Complex genetic effect of B10.D2 (M504) (H-2dm1) mutation

Serological and capping experiments show that the strain B10.D2 (M504) carrying the mutant haplotypeH-2 ^dm1 has two molecules in the products of theD region: H-2D^dm1 and H-2L^dm1 which are detectable by anti-H-2.4 and by anti-H-2.28 sera, respectively. Both these molecules differ serologically from the H-2D^d and H-2L^d molecules of the original (nonmutant) strain B10.D2. A third molecule, different from H-2D and H-2L, was detected inH-2 ^d ,H-2 ^dm2 but not inH-2 ^dm1 products.     

Stability of surface H-2K(b), H-2D(b), and peptide-receptive H-2K(b) on splenocytes.

We have used flow cytometry to study the stability and peptide-binding capability of MHC class I (MHC-I) on the surface of normal C57BL/6 mouse T lymphoblasts. The MHC-I molecules on each cell are nearly evenly divided into two populations with mean half-life values of approximately 1 and 20 h. Our observations suggest that members of the later contain peptide bound with medium to high affinity. Cell surface MHC-I molecules capable of binding exogenous peptide (thus, "peptide-receptive") belong almost entirely to the less stable population. Before exogenous peptide can bind, MHC-I must undergo a change, probably loss of a very low affinity peptide. For MHC-I-K(b), we found that the maximum rate for binding of exogenous peptide corresponds to a t(1/2) value of 12 min. To maintain the 50:50 steady-state distribution of long- vs short-lived MHC-I molecules on the cell surface, approximately 20 short-lived molecules must be exported to the cell surface for each long-lived molecule.     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

Capsid-specific cytotoxic T lymphocytes recognize three distinct H-2D(b)-restricted regions of the BeAn strain of Theiler's virus and exhibit different cytokine profiles

The role of virus-specific cytotoxic T lymphocytes (CTL) in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a viral model for multiple sclerosis, is not yet clear. To investigate the specificity and function of CTL generated in response to TMEV infection, we generated a panel of overlapping 20-mer peptides encompassing the entire capsid and leader protein region of the BeAn strain of TMEV. Binding of these peptides to H-2K(b) and H-2D(b) class I molecules of resistant mice was assessed using RMA-S cells. Several peptides displayed significant binding to H-2K(b), H-2D(b), or both. However, infiltrating cytotoxic T cells in the central nervous system of virus-infected mice preferentially lysed target cells pulsed with VP2(111-130/121-140) or VP2(121-130), a previously defined CTL epitope shared by the DA strain of TMEV and other closely related cardioviruses. In addition, at a high effector-to-target cell ratio, two additional peptides (VP2(161-180) and VP3(101-120)) sensitized target cells for cytolysis by infiltrating T cells or splenic T cells from virus-infected mice. The minimal epitopes within these peptides were defined as VP2(165-173) and VP3(110-120). Based on cytokine profiles, CTL specific for these subdominant epitopes are Tc2, in contrast to CTL for the immunodominant epitope, which are of the Tc1 type. Interestingly, CTL function towards both of these subdominant epitopes is restricted by the H-2D molecule, despite the fact that these epitopes bind both H-2K and H-2D molecules. This skewing toward an H-2D(b)-restricted response may confer resistance to TMEV-induced demyelinating disease, which is known to be associated with the H-2D genetic locus.     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

Crystal structures of murine MHC Class I H-2 D(b) and K(b) molecules in complex with CTL epitopes from influenza A virus: implications for TCR repertoire selection and immunodominance

Cytotoxic T lymphocyte (CTL) responses against influenza A virus in C57BL/6 mice are dominated by a small number of viral peptides among many that are capable of binding to major histocompatibility complex (MHC) class I molecules. The basis of this limited immune recognition is unknown. Here, we present X-ray structures of MHC class I molecules in complex with two immunodominant epitopes (PA(224-233)/D(b) and PB1(703-711)/K(b)) and one non-immunogenic epitope (HA(468-477)/D(b)) of the influenza A virus. The immunodominant peptides are each characterized by a bulge at the C terminus, lifting P6 and P7 residues out of the MHC groove, presenting featured structural elements to T-cell receptors (TCRs). Immune recognition of PA(224-233)/D(b) will focus largely on the exposed P7 arginine residue. In contrast, the non-immunogenic HA(468-477) peptide lacks prominent features in this C-terminal bulge. In the K(b)-bound PB1(703-711) epitope, the bulge results from a non-canonical binding motif, such that the mode of presentation of this peptide strongly resembles that of D(b)-bound peptides. Given that PA(224-233)/D(b), PB1(703-711)/K(b) and the previously defined NP(366-374)/D(b) epitopes dominate the primary response to influenza A virus in C57BL/6 mice, our findings indicate that residues of the C-terminal bulge are important in selection of the immunodominant CTL repertoire.     

Ligands presumed to label high affinity and low affinity ATP binding sites do not interact in an (alpha beta)2 diprotomer in duck nasal gland Na+,K+-ATPase, nor Do the sites coexist in native enzyme

The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same alphabeta protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each alphabeta protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITC-bound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITC-labeled enzyme, TNP-ADP and ErITC inhibit p- nitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC.