Generation of H-2-reactive T cell lines that bear the 5936 idiotype(s)

The present experiments showed 1) that it was possible to produce mouse T cell lines against MHC determinants with a relatively high success rate by stimulation of purified T cells with allogeneic cells in the presence of irradiated syngeneic spleen cells; 2) that these lines could be led to react against selected H-2 specificities; 3) that only T cell lines established from Ig-1b allotype mice contained 5936-Id+ T cells (5936-Idiotypes are defined by an antiserum against B6 anti-CBA IgG produced in rabbit no 5936, which was tolerant to mouse gamma-globulin); and 4) that antigenic determinants coded by IAk genes induce the 5936-Idiotype(s). The latter data are in accordance with the 5936-idiotype characteristics of primary MLC T blasts. All T cell lines contained both specific MLC-responding cells and cytolytic cells. However, studies on the functional capacity of 5936-Id+ T cells from both primary MLC and the T cell lines showed that neither MLC-responding cells nor cytolytic cells directed against H-2Kk, IAk, or H-2Dk were 5936-Id+. Thus, 5936-Id+ T cells may be regulator cells induced by IAk antigens.     

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1 ^b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.     

Genetic chasing of T helper cell idiotype and allotype genes

The present experiments were performed to study whether the genes responsible for the expression of T-cell idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 anti-B10.BR sera and on Lyt-1⁺, 2.3^–B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1⁺, 2.3^–B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 x C3H.OH) x C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1 ^b genes (B6) and not with Igh-1 ^b genes (C3H.OH, CBA). Our results will show, first, that 5936 idiotypes on Lyt-1⁺, 2.3^–B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1 ^b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1⁺, 2.3^–B6 T cells are produced by genes also linked to Igh-1 ^b -allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.Abbreviations used in this paper BCGF B cell growth factor - B6 C57B1/6 - C_H constant region of immunoglobulin heavy chain - Con A concanavalin A - FCS fetal calf serum - Id idiotype - Ig immunoglobulin - LPS lipopolysaccharide - M mouse - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MRBC mouse red blood cells - NMS normal mouse serum - NP nitrophenacetyl - NRS normal rabbit serum - PFC plaque forming cell - R rabbit - Tcf T cell factor - Tcr T cell receptor - TNP Trinitrophenol - V_H variable region of Ig heavy chain Definitions of terms used in this paper: T-cell idiotypes, structures on T-cell membranes or released T-cell molecules detected by an anti-idiotypic antiserum (5936) produced against specific immunoglobulin idiotypes. The 5936 T-cell idiotypes are related to the specific binding of IA^k gene products by certain Igh-1^b T cells. T cell allotypes, structures on T-cell membranes or released T-cell molecules detected by an antiserum (6036) produced against 5936 idiotype-bearing T-cell molecules. The 6036 T-cell allotypes are related to the binding by Igh-1^b T cells of all Ia gene products tested, and they are non-cross-reactive with immunoglobulin allotypes.     

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: Codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1–6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F₁ hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1^b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1^a) mice were low responders. The V_H recombinant strains BAB.14 and CB-8KN that possess the Ig-1^b allotype of C57BL, but have some of theV _H genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.     

H-2 restriction of cytolysis after immunization of minorH congenic pairs of mice

Mice of three congenic resistant lines differing from C57BL/10 at theH-3, H-13, H-7, andH-8 minor histocompatibility loci were used to immunize, and were immunized with, C57BL/10. Cytotoxic cells which were capable of causing rapid lysis of cells from the immunizing strain were generated at least one-way in all combinations tested. In order for a target to be susceptible to cytolysis, it had to carry both the sameH-2 ^b haplotype and the same differential minor histocompatibility alleles as the immunizing strain. That is, B10.C(47N) (H-2 ^b ,H-7 ^b ) anti-C57BL/10 (H-2 ^b ,H-7 ^a ) cytotoxic cells lysed C57BL/10 targets but not B10.BR (H-2 ^k ,H-7 ^a ) targets, nor BALB.B (H-2 ^b ,H-7 ^b ) targets. Crossreactions in the cytotoxic assay suggest that theH-3, H-13 congenic pair —B10.LP and C57BL/10 —may differ in at least two more minor histocompatibility loci which have not yet been defined. Although 6 x 10⁶6 C57BL/10 lymphoid cells primed B10.D2(57N) (H-8 ^b ) mice for a secondary in vitro cytotoxic response, a tenfold higher dose apparently made them tolerant. It is concluded that all minor histocompatibility differences can generate cytotoxic T cells which show specificity both for the minor and major histocompatibility alleles.     

Susceptibility to db gene and streptozotocin-induced diabetes in C57BL mice: control by gender-associated,MHC-unlinked traits

H-2 haplotype differences distinguish the related C57BL/KsJ (BKs) and C57BL/6J (B6) inbred strains. BKs mice are more susceptible to diabetes induction by a recessive obesity gene, diabetes (db), or by multi-dose streptozotocin (MSZ) administration. The purpose of this study was to evaluate whether the H-2 differences were the important genetic background modifiers determining inbred strain susceptibility or resistance to these diabetogenic stresses. Diabetes susceptibility of BKs.B6-H-2 ^b congenic mice was compared with that of the parental BKs and B6 stocks. In addition, diabetes severity was studied in (B6 × BKs)F₁ and F₂ db/db mice and an H-2 segregation analysis was performed. BKs susceptibility genes expressed in a dominant fashion in the F₁ generation, and were transmitted to F₂ db/db males without apparent segregation. No association between H-2 ^b haplotype and B6-type diabetes resistance was found in response to either the db mutation or to MSZ. Insulitis, associated with development of hyperglycemia in BKs males, also occurred in the H-2 ^b congenic stock. However, an apparent interaction between H-2 ^b haplotype, the db mutation (on chromosome 4), and male gender (Y chromosome?) was indicated by a segregation ratio distortion in recovery of this genotype. A more moderate diabetes in some F₂ db/db females suggested that non-MHC-linked genes controlling sex steroid metabolism were the important determinants of diabetogenic sensitivities in the C57BL stocks. In support of the latter, strain differences were demonstrated in activity levels of steroid sulfatase, which is regulated by a sex-linked gene likely expressed on both the X and Y chromosome, and which may control tissue levels of active androgens and estrogens. We show that the diabetes-susceptible F₁ hybrids exhibit the higher activity associated with the BKs strain.     

Bence jones proteins and light chains of immunoglobulins

The availability of antisera with specificity forλ light chains which have the Mcg- or the non-Mcg-associated amino acid C-region sequence alternations has made possible our immunochemical differentiation of humanλ chains as Mcg⁺ or Mcg^?. One antiserum, prepared against an Mcg⁺ λ chain having the Mcg-associated C-region amino acid residues (asparaginyl, threonyl, and lysyl at positions 116, 118, and 167, respectively), had specificity forλ chains with this C-region sequence. A second antiserum, prepared against an Mcg^? λ chain having the non-Mcg-associated C-region residues (alanyl, seryl, and threonyl at these same three respective positions), had specificity forλ chains with this alternative type of C-region sequence. Immunodiffusion analyses ofλ chains of known amino acid sequence confirmed their chemical classification as Mcg or non-Mcg in type. No association between a particular V-regionλ-chain subgroup and the Mcg factor was evident. Based on sequence and serological analyses, ～ 11 percent ofλ light chains have the Mcg-related C-region sequence alternations. The immunochemical recognition of both Mcg⁺ and Mcg^? light chains isolated from the IgG of normal individuals corroborated the isotypic nature of the Mcg factor. Despite the fact that the Mcg-related substitutions are in the C_λ, the loss of Mcg antigenicity upon cleavage of Mcg⁺ and Mcg^? λ chains into V_L and C_L indicates that the intact light polypeptide chain is essential for expression of the Mcg antigenic factor.     

Analysis ofH-2 mutants: Evidence for multiple CML target specificities controlled by theH-2K b gene

C57BL/6 (H-2 ^b ) mice and two mutants derived from this strain, B6.C-H-2 ^ba (Hz1) andE6-H-2 ^bd (M505), were studied in a number of functional tests, in vitro and in vivo, that assay for differences at theH-2 complex. All three strains give rise to reciprocal mixed lymphocyte reactivity (MLR) and cell-mediated lympholysis (CML) in vitro as well as graft-host reactivity (GVHR) and skin graft rejection in vivo. Analysis for cross-reactivity between these strains in CML revealed that the gained antigens in each mutant do not cross-react, and that Hz1 has lost an antigen shared by C57BL/6 and M505 strains. In addition, spleen cells from B10.A(4R) mice, which differ from theH-2 ^b haplotype only at theK end of theH-2 complex, recognize a common antigen shared by all three strains tested. Provided that the mutations occurred in theH-2K ^b gene, these data indicate that a) there are at least three antigenic specificities coded for by theH-2K ^b gene(s) that serve as targets for receptors on thymus-derived (T) cells in CML; b) since C57BL/6 strain mice and the mutants are serologically indistinguishable on a qualitative basis, the antigens recognized by the receptors on T cells and by humoral H-2 antibody are nonidentical; and c) mutation in theH-2K ^b locus itself can give rise to allogeneic recognition phenomena such as MLR and GVHR.     

Epitope-Specific Regulatory CD4 T Cells Reduce Virus-Induced Illness while Preserving CD8 T-Cell Effector Function at the Site of Infection

The role of epitope-specific regulatory CD4 T cells in modulating CD8 T-cell-mediated immunopathology during acute viral infection has not been well defined. In the murine model of respiratory syncytial virus (RSV) infection, CD8 T cells play an important role in both viral clearance and immunopathology. We have previously characterized two RSV epitope-specific CD4 T-cell responses with distinct phenotypic properties. One of them, the IA^bM₂₀₉-specific subset, constitutively expresses FoxP3 and modulates CD8 T-cell function in vitro. We show here that the IA^bM₂₀₉-specific CD4 T-cell response regulates CD8 T-cell function in vivo and is associated with diminished RSV-induced illness without affecting viral clearance at the site of infection. Achieving the optimal balance of regulatory and effector T-cell function is an important consideration for designing future vaccines.A subset of CD4 T cells with regulatory function (Treg) has been shown to play an important role in modulating adaptive immune responses. Natural Tregs are characterized by the expression of FoxP3 and participate in reducing the activation of CD8 T-cell responses in peripheral lymphoid organs (11, 20, 35). This modulation can diminish the ability of adaptive immune responses to control systemic infections (4). However, the presence of natural regulatory CD4 T cells can have a beneficial effect on immune-mediated pathology, particularly at the site of infection. Tregs have been shown to limit pulmonary inflammation and lung injury induced by pneumocystis infection (29) and to modulate herpes simplex virus-induced inflammatory lesions of the eye (46). Natural Tregs also reduce the symptoms of West Nile virus infections in both humans and mice; Treg-deficient mice were more likely to develop lethal infection (25). Viral infection can also induce antigen-specific CD4 T cells that express FoxP3 (27), and their role in protective immunity and immunopathology needs more detailed investigation.T lymphocytes are key components of adaptive immunity against respiratory syncytial virus (RSV) infection. Children with T-cell deficiencies have delayed virus clearance and are more susceptible to fatal RSV infection (10, 18). The absence of T cells infiltrating into lung is associated with fatal RSV infections in children without recognized underlying disease (49). In the murine model, CD8 T cells play a major role in RSV clearance, presumably through direct cytotoxicity to infected cells and the generation of immunocompetent molecules (2, 15, 43); depletion of CD8 T cells in mice results in delayed viral clearance (14). The CD8 T-cell response also induces immunopathology in primary infection of mice (15, 32, 48). Transferring high doses of CD8 T cells facilitates virus clearance but also causes hemorrhagic pneumonia and enhanced disease (6, 14). These studies demonstrate that while CD8 T cells are required for viral clearance, they are responsible for immunopathology. We have described the pattern of CD8 T-cell responses that occur in mice that are the F₁ hybrid (H-2^d/b) between BALB/c (H-2^d) and C57BL/6 (H-2^b) (39). These mice respond both to the well-characterized K^dM2₈₂ epitope (24) and to a more recently described D^bM₁₈₇ epitope (38). Both CD8 T-cell responses are dominant in the parent strains but assume a hierarchy (K^dM2₈₂ > D^bM₁₈₇) in the F₁ hybrid (39). This model infection allows the analysis of factors that determine T-cell response hierarchy and provides multiple endpoints for the assessment of factors that modify or regulate CD8 T-cell responses.We recently described epitope-specific CD4 T-cell responses distinguished by novel major histocompatibility complex (MHC) class II tetramers in RSV infection. The IA^bM₂₀₉-specific CD4 T cells have a high frequency of FoxP3 expression and suppress RSV-specific CD8 T-cell cytokine production in vitro (27). To investigate the regulatory role of IA^bM₂₀₉-specific CD4 T cells in vivo, we sought to determine how immunizing mice with the CD4 T-cell epitope peptide M₂₀₉ would affect the RSV-specific CD8 T-cell response. We show here that the IA^bM₂₀₉-specific CD4 T cells have a regulatory effect on the dominant CD8 T-cell response to RSV infection, reducing both the magnitude and effector cytokine production in peripheral lymphoid organs while allowing effector functions at the site of infection to clear virus with normal kinetics. Viral clearance was thus achieved with less illness.     

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2 bm6 , B6.C-H-2 bm7 , and B6.C-H-2 bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2 ^bm5 (bm5) and B6-H-2 ^bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2K^b molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2 ^bm6 (bm6), B6.C-H-2 ^bm7 (bm7), and B6.C-H-2 ^bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the K^b molecule.     

Biochemical studies on the H-2K antigens of the MHC mutantsbm3 andbm11

Biochemical analyses of the H-2K-gene products of the C57BL/6 mutant strains, bm3 and bm11, have been carried out in order to characterize the structural relationships among these antigens. From comparative tryptic peptide mapping of cyanogen bromide fragments from the mutant and parent K^b glycoproteins and from preliminary amino-acid sequence analyses, a number of discrete differences have been discerned. Two sites of difference at amino residues 77 and 89 in the K^bm3 glycoprotein are noted relative to K^b. An alteration at residue 77 similar or identical to that seen in K^bm3 is present in the K antigen of thebm11 mutant. Because our techniques sample only 75 to 80 percent of the extracellular portion of H-2K^b, other undetected changes are possible. However, our present findings are most consistent with the conclusion that only very limited differences exist between mutant and parent molecules. Further, taken together with CML (cell-mediated-lymphocytotoxicity) reaction patterns (Melief et al. 1980), the biochemical data support the hypothesis that the proposed structural alterations in theK ^b mutant glycoproteins are directly related to their observed immunological specificity.     

Genetic regulation of the expression of antigen specificity H-2K.31 on mouse erythrocytes

In titration of anti-H-2K.31 antiserum by hemagglutination, erythrocytes of some mice show “standard” titration curves without a detectable prozone, whereas erythrocytes of other mice show a marked prozone or midzone phenomenon. Genetic studies have shown that a single, dominant gene identical with or closely lined to theEa-4 ^b allele of C57BL-related mouse strains is responsible for this prozone. The anti-H-2K.31 serum (A anti-BALB/c sarcoma Meth A) also contains a low titer of anti-Ea-6.2 antibodies, the first reported occurrence of these antibodies.     

Multigenic control of immune responses of inbred mice against the terpolymers poly(Glu57Lys38Ala5) and poly(Glu54Lys36Ala10) and linkage withH-2 haplotype

Results of immunizations of recombinant inbred and congenic strains of mice with the random polymers poly(glu⁵⁷ lys³⁸ala⁵) or GLA⁵ and poly(glu⁵⁴lys³⁶ala¹⁰) or GLA¹⁰ indicate that there is an association of the responsiveness with theH-2 haplotype. Although the C57BL/6J mice (H-2 ^b haplotype) are “non responders”, the C57BL/6By originally derived from mice of the same haplotype are responders. The immune response pattern of recombinant strains carrying haplotypes derived by crossing over within theH-2 complex indicate that the responsiveness is under control of anIr gene which maps to the left of theIB subregion. Studies with the backcross mice indicated multigenic control of the responsiveness, with one locus beingH-2 linked and another locus segregating independently ofH-2.     

A new serologically defined locus,Qa-1, in theTla-region of the mouse

A new cell-surface antigen, specified by a gene betweenH-2D andTla is described. The provisional notationQa-1 is suggested for the locus determining this newly recognized cell surface component. Qa-1 is distinguished from known TL antigens by the following two criteria. Its expression is not confined to thymocytes — it occurs on lymph node cells (LNC) also; and the phenotypes of the new congenic recombinant strains B6.K1 and B6.K2, derived fromH-2D/Tla crossovers, are Qa-1⁺ Qa-2^–TL^– and Qa-l⁺Qa-2⁺TL^–. Qa-1 antigen is defined by reaction of the standard TL typing serum, (B6 × A -Tla ^b)F₁ anti-A strain leukemia ASL1, with lymph node cells (LNC) in the cytotoxicity assay. Qa-1 antigen evidently is expressed, at least, on a subpopulation of T cells as well as on thymocytes. The gene order isH-2D, Qa-1, Qa-2, Tla.Abbreviations used in this paper LNC lymph node cells pooled from inguinal, axillary, brachial, and mesentric nodes - BA⁺ (C57BL/6-Tla^axA)F1 - BA^– (C57BL/6 × A -Tla ^b)F₁ - PBS phosphate buffered saline pH 7.2 - Thy thymocytes - RMIg Rabbit anti-mouse immunoglobulin Please address proofs and communications concerning this paper to Dr. Thomas Stanton, Sloan Kettering Institute, 1275 York Ave., New York, N.Y. 10021     

H-2-linked murine factor B phenotypes

A hemolytic assay has been developed which is specific for Factor B (B) activity in murine EDTA-plasma. Three discrete levels of B activity were observed among B 10-congenic strains. Mice with standard H-2 haplotypes, b, d, k, r, f, q, s, and u, all exhibited the same mean level of activity. However, plasma from H-2 ^v (B10.SM) mice contained only 0.25 of that level, and those with standard haplotype H-2 ^ja (B10.WB) or wild haplotype H-2^wr7 (B10.WR) exhibited 2.5 times the H-2 ^b (1310) basal level of activity. These differences among B10 congenic lines suggested that the activity is H-2 controlled; further tentative mapping with intra-H-2 recombinants indicated that the gene is located in the S region. A fourth phenotype was found among progeny of backcross generations between B10.BR (H-2 ^k ) and mice of subspecies Mus musculus molossinus and M. m. bactrianus. This ultra-high activity was found also to be governed by a gene very closely linked to Ss, the primary S region marker. F₁ generations between disparate phenotypes yielded progeny with activity levels intermediate between the parents; progeny of parents of different strains with the same phenotype expressed B hemolytic titres equal to those of the parental strains. No differences in antigenic levels of the protein among the strains of different phenotypes could be detected by radial immunodiffusion. In mixing experiments, resultant activity levels were intermediate between the higher and the lower phenotype, ruling out independent inhibitors or activators of the reaction. These studies indicate that an H-2-linked S region-located single gene governs structural differences in allelic B molecules that lead to differences in specific activities.     

Interaction between MHC-encoded products and cloned T cells

The interaction between class I major histocompatibility complex (MHC) products and T cells was studied using H-2K^b-specific alloreactive T-cell lines and clones obtained by repeated in vitro stimulation with allogeneic cells. Induction of proliferation of these T cells appeared to involve two signals: the H-2K^b alloantigen and interleukins. Immunopurified liposome-inserted H-2K^b, which stimulates specific secondary in vitro cytotoxic T lymphocyte (CTL) responses, could not replace cell-associated H-2K^b in the stimulation of these T-cell lines, even in the presence of feeder cells and interleukins. When T-cell lines were initiated in vitro and repeatedly stimulated with H-2K^b liposomes and feeder cells, it was possible to obtain T cells that could proliferate in response to H-2K^b liposomes in the presence of feeder cells and interleukin-2-containing supernatants or on H-2K ^b -expressing cells. Only stimulation with cells permitted maintenance of these T cells in culture for more than 12 weeks. Analyses of cell surface markers and of patterns of inhibition of proliferation by monoclonal antibodies (mAb) of T-cell lines induced in vitro with cell- or liposome-associated H-2K^b indicated that T-cell stimulation by class I antigen can occur in at least two ways. In the first, the H-2K^b-induced proliferation of Lyt-1^- Lyt-2⁺ T4^- T cells is inhibited by H-2K^b- and by Lyt-2-specific mAb, but not by Ia or T4-specific mAb. In the second, both Lyt-2⁺ and T4⁺ T cells are involved and the H-2K^b-induced proliferation is inhibited by H-2K^b- and Lyt-2-specific mAb and by Ia- and T4-specific mAb.Abbreviations used in this paper Ab antibody - mAb monoclonal antibody - C complement - i.p. intraperitoneally - PBS phosphate-buffered saline - PBS-B-N PBS containing bovine serum albumin and NaN₃ - CTL cytotoxic T lymphocyte - Th T helper cell - MHC major histocompatibility complex - PMA 4-phorbol 12-myristate 13-acetate - SCA concanavalin A-stimulated rat spleen cell supernatant - SC16 EL4 clone 16 supernatant - IL-1 interleukin-1 - IL-2 interleukin-2 (T-cell growth factor) - FCS fetal calf serum - H-2K^b-lip. H-2K^b inserted in liposomes - C. E. cell equivalents     

Plasma membrane H+-ATPase in sorghum roots as affected by potassium deficiency and nitrogen sources

We studied the influence of inorganic nitrogen sources (NO₃ ^? or NH₄ ⁺) and potassium deficiency on expression and activity of plasma membrane (PM) H⁺-ATPase in sorghum roots. After 15 d of cultivation at 0.2 mM K⁺, the plants were transferred to solutions lacking K⁺ for 2 d. Then, K⁺ depletion assays were performed in the presence or absence of vanadate. Further, PMs from K⁺-starved roots were extracted and used for the kinetic characterization of ATP hydrolytic activity and the immunodetection of PM H⁺-ATPase. Two major genes coding PM H⁺-ATPase (SBA1 and SBA2) were analyzed by real-time PCR. PM H⁺-ATPase exhibited a higher V_max and K_m in NH₄ ⁺-fed roots compared with NO₃ ^? -fed roots. The optimum pH of the enzyme was slightly lower in NO₃ ^? -fed roots than in NH₄ ⁺-fed roots. The vanadate sensitivity was similar. The expressions of SBA1 and SBA2 increased in roots grown under NH₄ ⁺. Concomitantly, an increased content of the enzyme in PM was observed. The initial rate of K⁺ uptake did not differ between plants grown with NO₃ ^? or NH₄ ⁺, but it was significantly reduced by vanadate in NH₄ ⁺-grown plants.     

Genetic control of the immune response to a thymus independent synthetic polypeptide

Strain dependent differences were found in the ability of inbred mice to produce antibodies to a thymus independent synthetic polypeptide, poly (DTyr, DGlu)-polyDPro-polyDLys. These differences were detected after injecting the antigen either in complete Freund's adjuvant or in aqueous solution, and were significant already in the primary response. High- and low-responder mice produced mainly antibodies of the IgG class as deduced from their mercaptoethanol resistance and their elution in the second fraction of a Sephadex G-200 chromatography. Genetic analysis of the immune response potential to poly(DTyr,DGlu)-polyDPro-polyDLys has indicated that responsiveness to this immunogen is controlled by a dominant, quantitative gene(s) which is not linked to either the major histocompatibility complex (H-2) of the mouse or to the heavy chain locus and is not located on the X-chromosome.     

Assembly-dependent expression of antigenic epitopes by β2-microglobulinb

In this report we provide evidence for the expression of antigenic epitopes on mouse (₂-microglobulin^b _2m ^b) that result from assembly with cognate H-2 class I heavy chains. For the cell line 69.9.15 (₂m^a × ₂m^b), which expresses a mutant cytosolic form of H-2K^b and wild-type H-2D^b, flow cytometry with rabbit antiserum against mouse ₂m displayed ₂m expression by cells grown in the presence or absence of fetal calf serum. By contrast, the epitopes identified by the ₂m^b-specific monoclonal antibody (mAb) S19.8 and clone 23 were not expressed by 69.9.15 cells grown in serum-containing conditions, and although S19.8 reactivity was weakly recovered by culture in the absence of serum, no such reacitivity was observed with clone 23. Strong expression of these epitopes was achieved following transfection of 69.9.15 cells with the wild-type H-2K ^b gene, indicating that the ₂m^b epitopes defined by mAb S19.8 and clone 23 were expressed when ₂m^b was assembled with an appropriate heavy chain. In support of this conclusion, we observed the recovery of the S19.8 and clone 23 epitopes by in vitro assembly of H-2K^b heavy chains with ₂m^b in the presence of the VSV N protein p52–59; however, such epitopes were expressed neither by ₂m^b prior to heterodimer assembly nor by non-conformed ₂m^b present in tissue culture supernatants recovered from H-2 class I surface positive cells. Taken together, these data indicate that in addition to the property of ₂m to modify the antigenicity of the MHC class I heavy chains, ₂m epitopes are induced in a reciprocal manner by assembly with MHC class I heavy chain molecules. Correspondence to: R. A. Zeff.