A divergent transcriptional landscape underpins the development and functional branching of MAIT cells.

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing-based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 "mature" MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ-producing MAIT1 cells and interleukin-17-producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)-deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.

Relaxin binds to and elicits a response from cells of the human monocytic cell line, THP-1.

Relaxin is a 6-kDa peptide of the insulin family that is present at increased levels in the circulation during pregnancy. Its functions at that time are thought to include maintenance of myometrial quiescence, regulation of plasma volume, and release of neuropeptides, such as oxytocin and vasopressin. The protein also promotes connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis in various mammalian species. In this report, we provide evidence for a novel target of relaxin, the human monocytic cell line, THP-1. Relaxin bound with high affinity (Kd = 102 pM) to a specific receptor on THP-1 cells. Receptor density was low ( approximately 275 receptors/cell), but binding of relaxin triggered intracellular signaling events. Receptor density was not modulated by pretreatment with estrogen, progesterone, or a number of other agents known to induce differentiation of THP-1 cells. Cross-linking studies showed radiolabeled relaxin bound primarily to cell surface proteins with an apparent molecular mass of >200 kDa. Other members of the insulin-like family of proteins (insulin, insulin-like growth factors I and II, and relaxin-like factor) were unable to displace the binding of relaxin to THP-1 cells, suggesting that a distinct receptor for relaxin exists on this monocyte/macrophage cell line.

Functional and biochemical analysis of the cloned Duffy antigen: identity with the red blood cell chemokine receptor.

The Duffy blood group antigen has been postulated to be a receptor on red blood cells (RBCs) for the malarial parasite Plasmodium vivax and a promiscuous receptor for the chemokine superfamily of inflammatory proteins. Recently, the Duffy antigen glycoprotein D cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). We have analyzed the binding properties of the cloned Duffy antigen. Duffy-antigen cDNAs expressed in human embryonic kidney cells produced cell-surface proteins that reacted with two known anti-Duffy monoclonal antibodies. Direct ligand binding and displacement experiments using recombinant chemokine proteins also show that the cloned Duffy protein is the RBC chemokine receptor. Radiolabeled chemokines of both the C-C (RANTES and MCP-1) and C-X-C (IL-8 and MGSA/gro) subclasses bound reversibly to transfected cells with dissociation constants in the nanomolar range. Chemokines of either class displaced heterologous chemokines, indicating that they were competing for a single site on the transfected cells. Although the chemokines bound to the transfected cells with high affinity, there was no evidence for signal transduction, as measured by transient increases in intracellular calcium ion concentration, through the Duffy antigen/RBC chemokine receptor in transfected cells. Lastly, we have performed a computer analysis on the amino acid structure of the Duffy antigen/RBC chemokine receptor. Although the cloned Duffy antigen has been postulated to be a nine-transmembrane-spanning receptor, our analysis suggests that the molecule most likely belongs to the seven-transmembrane-spanning receptor superfamily and is therefore similar to other chemokine receptors previously identified.

HSP 70 synthesis in clinical hyperthermia patients: preliminary results of a new technique.

PURPOSE: Although thermotolerance may be an important variable in clinical hyperthermia, few means have been described to measure its effect or duration in the clinical setting. This study was undertaken to determine if heat shock protein 70 could be used as an assay to predict the presence of retained thermotolerance in human tumors. METHODS AND MATERIALS: Tissue samples were obtained from patients undergoing hyperthermia and assayed for heat shock protein 70 synthesis. Eight patients having advanced, persistent, or recurrent malignant tumors had open-ended thermometry catheters placed into the lesion being heated. Through these catheters, tissue samples were obtained using a fine needle aspiration technique. Attempts were made to obtain samples before and after the first three heat treatments. Some samples were labeled immediately with radioactive methionine (35S) at 37 degrees C for 4-8 hr, others were given a test heat dose in vitro and then labeled. Protein synthesis profiles were analyzed by gel electrophoresis and autoradiography. RESULTS: Preliminary results show that it is possible to obtain tissue from hyperthermia patients in a safe and practical manner, that the rate of heat shock protein 70 synthesis can be measured in a variety of tumors, and that the persistence of thermotolerance in the clinical setting can be shown by the inability to reinduce heat shock protein 70 synthesis. CONCLUSION: The measurement of heat shock protein 70 using the described technique may provide an assay for retained thermotolerance in clinical hyperthermia. Technical difficulties which need to be addressed include obtaining sufficient tissue in all patients, confirming the presence of tumor in the obtained tissue, and obtaining tissue at more frequent intervals to best determine the kinetics of thermotolerance.

Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor.

The immunoregulatory proteins C-C chemokines are potent chemoattractants of lymphocytes and monocytes, as well as activators and attractants of eosinophils and basophils. We have isolated a cDNA that encodes a seven transmembrane-spanning receptor, with homology to other chemoattractant receptors, that encodes a protein designated C-C CKR-1 that acts as a receptor for the C-C chemokines. Human and murine macrophage inflammatory protein 1 alpha (MIP-1 alpha), human human monocyte chemotactic protein 1 (MCP-1), and RANTES all bind to the C-C CKR-1 with varying affinities. Chemokine binding affinity does not predict how well the ligand will transmit a signal through the receptor: RANTES and human MIP-1 alpha induce a similar intracellular calcium flux while binding with disparate affinities, while MCP-1 and human MIP-1 beta induce calcium mobilization only at high concentrations. Finally, C-C chemokines were shown to bind a C-C CKR-1-related gene product encoded by cytomegalovirus, suggesting a role for C-C chemokines in viral immunity.

Molecular cloning and expression of the murine RANTES cytokine: structural and functional conservation between mouse and man.

The infiltration and activation of monocytes is a hallmark of chronic inflammation, including that associated with a variety of disease states such as rheumatoid arthritis, atherosclerosis, and various autoimmune conditions. Recently, a family of small molecular mass proteins has been described which appear to have inflammatory properties, including chemoattractant effects on monocytes. We report here on the molecular cloning, characterization, and functional expression of mu RANTES, a new murine member of this family. mu RANTES expressed in a mammalian expression system is an approximately 8-kDa protein exhibiting immune cross-reactivity with a rabbit polyclonal antiserum generated against human RANTES. Boyden chamber chemotaxis experiments reveal some lack of species specificity in monocyte chemoattractant potential, as recombinant mu RANTES attracts human monocytes in a dose-dependent fashion in vitro. mu RANTES and its human homolog share approximately 85% amino acid identity, a higher level of conservation than that seen with any other species homologs in this cytokine family, and second only to transforming growth factor-beta among reported immune cytokines.

Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene.

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. We have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, two-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45 degrees C and their thermal survivals were determined by colony-formation assay, we found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress.

Thermal response of oncogene-transfected rat cells.

Rat embryo cells or Rat-1 fibroblasts were transfected with either an activated c-myc or a c-Ha-ras from the T24/EJ bladder carcinoma, or they were cotransfected with both. A gene conferring neomycin or hygromycin resistance was also cotransfected so that independent cell lines could be selected by growth in medium containing the antibiotic. Certain isolates from cells transfected with only one type of oncogene were further transformed by exposure to 600 cGy of 250-kVp X-rays. Successful transfection and transformation were characterized by altered morphology, increased plating efficiency, shorter doubling time, longer life span, foci formation, anchorage-independent growth, and Southern and Northern hybridization analysis. The thermal response of these cells at different stages of oncogenic transformation was examined by exposing exponentially growing cells to 45 degrees C for 0 to 45 min and measuring cellular survivals using colony formation assay. We found that cells transfected with myc oncogene, singly or in combination with ras, were more sensitive to thermal stress. Aside from that, the cells' thermal sensitivity was not affected by the degree or the nature of transformation.

Re-induction of hsp70 synthesis: an assay for thermotolerance.

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp 70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41 degrees C, 4 h) and then incubated at 37 degrees C for 0-72 h. At various times after the first heat treatment, cells were either challenged with a 45 degrees C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [35S]methionine before or after an additional test heat dose (e.g. 43.5 degrees C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.

Chemically induced resistance to heat treatment and stress protein synthesis in cultured mammalian cells.

Short exposure (1-2 h) of cultured cells, derived from a transplantable murine mammary carcinoma, to sodium arsenite, 2,4-dinitrophenol (DNP), carbonylcyanide-3-chlorophenylhydrazone (CCP) or disulfiram, induced resistance to a subsequent heat treatment, similar to heat-induced thermotolerance. Optimum resistance to a test heat treatment of 45 min at 45 degrees C after sodium arsenite exposure was obtained at a concentration of 300 microM, after DNP exposure at 3mM, after CCP at 300 microM and after disulfiram exposure in the range 1-30 microM. Exposure of cells to CCP, sodium arsenite or disulfiram led to enhanced synthesis of some proteins with the same molecular weight as 'heat shock' proteins. The pattern of enhanced synthesis of these proteins was agent specific. We could not detect significantly enhanced synthesis of the proteins after DNP using one-dimensional gel electrophoresis. These results suggest that enhanced stress protein synthesis is not a prerequisite for the development of thermal resistance.

Induction of heat shock protein synthesis in murine tumors during the development of thermotolerance.

The function of one or more heat shock proteins (HSPs) may be to confer protection of cells against thermal damage. We examined the induction kinetics of thermotolerance and the synthesis of HSPs in murine tumor models. Squamous cell carcinomas (SCC VII/SF) or radiation-induced fibrosarcomas (RIF) were implanted in the flanks of C3H mice. These flank tumors were first exposed to an elevated temperature (41 degrees-45 degrees C) for a fixed duration, for example, 43 degrees C for 15 min. Some of the tumors were excised immediately, and tumor cell suspensions were made. The other mice with tumors were returned to the cages and left undisturbed for various times up to 72 h before being sacrificed. Again, tumors were then removed and tumor cell suspensions were prepared. These tumor cells were either challenged with a second heat treatment at 45 degrees C in vitro or labeled with [35S]methionine at 37 degrees C in vitro. The tumor cell survival after the combined heat treatments was measured using the in vitro cloning assay. The cellular proteins were analyzed by one- or two-dimensional gel electrophoresis. We found that mild heat shock induced thermotolerance in murine tumors, a result consistent with those of others. The kinetics of induction and decay of thermotolerance depended on the temperature and duration of the priming treatment. Mild heat shock also enhanced the rate of synthesis and accumulation of some HSPs during the development of thermotolerance. For example, after an initial treatment at 43 degrees C for 15 min, the rates of synthesis of HSPs with molecular weights 68,000, 70,000, and 88,000 were greatly enhanced in SCC VII/SF tumors when compared to unheated controls. Qualitatively similar results were seen with radiation-induced fibrosarcoma tumors. The rate of synthesis of Mr 68,000 to 70,000 HSPs reached maximum value (300% of control value) 2 to 4 h after heat shock and decreased to the control value 6 to 24 h later. On the other hand, the rate of synthesis of actin, a major structural cellular protein, remained relatively constant throughout the 72 h of experiments. We then determined the relationship between the synthesis and accumulation of these HSPs and the expression of thermotolerance in murine tumors after a priming heat treatment. The data indicate that the levels of Mr 68,000 to 70,000 HSPs correlate well with thermotolerance.(ABSTRACT TRUNCATED AT 400 WORDS)

Heat-induced protection of mice against thermal death.

The possibility that the exposure of organisms to whole-body hyperthermia may provide protection against subsequent thermal exposures is intriguing and may play an important role in the clinical scheduling of fractionated hyperthermia. We used C3H mice to investigate whether whole-body heating can be used as a conditioning treatment to induce protection of mice against thermal death from a subsequent heat treatment. Our data clearly show that a conditioning whole-body heat dose (41 degrees for 40 min), by itself nonlethal, can give substantial protection to animals against a later heat treatment. The heat-induced protection is transient in nature: it reaches a maximum by 6 to 24 hr following the 41 degrees conditioning dose and decays by approximately 60% by 72 hr. The data presented do not shed any light on the cause of death following whole-body hyperthermia. Our results show clearly that the response of a complex organism to heat can be altered by previous heat exposure.