alleles of two loci ( HLA-A,-DR; -B-DR and -C,-DR ) using the population data. Furthermore, linkage disequilibrium involving HLA-DR alleles was assessed for multiple loci by using haplotype data from 32 HLA-A,-B,-C,-D,-DR, and Bf typed Danish families.
The haplotype data showed that HLA-DR4 is strongly associated with HLA-B15 only in haplotypes together with HLA-Dw4. This indicates genetic heterogeneity of HLA—DR4 which, however, does not appear serologically in this study.     

Cell mediated cytotoxicity against HLA-D region products expressed in monocytes and B lymphocytes. III. Inhibition by monoclonal antibodies and study of an HLA-B/DR recombinant

Attempts to further define the antigens recognized by HLA-D region specific cytotoxic lymphocytes were undertaken using monocytes and transformed B cell lines as target cells. Monoclonal antibody against framework determinants of HLA-DR antigens partially blocked cell mediated lysis, suggesting that at least a portion of the D-region specific cytotoxic cells recognized the HLA-DR determinants. The study of a family with an HLA-B/DR recombinant showed that the determinants recognized by allogeneic anti-HLA-D-region cytotoxic lymphocytes are encoded outside of HLA-B. In addition, cytotoxic lymphocytes specific for the HLA-D region could be generated with cells identical throughout the interval from HLA-A to B and disparate only to the left of HLA-B.     

Tumor cell expression of HLA-DM associates with a Th1 profile and predicts improved survival in breast carcinoma patients

Studies aimed at elucidating the immunological and prognostic significance of HLA-DR expression on breast carcinoma cells have yielded contradictory results. To expand on previous studies, we have investigated the associations of tumor cell expression of HLA-DR and its related co-chaperones, invariant chain (Ii) and HLA-DM, with infiltrating inflammatory cells, in situ cytokine mRNA levels and prognosis and outcome in 112 breast carcinoma patients with a median follow-up of 59 months. While the majority of HLA-DR+ tumors co-express Ii, only a minority express HLA-DM. Tumor cell expression of HLA-DR and co-chaperones positively associated with both infiltrating CD4+ and CD8+ T-cell subsets (P < 0.01). Expression of HLA-DR and Ii associated with decreased estrogen receptor alpha levels and younger age at diagnosis, suggesting a role for hormones in the control of HLA class II expression in breast carcinoma. Patients with DR+Ii+DM- tumors had markedly decreased recurrence-free and disease-specific survival as compared with patients with DR+Ii+DM+ tumors (P < 0.05) and HLA-DR/co-chaperone expression was an independent predictor of survival by multivariate Cox regression analysis, controlling for standard prognostic indicators. Tumors that co-express HLA-DR, Ii and HLA-DM have increased levels of IFN-gamma, IL-2 and IL-12 mRNA, suggesting improved survival of patients with DR+Ii+DM+ tumors may be attributable to Th1-dominated immunity. We conclude that expression of determinants of the immune response by tumor cells may influence breast tumor progression and patient outcome.     

Non-co-ordinate expression of HLA-DR antigens and invariant chain

The expression of HLA-DR and MHC class II antigen-associated invariant chain (Ii) was studied in normal colorectal mucosa, adenomas and carcinomas using monoclonal antibodies and a sensitive immunoperoxidase technique on frozen sections. In six of 15 specimens of normal mucosa, epithelial cells expressed Ii, but were unreactive for HLA-DR antigens. In 15/20 adenomas and 51/70 carcinomas, Ii-positive tumour cells clearly outnumbered HLA-DR-positive tumour cells. Expression of Ii in non-neoplastic epithelium adjacent to carcinoma was much stronger than expression of HLA-DR. The results indicate that in certain tissues expression of these two antigens is not closely associated.     

T lymphocyte subsets in human intestinal mucosa: the distribution and relationship to MHC-derived antigens

T lymphocytes in the normal human intestinal tract have been analysed in tissue sections by a double-marker immunofluorescence technique, combining antiserum to T lymphocyte antigen (HuTLA) with a monoclonal antibody detecting T cells of suppressor-cytotoxic phenotype (OKT8). The distribution of HLA-A -B, -C and Ia-like antigens in intestinal mucosa was also examined by a similar method. In small and large intestine 67 to 90% (mean 70%) of intraepithelial T lymphocytes were of suppressor-cytotoxic phenotype (OKT8+). In contrast, only 27 to 56% (mean 39%) of lamina propria T cells were OKT8+. Intestinal epithelial cells demonstrated strong membrane staining for HLA-A, -B, -C antigens. Ia-like antigens were detected on the epithelial cells of small intestinal villi, but not on colonic epithelial cells. Lamina propria macrophages expressed both HLA-A, -B, -C and Ia-like antigens, the latter having strong membrane and cytoplasmic fluorescence. The distribution of T cells with suppressor-cytotoxic or inducer phenotype in the intestinal epithelium and lamina propria may be related to the differential expression of Ia-like and HLA-A, -B, -C antigens in intestinal mucosa.     

HLA—A, —B, and —D Antigens in Paralytic Poliomyelitis

Sixty-two unrelated Caucasian patients from the Munich area who had had paralytic poliomyelitis in the 1950s and the early 1960s were analyzed. HLA-A and -B typing was performed for 26 antigens. HLA-D tying was done using six different established homozygous typing cells defining the specificities Dw1-Dw5 and Dw11, plus one locally defined typing cell. None of the HLA-A, -B or -D determinants showed a significant deviation in frequency from control populations. Of interest may be a decrease of B8 and an increase of Bw16 (Bw38/39) and B27, but these deviations are not significant in their P values are corrected for the number of comparisons made.     

Sequential induction of MHC antigens on autochthonous cells of ileum affected by Crohn''s disease.

Changes were examined in the expression of Class I and II major histocompatibility complex (MHC) antigens by autochthonous cells of the terminal ileum affected by Crohn's disease. The study was based on the analysis of transmural specimens from terminal ileum segments obtained in the course of ileocolectomy for colon cancer and Crohn's disease. Serial sections were immunostained using monoclonal antibodies directed against monomorphic determinants of HLA-A,B,C, DR, DP, DQ, and the invariant chain (Ii) associated with Class II molecules. Compared with the normal state, the only change in Class I antigen expression occurring in Crohn's disease was the induction of HLA-A,B,C antigens in lymphatic endothelium. Changes in Class II antigen expression were more substantial. Enhancement of HLA-DR expression was found in enterocytes; DR induction was observed in glial cells of the visceral nervous plexus and in venular and venous endothelium. HLA-DP and DQ antigens were induced in enterocytes, glial cells, and capillary and venular endothelium, although this induction was restricted to areas of moderate or high inflammatory activity. The tissue distribution of Ii closely resembled that of HLA-DR, although this association was not strict: on the one hand, arterial endothelium contained low amounts of Ii in the absence of DR antigens; on the other hand, glial cells expressed Class II molecules in the absence of Ii. The extent of local enhancement/induction of MHC antigens was positively correlated with the local density of the cellular infiltrate. These data suggest that altered MHC antigen expression by autochthonous structures might be mediated by factors released from the lymphohistiocytic infiltrate, which is itself attracted by an unknown signal. In conjunction with an unknown antigen, the enhanced expression of Class II antigens might trigger an autoaggressive immune response.     

Differential expression of HLA-DR,HLA-DP,HLA-DQ and associated invariant chain (Ii) in normal colorectal mucosa,adenoma and carcinoma

Summary The expression of MHC class II antigens (HLA-DR, HLA-DP and HLA-DQ) and the associated invariant chain (Ii) was studied in epithelial cells of normal colorectal mucosae, colorectal adenomas and carcinomas, using a sensitive immunoperoxidase technique with monoclonal antibodies on frozen sections. In contrast to class II antigens, Ii was detected in some normal mucosae distant from the tumour. In residual non-neoplastic mucosa adjacent to carcinomas, Ii and class II antigens were induced in the order Ii HLA-DRHLA-DPHLA-DQ, the reactions being most pronounced in cases with inflammatory alteration of the crypts. In 22/37 adenomas and 77/123 carcinomas, Ii expression clearly exceeded class II antigen expression. Class II antigens were found in 20/37 adenomas and 62/123 carcinomas, mostly in a non-coordinate manner, following the above order. A detailed analysis of the expression patterns in normal and neoplastic colon epithelial cells revealed a closer association of HLA-DP with HLA-DQ than of HLA-DR with HLA-DP, or HLA-DQ.     

Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs I

Cytotoxic T lymphocytes were activated in primary one-way mixed lymphocyte cultures of cells matched for serologically defined HLA-A, -B and -C antigens. In 16 out of the 29 combinations mismatched for the HLA-D/DR antigens, cell-mediated lympholysis of the stimulator cells occurred. The specificity of 5 selected cytotoxic T lymphocytes was studied in detail. Three of these cytotoxic T lymphocytes recognize antigenic determinants associated with HLA-Bw35 (Breuning et al. 1984, II). The 2 other cytotoxic T lymphocytes failed to lyse T-target cells enriched by rosetting with sheep red blood cells, whereas target cells from the 'non-T' fraction were strongly lysed, indicating that antigenic determinants associated with Class-II HLA molecules were the targets recognized by these cytotoxic T lymphocytes. This notion was supported by a study of a panel of HLA-typed third-party target cells. One cytotoxic T-lymphocyte population preferentially lysed HLA-DR2-positive target cells. Family studies, including a family with a recombination between HLA-B and -D, showed that the target antigen recognized by the latter cytotoxic T lymphocyte segregated with DR2. The second cytotoxic T-lymphocyte population recognized a determinant associated with DRw8. However, in 13 of the 29 HLA-A-, -B- and -C-identical, D/DR-different combinations, cell-mediated lympholysis of stimulator target cells could not be detected, not even on enriched 'non-T' target cells. Thus, after primary mixed lymphocyte culture of HLA-A-, -B- and C-identical, HLA-D/DR-non-identical cells, cytotoxic T lymphocytes directed against sensitizing Class-II molecules can be detected in some combinations, but not in others.     

Serological identification of five HLA-D associated (Ia-like) determinants.

Five antisera raised within HLA-A and -B compatible, HLA-D disparate combinations were reactive in a modified NIH microcytotoxicity test with B lymphocytes, but not with T lymphocytes from the immunizing donor, as well as with B lymphocytes from most or all donors sharing his immunizing HLA-D phenotype. Four antisera recognized structures closely associated with the HLA-D determinants Dw2, Dw3, Dw4 and LD 108. One serum had a broad reactivity pattern including Dw3, Dw6 and some unknown specificity(ies). In population and family studies, these B lymphocyte antigens behaved as if they were governed by one genetic locus in the B-D region of the HLA complex. We conclude that the antisera produced by this method recognize Ia-like antigens identical to or very closely associated with the HLA-D determinants.     

HLA-DP antigens provide the proliferative impetus for the generation of cytotoxic T lymphocytes

Investigating HLA-A, -B, -C, -DR and -Dw antigens and MLR, CML, and PLT reactivity in two unrelated persons, it was found that, despite their HLA-D/DR identity, cytotoxic T lymphocytes (CTL) could be induced in the CML assay. The HLA-DP antigens proved to provide the proliferative impetus for the generation of CTL.     

HLA-A, -B, and -DR specificities on human monocytes

Monocyte-enriched cell suspensions obtained by a plate adherence method from 57 blood donors were typed by microcytotoxicity for HLA-A, -B and -DR determinants. Parallel assays were performed with autologous unfractionated lymphocytes for HLA-A and -B determinants and with autologous B-lymphocytes for HLA-DR determinants. Correlation coefficients (r values) were calculated for nine HLA-A specificities (r > 0.79), 16 HLA-B specificities (r > 0.56) and seven HLA-DR specificities (r > 0.81). For certain HLA-A and -B specificities detection was less readily achieved on monocytes than on autologous lymphocytes. Extra reactions were observed in some instances. With sera defining HLA—DR specificities, monocytes and B-cells showed reactivity of almost identical strength and reliability.