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Previously, we demonstrated that human anti-HSV CTL and allo-antigen-specific CTL were inhibited in lysing their normally sensitive target cells when they were exposed to human fibroblasts (FB) infected with herpes simplex virus (HSV). In this study, the mechanism of inhibition of CTL lytic function by FB infected with HSV-1 (HSV-FB) was studied. CTL exposed to HSV-FB early (2 h) in the infection cycle were inhibited by a mechanism that appears to be distinct from the inhibition of lytic function mediated by HSV-FB at late times (20 h) during the infection cycle. The inhibition of CTL-mediated lysis by FB infected with HSV-1 for 2 h required the expression of ICP4, an immediate-early protein of HSV-1, but not the production of infectious virus or virus-induced shut-off of host protein synthesis. In contrast, the expression of HSV-specific glycoproteins essential for viral infectivity (glycoproteins B, D, H, K, and L), and thus, infectious virus, was required for inhibition of CTL lytic function by FB infected with HSV-1 for 20 h. Further, CTL exposed to FB infected with HSV-1 for 20 h expressed HSV-specific proteins indicating that they were infected with HSV-1. Cell-to-cell spread of HSV-1 appeared to be the major mode of transmission because 1) an insufficient level of HSV-1 was present in the supernatant of HSV-FB to inhibit CTL lytic function; and 2) paraformaldehyde-fixed HSV-FB did not inhibit CTL-mediated lysis. The inhibition of CTL lytic function by HSV-FB may be an important mechanism of HSV-induced immunosuppression, permitting the virus to spread and persist in immunocompetent hosts after primary infection or reactivation of latent HSV.

In this study, we have examined the in vivo effects of interleukin-5 (IL-5) and IL-6 over-expression on systemic and mucosal immune responses using recombinant human type 5 adenoviruses capable of expressing these cytokines upon infection. A recombinant adenovirus containing the murine IL-5 gene within the E3 region was constructed and found to express high levels of IL-5 protein both in vitro and in vivo. Intranasal inoculation of mice with this vector or a vector expressing murine IL-6 increased adenovirus-specific immunoglobulin A (IgA) titres in lung lavage fluid threefold compared with those elicited by control virus. The simultaneous expression of both cytokines by co-inoculation altered the kinetics of the mucosal anti-adenovirus IgA response and resulted in a more than additive increase in antibody titres. The co-expression effect on IgA synthesis was not due to an increase in numbers of antigen-specific resident lung tissue lymphocytes. When mucosal IgG responses were examined, IL-6 expression had the largest impact on anti-adenovirus levels, whereas co-expression produced an intermediate response. Systemic immune responses were also affected by IL-6 expression as a twofold increase in serum IgG anti-adenovirus titres was observed after a secondary challenge with wild-type adenovirus. These results demonstrate a relevant role for IL-5 and IL-6 in the development of mucosal immune responses in vivo and suggest that the incorporation of either IL-5 and/or IL-6 into recombinant adenovirus vectors may be a useful tool in the development of mucosal vaccines.

To evaluate clinical, laboratory, radiographic, ultrasonographic, surgical, and histologic findings in ferrets with insulinoma and to determine their long-term outcome.Retrospective study.57 ferrets with a histopathologic diagnosis of pancreatic islet cell tumor.Medical records of ferrets with pancreatic islet cell tumors were reviewed.Lethargy, weakness, and collapse were the most common clinical signs. All ferrets had hypoglycemia, and hyperinsulinemia was documented in 39 of 47 (83%) ferrets. Ultrasonographic examination of the abdomen revealed pancreatic nodules in 5 of 23 ferrets. Surgical treatment was performed in 50 ferrets, 3 were treated by medical management alone, and 4 did not have treatment. At the time of surgery, 1 pancreatic nodule was found in 13 (26%) ferrets and multiple nodules were found in 37 (74%) ferrets. Pancreatic carcinoma alone was found in 34 ferrets. Whereas a combination of carcinoma and either hyperplasia or adenoma was found in 23 ferrets; 4 ferrets had metastasis to regional lymph nodes or liver. In 26 (53%) ferrets, hypoglycemia persisted after surgery, necessitating medical treatment with prednisone, diazoxide, or both. Sixteen (33%) ferrets had redevelopment of hypoglycemia at 1 to 23.5 months (median, 10.6 months) after surgery. Only 7 of the 50 (14%) ferrets remained euglycemic after surgery.In ferrets, surgical removal of insulin-secreting pancreatic islet cell tumors is recommended as definitive treatment; however, multiple pancreatic nodules are common, making complete excision of all tumor tissue difficult. Persistent hypoglycemia after surgical treatment indicates that lifelong medical management with prednisone or diazoxide or both may be necessary in many ferrets. Finally, because the insulin-secreting tumors are malignant, long-term cure and survival are not likely.

To determine plasma concentrations of steroid hormones in ferrets with hyperadrenocorticism associated with adrenocortical neoplasia or nodular hyperplasia of the adrenal gland, the effect of surgical removal of the affected adrenal gland on concentrations of these hormones, and whether any hormone concentrations could be used as a marker for the disease.Prospective case series.32 ferrets with hyperadrenocorticism associated with adrenocortical neoplasia or nodular hyperplasia of the adrenal gland.A blood sample was obtained from each ferret before adrenalectomy. In 26 of the 32 ferrets, a second blood sample was collected 24 to 48 hours after adrenalectomy. Plasma concentrations of 7 hormones were measured (cortisol, estradiol, testosterone, progesterone, 17-hydroxyprogesterone, androstenedione, and dehydroepiandrosterone sulfate).Median plasma concentrations of 17-hydroxyprogesterone, androstenedione, and dehydroepiandrosterone sulfate were significantly higher in ferrets with adrenal gland disease, compared with concentrations in clinically normal ferrets. After adrenalectomy, median concentrations of estradiol, 17-hydroxyprogesterone, and androstenedione decreased significantly. Of 23 ferrets in which concentrations of estradiol, 17-hydroxyprogesterone, and androstenedione all were measured, 22 (96%) had high concentrations of at least 1 of these 3 hormones, but only 5 (22%) ferrets had high concentrations of all 3 hormones.The condition that develops in ferrets with adrenal gland disease apparently is caused by excessive secretion of 1 or more steroids other than cortisol. Because concentration of a particular hormone was not high in all ferrets, we recommend determining plasma concentrations of several sex steroids, including androstenedione, 17-hydroxyprogesterone, and estradiol.

In HIV-1 infection, the appearance of syncytia-inducing (SI) isolates is associated with a more rapid decline of CD4+ cells and progression to AIDS. Agents that inhibit either virus infection or syncytia formation have the potential to be therapeutically useful. Lipophosphoglycan (LPG), the major glycoconjugate of Leishmania, was recently shown to be a potent nonspecific inhibitor of viral membrane fusion. In this study, LPG demonstrated a dose-dependent inhibition of HIV-1-induced syncytia formation in CD4+ MT-2 cells infected with distinct SI isolates. Fragments of LPG were used to show that inhibition of syncytia formation was dependent on the length of the LPG fragment. Treatment of CD4+ cells or HIV-1 isolates with LPG inhibited infection in vitro. Furthermore, LPG inhibited the replication of SI viral isolates in CD4+ T cells in vitro. LPG had no toxic effects on peripheral blood mononuclear cells at the highest concentrations used in these assays. Further, LPG rapidly associated with the surface membrane of a human T cell line and subsequently disassociated over a 24-h period. The development of compounds capable of inhibiting HIV-induced syncytia formation should provide novel therapeutic approaches to control the spread of virus and disease progression.

Previously, we demonstrated that human anti-HSV CTL and allo-antigen-specific CTL were inhibited in lysing their normally sensitive target cells when they were exposed to human fibroblasts (FB) infected with herpes simplex virus (HSV). In this study, the mechanism of inhibition of CTL lytic function by FB infected with HSV-1 (HSV-FB) was studied. CTL exposed to HSV-FB early (2 h) in the infection cycle were inhibited by a mechanism that appears to be distinct from the inhibition of lytic function mediated by HSV-FB at late times (20 h) during the infection cycle. The inhibition of CTL-mediated lysis by FB infected with HSV-1 for 2 h required the expression of ICP4, an immediate-early protein of HSV-1, but not the production of infectious virus or virus-induced shut-off of host protein synthesis. In contrast, the expression of HSV-specific glycoproteins essential for viral infectivity (glycoproteins B, D, H, K, and L), and thus, infectious virus, was required for inhibition of CTL lytic function by FB infected with HSV-1 for 20 h. Further, CTL exposed to FB infected with HSV-1 for 20 h expressed HSV-specific proteins indicating that they were infected with HSV-1. Cell-to-cell spread of HSV-1 appeared to be the major mode of transmission because 1) an insufficient level of HSV-1 was present in the supernatant of HSV-FB to inhibit CTL lytic function; and 2) paraformaldehyde-fixed HSV-FB did not inhibit CTL-mediated lysis. The inhibition of CTL lytic function by HSV-FB may be an important mechanism of HSV-induced immunosuppression, permitting the virus to spread and persist in immunocompetent hosts after primary infection or reactivation of latent HSV.

Recombinant adenovirus vectors expressing the entire gag (p55) or CA (p24) region of human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the appropriate HIV DNA sequences into the E3 region of human adenovirus type 5 (Ad5) with and without an exogenous SV40 early promoter. The infectious recombinant adenoviruses Adgag1, AdSVgag1, and AdSVCA1 were shown to express the appropriate HIV-1 antigens in human cells in vitro, as measured by immunoprecipitation and p24 antigen capture assays. Using the p24 antigen capture assay, HIV antigen expressed by AdSVCA1 was detected earlier in infection and in greater amounts than that produced by either Adgag1 or AdSVgag1. In studies concerning the immunogenicity of these vectors, Balb/c (H-2d) mice given a single intraperitoneal injection of 10(7) or 10(8) plaque-forming units of purified vector developed serum antibodies to p24, detected by Western blotting, by 2 weeks postinjection. In the preliminary test of the immunogenicity of the recombinant adenovirus vectors in primates, two of four rhesus macaque monkeys generated antibodies to HIV-1 p24 following two injections of AdSVCA1. As expected, monkeys injected with control adenovirus failed to show any anti-HIV response, and none of the monkeys showed any adverse reactions following infection with either recombinant or control adenoviruses. These results suggest that adenovirus vectors have considerable potential in the study of possible immune therapies for HIV infection.

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.

Antibodies specific for carcinoembryonic antigen (CEA) induced polar redistribution of CEA which was expressed on the surface of human intestinal carcinoma cells grown in tissue culture. Polar redistribution occurred at 23°C and 37°C but not at 4°C and was inhibited by sodium azide and cytochalasin B. Antibodies to isoantigen A did not react with CEA at the cell surface but did react with isoantigen A which was not redistributed by the antibody. Cells which expressed both CEA and isoantigen A were used to demonstrate that polar redistribution of CEA could be induced without altering the distribution of isoantigen A. The data indicate that CEA and isoantigen A can be expressed on the same cell as separate molecules.

Pichinde virus (PV), a member of the arenavirus group, was found to elicit strong cell-mediated immune responses in various strains of mice. After primary i.v. inoculation, augmentation of natural killer (NK) cell activity occurred and peaked 3 to 4 days after infection. The NK response was followed by a second peak of cytotoxic activity that was found to be H-2 restricted, virus specific, and mediated by Thy-1.2+, Lyt-2.2+ lymphocytes. This cytotoxic T lymphocyte (CTL) response peaked 7 days post infection. Neutralizing antibodies were not detectable after PV infection of the mice. In light of this, we investigated the generation and kinetics of secondary cell-mediated immune responses after reinjection of homologous virus in vivo. Slight but significant augmentation of NK activity was observed 1 day after secondary virus challenge. As in the primary response, effectors of this NK activity rapidly became sensitive to anti-Thy-1.2 and complement treatment. NK activity rapidly returned to background levels and was followed by an anamnestic CTL response that peaked 4 days after reinjection of the virus. Thus, cell-mediated immune responses appeared more rapidly after secondary challenge in vivo, and the temporal relationship between NK and CTL generation was maintained. Both secondary NK and CTL responses were generated in mice that had been pretreated with cyclophosphamide (CY), suggesting that memory cell-mediated immune responses can be reactivated in vivo without undergoing cell division. In contrast, treatment with CY before primary infection delayed the appearance of virus-induced NK activity and abrogated the generation of H-2-restricted virus-specific CTL. Rechallenge of these CY-treated NK-primed mice resulted in the rapid generation of a secondary NK response that was not followed by either a primary or secondary CTL response. The data suggest that cells mediating a nonspecific effector function may possess specific memory. We discuss our results with respect to possible NK-CTL relationships.

Natural killer (NK) activity is mediated by large granulated lymphocytes (LGL). Recently, the relationship of NK cells to mast cells and basophils has been suggested. We therefore examined three distinct interleukin 2-dependent cloned cell lines capable of mediating NK lysis. Virtually all the cells of each line contained membrane-bound granules. Interestingly, ultrastructural studies demonstrated that the granules in each cell line were morphologically distinct and thus heterogeneous. All three cloned, granulated NK cell lines were found to variably express low numbers (less than or equal to 1.3 X 10(4)) of low affinity plasma membrane IgE receptors (Fc epsilon R). In contrast to mast cells and basophils, however, none were found to express high numbers of high affinity Fc epsilon R. In addition, none of the three NK cell lines were found to contain histaminase-sensitive histamine. Our results suggest that NK cells are not related to mast cells or basophils.

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.

Cytotoxic T lymphocytes (CTL) generated in mice against a particular serotype of vesicular stomatitis virus (VSV) were previously shown to cross-reactively lyse syngeneic target cells infected with serologically distinct types of VSV. To analyze the antigenic basis of this T cell cross-reactivity, we generated CTL against VSV-Indiana (VSV-Ind) and established them by limiting dilution as cloned in vitro cell lines. The cells continuously proliferate in medium containing concanavalin A-induced T cell growth factors. All of the cells are Thy-1.2+ and Lyt-2.2+. Lysis by these cells is H-2Dd-restricted, no natural killer cell activity is detectable, and all the clones cross-reactively lyse target cells infected with either VSV-Ind or VSV-New Jersey (VSV-NJ). In addition, no specific blocking of primary, secondary, or cloned anti-VSV CTL was achieved with the use of several monoclonal antibodies specific for the glycoprotein of VSV and capable of neutralizing either VSV-Ind or VSV-NJ. These results suggest that VSV serotype-specific neutralizing antibodies may recognize immunodominant determinants of VSV glycoprotein that are distinct from those recognized by the majority of VSV-specific CTL.

Highly purified preparations of intraepithelial leukocytes (IEL) were obtained from the small intestinal mucosa. Leukocytes from the lamina propria (LPL) were isolated and phenotypically compared with IEL to verify that IEL were minimally contaminated by LPL. Because approximately 80% of IEL expressed the Lyt-2 antigen usually associated with cytotoxic/suppressor T lymphocytes, we wished to determine if precursors for cytotoxic T cells were present in this population. In order to generate cytotoxic cells, IEL and spleen cells from CBA/J mice (H-2k) were co-cultured with irradiated allogeneic spleen cells (H-2d or H-2b) in a one-way mixed leukocyte reaction (MLR). Four to six days later, the cultured cells were assayed against 51Cr-labeled H-2d or H-2b tumor or Con A-stimulated lymphoblast target cells, and the specificity of alloantigen-stimulated IEL and spleen cells was compared. The cytotoxic cells derived from both tissues displayed antigen-specific lysis of the allogeneic targets. Treatment of effector cells, generated from intraepithelial or splenic precursors, with monoclonal antibodies against Thy-1.2, Lyt-1.1, or Lyt-2.1 antigens plus complement, decreased cytotoxicity 85 to 100%, even though only 20 to 50% of the cells were lysed. The alloantigen specificity and surface antigen phenotype of the cultured IEL cells were identical to those of spleen cells and allowed us to conclude that IEL contained a cytotoxic T lymphocyte precursor (CTLp). Further characterization showed that, like spleen, the intraepithelial CTLp was Thy-1+ and Lyt-1+ and their sedimentation velocity was the same but differed from intraepithelial natural killer cells. Although 80% of IEL were Lyt-2+, the frequency of CTLp in the IEL population was estimated to be threefold to fivefold lower than in spleen, and the Lyt-2+ cells were shown not to be an enriched source of CTLp. Thus, the function of the majority of the IEL is still not known. However, there exists within this population CTLp, which may be capable of being stimulated with luminal antigens.

Initially the intraepithelial leukocytes (IEL) of specific pathogen free (SPF) mice were compared with those of mice held without isolation and were found to differ markedly in total number and distribution of cell surface antigens. The IEL from SPF mice expressed significantly less Thy-1, Lyt-1, and Lyt-2 antigens than their conventional counterparts. The local cell-mediated immune response of mucosal lymphocytes to an enteric murine coronavirus (MHV-Y) was studied in inbred strains of naive SPF mice. A potent in vitro cytotoxic activity was demonstrated by mucosal leukocytes, especially IEL, and spleen cells for MHV-Y-infected syngeneic and allogeneic target cells. The cytotoxicity was not restricted by the major histocompatibility complex. Targets infected with Pichinde virus, an enveloped nonenterotropic virus, were not lysed by these cells. The phenotype of the IEL effector cell was asialo GM1+, Thy-1-, Lyt-1-, Lyt-2-. This cell represents a small subpopulation of the total IEL. After the in vivo administration of anti-asialo GM1 sera, the virus-specific cytotoxic function of the IEL was markedly diminished in in vitro assays, and there was enhanced persistence of virus in gut tissues in vivo. The IEL effector population is defined as a natural killer-like cell that appears to be active in the defense of the gut epithelium to a murine enteric coronavirus.

The mammalian fetus expresses a variety of antigens against which the maternal immune system can react and which in an allogeneic mating bears paternal transplantation antigens. Although these antigens may be expressed on the fetal trophoblast cells that contact maternal uterine decidua, the "fetal allograft" is not usually rejected. Previous studies have demonstrated the presence of nonspecific non-thymus-derived suppressor cells in the lymph nodes draining the uterus and in decidua of laboratory mice undergoing first allogeneic pregnancy. These suppressor cells appeared to be small lymphocyte cells that inhibit the generation of cytotoxic T lymphocytes (CTL) in vitro and in vivo and elaborate a nonspecific non-MHC-restricted soluble suppressor activity when cultured for 48 hours at 37 degrees C in vitro. We now report that soluble suppressor activity obtained from the decidua (DS) of allopregnant C3H/HeJ mice inhibits both the primary and secondary (memory) CTL response in vitro but does not inhibit lysis of target cells by preformed CTL. DS did not suppress the proliferation of YAC lymphoma cells, P-815 cells, or a C3H placental trophoblastoma line. Suppressor activity was obtained from anti-thy-1.2 + complement-resistant cells in the decidua, could also be obtained from the decidua of allopregnant CD1 nu/nu mice, and was associated with a single peak of activity of approximately 100,000 daltons on Sephacryl 200 chromatography. Suppression could not be overcome by adding either crude or HPLC-purified IL 2 to the mixed lymphocyte cultures in vitro, and both crude and column-purified suppressor factor inhibited the IL 2-dependent proliferation of H-Y cells (a cloned T cell line with NK activity). Furthermore, DS inhibited the IL 2-dependent generation of cytotoxic effector cells in vitro in the absence of allogeneic stimulator cells. Thus, a soluble suppressor factor obtained from non-T cells present in the decidua of successfully allopregnant mice could block the response to IL 2 and inhibit the generation of both specific and nonspecific cytotoxic effector cells. The significance of this inhibition with respect to survival of the "fetal allograft" is discussed.

H-2k mice are unable to generate cytotoxic T lymphocytes (CTL) to vesicular stomatitis virus (VSV). This apparent unresponsiveness is found for both major serotypes of VSV, VSV-Indiana and VSV-New Jersey. CTL unresponsiveness occurs despite the ability of H-2k mice to generate a humoral immune response against VSV that is comparable to that found in responder (H-2b and H-2a) strains. All H-2k mice regardless of background genes, including various Ig allotypes, were found to be nonresponders. H-2k-linked unresponsiveness mapped to both H-2Kk and H-2Dk and occurred despite the presence of responder alleles in (responder x nonresponder)F1 mice. The unresponsiveness cannot be attributed to an inability of VSV-infected H-2k target cells to express viral surface antigens of H-2 molecules. Further, unresponsiveness cannot be overcome by using secondary stimulation in vivo or in vitro. H-2k-linked unresponsiveness does not appear to be due to suppression, and no complementation has been found in various (nonresponder x nonresponder)F1 mice. Thus unresponsiveness to VSV in association with H-2Kk or H-2Dk appears to represent an extensive defect of immune responsiveness that probably occurs because VSV is not a natural mouse pathogen.

Factors affecting the expression of carcinoembryonic antigen (CEA) at the surface of in vitro human colon carcinoma cells were determined using 125I-labeled antibodies. Binding of specific anti-CEA antibodies resulted in polar redistribution of CEA, followed by endocytosis of most of the CEA-anti-CEA complexes. These processes were temperature and energy dependent. CEA removed from the tumor cell surface by antibody was totally replaced within 6 hr at 37 degrees, and the reexpression of CEA required protein synthesis. Examination of clonally derived subpopulations of various strains of human colon cancer cells indicated that control over the level of cell surface CEA expressed was genetically stable in vitro. CEA expression was enhanced in a low-CEA-producing strain by incorporating theophylline in the culture medium, and the inclusions of bromodeoxyuridine enhanced the expression of a high-CEA-producing strain. The kinetics of enhancement of CEA expression by these two drugs differed. These findings suggest that CEA expression may be controlled by more than a single gene function.

Pichinde virus (PV), a member of the arenavirus group, was found to elicit strong cell-mediated immune responses in various strains of mice. After primary i.v. inoculation, augmentation of natural killer (NK) cell activity occurred and peaked 3 to 4 days after infection. The NK response was followed by a second peak of cytotoxic activity that was found to be H-2 restricted, virus specific, and mediated by Thy-1.2+, Lyt-2.2+ lymphocytes. This cytotoxic T lymphocyte (CTL) response peaked 7 days post infection. Neutralizing antibodies were not detectable after PV infection of the mice. In light of this, we investigated the generation and kinetics of secondary cell-mediated immune responses after reinjection of homologous virus in vivo. Slight but significant augmentation of NK activity was observed 1 day after secondary virus challenge. As in the primary response, effectors of this NK activity rapidly became sensitive to anti-Thy-1.2 and complement treatment. NK activity rapidly returned to background levels and was followed by an anamnestic CTL response that peaked 4 days after reinjection of the virus. Thus, cell-mediated immune responses appeared more rapidly after secondary challenge in vivo, and the temporal relationship between NK and CTL generation was maintained. Both secondary NK and CTL responses were generated in mice that had been pretreated with cyclophosphamide (CY), suggesting that memory cell-mediated immune responses can be reactivated in vivo without undergoing cell division. In contrast, treatment with CY before primary infection delayed the appearance of virus-induced NK activity and abrogated the generation of H-2-restricted virus-specific CTL. Rechallenge of these CY-treated NK-primed mice resulted in the rapid generation of a secondary NK response that was not followed by either a primary or secondary CTL response. The data suggest that cells mediating a nonspecific effector function may possess specific memory. We discuss our results with respect to possible NK-CTL relationships.

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.

Highly purified preparations of intraepithelial leukocytes (IEL) were obtained from the small intestinal mucosa. Leukocytes from the lamina propria (LPL) were isolated and phenotypically compared with IEL to verify that IEL were minimally contaminated by LPL. Because approximately 80% of IEL expressed the Lyt-2 antigen usually associated with cytotoxic/suppressor T lymphocytes, we wished to determine if precursors for cytotoxic T cells were present in this population. In order to generate cytotoxic cells, IEL and spleen cells from CBA/J mice (H-2k) were co-cultured with irradiated allogeneic spleen cells (H-2d or H-2b) in a one-way mixed leukocyte reaction (MLR). Four to six days later, the cultured cells were assayed against 51Cr-labeled H-2d or H-2b tumor or Con A-stimulated lymphoblast target cells, and the specificity of alloantigen-stimulated IEL and spleen cells was compared. The cytotoxic cells derived from both tissues displayed antigen-specific lysis of the allogeneic targets. Treatment of effector cells, generated from intraepithelial or splenic precursors, with monoclonal antibodies against Thy-1.2, Lyt-1.1, or Lyt-2.1 antigens plus complement, decreased cytotoxicity 85 to 100%, even though only 20 to 50% of the cells were lysed. The alloantigen specificity and surface antigen phenotype of the cultured IEL cells were identical to those of spleen cells and allowed us to conclude that IEL contained a cytotoxic T lymphocyte precursor (CTLp). Further characterization showed that, like spleen, the intraepithelial CTLp was Thy-1+ and Lyt-1+ and their sedimentation velocity was the same but differed from intraepithelial natural killer cells. Although 80% of IEL were Lyt-2+, the frequency of CTLp in the IEL population was estimated to be threefold to fivefold lower than in spleen, and the Lyt-2+ cells were shown not to be an enriched source of CTLp. Thus, the function of the majority of the IEL is still not known. However, there exists within this population CTLp, which may be capable of being stimulated with luminal antigens.

The mammalian fetus expresses a variety of antigens against which the maternal immune system can react and which in an allogeneic mating bears paternal transplantation antigens. Although these antigens may be expressed on the fetal trophoblast cells that contact maternal uterine decidua, the "fetal allograft" is not usually rejected. Previous studies have demonstrated the presence of nonspecific non-thymus-derived suppressor cells in the lymph nodes draining the uterus and in decidua of laboratory mice undergoing first allogeneic pregnancy. These suppressor cells appeared to be small lymphocyte cells that inhibit the generation of cytotoxic T lymphocytes (CTL) in vitro and in vivo and elaborate a nonspecific non-MHC-restricted soluble suppressor activity when cultured for 48 hours at 37 degrees C in vitro. We now report that soluble suppressor activity obtained from the decidua (DS) of allopregnant C3H/HeJ mice inhibits both the primary and secondary (memory) CTL response in vitro but does not inhibit lysis of target cells by preformed CTL. DS did not suppress the proliferation of YAC lymphoma cells, P-815 cells, or a C3H placental trophoblastoma line. Suppressor activity was obtained from anti-thy-1.2 + complement-resistant cells in the decidua, could also be obtained from the decidua of allopregnant CD1 nu/nu mice, and was associated with a single peak of activity of approximately 100,000 daltons on Sephacryl 200 chromatography. Suppression could not be overcome by adding either crude or HPLC-purified IL 2 to the mixed lymphocyte cultures in vitro, and both crude and column-purified suppressor factor inhibited the IL 2-dependent proliferation of H-Y cells (a cloned T cell line with NK activity). Furthermore, DS inhibited the IL 2-dependent generation of cytotoxic effector cells in vitro in the absence of allogeneic stimulator cells. Thus, a soluble suppressor factor obtained from non-T cells present in the decidua of successfully allopregnant mice could block the response to IL 2 and inhibit the generation of both specific and nonspecific cytotoxic effector cells. The significance of this inhibition with respect to survival of the "fetal allograft" is discussed.

A variant, HCT-8R, of the colon adenocarcinoma cell line HCT-8 was located and cloned. The variant and parent cell line were characterized with respect to morphology, growth characteristics, karyotype, production of Cea, and ability to form tumors in nude mice. The variant cells differed from the parent cells in morphology, marker chromosomes, and ability to form colonies in soft agar and produced more carcinoembryonic antigen. The two cell strains were equally oncogenic in nude mice, although HCT-8R cells produced poorly differentiated tumors while HCT-8 cells produced tumors with both differentiated and poorly differentiated areas. Thus, in nude mice, no correlation was observed between carcinoembryonic antigen production by cells and their oncogenicity.

H-2k mice are unable to generate cytotoxic T lymphocytes (CTL) to vesicular stomatitis virus (VSV). This apparent unresponsiveness is found for both major serotypes of VSV, VSV-Indiana and VSV-New Jersey. CTL unresponsiveness occurs despite the ability of H-2k mice to generate a humoral immune response against VSV that is comparable to that found in responder (H-2b and H-2a) strains. All H-2k mice regardless of background genes, including various Ig allotypes, were found to be nonresponders. H-2k-linked unresponsiveness mapped to both H-2Kk and H-2Dk and occurred despite the presence of responder alleles in (responder x nonresponder)F1 mice. The unresponsiveness cannot be attributed to an inability of VSV-infected H-2k target cells to express viral surface antigens of H-2 molecules. Further, unresponsiveness cannot be overcome by using secondary stimulation in vivo or in vitro. H-2k-linked unresponsiveness does not appear to be due to suppression, and no complementation has been found in various (nonresponder x nonresponder)F1 mice. Thus unresponsiveness to VSV in association with H-2Kk or H-2Dk appears to represent an extensive defect of immune responsiveness that probably occurs because VSV is not a natural mouse pathogen.

Cytotoxic T lymphocytes (CTL) generated in mice against a particular serotype of vesicular stomatitis virus (VSV) were previously shown to cross-reactively lyse syngeneic target cells infected with serologically distinct types of VSV. To analyze the antigenic basis of this T cell cross-reactivity, we generated CTL against VSV-Indiana (VSV-Ind) and established them by limiting dilution as cloned in vitro cell lines. The cells continuously proliferate in medium containing concanavalin A-induced T cell growth factors. All of the cells are Thy-1.2+ and Lyt-2.2+. Lysis by these cells is H-2Dd-restricted, no natural killer cell activity is detectable, and all the clones cross-reactively lyse target cells infected with either VSV-Ind or VSV-New Jersey (VSV-NJ). In addition, no specific blocking of primary, secondary, or cloned anti-VSV CTL was achieved with the use of several monoclonal antibodies specific for the glycoprotein of VSV and capable of neutralizing either VSV-Ind or VSV-NJ. These results suggest that VSV serotype-specific neutralizing antibodies may recognize immunodominant determinants of VSV glycoprotein that are distinct from those recognized by the majority of VSV-specific CTL.

Initially the intraepithelial leukocytes (IEL) of specific pathogen free (SPF) mice were compared with those of mice held without isolation and were found to differ markedly in total number and distribution of cell surface antigens. The IEL from SPF mice expressed significantly less Thy-1, Lyt-1, and Lyt-2 antigens than their conventional counterparts. The local cell-mediated immune response of mucosal lymphocytes to an enteric murine coronavirus (MHV-Y) was studied in inbred strains of naive SPF mice. A potent in vitro cytotoxic activity was demonstrated by mucosal leukocytes, especially IEL, and spleen cells for MHV-Y-infected syngeneic and allogeneic target cells. The cytotoxicity was not restricted by the major histocompatibility complex. Targets infected with Pichinde virus, an enveloped nonenterotropic virus, were not lysed by these cells. The phenotype of the IEL effector cell was asialo GM1+, Thy-1-, Lyt-1-, Lyt-2-. This cell represents a small subpopulation of the total IEL. After the in vivo administration of anti-asialo GM1 sera, the virus-specific cytotoxic function of the IEL was markedly diminished in in vitro assays, and there was enhanced persistence of virus in gut tissues in vivo. The IEL effector population is defined as a natural killer-like cell that appears to be active in the defense of the gut epithelium to a murine enteric coronavirus.

The mammalian fetus expresses a variety of paternal histocompatible, oncofetal, and trophoblast antigens against which the mother can mount an immune response. Survival of the "fetal graft" appears to depend upon local immunosuppressive mechanisms in lymph nodes draining the uterus and at the intrauterine implanation site itself. Nonspecific not-T-Fc-receptor-bearing small lymphocytes containing cytoplasmic granules present in successfully allopregnant mice can suppress both the generation of maternal-antipaternal killer T cells and the infiltration of cytotoxic T lymphocytes into sponge-matrix allografts during the effector phase of the immune response. These suppressor cells are deficient at the implantation sites of xenogeneic and allogeneic mouse embryos that are susceptible to maternal immunity and are destined to resorb. A soluble suppressor factor of approximately 100,000 daltons in size can be obtained from the suppressor cells and acts to block the response of T cells to interleukin-2 by interfering with IL-2 receptors. The development of the suppressor cells in the decidua requires certain hormonal signals as well as signals provided by trophoblast cells. Freshly explanted or cultured murine trophoblast cell lines elaborate soluble factor(s) that are active in recruitment or activation of suppressor cells. Since suppressor cells may be isolated from decidua of successfully allopregnant humans, the suppressor cell mechanism and its regulation may represent a key factor in the protection of the "fetal allograft" from rejection by maternal immunity.

Antibodies specific for carcinoembryonic antigen (CEA) induced polar redistribution of CEA which was expressed on the surface of human intestinal carcinoma cells grown in tissue culture. Polar redistribution occurred at 23 degrees C and 37 degrees C but not at 4 degrees C and was inhibited by sodium azide and cytochalasin B. Antibodies to isoantigen A did not react with CEA at the cell surface but did react with isoantigen A which was not redistributed by the antibody. Cells which expressed both CEA and isoantigen A were used to demonstrate that polar redistribution of CEA could be induced without altering the distribution of isoantigen A. The data indicate that CEA and isoantigen A can be expressed on the same cell as separate molecules.