The roles of homologous recombination and the immune system in the genomic evolution of cancer.

A variety of factors, whether extracellular (mutagens/carcinogens and viruses in the environment, chronic inflammation and radiation associated with the environment and/or electronic devices/machines) and/or intracellular (oxidative metabolites of food, oxidative stress due to inflammation, acid production, replication stress, DNA replication/repair errors, and certain hormones, cytokines, growth factors), pose a constant threat to the genomic integrity of a living cell. However, in the normal cellular environment multiple biological pathways including DNA repair, cell cycle, apoptosis and the immune system work in a precise, regulated (tightly controlled), timely and concerted manner to ensure genomic integrity, stability and proper functioning of a cell. If damage to DNA takes place, it is efficiently and accurately repaired by the DNA repair systems. Homologous recombination (HR) which utilizes either a homologous chromosome (in G1 phase) or a sister chromatid (in G2) as a template to repair the damage, is known to be the most precise repair system. HR in G2 which utilizes a sister chromatid as a template is also called an error free repair system. If DNA damage in a cell is so extensive that it overwhelms the repair system/s, the cell is eliminated by apoptosis. Thus, multiple pathways ensure that genome of a cell is intact and stable. However, constant exposure to DNA damage and/or dysregulation of DNA repair mechanism/s poses a risk of mutation and cancer. Oncogenesis, which seems to be a multistep process, is associated with acquisition of a number of genomic changes that enable a normal cell to progress from benign to malignant transformation. Transformed/cancer cells are recognized and killed by the immune system. However, the ongoing acquisition of new genomic changes enables cancer cells to survive/escape immune attack, evolve into a more aggressive phenotype, and eventually develop resistance to therapy. Although DNA repair (especially the HR) and the immune system play unique roles in preserving genomic integrity of a cell, they can also contribute to DNA damage, genomic instability and oncogenesis. The purpose of this article is to highlight the roles of DNA repair (especially HR) and the immune system in genomic evolution, with special focus on gastrointestinal cancer.

MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells.

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.

miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

Targeting IL-17A in multiple myeloma: a potential novel therapeutic approach in myeloma.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Enhancement of the expression of activation markers on human peripheral blood mononuclear cells by in vitro culture with retinoids and carotenoids.

Retinoids (retinol, retinal, retinoic acid, retinyl beta-glucuronide, and 13-cis retinoic acid) and carotenoids (beta-carotene and canthaxanthin) were evaluated for their immunomodulatory effects on human peripheral blood T-lymphocyte subpopulations and natural killer (NK) cells. Peripheral blood mononuclear cells (PBMC) from healthy young volunteers were isolated and incubated for 72 hours at various levels of retinoids and carotenoids including a physiological concentration (10(-8) M). Expression of surface antigens for total T cells, T-helper and T-suppressor cells, and activation markers (transferrin receptor, HLA-Dr antigen, and interleukin 2 receptor) were analyzed with an EPICS V flow cytometer. Retinoic acid and 13-cis retinoic acid (13-cRA) produced significant increases in the percentage of cells with markers for total T-helper cells, with a minimal effect on percentage of lymphocytes with markers for NK cells. However, beta-carotene (BC), canthaxanthin (CTX), and retinyl beta-glucuronide (RBG) dramatically increased the percentage of PBMC with markers for NK cells and produced a smaller increase in lymphocytes with surface antigens identifying them as T-helper cells. Furthermore, retinol and retinal did not show significant change either in the percentage of lymphocytes with markers for T-helper cells or in the helper/suppressor ratio. An increase in the expression of HLA-Dr antigen and transferrin receptors was greater when cells were incubated with 13-cRA than with either BC, CTX, or RBG, while carotenoids produced a greater increase in the expression of IL-2 receptors than 13-cRA. Our study indicates that both retinoids and carotenoids might be activating different subpopulations of immune cells.

A multiepitope of XBP1, CD138 and CS1 peptides induces myeloma-specific cytotoxic T lymphocytes in T cells of smoldering myeloma patients.

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL), for their ability to elicit multipeptide-specific cytotoxic T lymphocytes (MP-CTLs) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTLs generated from SMM patients' T cells show effective anti-MM responses including CD137 (4-1BB) upregulation, CTL proliferation, interferon-γ production and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically, we observed increased total CD3(+)CD8(+) T cells (>80%) and cellular activation (CD69(+)) within the memory SMM MP-CTL (CD45RO(+)/CD3(+)CD8(+)) subset after repeated multipeptide stimulation. Importantly, SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and antitumor activity. In high responders, the effector memory (CCR7(-)CD45RO(+)/CD3(+)CD8(+)) T-cell subset was enriched, whereas the remaining responders' CTL contained a higher frequency of the terminal effector (CCR7(-)CD45RO(-)/CD3(+)CD8(+)) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore, our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease.

Cytokine regulation of the mucosal immune system: in vivo stimulation by interferon-gamma of secretory component and immunoglobulin A in uterine secretions and proliferation of lymphocytes from spleen.

The secretory immune system in the female reproductive tract is known to be regulated by sex hormones and antigen. The purpose of the present study was to examine the control by interferon-gamma (IFN gamma) of the secretory component (SC), the polymeric immunoglobulin A (IgA) receptor, and IgA in uterine secretions and to determine whether IFN gamma influences the proliferation of spleen cells in response to mitogens. When measured by RIA, SC levels in uterine secretions were elevated when increasing doses of IFN gamma (1000-5000 U/uterus) were placed in the uterine lumen of ovariectomized rats. In contrast, IFN alpha-beta (5000 U/uterus) placed in the uterine lumen had no effect on uterine SC levels. To determine whether IgA movement increases in response to IFN gamma, animals were treated with estradiol to increase uterine tissue IgA levels without stimulating the accumulation of IgA or SC in uterine secretions. Under these conditions, intrauterine IFN gamma increased SC and IgA levels in uterine secretions, suggesting that in vivo IFN gamma stimulation of uterine SC increases the transport of IgA from tissue to lumen. Analysis of uterine tissues indicated that intrauterine IFN gamma had no apparent effect on epithelial cell morphology. In contrast, intraepithelial lymphocytes and polymorphonuclear leucocytes, which were sparse in control tissues, increased markedly with IFN gamma treatment. This increase was antagonized when estradiol was administered along with IFN gamma. In other studies, IFN gamma placed in the uterine lumen significantly increased spleen cell proliferation in response to Concanavalin-A, phytohaemagglutinin, and lipopolysaccharide mitogens relative to those in spleen cells from control animals. These studies demonstrate that in vivo treatment with IFN gamma stimulates the mucosal immune system in the female reproductive tract by increasing SC and IgA levels in the uterine lumen and promoting the infiltration of intraepithelial lymphocytes and polymorphonuclear leucocytes into uterine tissue. Further, it suggests that antigen, in stimulating a local uterine response, may act through cytokines, particularly IFN gamma, to regulate the transport of IgA into uterine secretions as well as modulate lymphocyte proliferation at sites distal to the uterus.

Effect of beta-carotene on lymphocyte subpopulations in elderly humans: evidence for a dose-response relationship.

The effects of various doses (0, 15, 30, 45, and 60 mg/d) of supplementary beta-carotene were evaluated. The percentage of lymphoid cells with surface markers for T-helper and natural killer (NK) cells and cells with interleukin 2 (IL-2) and transferrin receptors were significantly and substantially increased in peripheral blood mononuclear cells collected from older human adult volunteers after supplementation with greater than or equal to 30 mg beta-carotene/d for 2 mo. The increase in the percentage of cells with markers of NK cells and in expression of IL-2 receptors was dose dependent. The plasma concentrations of beta-carotene were also elevated significantly; however, there was no increase in the amount of retinol present in plasma. This indicated that immunomodulation induced by beta-carotene may be due to the carotenoid rather than to an increased amount, and hence actions, of vitamin A. These results support the role of immunostimulation as a potential mechanism of action of beta-carotene with cancer-prevention potential.

The mucosal immune system in the human female reproductive tract: potential insights into the heterosexual transmission of HIV.

Using isolated cell suspensions and in situ techniques, we have partially characterized the organization, functional capacity, and sex hormone regulation of the mucosal immune system in the human female reproductive tract. Isolated cells suspensions have been used to demonstrate that the uterus contains antigen-presenting cells that are functionally able to present antigen to autologous tetanus toxoid-specific T cells. Immunophenotypic analyses of the female reproductive tract by three-color immunofluorescent staining has been used to show that lymphoid aggregates, which are absent in postmenopausal women, develop in the uterine endometrium during the menstrual cycle in premenopausal women. Lymphoid aggregates are composed of a B lymphocyte core surrounded by numerous CD8+CD4- T lymphocytes and an outer halo of macrophages. Macrophages, CD4+ and CD8+ T cells, and CD56+ NK cells are distributed throughout the uterine endometrium. In contrast, the Fallopian tube, cervix, and vagina, which lack lymphoid aggregates, contain CD8+ and CD4+ T cells as well as macrophages. The female reproductive tract has also been analyzed for the presence of antigen-independent CD3+ T lymphocyte cytolytic function by an anti-CD3 MAb-mediated redirected lysis assay. High levels of CD3+ T lymphocyte cytolytic activity were demonstrated in cervix and vagina and independent of stage of the menstrual cycle. In the uterus, cytolytic activity changed with endocrine state. In postmenopausal women the uterine endometrium had CD3+ T lymphocytes with high cytolytic activity, whereas premenopausal women had CD3+ T lymphocytes with moderate cytolytic potential during the proliferative phase to low/no cytolytic activity during the secretory phase of the menstrual cycle. In studies to determine whether the upper reproductive tract could be infected with HIV-1, we found on the basis of nef expression and p24 release that epithelial cells from the Fallopian tube, and from the uterus and cervix, are infectable. These studies demonstrate that the human female reproductive tract is an inductive site for immune responses and the cell-mediated immunity is present throughout the female reproductive tract. These studies further indicate that the Fallopian tube and uterus are potential entry sites for HIV-1 infection and that uterine immune cell architecture as well as cytolytic activity are under hormonal control.

Regulation by human uterine cells of PBMC proliferation: influence of the phase of the menstrual cycle and menopause.

To determine the influence of human uterine cells recovered at different stages of the menstrual cycle and following menopause on the proliferation of peripheral blood mononuclear cells (PBMC), whole cell suspensions of uterine tissues were co-cultured with autologous and heterologous PBMC. PBMC proliferation in response to tetanus toxoid (TT) or Con A was inhibited by uterine endometrial cells and was dependent on the phase of the menstrual cycle. Inhibition by cells from the proliferative phase was significantly greater than by cells from the secretory phase. Uterine cells isolated from post-menopausal women also inhibited proliferation of PBMC. Cell fractionation studies indicated that epithelial cells are the primary source of uterine inhibitory activity. When epithelial cells and PBMC were cultured in separate compartments, epithelial cells released a soluble factor(s) that inhibited the PBMC proliferation. These results suggest that uterine epithelial cells produce cytokines that down-regulate the proliferation of PBMC in response to antigens and mitogens. This may be important for the control of uterine immune responses, as well as the growth of the reproductive tract in preparation for implantation during the secretory phase of the menstrual cycle.

Influence of beta-carotene on immune functions.

Recent reports have demonstrated that beta-carotene, a nontoxic carotenoid, is able to stimulate immune functions in humans. The purpose of this study is to understand the mechanisms of immunoenhancement by carotenoids in order to explain their anticancer effects. We have evaluated the clinical efficacy of beta-carotene, given 30 mg/day orally, for treatment of oral leukoplakia patients. Patients who responded to beta-carotene treatment showed increased plasma levels of TNF-alpha. Epithelial cells from these patients were characterized in vitro. These results may lead to a better understanding of the therapeutic use of beta-carotene in humans.

Changes in lymphocyte subsets and macrophage functions from high, short-term dietary ethanol in C57/BL6 mice.

Chronic administration of a diet containing 7% ethanol (36% of total calories) for 8 days to male C57/BL6 mice resulted in significant changes in functioning of macrophages. Peritoneal exudate macrophages from the ethanol-fed mice released more tumor cell cytotoxic materials upon culturing in vitro than cells from controls. However, peritoneal exudate cells continued to respond to exogenous beta carotene in vitro to produce additional cytotoxic materials. Phagocytosis of sheep red blood cells in vitro was suppressed in cells from ethanol treated mice. The number of splenic lymphocytes of various subsets was significantly changed by the ethanol exposure. Total T cells and T suppressor cells were lower, with a significant decrease in B cells containing IgM on their surface. The percentage of spleen cells showing markers for macrophage functions and their activation were significantly reduced. It is concluded that short-term chronic consumption of dietary ethanol, which was sufficient to produce physical dependence, results in significant alterations in lymphocyte subtypes and suppression of some macrophage functions.

Changes in lymphocyte and macrophage subsets due to morphine and ethanol treatment during a retrovirus infection causing murine AIDS.

Infection by LP-BM5 murine leukemia virus (MuLV) suppressed significantly the percentage of peripheral blood cells showing surface markers for macrophages, lymphocytes and activated lymphoid cells. Chronic administration of a 7% (36% calories) ethanol diet or injection of 1.9 mg/mouse/day of morphine for a 7 day period were followed by 3 week periods of abstinence and then 1 week periods of consumption of 5% ethanol diets or morphine injection to female C57BL/6 mice resulted in changes in the numbers of macrophages and lymphocyte subsets. The number of lymphocytes of various subsets were not significantly changed by the ethanol exposure except those showing activation markers which were reduced. The percentage of peripheral blood cells showing markers for macrophage functions and their activation were significantly reduced after "binge" use of ethanol. Ethanol retarded suppression of cells by retroviral infection. However by 25 weeks of infection there was a 8.6% survival in the ethanol fed mice infected with retrovirus which was much less than virally infected controls (45.0%). Morphine treatment also increased the percentage of cells with markers for macrophages and activated macrophages in virally infected mice, while suppressing them in uninfected mice. The second and third morphine injection series suppressed lymphocyte T-helper and T-suppressor cells, but not total T cells. However, suppression by morphine was significantly less during retroviral disease than suppression caused by the virus only. At 25 weeks of infection 44.8% of morphine treated, infected mice survived. Morphine treatment also caused deaths such that the survival in morphine treated, retrovirally infected was higher than would have been expected if the death rate in virally infected, and morphine injected animals were combined during combined treatment. Thus these drugs of abuse can modulate peripheral blood lymphoid subsets, suppression caused by retroviral infection, and survival.

Enhanced survival by vitamin A supplementation during a retrovirus infection causing murine AIDS.

Infection by LP-BM5 murine leukemia virus (MuLV) produces an AIDS-like condition in mice. The viral infection suppressed the percentage of peripheral blood cells showing surface markers for macrophages, activated macrophages, T lymphocytes and activated lymphoid cells. High dietary vitamin A (retinyl palmitate) caused increased numbers of activated macrophages. It also increased the percentage of cells with markers for Ia+ cells and macrophages in the retrovirally infected mice compared to infected controls. In uninfected mice retinyl palmitate stimulated the percentage of cells with activated lymphocytes bearing IL-2R, and T cytotoxic cells. These were associated with a retarded death rate during infection with LP-BM5 murine leukemia in C57BL/6 mice. By 25 weeks of infection and 20 weeks of retinyl palmitate supplementation 71.3% survived, while 45.0% virally infected controls survived. The mice also had elevated numbers of B cells measured in the blood after 4 and 8 weeks of dietary treatment. Vitamin A stimulation may play a role in the slower death rate for retrovirally infected mice.

Human intestinal tissue antibiotic concentrations. Clindamycin, gentamicin, and mezlocillin.

An antibiotic, to be effective for prophylaxis in abdominal trauma, should quickly achieve high concentrations in the intestinal wall and at enough inhibitory levels to kill most aerobic and anaerobic bacteria that are potential contaminants at the site of surgical incision. Therefore, we studied the intestinal tissue levels of clindamycin, gentamicin, and mezlocillin to see whether the tissue levels achieved by these antibiotics in the intestinal tissue were adequate. A single dose of mezlocillin, 4 grams; clindamycin, 600 mg and gentamicin, 80 mg; quickly reached the desired concentrations, i.e., 52.3, 9.69 and 6.1 micrograms/gram of intestinal tissue respectively. These levels were high enough to inhibit the growth of most isolates of E. coli and B. fragilis, common pathogens involved in intra-abdominal abscess.

Influence of estrous cycle and estradiol on mitogenic responses of splenic T- and B-lymphocytes.

Previous studies from our laboratory have shown that the stage of the estrous cycle and hormone treatment regulates the presence of immune cells in the female reproductive tract. The purpose of the present study was to determine whether sex hormones influence spleen cell responses to mitogens. The present study demonstrates that spleen cell responses to mitogens vary with the stage of the estrous cycle in intact rats. Further, our findings indicate that estradiol administered to ovariectomized animals increases the mitogenic response of spleen cells to both B and T lymphocyte mitogens.

Identification of novel myeloma-specific XBP1 peptides able to generate cytotoxic T lymphocytes: a potential therapeutic application in multiple myeloma.

The purpose of these studies was to identify human leukocyte antigen (HLA)-A2(+) immunogenic peptides derived from XBP1 antigens to induce a multiple myeloma (MM)-specific immune response. Six native peptides from non-spliced XBP1 antigen and three native peptides from spliced XBP1 antigen were selected and evaluated for their HLA-A2 specificity. Among them, XBP1(184-192), XBP1 SP(196-204) and XBP1 SP(367-375) peptides showed the highest level of binding affinity, but not stability to HLA-A2 molecules. Novel heteroclitic XBP1 peptides, YISPWILAV or YLFPQLISV, demonstrated a significant improvement in HLA-A2 stability from their native XBP1(184-192) or XBP1 SP(367-375) peptide, respectively. Cytotoxic T lymphocytes generated by repeated stimulation of CD3(+) T cells with each HLA-A2-specific heteroclitic peptide showed an increased percentage of CD8(+) (cytotoxic) and CD69(+)/CD45RO(+) (activated memory) T cells and a lower percentage of CD4(+) (helper) and CD45RA(+)/CCR7(+) (naïve) T cells, which were distinct from the control T cells. Functionally, the cytotoxic T lymphocytes (CTL) demonstrated MM-specific and HLA-A2-restricted proliferation, interferon-γ secretion and cytotoxic activity in response to MM cell lines and importantly, cytotoxicity against primary MM cells. These data demonstrate the distinct immunogenic characteristics of unique heteroclitic XBP1 peptides, which induce MM-specific CTLs and highlights their potential application for immunotherapy to treat the patients with MM or its pre-malignant condition.

Biological pathways and in vivo antitumor activity induced by Atiprimod in myeloma.

Atiprimod (Atip) is a novel oral agent with anti-inflammatory properties. Although its in vitro activity and effects on signaling in multiple myeloma (MM) have been previously reported, here we investigated its molecular and in vivo effects in MM. Gene expression analysis of MM cells identified downregulation of genes involved in adhesion, cell-signaling, cell cycle and bone morphogenetic protein (BMP) pathways and upregulation of genes implicated in apoptosis and bone development, following Atip treatment. The pathway analysis identified integrin, TGF-beta and FGF signaling as well as Wnt/beta-catenin, IGF1 and cell-cycle regulation networks as being most modulated by Atip treatment. We further evaluated its in vivo activity in three mouse models. The subcutaneous model confirmed its in vivo activity and established its dose; the SCID-hu model using INA-6 cells, confirmed its ability to overcome the protective effects of BM milieu; and the SCID-hu model using primary MM cells reconfirmed its activity in a model closest to human disease. Finally, we observed reduced number of osteoclasts and modulation of genes related to BMP pathways. Taken together, these data demonstrate the in vitro and in vivo antitumor activity of Atip, delineate potential molecular targets triggered by this agent, and provide a preclinical rational for its clinical evaluation in MM.

Sex hormone and IL-6 regulation of antigen presentation in the female reproductive tract mucosal tissues.

The purpose of this study was to determine whether Ag presentation takes place in the female reproductive tract and if the stage of the reproductive cycle, sex hormones, and IL-6 regulate uterine Ag presentation. Purified epithelial and mixed stromal cells were incubated with sensitized lymph node T lymphocytes in the presence of OVA. Ag presentation was highest at proestrus, the stage of the reproductive cycle that immediately precedes ovulation when estradiol levels peak, and lowest at estrus, when sperm enter the uterus in preparation for fertilization. Further, these studies demonstrate that estradiol given to ovariectomized rats increases uterine cell Ag presentation relative to that in controls. In the presence of mouse anti-rat Ia Ab, Ag presentation was blocked, indicating that Ag presentation by uterine cells is MHC class II restricted. In studies to analyze Ag presentation at sites distal to the female reproductive tract, we found that Ag presentation by adherent cells from the spleen is elevated at proestrus relative to that at other stages of the estrous cycle. When IL-6 was placed in the uterine lumen of ovariectomized rats, Ag presentation by epithelial and mixed stromal cells as well as mitogenesis by spleen cells were increased significantly relative to those in controls. These studies demonstrate that the female reproductive tract is an inductive site for immune responses and that mucosal immune protection may be either enhanced or suppressed depending on the endocrine balance when the female reproductive tract is exposed to pathogens.