Expression of major histocompatibility complex antigens on the bovine corpus luteum during the estrous cycle, luteolysis, and early pregnancy.

Treatment of cultured bovine luteal cells with the cytokine, interferon-gamma, induces the expression of Class II major histocompatibility complex antigens (MHC Ags). To determine if Class II MHC Ags are present on the CL in vivo and if the degree of Ag expression changes during luteal life span, bovine corpora lutea were obtained on Day 6, Days 10-12, and Day 18 of the estrous cycle and MHC Ag expression was evaluated via indirect immunofluorescence. Flow cytometry was used to determine the percentage of MHC Ag-positive cells on cell populations distinguished by cell size and intracellular density. Minimal Class II MHC Ag expression was detected on Day 6 CL (approximately 25%), which consisted primarily of smaller cells. The midcycle and late CL consisted of these small cells (SC) and two populations of large cells that differed in intracellular density, or right-angle light scatter. In midcycle CL, few (less than 25%) SC or large, dense cells (LDC) expressed the Class II MHC Ag whereas a high percentage (75%) of the large, less-dense cells (LLDC) were Class II MHC Ag-positive. Class II MHC Ag expression remained negligible on the LDC of the Day 18 CL; however, there was an elevation in the percentage of SC and LLDC expressing Class II Ag (p less than 0.05). To determine if Class II MHC Ag expression also varied with different functional states of the CL, bovine CL were collected after prostaglandin (PG) F2 alpha-induced regression and on Day 18 of early pregnancy. When luteolysis was allowed to progress in vivo, the percentage of Class II MHC Ag-positive cells was increased in all cell populations (p less than 0.05). Class II MHC Ag expression was significantly lower (p less than 0.05) on the three cell populations comprising the CL of pregnancy as compared to the Day 18 cyclic CL. It is hypothesized that enhanced expression of Class II MHC Ags on the late CL and during PGF2 alpha-induced regression may potentiate immune response mechanisms for luteolysis.     

The RAG cell line defines a new complementation group of MHC class II deficiency

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN- ofDMA, DMB, and theinvariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designatedF16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding humanCIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.     

Immunolocalization of beclin 1, a bcl-2-binding,autophagy-related protein,in the human ovary: possible relation to life span of corpus luteum

Ovarian tissue homeostasis is maintained by highly regulated cyclic phases of cell proliferation/differentiation and programmed cell death. Compelling evidence indicates that both apoptotic and autophagic types of programmed cell death are involved in the regression of the corpus luteum (CL) in primate species. Beclin 1 is an autophagy-related protein that is involved in the inter-relationships between apoptosis and autophagy, through interaction with the anti-apoptotic protein bcl-2. We studied the presence and expression pattern of beclin 1 in the adult human ovary. In ovarian follicles, beclin 1 immunostaining was found in the theca layer, whereas granulosa cells were negative. After ovulation, beclin 1 immunostaining was present in both theca-lutein and granulosa-lutein areas. The expression of beclin 1 in granulosa-lutein cells was related to the functional and structural status of the CL, being strong at the early and mid luteal phases, barely detectable at the late luteal phase, and absent in granulosa-lutein cells in subsequent cycles. Our results indicated that beclin 1 expression was related to luteal cell survival rather than to cell death. Accordingly, persistent beclin 1 expression was found in granulosa-lutein cells under either physiological (i.e., CL of pregnancy) or pathological (irregularly regressing CL in climacteric women) conditions involving prolonged CL life span. Strong beclin 1 immunostaining was also found in ovarian androgen-producing cells (i.e., secondary interstitial and hilus cells). Our data thus suggest that beclin 1 plays important roles in the regulation of the life span of human CL and ovarian androgen-secreting cells, by maintaining autophagy at levels promoting cell survival rather than cell death. This study was subsidized by grant BFU 2005-01443 from the DGICYT (Spain).     

B-L antigens (Class II) of the chicken major histocompatibility complex control T-B cell interaction

The detailed study of the genetic control of T-B cell interactions in the chicken has been hampered by the lack of defined major histocompatibility complex (MHC) recombinant chicken lines. In the present study we have used some recently described MHC recombinant chicken lines separating regions encoding antigens that are homologous to class I and class II antigens of mammals in adoptive bursa cell transfer experiments, in which bursa cells from newly hatched chicks were transplanted into cyclophosphamide (Cy)-treated chicks. Subsequent immunizations of the recipients with a thymus-dependent antigen (SRBC) and a thymus-independent antigen (Brucella abortus) showed that the generation of germinal centers in the spleen and the production of antibodies to SRBC required identity between donor and recipient class II antigens (B-L antigens), whereas response to Brucella antigen did not require identity at any of the known MHC loci of the chicken. The results thus reveal that also in the chicken class II (B-L) region genes encode cell-surface glycoproteins that serve as restriction elements in T-B cell cooperation.This work has been presented in part at the Ninth International Congress of the Transplantation Society (Vainio et al. 1983a).     

The influence of embryo presence on prostaglandins synthesis and prostaglandin E2 and F2alpha content in corpora lutea during periimplantation period in the pig

We determined the expression of PGE2 synthase (mPGES-1), PGF synthase (PGFS), carbonyl reductase/prostaglandin 9-ketoreductase (CBR1) genes and the content of PGE2, PGF2alpha in porcine corpora lutea on Days 12-14 of pregnancy and Days 12-14 of the estrous cycle. For this study we used a surgically-generated model in which one of the uterine horns was cut transversely and a part of this horn was detached from the uterine corpus. The expression of mPGES-1, PGFS, and CBR1 genes and mPGES-1/PGFS ratio were significantly higher in corpora lutea of the pregnant gilts compared to the corpora lutea from the parallel ovaries of the cyclic gilts. There was no difference in mPGES-1, PGFS, CBR1 genes expression and mPGES-1/PGFS ratio between corpora lutea ipsi-(CL1) and contralateral (CL2) to the uterine horn with the developing embryos. The highest content of PGE2 was found in CL1 of the pregnant gilts. The PGE2/PGF2alpha ratio was significantly higher in CL1 of the pregnant gilts compared to corpora lutea from parallel ovary of the cyclic gilts. We suggest that the activity of the investigated genes is induced by compounds of embryonic origin which are not distributed only to the ipsilateral ovary but are transported within the mesometrium to both ovaries in a more systemic manner.     

Immunohistochemical localization of PGF2 alpha receptor in the rat ovary.

As a step towards understanding the role of prostaglandin F2 alpha (PGF2 alpha) in ovarian function, a rabbit antiserum against purified PGF2 alpha receptor (PGF2 alpha-R) was produced. This report details the use of this antiserum in immunohistochemical staining of ovaries of non-pregnant and pregnant rats to ascertain which cell types, in vivo, possess PGF2 alpha-R. In non-pregnant rats, three ovarian cell subpopulations contain immunoreactive PGF2 alpha-R. These include: a subpopulation of the cells found in corpora lutea, a subpopulation of the thecal cells surrounding secondary and mature (Graafian) follicles, and a subpopulation of primary and secondary interstitial cells. The ovarian tissues and cell types in which immunoreactive PGF2 alpha-R cannot be demonstrated include: the serosa overlying the ovary and its vessels, the coelomic epithelium and its underlying cortical stroma, medullary stroma and vessels, granulosa cells of primary, secondary and mature follicles, the oocyte, and the blood vessels and stroma within corpora lutea. PGF2 alpha-R immunohistochemical staining of corpora lutea from non-pregnant animals was examined both prior to the start of luteolysis and during luteolysis. During luteolysis, cells undergoing apoptosis stained for the presence of PGF2 alpha-R. PGF2 alpha-R immunohistochemical staining was also examined in corpora lutea during pregnancy and until 4 days postpartum. The major findings here were the apparent large increase in staining intensity of granulosa-lutein cells during pregnancy, and the loss of PGF2 alpha-R immunopositivity of the granulosa-lutein cells during the postpartum period. In summary, three ovarian cell subpopulations, all of which can secrete steroids, possess immunoreactive PGF2 alpha-R.     

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries.     

Targeting of superantigens

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on MHC class II antigens. In order to induce T lymphocytes to reject a tumor, we substituted the specificity of SEA for MHC class II molecules with specificity for tumor cells by combining SEA with a MAb recognizing colon carcinomas. Chemical conjugates or recombinant fusion proteins of the MAb C215 and SEA retained excellent antigen binding properties whereas the binding to MHC class II was markedly reduced. The hybrid proteins directed SEA responsive T cells to tumors with specificity determined by the specificity of the MAb. Significant tumor cell killing was obtained at picomolar concentrations of the hybrid proteins and was the result of direct cell mediated by cytotoxicity as well as production of tumoricidal cytokines by T cells. Targeting of superantigens represents a novel approach to specific immunomodulation and deserves further study as a potential therapy for malignant disease.     

Expression of invariant chain (CD 74) and major histocompatibility complex (MHC) class II antigens in the human fetus.

During the initiation of an immune response, antigen-presenting cells employ MHC class II antigens as key molecules to present small peptides to CD4-positive lymphocytes. The invariant chain (Ii; CD74) plays a critical role in this process by influencing the expression and peptide loading of the MHC class II molecules. Therefore, coordinate expression of these molecules is believed to play an important role in antigen presentation. This study explores the expression of these molecules in fetal tissues. Formalin-fixed, paraffin-embedded multi-organ tissue blocks from aborted fetuses (age range 7-22 weeks) were immunostained for Ii/CD74 and MHC class II antigens using commercially available monoclonal antibodies for Ii/CD74 (LN2) and MHC class II antigens (LN3), respectively. Coordinate staining for Ii/CD74 and MHC class II antigens was seen in the skin, proximal renal tubules, tips of small intestinal mucosa, and cells of the reticuloendothelial system, including the spleen and thymus. Expression of Ii/CD74, but not of MHC class II antigens, was seen in pulmonary alveolar epithelium in all cases and in testicular Leydig cells (11 of 11 testes examined). The distribution and intensity of staining did not change significantly with age. In conclusion, this study describes distribution of Ii/CD74 and MHC class II antigens in human fetal tissues. Coordinate expression of Ii/CD74 and MHC class II antigens was identified in most fetal tissues, but there were also notable exceptions. In all cases this took the form of expression of Ii/CD74 in the absence of MHC class II expression. Discordance was particularly striking in pulmonary alveolar epithelium and testicular Leydig cells. This suggests that the Ii/CD74 molecule has functional roles in addition to its role in antigen presentation.     

Immune Surveillance via Self Digestion

The adaptive immune system is orchestrated by CD4⁺ T cells. These cells detect peptides presented on Major Histocompatiblity Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4⁺ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4⁺ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.

Addendum to:

MHC Class II Antigen Loading Compartments Continuously Receive Input from Autophagosomes

Dorothee Schmid, Marc Pypaert and Christian Münz

Immunity 2006; In press     

Monoclonal Antibodies againstAntigens of Hydra vulgaris and Hydra oligactis

The goal of the study was to obtain a panel of monoclonal antibodies (MAb) against antigens of freshwater polyps of the genus Hydra. Hybrid mice F₁(Balb/c × SJL/J) were immunized with cell membrane fraction of H. vulgaris and three months later their splenocytes were fused with cultured mouse myeloma cells 653A. Testing of culture fluids in ELISA with immobilized H. vulgaris cells, 82 hybridomas producing MAb were revealed. Study of MAb specificity in ELISA with H. vulgaris and H. oligactis cells indicated that 22% of them recognized only H. vulgaris antigens. About 50% of MAb recognized equally antigens of the both species. The rest of MAb reacted with H. vulgaris and H. oligactis antigens to different degree. Eight hybridomas producing MAb of all three above groups were adapted for growth as ascitic tumors. The distribution of antigens binding these MAb was studied in indirect immunofluorescence on fixed polyps, living or fixed cells, and on paraffin- embedded sections. Among the best studied MAb, of the greatest interest were the following reagents. One of them (1A10) revealed an antigen on surface membranes of ectodermal epithelial cells of H. vulgaris. The second one (1G10) was specific of the antigen located in mesoglea and basal cytoplasmic areas of ectodermal and entodermal epithelial cells of the both hydra species. The MAb 4G3 interacted with cytoplasmic antigen of ectodermal epithelia-muscular cells of the both hydra species. MAb 4H1 revealed nematocytes in H. vulgaris and H. oligactis. The data obtained indicate that in two species of hydra the epitopes binding the same MAb might be located in cells of different types.     

Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy

Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4⁺ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluoresence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37°C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37°C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1)T=0, continuous peripheral staining; (2)T=15 min, patchy peripheral staining; (3)T=30 min, patches or intracellular vesicular staining; (4)T=45 min, intracellular vesicular staining; (5)T=60 min, diffuse intracellular staining; (6)T=90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules. The use of the two complementary techniques allows us to characterize the spontaneous endocytosis of HLA-DR molecule by freshly isolated LC. Thisin vitro study model might be useful for testing the sensitizing potential of different chemical substances.Abbreviations Ab antibodies - APC antigen-presenting cells - BG Birbeck granules - DNCB 1-chloro-2,4-dinitrobenzene - DNFB 2,4-dinitrofluorobenzene - DTAF dichlorotriazinylfluorescein - FSC forward light scatter - LC Langerhans cells - LSCM laser scanning confocal microscopy - MHC major histocompatibility complex - MAb monoclonal antibodies - PFA paraformaldehyde - SSC side light scatter     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG,and in pregnant rat ovaries

Summary Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection.At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts.In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera. The luteal cells were increased in size during pregnancy. And weakly positive reaction was detected on day 7 of pregnancy, then the immunoreaction became stronger in the corpora lutea on day 15 and 19 of pregnancy.The localization of aromatase was immunocytochemically examined in immature rat ovaries treated with PMSG and hCG injection, and the reaction of the granulosa cells of the antral follicles against anti-aromatase antibody became strongly positive about 12 h before ovulation and the became very weak suddenly after ovulation. In rat-ovaries, the pregnant corpora lutea was positively stained for aromatase after day 7 of pregnancy.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA     

Role of oncogenes in the regulation of MHC antigen expression.

Class I and class II major histocompatibility complex (MHC) antigens are required for CD8+ cytotoxic T cells and CD4+ helper T-cells, respectively, to recognize foreign antigen. Regulating the levels of expression of these MHC antigens regulates the T-cell responses [1]. This regulation is mainly carried out by the interferons (IFN), which are produced in the disease state. Type I IFN (IFN alpha or IFN beta; collectively 'IFN alpha beta) up-regulates class I MHC and IFN gamma up-regulates class I and class II MHC. We and others [1-3] have shown that transfection of cells with a variety of oncogenes including ras and myc affects the level of MHC antigen expression. This and other data provide evidence for a scheme in which the signal transduction mechanisms whereby IFN up-regulates MHC antigens involve several (proto) oncogenes.     

Proteasome and class I antigen processing and presentation

The recent discovery of two proteasome homologous genes,LMP2 andLMP7, in the class II region of the human MHC, has implicated this multi-subunit protease in an early step of the immune response; the degradation of intracellular and viral proteins. Short peptides produced by the proteasome are transported into the ER by the product of another set of MHC class II genes,TAP1 andTAP2, where they bind and stabilise HLA class I molecules. Antigenic peptides displayed at the cell surface by HLA class I molecules mark cells for destruction by cytotoxic T lymphocytes. The role of the proteasome in antigen processing was questioned when mutant cells, which lack theLMP genes, were able to process and present antigens normally. The discovery that two proteasome -subunits, delta andMB1, highly homologous toLMP2 andLMP7 and expressed in reciprocal manner, is now consistent with a role for the proteasome in antigen processing. The incorporation of different -subunits into the proteasome may be a mechanism to modulate catalytic activity of the proteasome complex, allowing production of peptides that are more suitable to enter into the ER by the TAP transporters and to bind HLA class I molecules. But, in the absence of the LMPs, the other subunits permit processing of most antigens reasonably efficiently.Abbreviations ABC ATP-binding cassete - 2m 2-microglobulin - ER endoplasmic reticulum - IFN interferon - LMP low molecular weight peptide - MHC major histocompatibility complex - TAP transporter associated with antigen processing     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Immunocytochemical localization of oxytocin and neurophysin in human corpora lutea

Corpora lutea, corpora albicantia, and ovarian stroma from normal human premenopausal ovaries were examined for the presence of oxytocin and neurophysin by using highly specific antisera and peroxidase-antiperoxidase light-microscopic immunohistochemistry. Oxytocin and neurophysin immunoreactivity was found in some but not all cells of the corpora lutea obtained on days 19 to 24 of the menstrual cycle. Stromal tissue and corpora albicantia did not give a positive reaction for either of these peptides, and negative results were also obtained with corpora lutea of mid- and term-pregnancy and preovulatory follicles. Specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive oxytocin and neurophysin in corpora lutea of the human menstrual cycle directly demonstrates the presence of oxytocin- and neurophysin-positive cells within the human corpus luteum.     

Expression of major histocompatibility complex antigens on the bovine placenta

Immunohistochemistry was utilized to determine expression of the major histocompatibility complex (MHC) antigens on Day 8-9 hatched blastocysts and fetal membranes of mid- to late gestation cows and to examine the pattern of leucocytic infiltration into the gravid uterus. Hatched blastocysts were weakly positive for MHC class I antigens. In the mature placenta, chorioallantoic membranes in the interplacentomal area showed positive immunostaining for class I antigens on the chorionic epithelium but had no staining for class II antigens. There was an accumulation of lymphoid cells expressing class II antigens directly beneath the luminal epithelium of the endometrium. In addition, cells staining for leucocyte common antigen were present both within and beneath the luminal epithelium. Some cells positive for class II and leucocyte common antigen (CD45) were also associated with uterine glands. In the placentomes, class I antigens were expressed only on maternal caruncular septa. Fetal cotyledonary villi had no detectable immunostaining for class I and II antigens. No distinct pattern of leucocyte infiltration in the maternal caruncular tissue was observed; the caruncular septa contained some cells that were labelled for CD45 and a few class II-positive cells around blood vessels. The results indicate that the fetal placenta of the cow expresses MHC class I antigens in a regionally defined manner and there is a differential accumulation of lymphoid cells in the uterus.     

MHC class II and CD80 tumor cell–based vaccines are potent activators of type 1 CD4<Superscript>+</Superscript> T lymphocytes provided they do not coexpress invariant chain

We are developing vaccines that activate tumor-specific CD4⁺ T cells. The cell-based vaccines consist of MHC class I⁺ tumor cells that are genetically modified to express syngeneic MHC class II and costimulatory molecules. Previous studies demonstrated that treatment of mice with established tumors with these vaccines resulted in regression of solid tumors, reduction of metastatic disease, and increased survival time. Optimal vaccines will prime naïve T cells and activate T cells to tumor peptides derived from diverse subcellular compartments, since potential tumor antigens may reside in unique cellular locales. To determine if the MHC class II / costimulatory molecule vaccines fulfill these conditions, the vaccines have been tested for their ability to activate antigen-specific, naïve, transgenic CD4⁺ T lymphocytes. MHC class II⁺CD80⁺ vaccine cells were transfected with hen eggwhite lysozyme targeted to the cytosol, nuclei, mitochondria, or endoplasmic reticulum, and used as antigen-presenting cells to activate I-A^k–restricted, lysozyme-specific CD4⁺ 3A9 transgenic T cells. Regardless of the cellular location of lysozyme, the vaccines stimulated release of high levels of IFN- and IL-2. If the vaccines coexpressed the MHC class II accessory molecule invariant chain, then IFN- and IL-2 release was significantly reduced. These studies demonstrate that in the absence of invariant chain the MHC class II and CD80 tumor cell vaccines (1) function as antigen-presenting cells to activate naïve, tumor-specific CD4⁺ cells to endogenously synthesized tumor antigens; (2) polarize the activated CD4⁺ T cells toward a type 1 response; and (3) present epitopes derived from varied subcellular locales.Abbreviations APC antigen-presenting cells - CIITA MHC class II transactivator - CytoHEL HEL targeted to cytoplasm - ER endoplasmic reticulum - ErHEL HEL targeted to ER - HEL hen eggwhite lysozyme - 3A9 HEL_46–61–specific, I-A^k–restricted TCR - Hph hygromycin - Ii invariant chain - MAb monoclonal antibody - MitoHEL HEL targeted to mitochondria - NucHEL HEL targeted to nucleus - Puro puromycin - TG transgenic - Zeo Zeocin