Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

The Qa-1 alloantigens. I. Identification and molecular weight characterization of glycoproteins controlled by the Qa-1a and Qa-1b alleles

Splenocytes from the Qa-Tla congenic strain pairs, A and A-Tlab or B6 and B6-Tlaa, were biosynthetically labeled with 3H-amino acids or cell surface labeled with 125I. Membrane proteins were solubilized with detergent and chromatographed on lentil lectin-Sepharose, and the resulting adherent pools were immunoprecipitated with antisera specific for determinants controlled by the Qa-1a and Qa-1b alleles, Qa-1.1 and Qa-1.2, respectively. Polyacrylamide gel electrophoresis analysis of immunoprecipitates from biosynthetically labeled preparations indicated that both the Qa-1.1 and Qa-1.2 antigens were glycoproteins with a m.w. of approximately 46,000. Qa-1.2 isolated from radioiodinated spleen cells similarly had a m.w. of 46,000. Analysis of anti-Qa-1.1 precipitates from 125I-labeled Qa-1a lysates demonstrated in addition to the 46,000 m.w. component, an electrophoretically heterogeneous protein or series of proteins in the m.w. range of 55,000 to 75,000. The specificity of these reactivities was shown by both antiserum and genetic control immunoprecipitations. These findings indicate that the Qa-1.1 and Qa-1.2 antigens are cell surface glycoproteins that are distinct from the TL antigens, and suggest a further complexity at the Qa-1--Tla locus.     

Characterization of determinants encoded by four Qa-1 genotypes and their recognition by cloned cytotoxic T lymphocytes

The alloantigens encoded by the four defined Qa-1 genotypes were characterized by cloned cytotoxic T lymphocyte (CTL) recognition. CTL clones specific for Qa-1a- and for Qa-1b-encoded antigens were generated. Examination of the reactivity of these clones with target cells from H-2r and H-2f strains provided the strongest evidence to date for the designation of the Qa-1c and Qa-1d genotypes, respectively, for these strains. Qa-1c-encoded antigens were recognized by most, but not all CTL clones that specifically lysed Qa-1b target cells, thus demonstrating that these antigens lack a Qa-1b-associated determinant. Similarly, Qa-1d encoded antigens were recognized by only half of the CTL clones that lysed Qa-1a target cells. In addition, one CTL clone that was cytotoxic for Qa-1b and Qa-1c target cells demonstrated low affinity, cross-reactive recognition of a Qa-1d encoded antigen. The reactivity patterns of the monoclonal CTL defined five Qa-1 determinants. Qa-1a, Qa-1b, and Qa-1d each encode multiple determinants. Two Qa-1d encoded determinants probably reside on different molecular species. Finally, large numbers of CTL clones tested on panels of target cells indicated that the Qa-1a strains expressed indistinguishable Qa-1.1 antigens and the Qa-1b strains expressed indistinguishable Qa-1.2 antigens. Therefore, additional polymorphism among these strains is improbable.     

New complexities at the Qa-1 locus

Mouse strain and tissue distribution analyses indicate that the new antiserum A anti-A-Tla ^b recognizes the cell-surface product governed by the previously serologically undetectable Qa-I ^b allele. This cell-surface product has therefore been called Qa-1.2. Three levels of anti-Qa-1.2 cytotoxicity in the presence of complement have been observed: high, intermediate, and zero lysis. In general, high levels of lysis correlate with the presence of the Qa-1 b allele, while zero levels of lysis correlate with the presence of the Qa-1 ^aallele. The A.CA strain reacts with both anti-Qa-1.1 and anti-Qa-1.2 and may possess a third allele, Qa-1 ^d. Several strains including B6-H-2 ^k react in an intermediate fashion. Recombinant strain analyses indicate that this intermediate reaction may be due to modifying genes within the H-2D region.     

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.     

The Qa-1 alloantigens. III. Biochemical analysis of the structure and extent of polymorphism of the Qa-1 allelic products

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to examine and compare the products of the Qa-1 locus. Analysis of Qa-1 isolated from detergent lysates of surface labeled cells indicated this molecule was a slightly acidic 48,000 to 50,000 dalton glycoprotein that displayed little charge heterogeneity on resting lymphocytes. The level of expression and degree of charge heterogeneity were both increased on activated lymphocytes. Direct comparison of the Qa-1b, Qa-1c, and Qa-1d allelic products by 2-D PAGE revealed that these three molecules could be distinguished from one another on the basis of isoelectric point, indicating that they were distinct at the molecular level. Comparison of Qa-1 isolated from several Qa-1b strains did not detect additional polymorphism. Removal of asparagine-linked oligosaccharides by treatment with endoglycosidase F indicated that carbohydrate contributed 10,000 to 12,000 to the m.w. of these allelic products. Comparative 2-D PAGE analysis could not distinguish between the deglycosylated Qa-1b, Qa-1c, and Qa-1d allelic products, implying that these molecules have similar primary structures. Peptide mapping supported this conclusion. Proteolytic digestion of the deglycosylated Qa-1b and Qa-1c allelic products resulted in identical peptide map patterns; such treatment of the deglycosylated Qa-1d allelic product produced a slightly different pattern. Peptide mapping analysis also demonstrated that the Tlaa and Qa-1a allelic products were distinct from one another, as well as being very different from the other three Qa-1 allelic products.     

Clonal analysis of the anti-Qa-1 cytotoxic T lymphocyte repertoire: definition of the Qa-1d and Qa-1c alloantigens and cross-reactivity with H-2

To characterize the four common Qa-1 allelic products, we examined in detail the CTL-defined determinants encoded by Qa-1. In previous studies with anti-Qa-1 CTL and alloantisera, investigators have described antigenic determinants present on Qa-1a and Qa-1b antigens, but they have defined Qa-1c and Qa-1d exclusively by their cross-reactivity with Qa-1a and/or Qa-1b determinants. To delineate further the CTL-defined determinants encoded by Qa-1d, we generated CTL clones with Qa-1d specificity and demonstrated that the Qa-1d molecule expressed determinants that were not detected on Qa-1a, Qa-1b, or Qa-1c target cells. Other CTL clones derived from anti-Qa-1d MLC recognized new antigenic determinants on Qa-1c that cross-reacted with Qa-1d. Each of the four common Qa-1 phenotypes was shown to exhibit unique antigenic determinants. In addition, Qa-1d anti-Qa-1a and Qa-1d anti-Qa-1b CTL confirmed extensive cross-reactivity among these Qa-1 alloantigens. Analysis of CTL from these four immunizations also resulted in the isolation of Qa-1a-specific and Qa-1d-specific CTL clones that cross-reacted with H-2Df and H-2Ks, respectively.     

Wild-type alleles of Rht-B1 and Rht-D1 as independent determinants of thousand-grain weight and kernel number per spike in wheat

The utilization of dwarfing genes Rht-B1b and Rht-D1b in wheat significantly increased grain yield and contributed to the “green revolution”. However, the benefit of Rht-B1b and Rht-D1b in drought environments has been debated. Although quantitative trait loci (QTL) for kernel number per spike (KN) and thousand-grain weight (TGW) have been found to be associated with Rht-B1 and Rht-D1, the confounding effect of environmental variation has made a direct association difficult to find. In this study, we used a doubled haploid population (225 lines) of Westonia × Kauz, in which both Rht-B1b (Kauz) and Rht-D1b (Westonia) segregated. The purpose of the study was to determine the interaction of Rht-B1 and Rht-D1 with grain yield components, namely KN and TGW, and to investigate genotype-by-environment interactions in glasshouse and field trials conducted in 2010 and 2011 in Western Australia. A genetic map of 1,156 loci was constructed using 195 microsatellite markers, two gene-based markers for Rht-B1 and Rht-D1, and 959 single nucleotide polymorphisms. The major QTL for TGW and KN were strongly linked to Rht-B1 and Rht-D1 loci and the positive effects were associated with the wild-type alleles, Rht-B1a and Rht-D1a. The major QTL of TGW were on chromosome 2D and 4B. The significant genetic effects (14.6–22.9 %) of TGW indicated that marker-assisted selection for TGW is possible, and markers gwm192a (206 bp) or gwm192b (236 bp) can be used as indicators of high TGW. For KN, one major QTL was detected on chromosome 4D in the analysis across three environments. The association of the wild-type alleles Rht-B1a and Rht-D1a in drought environments is discussed.     

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes

The MHC class Ib molecule Qa-1 is the primary ligand for mouse CD94/NKG2A inhibitory receptors expressed on NK cells, in addition to presenting Ags to a subpopulation of T cells. CD94/NKG2A receptors specifically recognize Qa-1 bound to the MHC class Ia leader sequence-derived peptide Qdm. Qdm is the dominant peptide loaded onto Qa-1 under physiological conditions and this peptide has an optimal sequence for binding to Qa-1. Peptide dissociation experiments demonstrated that Qdm dissociates from soluble or cell surface Qa-1(b) molecules with a t(1/2) of approximately 1.5 h at 37 degrees C. In comparison, complexes of an optimal peptide (SIINFEKL) bound to the MHC class Ia molecule H-2K(b) dissociated with a t(1/2) in the range from 11 to 31 h. In contrast to K(b), the stability of cell surface Qa-1(b) molecules was independent of bound peptides, and several observations suggested that empty cell surface Qa-1(b) molecules might be unusually stable. Consistent with the rapid dissociation rate of Qdm from Qa-1(b), cells become susceptible to lysis by CD94/NKG2A(+) NK cells under conditions in which new Qa-1(b)/Qdm complexes cannot be continuously generated at the cell surface. These results support the hypothesis that Qa-1 has been selected as a specialized MHC molecule that is unable to form highly stable peptide complexes. We propose that the CD94/NKG2A-Qa-1/Qdm recognition system has evolved as a rapid sensor of the integrity of the MHC class I biosynthesis and Ag presentation pathway.     

The differentiation of cytotoxic T cells in vitro. IV. Interleukin-2 production in primary mixed lymphocyte cultures involves cooperation between Qa-1+ and Qa-1- helper T cells

Studies with C57BL/6-TIaa mice have established that both Qa-1+ and Qa-1- helper T cells are required for the optimal production of Interleukin 2(IL-2) activity in primary MLC. This was established both by depletion of Qa-1 bearing cells by treatment of responder cells with anti-Qa-1 serum in the presence of complement and by positive selection (using FACS II analysis) of those cells displaying Qa-1. Furthermore, microfluoremetry revealed that the great majority of C57BL/6-TIaa and of A/J splenic T cells bore Qa-1 alloantigen but that only those with the highest antigen density were susceptible to complement-mediated lysis.     

The varicellovirus-encoded TAP inhibitor UL49.5 regulates the presentation of CTL epitopes by Qa-1b1

Impairment of MHC class I Ag processing is a commonly observed mechanism that allows viruses and tumors to escape immune destruction by CTL. The peptide transporter TAP that is responsible for the delivery of MHC class I-binding peptides into the endoplasmic reticulum is a pivotal target of viral-immune evasion molecules, and expression of this transporter is frequently lost in advanced cancers. We recently described a novel population of CTL that intriguingly exhibits reactivity against such tumor-immune escape variants and that recognizes self-peptides emerging at the cell surface due to defects in the processing machinery. Investigations of this new type of CTL epitopes are hampered by the lack of an efficient inhibitor for peptide transport in mouse cells. In this article, we demonstrate that the varicellovirus protein UL49.5, in contrast to ICP47 and US6, strongly impairs the activity of the mouse transporter and mediates degradation of mouse TAP1 and TAP2. Inhibition of TAP was witnessed by a strong reduction of surface MHC class I display and a decrease in recognition of conventional tumor-specific CTL. Analysis of CTL reactivity through the nonclassical molecule Qa-1(b) revealed that the presentation of the predominant leader peptide was inhibited. Interestingly, expression of UL49.5 in processing competent tumor cells induced the presentation of the new category of peptides. Our data show that the varicellovirus UL49.5 protein is a universal TAP inhibitor that can be exploited for preclinical studies on CTL-based immune intervention.     

Further biochemical data on Qa-2

The Qa-2 differentiation alloantigen is coded by a gene situated between the D and Tla loci of the murine major histocompatibility complex (H-2). Qa-2-bearing protein was isolated by immunoprecipitation and found to be composed of subunits of 40 000 and 12 000 daltons by SDS polyacrylamide gel electrophoresis (PAGE). The 12 000 dalton material was identified as 2-microglobulin (2M) by its molecular weight (SDS PAGE), charge (isoelectric focusing), antigenicity (reactivity with xenogenic anti- 2M), and genetics. The 40 000 dalton mol. wt. of Qa-2 heavy chain is 5 000 daltons less than that of D and K molecules (45 000 daltons). The quantity of Qa-2 isolated by immunoprecipitation was found to vary in a strain-specific fashion and as much as a 15-fold difference was observed.Abbreviations used in this paper B6 C57BL/6 strain mice - B10 C57BL/10 mice - 2M beta 2-microglobulin - IEF isoelectric focusing - K 1000 daltons - MHC major histocompatibility complex - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TL thymusleukemia antigen     

A new Tla region antigen Qa-11, similar to Qa-2 and associated with B-type beta 2-microglobulin

A new antigen, Qa-11, is detected as a 40,000 dalton band in the SDS-PAGE of immunoprecipitates of radiolabeled lymphocyte membrane preparations. In C57BL H-2 congenic strains, its presence is controlled by a gene in the Tla region. In strains with genetic background other than C57BL it is not expressed. Tests with recombinant inbred strains and with H-3 congenic strains show that, in addition to the Tla region, a gene linked to or identical with the beta 2-microglobulin-b-allele is required for the expression of Qa-11 as well. The mobility of the Qa-11 antigen in SDS-PAGE and in isoelectrofocusing is the same as that of Qa-2 antigen. The Cleveland peptide maps of Qa-2 and Qa-11 are identical as well. This finding, that the Tla region controlled Qa-11 antigen is structurally very similar to the Qa-2 antigen, contrasts with the fact that Tla region products do not react with anti-Qa-2 sera. This paradox could be explained by a separate Qa-11 region between Qa-2 and Tla. Alternatively, it is possible that the Qa-11 antigen is the result of the action of a modifying gene in the Tla region upon a Qa-2 gene product, or that the structural gene for Qa-11 is located in the Qa-2 region and a Tla region gene controls its expression.     

Mapping of independent V3 envelope determinants of human immunodeficiency virus type 1 macrophage tropism and syncytium formation in lymphocytes.

The V3 region of the human immunodeficiency virus type 1 (HIV-1) envelope protein is known to have a major influence on macrophage tropism as well as the ability to cause syncytium formation or fusion in CD4-positive lymphocyte cultures. Using infectious molecular HIV-1 clones, a series of mutant clones was created which allowed detailed mapping of V3 amino acid positions involved in these properties. In these experiments the non-syncytium-inducing phenotype in T cells did not always correlate with macrophage tropism. Macrophage tropism appeared to depend on the presence of certain combinations of amino acids at five specific positions within and just outside of the V3 loop itself, whereas syncytium formation in lymphocytes was influenced by substitution of particular residues at two to four positions within V3. In most cases, different V3 amino acid positions were found to independently influence macrophage tropism and syncytium formation in T cells and position 13 was the only V3 location which appeared to simultaneously influence both macrophage tropism and syncytium formation in lymphocytes.