Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity

In preparation for functional analyses, a study of the binding of H-2K^b-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2^b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2K ^bm mutant cells and on FOR-treated H-2K^b cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2KP^b-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2^b cells as indicated by cold target inhibition studies. The H-2K^b-specific CTL were probably reactive with conformational determinants of H-2K^b, which are dependent on the integrity of both the 1 and the 2 domains of the H-2K^b molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the 1 domain without affecting conformational-type CTL determinants.Abbreviations used in this paper CTL cytotoxic T lymphocyte - FOR formaldehyde - PBS phosphatebuffered saline - FCS fetal calf serum - mAb monoclonal antibody - TNBS trinitrobenzene sulfonate     

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy

To study in vivo activated cytolytic T cells, CD8⁺ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8⁺ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8⁺ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell lines, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8⁺ and 2 CD4⁺ CD8⁺ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V and V gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V17/V7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V17/V7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8⁺ CTL. CTL with a range of melanoma specificities and different TCR dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8⁺ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.Supported by National Institutes of Health research grants CA 36233 and EY 9031, the Lucy Adams Memorial Fund and a grant from the Concern Foundation     

Optimization of cell density and dilution rate in <Emphasis Type="Italic">Pichia pastoris</Emphasis> continuous fermentations for production of recombinant proteins

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon (INF-). The objective was to maximize the volumetric productivity (Q, mg INF- l^–1 h^–1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150 X_c 450 g cells (wet weight) l^–1 and 0.1 _mD0.9 _m (_m=0.0678 h^–1, the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c and D were 328.9 g l^–1 and 0.0333 h^–1 for a maximum Q of 2.73 mg l^–1 h^–1. The model of specific production rate (, mg INF- g^–1 cells h^–1) was also established and showed the optimal X_c=287.7 g l^–1 and D=0.0361 h^–1 for the maximum (predicted to be 8.92×10^–3 mg^–1 g^–1 h^–1). The methanol specific consumption rate (, g methanol g^–1 cells h^–1) was calculated and shown to be independent of the cell density. The relationship between and (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.     

Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes

Alterations in intracellular Zn²⁺ concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn²⁺ deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn²⁺ for Ca²⁺ and Mg²⁺ within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential ( m). Therefore, cytoplasmic and mitochondrial events are critical to this process.     

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     

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.     

Cytotoxic T lymphocytes recognize determinants on the BALB/c-H-2Ld molecule controlled by α1 and α2 but not α3 external domains

We have shown that cytotoxic T lymphocytes (CTL) raised in H-2 ^dmice use H-2L^d but not H-2D^d or H-2K^d antigens as restricting elements in lymphocytic choriomeningitis virus (LCMV) and vesicular stomatis virus (VSV) infections. To localize the regions of H-2L^d protein recognized by CTL, we constructed a recombinant H-2L ^d/D ^dgene encoding a hybrid antigen with 1 and 2 external domains of H-2L^d and 3, transmembrane and cytoplasmic domains of H-2D^d. The recombinant gene was transfected into mouse cells and the hybrid molecules were characterized serologically, biochemically and functionally. In all assays, H-2L^d/D^d molecules were recognized by LCMV- and VSV-specific H-2L^d-restricted CTL in a manner similar to that of wild-type H-2L^d antigens. Analogous results were obtained with alloreactive CTL. Hybrid antigens containing the 3 domain of H-2L^d fused to 1 and 2 domains of a Qa-2,3 region-encoded antigen were not used as restricting elements by LCMV-specific CTL. These results suggest that H-2L^d-restricted CTL directed against LCMV and VSV recognize determinants controlled by the 1 and/or 2 domains of the H-2L^d molecule.Abbreviations used in this paper CTL cytotoxic T lymphocytes - VSV vesicular stomatitis virus - LCMV lymphocytic choriomeningitis virus - tk thymidine kinase - HAT hypoxanthine, aminopterine, thymidine - HSV herpes simplex virus - FCS fetal calf serum - SAC Staphylococcus aureus Cowan I strain - TM transmembrane - CYT cytoplasmic     

Electrochemical potential of protons in vesicles reconstituted from purified,proton-translocating adenosine triphosphatase

Summary Measurements were made of the difference in the electrochemical potential of protons ( ) across the membrane of vesicles reconstituted from the ATPase complex (TF ₀ ·F ₁) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential () and pH difference across the membrane ( pH), respectively.In the presence of Tris buffer the maximal and no pH were produced, while in the presence of the permeant anion NO ₃ ^– the maximal pH and a low were produced by the addition of ATP. When the ATP concentration was 0.24mm, the was 140–150 mV (positive inside) in Tris buffer, and the pH was 2.9–3.5 units (acidic inside) in the presence of NO ₃ ^– . Addition of a saturating amount of ATP produced somewhat larger and pH values, and the attained was about 310 mV.By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Phylogenetic relationships of the stromateoid fishes (Perciformes)

The phylogenetic relationships of all 16 genera (plus Psenes pellucidus) of the suborder Stromateoidei were estimated cladistically based on 43 osteological, myological, and external characters. Thirty equally parsimonious trees were obtained. Based on the strict consensus tree, Centrolophidae was nonmonophyletic, Psenopsis being placed as a sister group of a clade comprising Amarsipus, Ariomma, nomeids, Tetragonurus, and stromateids. Schedophilus formed a sister group relationship with Seriolella. The relationships among the Centrolophus, Hyperoglyphe, Icichthys, Tubbia, Schedophilus+Seriolella clade, and Psenopsis+Amarsipus+Ariomma+nomeids+Tetragonurus+stromateids clade were unresolved. Amarsipus, which is unique within the suborder in lacking a pharyngeal sac, was nested within the stromateoid clade, being a sister group of the clade including Ariomma, nomeids, Tetragonurus, and stromateids. The absence of a pharyngeal sac in Amarsipus was interpreted as a reversal, its presence in the Stromateoidei therefore being considered as a synapomorphy. Ariomma was placed as the sister group of a clade comprising nomeids, Tetragonurus, and stromateids. Monophyly of the Nomeidae and Stromateidae were supported by 2 and 11 synapomorphies, respectively.     

Poor induction of interleukin-2 receptor expression on CD8bright+ cells in whole blood cell cultures with CD3 mAb. Implications for immunotherapy with CD3 mAb

To induce better stimulation of T cells during recombinant interleukin-2 (rIL-2) therapy of renal cell carcinoma patients, pretreatment with low-dose CD3 monoclonal antibody (mAb) has been proposed. However, in our clinic, such a treatment did not induce additional activation of T cells. To investigate this we performed whole blood cell cultures with rIL-2 or CD3 mAb as a stimulant. Cultures using isolated blood mononuclear cells were used as a control. When stimulated by the addition of rIL-2, the lymphocyte composition and activation of whole blood cultures did not differ from those of mononuclear cell (MNC) cultures. However, when stimulation was performed with CD3 mAb, CD8bright⁺ cells in whole blood cultures were not or only minimally induced to express CD25 or IL-2 receptor (IL-2R\). This is in contrast to the situation found in MNC cultures where all CD8bright⁺ cells expressed CD25 or IL-2Rß to a high extent at the end of culture. When rIL-2 or recombinant interferon (rIFN) was added to whole blood cultures together with CD3 mAb, significantly more CD8bright⁺ cells were induced to express CD25 or IL-2Rß. These results suggest that whole blood cultures represent the in vivo situation better than MNC cultures. In addition, the results suggest that, also in vivo, administration of low-dose CD3 mAb alone might not be sufficient to induce IL-2R expression on CD8bright⁺ cells, and would therefore not induce additional specific T cell activation in rIL-2-based immunotherapy. The presented results suggest that in vivosimultaneous administration of rIFN or rIL-2 with low-dose CD3 mAb might induce better stimulation of CD8⁺ T cells than CD3 mAb only.     

The expression ofI-region gene products on lymphocytes

Mixed lymphocyte reaction (MLR) stimulation by purified T and B lymphocytes and thymocytes was studied. The MLR gene products involved were localized to theH-2 complex by the use of congenic mice differing atH-2, and to loci within theH-2 complex through the use of congenic mice bearing recombinant chromosome 17. Stimulation by T cells was investigated in detail. The role of small amounts of contaminating B lymphocytes, and that of backstimulation, was found to be of minor importance. T cells and thymocytes stimulated as well as or better than B cells in combinations differing in theI, S, and possibly parts of theD end, thus suggesting that these genetic regions control cell-surface products expressed on both T and B lymphocyte populations.Abbreviations used in this paper are MLR mixed lymphocyte reaction - GVHR graft-versus-host reaction - CML cell-mediated lympholysis - Thy-1 the gene for the T-cell antigens, synonymous with - Thy-1.1 synonym for ^AKR - Thy-1.2 synonym for ^C3H - MHC major histocompatibility complex - Ir genes immune response genes linked to the MHC - LPS E. coli 055.35 lipopolysaccharide For the genetic nomenclature of theH-2 complex (H-2K, H-2D, I, S, D regions,Ia, etc.) see Kleinet al. 1974, and Shreffleret al. 1974.     

Analysis of D2 d : a D-region class I gene

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2 ^d , D3 ^d , and D4 ^d , in addition to H-2D ^d and H-2L ^d . We have determined the DNA sequence of the D2 ^d gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2 ^d gene is similar to other class I genes. It also contains similar 5 regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2^d molecule, we generated an exon-shuffled construct containing the promoter and exons 1–3, encoding the signal peptide, 1, and 2 external domains of the D2 ^d gene linked to exons 4–8, encoding the 3, transmembrane and cytoplasmic domains, of the H-2D ^d gene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the 3 domain of H-2D^d, as well as by other class I-specific mAbs. Although D2^d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.     

E infα supk transgene in B10 mice suppresses the development of myasthenia gravis

Mice bearing the H-2 ^bhaplotype are susceptible to the development of experimental autoimmune myasthenia gravis (EAMG), induced by acetylcholine receptor (AChR) autoimmunity. One of the genes influencing EAMG susceptibility has been mapped to the A ^blocus of the major histocompatibility complex, and the A chain has been implicated in the pathogenesis. Mice of the H-2 ^bhaplotype, including C57BL/10 (B10), have a genomic deletion of the E gene and therefore fail to express the E molecule on their cell surface. To test the hypothesis that failure to express the cell surface E molecule in B10 mice contributes to EAMG pathogenesis, E _inf ^supk transgenic B10 mice expressing the T molecule were examined. Expression of the E molecule in E _inf ^supk transgenic B10 mice partially prevented the development of EAMG.     

Molecular Properties of Purified Human Uncoupling Protein 2 Refolded from Bacterial Inclusion Bodies

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (K _d) of 0.3–0.5 and 23–92 M. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a K _d of greater than 100 M, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-¹⁴C] ATP and [8-¹⁴C] ADP with K _ds of 4–5 and 16–18 M, respectively. Mg²⁺ (2 mM) reduced the apparent ATP affinity to 53 M, an effect entirely explained by chelation of ATP; with Mg²⁺, K _d using calculated free ATP was 3 M. A combination of gel filtration, Cu²⁺-phenanthroline cross-linking, and ultracentrifugation indicated that 75–80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg²⁺-free nucleotides: the K _d for ATP is about 3–5 M and for Mant-ATP it is about 10 times lower; and (c) in C₁₂E₉ detergent, the monodisperse protein may be in dimeric form.     

Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fragment in mice with tumour xenografts revealed by the use of a residualizing radiolabel (dilactitol-125I-tyramine) and autoradiography

Summary Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (¹²⁵I) or by dilactitol-¹²⁵I-tyramine (¹²⁵I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with¹²⁵I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a binding site barrier effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.     

Biochemical and cytological characterization of wheat/Aegilops ventricosa addition and transfer lines carrying chromosome 4MV

Summary The gene encoding a variant of alcohol dehydrogenase, Adh-, has been found to be associated with the chromosome of the M^v genome which is present in type 9 wheat/Aegilops ventricosa addition line, to which the genes for protein CM-4 and for a phosphatase variant, Aph-v, had been previously assigned. Transfer line H-93-33, which has 42 chromosomes and has been derived from the cross (Triticum turgidum x Ae. ventricosa) x T. aestivum, carries genes encoding all three biochemical markers. Linkage between these genes has been demonstrated by analysis of individual kernels of the F2 (H-93-33 x T. aestivum cv. Almatense H-10-15). A study of the hybrids of line H-93-33 with T. aestivum H-10-15 and with the 4DS ditelosomic line has confirmed that, as suspected, the linkage group corresponds to chromosome 4M^v from Ae. ventricosa. Additionally, it has been found that the previously reported resistance of line H-93-33 to powdery mildew (Erysiphe graminis) is also linked to the biochemical markers; this indicates that either the gene responsible for it is different from that in lines H-93-8 and H-93-35, or that a translocation between two different M^v chromosomes has occurred in line H-93-33.     

Complexity at the mouse minor histocompatibility locusH-4

The fine immunogenetics of the chromosome 7 mouse minor histocompatibility (H) locusH-4 was investigated. Both class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and class II MHC-restricted helper T cells (TH) specifically reactive with H-4 antigens were isolated as clones and were used as genetic probes for classical backcross segregation analysis. Results of a four point cross indicated that theH-4 locus was actually comprised of two genes, that have been designatedH-46 andH-47. The former encodes antigens recognized by the TH and the latter encodes antigens recognized by the CTL. Moreover, these two genes could be separated from the gene pink-eyed dilution (p) which was found to be sandwiched between them. The functional significance of a minor H congenic strain differing by both TH-definedH-46 and CTL-definedH-47 was addressed using F₁ complementation tests. Such studies indicated that immune responses against H-46 antigens was required for generation of H-47-specific CTL. Altogether, these results suggest selective presentation of different minor H gene products by class I or class II MHC proteins and that the minor H locusH-4 may have necessarily included both TH and CTL-defined genes because of requisite TH-CTL collaboration. Address correspondence and offprint requests to: D. C. Roopenian.     

CD8+T cells from a patient with colon carcinoma,specific for a mutant p21-Ras-derived peptide (GLY13→ASP), are cytotoxic towards a carcinoma cell line harbouring the same mutation

Several T lymphocyte clones (TLC), specific for a p21-Ras-derived peptide expressing a Gly¹³Asp mutation and of the CD8⁺ subtype, were generated from peripheral blood of a colon carcinoma patient. The TLC exerted cytotoxicity against an interferon- (IFN)-pretreated colon carcinoma cell line, HCT116, which harbours the Gly¹³Asp mutation and shares both HLA-A2 and HLA-B12(44) with the patient. This cytotoxic effect could be blocked by a monoclonal antibody (mAb) against CD8 molecules, as well as with a mAb against HLA class I molecules and a polyclonal antiserum against HLA-B12, identifying B12(44) as the antigen-presenting molecule. In growth-inhibition experiments, the growth of both IFN-pretreated and untreated target cells were strongly inhibited by the presence of the CD8⁺TLC. Together these data indicate that human cancer cells harbouring a spontaneousras mutation can process aberrant p21 Ras and express peptide/HLA-class-I complexes on their surface in sufficient density to be recognized by Ras-specific cytotoxic T lymphocytes.     

Analysis of local conformation of membrane-bound and polycrystalline peptides by two-dimensional slow-spinning rotor-synchronized MAS exchange spectroscopy

2D slow-spinning, rotor-synchronized MAS exchange spectroscopy (SSRS-MASE) was applied to study local secondary structure of three structurally different peptides, two of which were membrane-bound. Each peptide was ¹³C carbonyl labeled at two adjacent residues in the peptide backbone. In general, this methodology is attractive for membrane-bound peptides because of its lenient spinning, decoupling, and RF homogeneity requirements.For a single set of raw SSRS-MASE data, two linearly independent methods exist for obtaining a 2D spectrum and each spectrum can be fit to obtain conformational constraints. An approach is described for combining the results of these two fits and this method is shown to work for spectra with both resolved and unresolved labeled site resonances. A spectrum is often fit well to a few different conformations which have somewhat different values of the fitting parameter ². A simple statistical theory is developed which relates the ² difference between a local minimum and the global minimum ² to the likelihood that the local minimum conformation is the correct structure. Because uncertainty in the simulated data can also contribute to the overall fitting uncertainty, an empirical method is described for incorporating the simulation uncertainty into the ² analysis.These data analysis methods were tested on polycrystalline Ala-Gly-Gly and then applied to the membrane-bound melittin and HIV-1 fusion peptides. Melittin gave a best-fit helical structure at Ala-4 while the fusion peptide gave a good-fit strand structure at Phe-8. The melittin analysis is in agreement with the known overall structure of this peptide.     

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2^k antigen coded by the Q5^k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2^– mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (L^Q7b/Kb), which expressed the 1 and 2 domains of the Qa-2 antigen (Q7^b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5 half of the Q7^b gene and the 3 half of the H-2K^b gene (pQ7^b/K^b). Using L^Q7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2^k, Qa-2^–) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the 1/2 region of the Q7^b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the / type (TCR/). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2^k+) derived from AKR mice (Qa-2^–, H-2^k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed L^Q7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the 1/2 region of the Qa-2^k tumor antigen by TCR/.