Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

Biochemical evidence for expression of a semi-allogeneic, H-2 antigen by a murine adenocarcinoma

LT-85 is an alveolegenic adenocarcinoma induced in mutant C3HfB/HeN (C3Hf) mice. This tumor, however, grows preferentially in allogeneic, wild-type C3H/HeN (C3H) mice. The tumor-associated transplantation antigen has been mapped to the K end of the major histocompatibility complex. H-2K antigens were isolated from detergent extracts of LT-85 cells by immunoprecipitation with monoclonal antibody. The tryptic peptides of these antigens were compared, by using high-pressure liquid chromatography, with the tryptic peptides of H-2K antigens isolated from syngeneic mutant C3Hf and ancestral wild-type C3H spleen cells. We found that the H-2K antigens of the LT-85 tumor cells were very similar to, but distinct from, those present on syngeneic C3Hf lymphoid cells. We also found, however, that the H-2K antigens of LT-85 tumor cells were clearly different from the H-2K antigens of allogeneic C3H spleen cells. The H-2K antigens of LT-85 cells are therefore foreign to syngeneic C3Hf cells, but do not represent expression by the tumor cells of the allogeneic H-2K antigens expressed by normal C3H cells. Furthermore, the nature of the differences observed between the H-2K antigens of LT-85 cells and C3Hf and C3H spleen cells strongly suggests that the structure of the H-2K molecule of LT-85 cells is identical in some regions to the H-2K molecule of C3Hf cells, and in other regions to the H-2K molecule of C3H cells.     

Transfection and expression of syngeneic H-2 genes does not reduce malignancy of H-2 negative teratocarcinoma cells in the autologous host

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.     

Intraspecies identification of cultured murine cells by using H-2 antigen typing

17 mouse cell lines have been screened with specific sera against H-2 antigens. All the cell lines tested expressed H-2 antigens characteristic of the donor haplotype. The data obtained indicate that H-2 typing of cultured mouse cells can be used as an approach to control their intraspecies diversity.     

IFN-alpha is not sufficient to drive Th1 development due to lack of stable T-bet expression

During inflammatory immune responses, the innate cytokine IL-12 promotes CD4+ Th-1 development through the activation of the second messenger STAT4 and the subsequent expression of T-bet. In addition, type I IFN (IFN-alphabeta), secreted primarily during viral and intracellular bacterial infections, can promote STAT4 activation in human CD4+ T cells. However, the role of IFN-alphabeta in regulating Th1 development is controversial, and previous studies have suggested a species-specific pathway leading to Th1 development in human but not mouse CD4+ T cells. In this study, we found that although both IFN-alpha and IL-12 can promote STAT4 activation, IFN-alpha failed to promote Th1 commitment in human CD4+ T cells. The difference between these innate signaling pathways lies with the ability of IL-12 to promote sustained STAT4 tyrosine phosphorylation, which correlated with stable T-bet expression in committed Th1 cells. IFN-alpha did not promote Th1 development in human CD4+ T cells because of attenuated STAT4 phosphorylation, which was insufficient to induce stable expression of T-bet. Further, the defect in IFN-alpha-driven Th1 development was corrected by ectopic expression of T-bet within primary naive human CD4+ T cells. These results indicate that IL-12 remains unique in its ability to drive Th1 development in human CD4+ T cells and that IFN-alpha lacks this activity due to its inability to promote sustained T-bet expression.     

Membrane vesicles shed by murine melanoma cells selectively inhibit the expression of Ia antigen by macrophages

We previously demonstrated that membrane vesicles shed by the F10 variant of the murine B16 melanoma cell line inhibited the induction by interferon-gamma (IFN) of murine macrophage immune response region-associated (Ia) antigen expression. In this paper we present evidence that the inhibition of macrophage Ia antigen expression is a selective effect of vesicles and characterize its temporal requirements. Membrane vesicles shed from F10 cells did not affect the expression of macrophage H-2K or H-2D antigens under conditions shown to profoundly inhibit Ia antigen expression. Similarly, the induction of plasminogen activator and interleukin 1 from macrophages was not inhibited by the vesicles. The vesicles did not measurably decrease total cellular RNA or protein synthesis. Macrophages were sensitive to the inhibitory effects of the vesicles during the induction and maintenance phases of Ia expression. Pretreatment of macrophages with vesicles before culture with IFN did not reduce the induction of Ia. The rate of decline of Ia expression after removal of IFN was unaffected by the presence of vesicles. Removal of vesicles from cultures of IFN-treated macrophages resulted in only a partial recovery of Ia expression, suggesting that the inhibition of Ia expression may be a slowly reversible process. The selective and partially reversible inhibition of Ia expression by vesicles shed from the plasma membrane of tumor cells is a possible mechanism whereby tumor-bearing hosts may become immunocompromised.     

Differential induction of H-2K vs H-2D class I major histocompatibility complex antigen expression by murine recombinant interferon-gamma

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.     

Growth regulation of transformed T cells by nonactivated macrophages: the role of Ia expression

We studied the influence of unactivated mouse peritoneal macrophages on the proliferative capacity of a spontaneously transformed MRL-lpr/lpr T cell clone. Macrophages, 25%, induced a reduction in proliferative rate from 20% to 95% measured by [3H]thymidine incorporation and microscopic cytometry. MHC-compatible (H-2k) macrophages caused growth inhibition reciprocal to the amount of Ia expression on the macrophage. Thus, with increasing preculture of the macrophages there was both decreasing Ia and increasing suppression. H-2-incompatible macrophages had maximal inhibitory capacity without preincubation. Macrophages derived from the peritoneum of MRL-lpr/lpr mice were less suppressive than macrophages from other H-2k mice. In contrast to the case of activated macrophages in other studies, in the present system there was no killing of T cells, only reduction in proliferation. The inhibitory effect of the macrophages correlated with the spontaneous formation of rosettes between the macrophages and the T cell clone. The number of rosettes forming a single layer of T cells around the macrophages, but not the number of rosettes with multiple layers of cells, was reciprocally related to the amount of Ia expression. The results suggest that macrophages bear a surface structure that influences and modulates the growth of T cells.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.     

Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2^b) and BALB/c mouse (H-2^d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions ( light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2K^b, H-2K^d, and H-2D^d) but not the H-2D^b antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2K^b antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.     

Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells

A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4- to 8-cell-stage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cell-surface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important cross-reactivities is also emphasized.     

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-beta was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-kappaB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.     

Regulation of murine macrophage function by IL-4. I. Activation of macrophages by a T-T cell hybridoma is due to IL-4

A T-cell hybridoma produced by fusion of concanavalin A-stimulated murine splenocytes produced a factor (MAFH) capable of activating tumoricidal capacity by responsive murine peritoneal macrophages. Macrophages treated with the MAFH required an additional trigger signal of bacterial lipopolysaccharide (LPS) for maximal activity. In contrast to interferon-gamma (IFN gamma), which induced tumoricidal activity against all tumor cells tested, MAFH only induced macrophage-mediated kill of the BI6P51 and 168 lines, and not of the P815 or B16BL6 lines. An identical pattern of tumoricidal activity was obtained by treating macrophages with recombinant interleukin-4 (IL-4). The active moiety of MAFH appeared to be IL-4 as (i) monoclonal antibody against IL-4 blocked MAFH, but not IFN gamma, activity, and (ii) the T-cell hybridoma contained large amounts of mRNA for IL-4 and no detectable mRNA for IFN gamma (as determined by Northern dot analysis). The pattern of tumoricidal activity observed may be due to an IL-4 mediated enhancement of tumor necrosis factor production by LPS-triggered macrophages.     

The unique antigen receptor signaling phenotype of B-1 cells is influenced by locale but induced by antigen

Normal animals contain an autoreactive B lymphocyte subset, the B-1 subset, which is controlled by undefined mechanisms to prevent autoimmunity. Using a V(H)11V(kappa)9 Ig transgenic mouse, with a specificity prototypic of the subset, we have explored conditions responsible for the previously reported Ag hyporesponsiveness of these cells. We report that peritoneal V(H)11V(kappa)9 B cells exhibit typical B-1 behavior with high basal intracellular free Ca(2+) and negligible receptor-mediated calcium mobilization. However, splenic B cells from this mouse, while phenotypically similar to their peritoneal counterparts, including expression of CD5, mount robust B-2-like responses to Ag as measured by calcium influx and altered tyrosine phosphorylation responses. When these splenic cells are adoptively transferred to the peritoneal cavity and encounter their cognate self-Ag, they acquire a B-1 signaling phenotype. The ensuing hyporesponsiveness is characterized by increases in both basal intracellular calcium and resting tyrosyl phosphorylation levels and is highlighted by a marked abrogation of B cell receptor-mediated calcium mobilization. Thus, we show that self-Ag recognition in specific microenvironments such as the peritoneum, and we would propose other privileged sites, confers a unique form of anergy on activated B cells. This may explain how autoreactive B-1 cells can exist while autoimmunity is avoided.     

Molecular analysis of deficient class I H-2 antigen expression by mouse lung carcinoma cells

We have continued our investigations of line lung carcinoma cells to understand the molecular basis of decreased expression of class I H-2 Ag and class I Ag induction with DMSO. We show that line 1, a murine lung carcinoma cell line, has low levels of class I Ag (H-2K, D, and L) because it is deficient in both class I and beta 2-microglobulin (B2M) RNA, and that these mRNA can be coordinately induced with DMSO. Evidence presented herein also shows that IFN-gamma can induce surface expression of class I Ag and suggests that it may act through a different mechanism than DMSO in inducing class I Ag. To further evaluate the regulation of class I expression, H-2Dp genes were transfected into line 1 cells. The transfected H-2 genes appear to be constitutively expressed at much higher levels than are the endogenous class I genes because surface expression of the foreign Dp Ag on the transfectants is elevated relative to the endogenous H-2d haplotype class I Ag. Both Dp surface expression and Dp mRNA are induced after treatment with DMSO. In all the Dp transfectants, we observed higher constitutive levels of class I mRNA as well as increased constitutive levels of endogenous B2M mRNA when compared to control or untransfected line 1 cells, however, we could not correlate these constitutive levels with Dp copy number. These results suggest that the regulation of class I and B2M genes is linked and that expression of class I genes can affect the expression of B2M genes.     

Sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle

Sarcopenia, loss of skeletal muscle mass, is a hallmark of aging commonly attributed to a decreased capacity to maintain muscle tissue in senescence, yet the mechanism behind the muscle wasting remains unresolved. To address these issues we have explored a rodent model of sarcopenia and age-related sensorimotor impairment, allowing us to discriminate between successfully and unsuccessfully aged cohort members. Immunohistochemistry and staining of cell nuclei revealed that senescent muscle has an increased density of cell nuclei, occurrence of aberrant fibers and fibers expressing embryonic myosin. Using real-time PCR we extend the findings of increased myogenic regulatory factor mRNA to show that very high levels are found in unsuccessfully aged cohort members. This pattern is also reflected in the number of embryonic myosin-positive fibers, which increase with the degree of sarcopenia. In addition, we confirm that there is no local down-regulation of IGF-I and IGF-IR mRNA in aged muscle tissue; on the contrary, the most sarcopenic individuals showed significantly higher local expression of IGF-I mRNA. Combined, our results show that the initial drive to regenerate myofibers is most marked in cases with the most advanced loss of muscle mass, a pattern that may have its origin in differences in the rate of tissue deterioration and/or that regenerating myofibers in these cases fail to mature into functional fibers. Importantly, the genetic background is a determinant of the pace of progression of sarcopenia.