Cre recombinase-mediated inactivation of H-2Dd transgene expression: evidence for partial missing self-recognition by Ly49A NK cells.

We have established H-2D(d)-transgenic (Tg) mice, in which H-2D(d) expression can be extinguished by Cre recombinase-mediated deletion of an essential portion of the transgene (Tg). NK cells adapted to the expression of the H-2D(d) Tg in H-2(b) mice and acquired reactivity to cells lacking H-2D(d), both in vivo and in vitro. H-2D(d)-Tg mice crossed to mice harboring an Mx-Cre Tg resulted in mosaic H-2D(d) expression. That abrogated NK cell reactivity to cells lacking D(d). In D(d) single Tg mice it is the Ly49A+ NK cell subset that reacts to cells lacking D(d), because the inhibitory Ly49A receptor is no longer engaged by its D(d) ligand. In contrast, Ly49A+ NK cells from D(d) x MxCre double Tg mice were unable to react to D(d)-negative cells. These Ly49A+ NK cells retained reactivity to target cells that were completely devoid of MHC class I molecules, suggesting that they were not anergic. Variegated D(d) expression thus impacts specifically missing D(d) but not globally missing class I reactivity by Ly49A+ NK cells. We propose that the absence of D(d) from some host cells results in the acquisition of only partial missing self-reactivity.     

MISC: missing imputation for single-cell RNA sequencing data

Background

Single-cell RNA sequencing (scRNA-seq) technology provides an effective way to study cell heterogeneity. However, due to the low capture efficiency and stochastic gene expression, scRNA-seq data often contains a high percentage of missing values. It has been showed that the missing rate can reach approximately 30% even after noise reduction. To accurately recover missing values in scRNA-seq data, we need to know where the missing data is; how much data is missing; and what are the values of these data.

Methods

To solve these three problems, we propose a novel model with a hybrid machine learning method, namely, missing imputation for single-cell RNA-seq (MISC). To solve the first problem, we transformed it to a binary classification problem on the RNA-seq expression matrix. Then, for the second problem, we searched for the intersection of the classification results, zero-inflated model and false negative model results. Finally, we used the regression model to recover the data in the missing elements.

Results

We compared the raw data without imputation, the mean-smooth neighbor cell trajectory, MISC on chronic myeloid leukemia data (CML), the primary somatosensory cortex and the hippocampal CA1 region of mouse brain cells. On the CML data, MISC discovered a trajectory branch from the CP-CML to the BC-CML, which provides direct evidence of evolution from CP to BC stem cells. On the mouse brain data, MISC clearly divides the pyramidal CA1 into different branches, and it is direct evidence of pyramidal CA1 in the subpopulations. In the meantime, with MISC, the oligodendrocyte cells became an independent group with an apparent boundary.

Conclusions

Our results showed that the MISC model improved the cell type classification and could be instrumental to study cellular heterogeneity. Overall, MISC is a robust missing data imputation model for single-cell RNA-seq data.

     

LncRNAs: the missing link to senescence nuclear architecture

During cellular senescence and organismal aging, cells display various molecular and morphological changes. Although many aging-related long noncoding RNAs (lncRNAs) are highly associated with senescence-associated secretory phenotype, the roles of lncRNAs in senescence-associated nuclear architecture and morphological changes are just starting to emerge. Here I review lncRNAs associated with nuclear structure establishment and maintenance, their aging-related changes, and then focus on the pervasive, yet underappreciated, role of RNA double-strand DNA triplexes for lncRNAs to recognize targeted genomic regions, making lncRNAs the nexus between DNA and proteins to regulate nuclear structural changes. Finally, I discuss the future of deciphering direct links of lncRNA changes to various nuclear morphology changes assisted by artificial intelligence and genetic perturbations.     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

Induction of linked suppression in addition to the donor H-2 class I-specific unresponsiveness in recipient T cells by transfusing class I plus class II-disparate, but not class I alone-disparate, bone marrow cells

This study was undertaken to determine whether bone marrow (BM) cells contain a cell population with the capacity to induce an unresponsiveness of T cells specific to the BM self-H-2 class I antigens in vivo, i.e., veto cell population. Recombinant or congenic mice were infused intravenously with H-2-incompatible BM cells. One to several weeks later, donor H-2-and irrelevant H-2-specific responses in mixed lymphocyte reaction cultures of recipient T cells were assessed. Transfusion of H-2-incompatible BM of C57BL/10 (B10) recombinant strains caused a long-lasting cytotoxic T lymphocyte (CTL) unresponsiveness to the donor class I antigens in recipient lymph node cells. When class I plus class II-disparate BM cells were transfused, an anti-donor class I CTL response and a response against a third-party class I antigen, which was presented on the stimulator cells coexpressing the donor class I and class II, were significantly suppressed. This linked suppression lasted for less than 2 weeks after transfusion. Transfusion of class I-alone-disparate BM induced the donor class I-specific CTL unresponsiveness, but not the linked suppression. The induction of linked suppression was prevented considerably by transfusing nylon wool-nonadherent BM or by treating recipients with cyclophosphamide 2 days before transfusion. An anti-third-party class I CTL response, stimulated in vitro with fully allogeneic spleen cells, was not hampered by the BM transfusion. Coculturing the lymph node (LN) cells obtained from the class I plus class II-disparate BM recipient with normal LN cells interfered with the generation of both anti-donor class I and anti-linked third-party class I CTL, whereas, coculturing LN cells from the class I alone-disparate BM recipient inhibited neither specificity of CTL generation. Transfusion of class I plus class II-disparate BM resulted in a significant suppression of the donor class II-specific proliferative response. In contrast, transfusion of class I alone-disparate BM did not suppress any proliferative responses, including even a "linked" third-party class II-specific response. Transfusion of bm 1, (B6 X bm 1)F1, or (bm 1 X bm 12)F1 BM to B6 did not induce unresponsiveness in bm 1-specific CTL responses. However, the transfusion resulted in a significant suppression of bm 1-reactive proliferative response of recipient LN cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenovirus E1A-mediated regulation of class I MHC expression.

Expression of class I MHC transplantation antigens has been shown to be reduced in baby rat kidney (BRK) cells transformed by highly oncogenic adenovirus type 12 (Ad12), as compared with untransformed cells and cells transformed by non-oncogenic Ad5. Here we show that this reduction of class I expression also occurs in a variety of other primary cell cultures transformed by Ad12, and that reduction of class I gene expression occurs for all class I loci. Transfection of Ad5E1 into class I-negative Ad12-transformed BRK cells leads to complete restoration of class I expression. Introduction of Ad12E1 into most class I-positive established cell lines does not result in suppression of class I expression. However, transfection of the Ad12E1A region into a class I-positive cell line which was immortalized by a mutant Ad12E1A region resulted in suppression of class I gene expression, implying that the suppression of class I activity in Ad12-transformed cells is due to an active switching-off process.     

Amnion membrane epithelial cells express class I HLA and contain class I HLA mRNA

Amnion epithelial cells in membranes from term deliveries, which have been reported not to express histocompatibility Ag, were evaluated for HLA by using an avidin-biotin immunoperoxidase staining system and for class I HLA mRNA by Northern blotting and in situ hybridization. There were three major findings from these studies. 1) Amnion cells frequently expressed class I HLA. Three mAb to monomorphic determinants of class I HLA were used: 61D2, PA2.6, and W6/32. 61D2 identified 1 of 8 fresh amnion membranes as class I positive whereas PA2.6 identified 4/8 and W6/32 identified 5/8. 2) Amnion cells contained class I HLA mRNA. RNA extracted from amnion membranes hybridized to a class I HLA probe (pHLA1.1) in Northern blotting. In situ hybridization procedures with pHLA1.1 showed that essentially all amnion cells contained class I HLA mRNA. 3) Levels of class I HLA mRNA in amnion cells could be modulated. Exposure of amnion explants to medium containing IFN-gamma enhanced levels of class I HLA mRNA in amnion cells, whereas epidermal growth factor diminished those levels. The results suggest that amnion cells transcribe class I HLA genes and are capable of synthesizing class I H chains but that expression may be modulated by extrinsic regulatory molecules.     

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.     

Expression of class I HLA genes by trophoblast cells. Analysis by in situ hybridization

Evaluation of trophoblast cells by immunohistology has shown that subpopulations of trophoblast cells express class I HLA differently from one another and differently from embryonic and adult cells. Placental syncytial trophoblast does not express detectable levels of class I HLA; chorion membrane cytotrophoblasts bind one mAb to monomorphic determinants of class I Ag, W6/32, but not a second, 61D2. In the present study, sections of normal term placentae and matching extraplacental membranes were evaluated by in situ hybridization procedures for cells containing class I HLA mRNA using pHLA1.1, which is complementary to HLA-B. Class I Ag expression was identified by immunohistology using two mAb to class I HLA (W6/32, 61D2) and the mAb 4E to identify HLA-B. Placental syncytial trophoblast contained low to undetectable levels of class I mRNA and failed to bind all three mAb. Chorion membrane cytotrophoblast cells contained moderate levels of class I HLA mRNA and were positive with the mAb W6/32 but were negative with 61D2 and 4E. In adjacent tissues, fetal mesenchymal cells and maternal decidual cells contained high levels of class I mRNA and were positive with all three mAb. The results suggest that syncytial trophoblast may not express class I HLA because of low steady-state levels of class I HLA mRNA. In contrast, chorionic cytotrophoblast cells may express truncated versions of class I HLA or nonclassical HLA-A,B,C-like Ag. Regulation of the expression of class I HLA gene products may be essential to the development of a satisfactory immunologic relationship between the mother and her semiallogeneic fetus during pregnancy.     

SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells

Background

HIV-1 infection is associated with profound dysfunction of myeloid dendritic cells, for reasons that remain ill-defined. Soluble HLA class I molecules can have important inhibitory effects on T cells and NK cells, but may also contribute to reduced functional properties of professional antigen-presenting cells. Here, we investigated the expression of soluble HLA class I isoforms during HIV-1 infection and assessed their functional impact on antigen-presenting characteristics of dendritic cells.

Results

Soluble HLA class I molecules were highly upregulated in progressive HIV-1 infection as determined by quantitative Western blots. This was associated with strong increases of intracellular expression of HLA class I isoforms in dendritic cells and monocytes. Using mixed lymphocyte reactions, we found that soluble HLA class I molecules effectively inhibited the antigen-presenting properties of dendritic cells, however, there was no significant influence of HLA class I molecules on the cytokine-secretion properties of these cells. The immunomodulatory effects of soluble HLA class I molecules were mediated by interactions with inhibitory myelomonocytic MHC class I receptors from the Leukocyte Immunoglobulin Like Receptor (LILR) family.

Conclusions

During progressive HIV-1 infection, soluble HLA class I molecules can contribute to systemic immune dysfunction by inhibiting the antigen-presenting properties of myeloid dendritic cells through interactions with inhibitory myelomonocytic HLA class I receptors.     

Identification of class I MHC mRNA in human first trimester trophoblast cells by in situ hybridization

Special gestation-related regulatory mechanisms for the expression of class I Ag by trophoblast cells directly exposed to maternal blood and tissues may be required for semiallogeneic pregnancy to be successful. Analysis of class I MHC mRNA by in situ hybridization and class I MHC Ag by immunohistology has revealed two phenotypically distinct subpopulations of trophoblast cells in term placentas and extraplacental membranes. Trophoblast cells external to the placenta are mRNA +/Ag+. They contain class I mRNA and express class I Ag that differ serologically from HLA-A,B,C. In contrast, trophoblast cells forming the syncytial layer of placental villi are mRNA-/Ag-. By immunohistology, trophoblast cells in 1st trimester placental tissues are similar to those in term tissues. In our study, in situ hybridization was used to determine if patterns of trophoblast cell class I mRNA were the same or different. Trophoblast cells external to the placental villi in 1st trimester tissues contained class I mRNA as would be predicted from the results with term tissues. Unexpectedly, class I mRNA was found in villous trophoblast cells. Thus, these studies identified an mRNA+/Ag- trophoblast cell subpopulation. The results suggest that tissue-specific mechanisms may interfere with translation of class I mRNA in 1st trimester villous trophoblast cells and/or that the protein products of the mRNA are not identified by available mAb.     

Invasive equine trophoblast expresses conventional class I major histocompatibility complex antigens

Monoclonal antibodies and alloantisera were used in an indirect immunohistochemical assay to determine the expression of class I and class II Major Histocompatibility Complex (MHC) antigens by equine placental cells and the endometrial tissues at the fetal-maternal interface. MHC class I antigens were expressed at high density on the surface of the trophoblast cells of the chorionic girdle at days 32-36, just prior to their invasion of the endometrium. The mature gonadotrophin-secreting cells of the endometrial cups, which are derived from the chorionic girdle cells, had greatly reduced levels of MHC class I antigen expression while no MHC class I antigens were detectable on the non-invasive trophoblast cells of the allantochorion, except in small isolated patches. MHC class I antigens immunoprecipitated from chorionic girdle cells with either monoclonal antibodies or alloantisera had a relative molecular mass of 44,000, which was identical to that of MHC class I antigens precipitated from lymphocytes with the same reagents. MHC class II antigens were not detected on any trophoblast cells, although they were expressed at high levels by the endometrial glandular and lumenal epithelium immediately bordering the endometrial cups. MHC class I antigens were also expressed at high levels by endometrial tissues in the area of the cups. The high level of MHC class I antigen expression by endometrial glands within and bordering the cups was in sharp contrast to the greatly reduced class I antigen expression by the mature endometrial cup cells themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide-induced modulation of target cell sensitivity to natural killing.

We have previously shown that the capacity of class I molecules to confer resistance to NK in transfected target cells maps to the Ag-binding site (ABS) of the HLA class I structure. Here we examine the effect of peptide (reagents specific for the ABS) pretreatment on the NK sensitivity of class I+ target cells. Synthetic peptides (10-17 amino acids in length) were used to pretreat C1R target cells expressing either no serologically detectable HLA-A, B class I molecules, or C1R transfectants expressing individual HLA-A or -B locus class I molecules. In each case in which the class I allele had previously been shown to directly bind a given peptide, peptide-pulsing of target cells resulted in increased sensitivity to NK-mediated conjugation and cytolysis. The NK susceptibility of C1R target cells expressing no HLA-A, B class I molecules or the nonprotective HLA-A2.1 or HLA-A2M70 mutant class I molecules was unaffected by pretreatment with HLA-A2-binding peptides. These results support the intimate involvement of the HLA class I ABS and potentially ABS-bound peptides in determining target cell sensitivity to NK. Furthermore, these findings form the basis of an effective screening procedure for discerning peptide class I allele-specific interactions.     

A quantitative analysis of lymphokine release from activated T cells. Evidence for a novel form of T-T collaboration in vitro

We previously developed a simple mathematical model describing Ag-triggered lymphokine release from activated T cells. Previous test of this model revealed qualitative differences in the antigenic requirement for lymphokine release between activated T cell populations with the same apparent specificity when activated under different conditions. We now have found a case where class I MHC-reactive T cells (class I T cells) can modulate the nature of Ag-triggered lymphokine release from class II MHC-reactive T cells (class II T cells). Two significant requirements for this modulation event are: 1) Linked recognition/presentation of class I and class II Ag; that is, class I and class II MHC alloantigens must be presented on the same APC, and 2) active participation of the APC in this process; metabolic inactivation of the APC abrogates the class I T cell modulation of the class II activated T cell. These results suggest a novel form of T-T collaboration that involves the active participation of the APC, and provides evidence that T cells of one MHC specificity (class I) can influence the function of T cells of another MHC specificity (class II).     

Analysis of mechanisms by which NK cells acquire increased cytotoxicity against class I MHC-eliminated targets

Acid treatment, where cells are exposed to 0.2 M citric acid buffer at pH 3 for 2 min, was described in a previous paper to be a method which specifically eliminates class I MHC antigens from the membrane of viable cells. We applied this method to characterize functional roles of class I MHC antigens on the target cells in NK cell cytotoxicity. When NK target cells, U937, Molt-4, and Raji, were subjected to acid treatment, the treated cells lost their surface class I MHC antigens and became more sensitive to NK cell killing. On the other hand, the susceptibility of K562 cells which initially lacked class I MHC antigens did not significantly change after such treatment. We then examined the mechanism which enables NK cells to become more cytotoxic against class I MHC antigen-eliminated target cells. Single cell binding assay and cold target inhibition assay demonstrated that class I MHC antigen-eliminated target cells did not acquire high binding affinity to NK cells. However, the interaction between NK cells and class I MHC antigen-eliminated targets resulted in a greater increase in production of NKCF-like factor than did the interaction between NK cells and untreated targets. Class I MHC antigen-eliminated targets did not acquire high killer susceptibility to NKCF-like factor. The present study utilizing the acid treatment method confirmed that surface class I MHC antigens on the targets are important immunoregulatory molecules not only for cytotoxic T lymphocytes but also for NK cells and elucidated some of the underlying mechanisms.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.     

Susceptibility to natural killer cell-mediated cytolysis is independent of the level of target cell class I HLA expression

To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.