FLEXamers: A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers.

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.

Rotavirus Vaccine Take in Infants Is Associated With Secretor Status.

Rotaviruses bind to enterocytes in a genotype-specific manner via histo-blood group antigens (HBGAs), which are also detectable in saliva. We evaluated antirotavirus immunoglobulin A seroconversion ('vaccine take") among 166 Ghanaian infants after 2-3 doses of G1P[8] rotavirus vaccine during a vaccine trial, by HBGA status from saliva collected at age 4.1 years. Only secretor status was associated with seroconversion: 41% seroconversion for secretors vs 13% for nonsecretors; relative risk, 3.2 (95% confidence interval, 1.2-8.1; P = .016). Neither Lewis antigen nor salivary antigen blood type was associated with seroconversion. Likelihood of "take" for any particular rotavirus vaccine may differ across populations based on HBGAs.

Whole-proteome analysis of mesonephric-derived cancers describes new potential biomarkers.

Mesonephric carcinomas (MEs) and female adnexal tumors of probable Wolffian origin (FATWO) are derived from embryologic remnants of Wolffian/mesonephric ducts. Mesonephric-like carcinomas (MLCs) show identical morphology to ME of the cervix but occur in the uterus and ovary without convincing mesonephric remnants. ME, MLC, and FATWO are challenging to diagnose due to their morphologic similarities to Müllerian/paramesonephric tumors, contributing to a lack of evidence-based and tumor-specific treatments. We performed whole-proteomic analysis on 9 ME/MLC and 56 endometrial carcinomas (ECs) to identify potential diagnostic biomarkers. Although there were no convincing differences between ME and MLC, 543 proteins showed increased expression in ME/MLC relative to EC. From these proteins, euchromatic histone lysine methyltransferase 2 (EHMT2), glutathione S-transferase Mu 3 (GSTM3), eukaryotic translation elongation factor 1 alpha 2 (EEF1A2), and glycogen synthase kinase 3 beta were identified as putative biomarkers. Immunohistochemistry was performed on these candidates and GATA3 in 14 ME/MLC, 8 FATWO, 155 EC, and normal tissues. Of the candidates, only GATA3 and EHMT2 were highly expressed in mesonephric remnants and mesonephric-derived male tissues. GATA3 had the highest sensitivity and specificity for ME/MLC versus EC (93% and 99%) but was absent in FATWO. EHMT2 was 100% sensitive for ME/MLC & FATWO but was not specific (65%). Similarly, EEF1A2 was reasonably sensitive to ME/MLC (92%) and FATWO (88%) but was the least specific (38%). GSTM3 performed intermediately (sensitivity for ME/MLC and FATWO: 83% and 38%, respectively; specificity 67%). Although GATA3 remained the best diagnostic biomarker for ME/MLC, we have identified EHMT2, EEF1A2, and GSTM3 as proteins of interest in these cancers. FATWO's cell of origin is uncertain and remains an area for future research.

Three HLA-D region antigens defined by primed LD typing.

We have recently described a new method, primed LD typing or PLT, for specific identification of HLA-D antigens. Highly discriminatory PLT cells have been developed which clearly differentiate between cells of individuals that restimulate strongly and those that restimulate weakly. Seven such discriminatory PLT cells have been used to define three antigens called PL1, PL2, and PL3; two more PLT cells may define antigen(s) PL4.

Localization of H-2 antigens on mouse trophoblast cells.

The presence of H-2 antigens of the paternal and maternal haplotypes on mouse trophoblast cells was examined at several stages of pregnancy by using a sensitive immunolabeling technique followed by quantitative radioautography. Results revealed the presence of H-2 antigens (determined by the K or D loci) of both parental haplotypes on the F1 trophoblast cells. At 14-16 d of gestation, the antigen density was equivalent to that on adult thymocytes and there was a further 50% increase on day 18. H-2 antigens of both parental haplotypes are also found to be expressed on 11-13 d trophoblast cells.

Immunochemical evidence for an additional H-2 region closely linked to H-2D.

Anti-H-2 reagents have been tested on solubilized spleen cell preparations in combinations expected to be specific for D region products. Two different types of molecules were detected. One showed the expected reactivity with both antisera to private and antisera to public specificities. However, an additional molecule was detected which reacted only with antisera to public specificities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration patterns indicated that both products have a similar molecular size of approximately 45,000 daltons. The data therefore present chemical evidence for the existence of a third H-2-associated gene product of 45,000 mol wt in addition to the classical H-2K and H-2D antigens.

Properties of cell lines derived from tumors induced by Friend virus in BALB/c and BALB/c-H-2b mice.

Cell lines have been established in culture from Friend virus-induced tumors of BALB/c (H-2d) and congenic BALB/c-H-2b (BALB.B) origin. Spleens from virus-infected hosts in the terminal stages of erythroleukemic disease provided tissues for the establishment of subcutaneously transplantable tumors of both strains. Subsequently cells of these tumors were introduced into culture and passed serially. Complete, infectious Friend virus (FV) has been routinely recovered from culture supernates of BALB.B tumor cells (HFL/b) throughout its 2-yr passage history. However, after only a few transfer generations in culture BALB/c tumor cells (HFL/d) became nonproducers of virus detectable in either the spleen focus assay in vivo or the XC assay in vitro. Nonproducer HFL/d cells possessed the complete genomes of the components of the FV complex, since FV could be recovered from them either by cocultivation with helper virus-infected syngeneic embryo fibroblasts or by serial passage in the ascitic form in normal, syngeneic adult hosts.

Carbohydrate moieties of major histocompatibility complex class I alloantigens are not required for their recognition by T lymphocytes.

The ability to generate specific cytotoxic responses using purified major histocompatibility complex (MHC) antigen in liposomes has made it possible to directly assess the importance of class I carbohydrate moieties in T cell recognition of alloantigen. Deglycosylation of affinity-purified H-2Kk to yield a single glycan-free product did not alter the specificity, the magnitude, nor the dose range of the cytotoxic T lymphocyte (CTL) response to the class I antigen. It can be concluded that carbohydrate moieties are not required to maintain the necessary conformation of the MHC protein, nor to interact with either the antigen-specific receptor or accessory proteins on precursor CTL.

Serologic evidence for antigens controlled by the Ir region in mice.

Antibodies produced in B10.D2 mice against soluble lymphocyte membrane antigens of B10.A (H-2(a)) mice reacted only with lymphocytes of the strains carrying the Ir(k) region, i.e., B10.A(2R), B10.K, B10.BR, B10.HTT, AQR, A.TE, C3H, and CBA; they did not react with cells of strains carrying different Ir regions, i.e., B10.A(4R), B10, B10.M, A.SW, DBA/1. It is therefore concluded that the antigen detected with these antibodies is apparently controlled by the Ir region of the H-2 complex. The antigen is present on some T lymphocytes and absent on B lymphocytes. Its presence or absence seems to correlate with MLC and GVH reactivity.

Analysis of H-2 and Ia molecules by two-dimensional gel electrophoresis.

Mouse lymphocyte H-2 and Ia glycoproteins have been analyzed with a two-dimensional (2-D) acrylamide gel electrophoresis technique, in which proteins are separated first according to their charge in isoelectrofocusing gels and then according to their size in sodium dodecyl sulfate gels. Individual polypeptide chains from radiolabeled cells are resolved as discrete spots on autoradiograms of the gels, forming patterns which are characteristic of the proteins in the sample. 2-D gels of H-2K, H-2D, and Ia glycoproteins immunoprecipitated from 35S-methionine-labeled cells reveal that these proteins exist in the cells as complex arrays of molecules heterogeneous in both size and charge. Lactoperoxidase-catalyzed radioiodination of lymphocyte surfaces labels only subsets of the total H-2 and Ia molecules with 125I, indicating that some of the molecules may represent cytoplasmic precursors of the cell surface proteins. This theory is supported by the kinetics of labeling of various spots in 35S-methionine pulse-chase experiments. The 2-D gel patterns obtained for both H-2 and Ia antigens have also been shown to be haplotype-specific and independent of the genetic background.

Sharing of Ia antigens between species. I. Detection of Ia specificities shared by rats and mice.

A mouse anti-rat xenogeneic antiserum, B10.D2 anti-BN, has been found to react with a subpopulation of lymphoid cells of certain mouse strains. The corresponding alloantiserum, B10.D2 anti-B10.BR, reacted in analogous fashion with lymphoid cells of BN rats. In the case of the cross-reaction on mouse cells, mapping studies indicated that at least part of the reactivity was with the product of gene(s) determined by the I-A subregion of the H-2 complex. Chemical isolation studies with radiolabeled cell surface preparations indicated that the antigens detected in both mouse and rat had mol wt characteristic of Ia antigens (35,000 and 28,000 dalton molecules). Testing of fractionated spleen cell populations revealed that the cross-reactive antigens were expressed predominatly on B cells, but that a subpopulation of T cells were also reactive. Wider strain and species distribution studies are in progress to determine the extent of such Ia cross-reactions between species and to further assess the practical and theoretical importance of such cross-reactions.

Identification in rat liver and serum of water-soluble class I MHC molecules possibly homologous to the murine Q10 gene product.

We have identified large quantities of a water-soluble, non-RT1.A class I MHC molecule in the serum of the DA rat strain, with a similar molecule being found in aqueous extracts of DA liver. The non-RT1.A class I molecules have heavy chains of 41 kD, which is smaller than RT1.A class I molecules isolated from liver membranes (45 kD) but larger than water-soluble RT1.A class I molecules previously identified in serum and aqueous extracts of liver and kidney (40 kD). NH3-terminal amino acid sequencing of bulk-purified RT1.A class I molecules and of this novel non-RT1.A class I molecule revealed two substitutions, in the first 25 amino acids, Tyr----His at position 9, and Ala----Ser at position 24. The non-RT1.A class I molecule did not react with any of the well-characterized polymorphic and monomorphic antibodies directed against RT1.Aa class I molecules, but did react with the MRC OX18 antibody. A similar class I molecule could not be identified on liver membranes. The non-RT1.A class I molecule was found in large quantities (approximately 20 micrograms/ml) in the serum of the DA rat strain, and similarly large quantities appeared to be present in the sera of BN, PVG, and LEW.RT1a rats. WAG and LEW.RT1u rats had readily detectable but lower amounts of this molecule in their serum, while LEW and SHR rats had little if any present. This molecule probably represents the rat homologue of the murine Q10 gene product, and is the major class I product in the serum of the DA rat strain.

Class I transplantation antigens in solution in body fluids and in the urine. Individuality signals to the environment.

Classical class I transplantation antigens present in solution in the body fluids have been studied. These antigens have been found in a monomeric, soluble form in blood, lymph, and urine, and a major source is the hemopoietic system which gives rise to cells that secrete these molecules into the blood. The cell types most probably involved in their secretion are of the macrophage/dendritic cell lineage. The serum molecule is a heterodimer with a heavy chain of 39,000 mol wt associated noncovalently with beta 2-microglobulin and is present in serum at a concentration between 350 and 390 ng/ml. These molecules have a short half-life of 2.7 h and are excreted into the environment via the kidneys in the urine. In the urine, greater than 90% of the molecules are degraded into smaller fragments. This finding that normal metabolic processes lead to the excretion of classical highly polymorphic class I molecules in the urine provides a direct explanation in molecular terms of the ability of animals to identify individuals on the basis of urinary odor. Since intact class I molecules are unlikely to be the odoriferous component in the urine, two hypotheses have been suggested. Either small fragments of class I molecules are detected or the molecule acts as a carrier that transports volatiles from the serum into the urine where they are released, giving rise to the class I-associated odor.

Identification of a unique tumor antigen as rejection antigen by molecular cloning and gene transfer.

Tumor-specific transplantation antigens are antigens that can lead to complete immunological destruction of a transplanted cancer by the syngeneic host. When such antigens are expressed on cancers induced by chemical or physical carcinogens, then they are usually unique, i.e., antigenically different for each independently induced tumor. In this study, we show that the product of a gene encoding a novel MHC class I molecule and isolated from the murine UV light-induced regressor tumor 1591 represents one such unique tumor-specific transplantation antigen that causes tumor rejection. The major evidence comes from our finding that 1591 progressor variants regularly lost the gene encoding this antigen that is expressed in the parental tumor that regresses in normal mice; furthermore, reintroduction of this gene into a 1591 progressor variant by DNA transfection caused the progressor variant to regress in normal immunocompetent mice. Thus, the progressor tumor reverted to the parental regressor phenotype following transfection. Consistent with the conclusion that the expression of the novel MHC class I gene following transfection was responsible for the regressor phenotype is also our finding that a variant of the transfected tumor that had lost expression of the transfected gene resumed its progressive growth behavior. Finally, we show that the molecule encoded by the novel class I gene is specifically recognized by a syngeneic tumor-specific cytolytic T cell clone that we have previously shown to select in vitro for progressor variants from the parental regressor tumor cell line. It remains to be determined to what extent unique tumor-specific rejection antigens of other highly immunogenic regressor tumors are encoded by novel MHC class I genes and whether these genes represent germline mutations or somatic mutations caused by the carcinogen treatment.

Structure and function of three novel MHC class I antigens derived from a C3H ultraviolet-induced fibrosarcoma.

The UV-induced, C3H fibrosarcoma, 1591, expresses at least three unique MHC class I antigens not found on normal C3H tissue. Here we report the complete DNA sequence of the three novel class I genes encoding these molecules, and describe in detail the recognition of the individual products by tumor-reactive and allospecific CTL. Remarkably, although C3H does not appear to express H-2L locus information, this C3H tumor expresses two distinct antigens, termed A149 and A166, which are extremely homologous to each other and to the H-2Ld antigen from BALB/c. The gene encoding the third novel class I antigen from 1591, A216, is quite homologous to H-2Kk) throughout its 3' end. Since all three of these genes account for polymorphic restriction fragments not found in C3H, it is likely that they were derived by recombination from the endogenous class I genes of C3H. The DNA sequence homology of A149, A166, and H-2Ld is especially significant given the functional conservation observed between the products of these genes. Limited sequence substitutions appear to correlate with some of the discrete serological differences observed between these molecules. In addition, both A149 and A166 crossreact, but to differing extents, with H-2Ld at the level of T cell recognition. Our results are consistent with the view that CTL recognize complex conformational determinants on class I molecules, but extend previous observations by comparing a set of antigens with discrete and overlapping structural and functional differences.

Dendritic cell-lymphoid cell aggregation and major histocompatibility antigen expression during rat cardiac allograft rejection.

To determine the pattern of cellular expression of donor MHC class I and class II antigens during the course of rat cardiac allograft rejection, ACI cardiac allografts transplanted to BN recipients were examined from day 2 to day 6 using immunohistologic and immunoelectron microscopic methods. We used both monomorphic and donor-specific mouse anti-rat MHC class I and class II mAbs in this study. In normal ACI hearts, MHC class I reactivity was confined to the vascular endothelium and to interstitial cells. Ongoing rejection was characterized by an increased donor MHC class I staining intensity of microvascular endothelium and induction of donor class I surface reactivity on cardiac myofibers. Donor MHC class II reactivity was exclusively confined to interstitial dendritic cells (IDC) in both normal ACI hearts and in rejecting allografts, although rejection was associated with marked fluctuations in class II IDC frequency. An early numerical depression in class II IDC present in both allografts and syngeneic heart grafts was attributed to a direct effect of the transplantation procedure. By days 3-4, allografts showed an absolute overall increase in donor class II IDC frequency, which was associated with the presence of multiple localized high-density IDC-lymphocyte aggregates. The lymphocytes present in the focal areas were predominantly of the class II-reactive Th cell subpopulation. These aggregates may thus represent the in vivo homologue of dendritic cell-lymphocyte clustering, which has been shown to be required for primary class II allosensitization in the rat and mouse in vitro. During the late phase of rejection, there was a marked numerical fall in donor class II IDC, which correlated with extensive overall graft destruction. This study has shown that acute rat cardiac allograft rejection can occur in the absence of donor MHC class II expression by allograft vascular endothelium and cardiac myofibers. The IDC, which are believed to represent the principal class II alloantigen presenting cells in the rat heart, remain the sole class II-expressing cellular constituents of the graft throughout the course of rejection.

Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface.

To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

Two genes required for diabetes in BB rats. Evidence from cyclical intercrosses and backcrosses.

The BB rat develops a syndrome of autoimmune diabetes similar to Type I diabetes of man. It also has a severe T cell lymphopenia. As part of an ongoing breeding program to transfer the diabetogenic genes of the BB rat onto inbred rat strain backgrounds, diabetic animals were used in a backcross (BC)- intercross (IC)-backcross breeding scheme with Brown Norway (BN), Lewis (L), and Wistar-Furth (WF) inbred rats. We have used monoclonal antibodies to analyze both lymphopenia and major histocompatibility (MHC) antigens (the RT1 locus in the rat) in relation to the development of diabetes. To examine T cell subsets we used a panel of monoclonal antibodies, in particular W3/25 and OX19 , which discriminate the abnormal phenotype better than W3/13. In our breeding program, at least two independent genes or gene complexes are required for the expression of diabetes. One gene determines the lymphopenia, is inherited by simple autosomal recessive genetics and is not linked to the MHC. The second gene is linked to the MHC. Both genes are necessary, but neither gene is sufficient by itself for the development of diabetes.

Detection of alloantigens during preimplantation development and early trophoblast differentiation in the mouse by immunoperoxidase labeling.

An immunoperoxidase-labeling technique allowing visualization of antibody binding to the cell surface at the electron microscopical level has been employed an an analysis of H-2 and non-H-2 alloantigen expression on the early mouse embryo. The presence of non-H-2 antigenic determinants has been confirmed on eight-cell, morula, and blastocyst stages of development. Contrary to previous reports, however, low levels of H-2 antigen have also been detected on the blastocyst. This is the earliest stage at which H-2 has been shown to be expressed on the fertilized mouse egg and may reflect the greater resolution of the immunoperoxidase technique. Using two different models to study the critical peri-implantation stages, those of experimentally induced blastocyst activation and blastocyst outgrowth in vitro, it has been demonstrated that antigen loss occurs on the trophectoderm at the time of implantation, and that this is not necessarily dependent upon maternal influence. It is suggested that the loss may be an important factor in the prevention of maternal immune rejection during the establishment of the fetal allograft. The two major components of the early postimplantation conceptus display a striking differential in antigenic status. The embryonic sac shows a high degree of peroxidase labeling, while the ectoplacental cone trophoblast is unlabeled. These findings add support to the concept of antigenic neutrality of the early trophoblast and its role in the maintenance of a normal fetomaternal immunological equilibrium.

Association of immunity and tolerance to host H-2 determinants in irradiated F1 hybrid mice reconstituted with bone marrow cells from one parental strain.

Semiallogenetic radiation chimeras were prepared by injecting heavily irradiated F1 hybrid mice with bone marrow cells from one parental strain; the bone marrow cells were treated with anti-theta serum and complement to remove T cells and injected in large numbers (2 times 10-7 cells). The mice survived in excellent health until sacrifice 6 mo later. Thoracic duct cannulation at this stage showed that the mice possessed normal numbers of recirculating lymphocytes. Close to 100% of thoracic duct lymphocytes and lymph node cells were shown to be of donor strain origin. The capacity of lymphocytes from the chimeras to respond to host-type determinants was tested in mixed leukocyte culture and in an assay for cell-mediated lympholysis (CML). Mixed leukocyte reactions (MLR) were measured both in vitro and in vivo; tumor cells and phytohemmaglutinin-stimulated blast cells were used as target cells for measuring CML. While responding normally to third party determinants, cells from the chimeras gave a definite, though reduced MLR when exposed to host-type determinants. However, this proliferative response to host-type determinants, unlike that to third party determinants, was not associated with differentiation into cytotoxic lymphocytes. No evidence could be found that unresponsiveness in this situation was due to blocking serum factors or suppressor T cells. It is argued that the results support the concept that lymphocytes responsive in mixed leukocyte culture have a different specificity to those exerting cell-mediated lympholysis.