bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial purification and characterization of a factor from a cloned thymic epithelium cell line.
Authors: Pfeifer J D , Wetzel G D , Dutton R W
Published: December 2022

The culture supernatant from a cloned line of thymic epithelium (TEPI) is shown to enhance the response of thymocytes to alloantigen as measured by cell-mediated lympholysis. The supernatant has no effect on the spleen cell response to alloantigen as measured by cell-mediated lysis and does not contain interleukin 1, interleukin 2, interleukin 3, or interferon-gamma activity. The activity is shown to have an apparent m.w. of 160,000 by Sephacryl S-200 gel permeation chromatography, to have an isoelectric point of 6.5, and to elute from DEAE-Sepharose at 0.07 M NaCl.
Human T cell lines differing in phenotype and specificity are reactive with the same anti-idiotypic antibody.
Authors: Borst J , Boylston A W , de Vries J E , Spits H
Published: December 2022

3D6, a monoclonal antibody selected for reactivity with the T cell antigen receptor on the T leukemic cell line HPB-ALL, was found to react with 3 to 13% of peripheral blood T lymphocytes of 10 out of 15 normal donors. Peripheral T cells of two donors were stimulated with allogeneic cells, and the 3D6+ cells were enriched by rosetting 3D6-coated cells with goat anti-mouse-coupled human red blood cells and were expanded in interleukin 2-containing medium. In this way, 90 to 100% 3D6+ cell lines were obtained that were cytotoxic for the allogeneic stimulator cells. 3D6 antibody could block antigen-specific cytotoxicity, as well as induce nonspecific cytotoxicity toward target cells that could not be killed in the absence of the 3D6 antibody. The 3D6+ cell populations contained T4+, as well as T8+ cells, indicating that 3D6 antibody defined a T cell receptor population that might harbor various antigenic specificities. One 3D6+ cell line was separated into T4+ T8- and T4- T8+ populations. 3D6 reactive T cell receptors isolated from HPB-ALL and normal cell lines were analyzed biochemically by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and V8 protease peptide mapping. Isoelectric focusing analysis provided additional evidence for the idea that 3D6 antibody detected a number of structurally distinct T cell receptors, because the T cell receptor alpha-chain was homogeneous in charge after desialation on the clonal tumor line HPB-ALL, but remained heterogeneous in charge on the 3D6+ normal cell lines. No great differences in charge were found between T cell receptors isolated from T4+ and T8+ 3D6+ lines, but their isoelectric focusing patterns were not identical. V8 protease peptide mapping revealed structural differences between the T cell receptor alpha-chain isolated from HPB-ALL on one hand and from the normal 3D6+ lines on the other, whereas the beta-chains did not differ greatly in primary structure according to this analysis. In addition, the peptide mapping suggested differences in primary structure between T cell receptors present on the T4+ population vs those present on the T8+ populations.
Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen.
Authors: Rabinovsky E D , Yang T J
Published: December 2022

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.
Purification to homogeneity and NH2-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line.
Authors: Rimsky L , Wakasugi H , Ferrara P , Robin P , Capdevielle J , Tursz T , Fradelizi D , Bertoglio J
Published: December 2022

A subclone, referred to as 3B6, derived from a DR-negative EBV-transformed B cell line, has been found to spontaneously produce IL 1. 3B6-IL 1 displays a pI of 5 on FPLC chromatofocusing. It has been purified to homogeneity by a sequence of ion-exchange chromatography and affinity chromatography on procion red agarose. The homogeneous material migrated with an apparent m.w. of 13,500 on SDS-PAGE. The overall recovery of IL 1 activity was estimated at 57%. The final material had a specific activity of 7.8 X 10(6) half-maximal units/mg and represented a 50,000-fold purification. A partial NH2-terminal amino acid sequence has been obtained that is different from those reported from monocytic IL 1. However, this molecule can formally be identified as IL 1 on its spectrum of biologic activities. In addition to inducing the proliferation of murine thymocytes in the co-stimulator assay. 3B6-IL 1 is active on both human T and B cells, respectively, in inducing IL 2 synthesis by cells from a subcloned HSB 2 line and promoting the proliferation of anti-IgM-stimulated human peripheral blood B lymphocytes. Furthermore, 3B6-IL 1 acts as a growth factor for normal human fibroblasts and for the 3B6 line itself. However, 3B6-IL 1 is not pyrogenic in rabbits. Thus, the 3B6 cell line was shown to produce a new molecular species of IL 1, with respect to its NH2-terminal sequence, which shared all of the studied biologic activities of monocytic IL 1 except for pyrogenicity.
The role of class II MHC molecules in the activation of class I-reactive T cell hybridomas.
Authors: Minami M , Nariuchi H , Kawasaki H , Dorf M E
Published: December 2022

To examine the role of Ia molecules in T cell responses to allo-class I major histocompatibility antigens, a series of allo-class I-reactive T cell hybridomas was established. Of 134 T cell hybridomas obtained from the fusion of C3H/HeJm or B10.HTT T cells stimulated with C57BL/6 splenocytes, nine T cell hybridomas were reactive to class I antigens and 126 T cell hybridomas were reactive to class II antigens. Six of the nine IL 2-producing T cell hybridomas were further analyzed: five mapped to H-2Kb and the other mapped to H-2Db. Three of these T cell hybridomas, HTB-157.7, HTB-176.10, and HTB-177.2, could react to the EL-4 cell line that expresses H-2Kb and H-2Db class I antigens but lacks class II I-Ab molecules. Furthermore, the activation of these three T cell hybridomas with C57BL/6-derived splenocytes was not blocked by either anti-I-A or anti-L3T4 antibody. In contrast, the other three T cell hybridomas, CB-127.6, CB-221.7, and HTB-102.7, failed to react with EL-4 but reacted with the LB cell line which expresses class I (H-2Kb, H-2Db) and class II (I-Ab) molecules. Although class II molecules were required for activation of the latter clones, there was no apparent I-A allele specificity, suggesting that a relatively nonpolymorphic Ia determinant was involved. The activation of the three latter T cell hybridoma clones with C57BL/6 splenocytes could be blocked completely by either anti-I-A or anti-L3T4 antibody. The data are interpreted in terms of possible T cell receptor models for recognition of class I with nonpolymorphic class II determinants.
Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation.
Authors: Ceuppens J L , Meurs L , Baroja M L , Van Wauwe J P
Published: December 2022

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.
Molecular basis of a unique tumor antigen of radiation leukemia virus-induced leukemia B6RV2: its relation to MuLV gp70 of xenotropic class.
Authors: Nakayama E , Uenaka A , DeLeo A B , Stockert E , Obata Y , Ueda R , Inui Y
Published: December 2022

Hybridomas secreting monoclonal antibodies that reacted with the B6 radiation leukemia virus (RadLV)-induced leukemia B6RV2 were produced by fusion of BALB/c NS-1 myeloma cells with spleen cells from (BALB/c X B6)F1 mice immunized with B6RV2. By direct and absorption analyses with 28 B6 and BALB/c leukemias, the monoclonal antibodies NU7-4 and NU7-99 were shown to react only with B6RV2, indicating that they recognized an individually distinct antigen on B6RV2 that was identified previously with conventional (BALB/c X B6)F1 anti-B6RV2 serum. Another monoclonal antibody, NU1-132, showed relatively restricted reactivity with B6 RadLV leukemias. These three monoclonal antibodies all precipitated material of approximately 80,000 daltons, which is the same size as that precipitated by anti-xenotropic MuLV gp70 serum. Sequential immunoprecipitation analysis revealed that the molecules precipitated by NU7-4 were not removed by pretreatment of NU7-99 or NU1-132 and that the molecules precipitated by NU7-99 were not removed by NU7-4 or NU1-132. The molecules precipitated by NU1-132 were partially removed by pretreatment with NU7-4, but not with NU7-99. The molecules precipitated by these three monoclonal antibodies were removed by pretreatment with anti-xenotropic gp70. These results suggested heterogeneity of the xenotropic MuLV gp70-related molecules expressed on B6RV2 and a possible relation between serologically defined unique tumor antigens and gp70-related molecules.
The contribution of constant region domains to the binding of murine IgM to Fc mu receptors on T cells.
Authors: Mathur A , Lynch R G , Köhler G
Published: December 2022

IgM hybridoma constant region domain deletional mutants were used to investigate the domain requirements for binding of murine IgM to Fc mu receptors (Fc mu R) on normal murine T lymphocytes. Parental Sp 6:18 (mu, kappa; anti-trinitrophenyl) and its mutant proteins or their trinitrophenyl-antigen immune complexes were tested for their ability to inhibit the binding of pentameric IgM to Fc mu R on T lymphocytes. Inhibition was observed with ligands containing multiple copies of the third constant region domain. Inhibition did not occur with ligands missing the third constant region domain. In addition, a battery of rat monoclonal antibodies specific for individual murine IgM constant region domains was tested for the ability to inhibit the binding of pentameric murine IgM to Fc mu R on normal murine T lymphocytes. Total inhibition was observed with the antibodies directed to different epitopes located in C mu 3, but significant inhibition was not observed with antibodies directed to C mu 1, C mu 2, or C mu 4. Studies with domain deletional mutants and anti-domain antibodies have independently provided strong evidence that the C mu 3 domain plays a major role in the binding of IgM to Fc mu R on T lymphocytes and that C mu 1, C mu 2, and C mu 4 are not essential for binding. These studies have also provided evidence that valency and avidity influence the binding of IgM to T lymphocytes that express Fc mu R.
Structural characterization of the human B lymphocyte-restricted differentiation antigen CD22. Comparison with CD21 (complement receptor type 2/Epstein-Barr virus receptor).
Authors: Boué D R , Lebien T W
Published: December 2022

CD22 and CD21 are glycoproteins primarily expressed on normal and neoplastic human B cells. The surface expression of these two molecules parallel each other during normal B cell differentiation, and the reported relative mobilities for CD22 and CD21 are 130/140 kDa and 140 kDa, respectively. Herein we present a detailed analysis of the biosynthesis and structure of CD22 and also compare it directly to CD21. Electrophoresis under reducing and nonreducing conditions suggested that CD22 and CD21 may have similarities in intra-chain disulfide bond formation. Biosynthesis and processing of CD22 and CD21 were very similar with respect to kinetics and post-translational modification, and both could be phosphorylated. However, endoglycosidase digestion (using N-glycanase and endoglycosidase H) and peptide mapping (using V8 protease and N-chlorosuccinimide) strongly suggested that CD22 and CD21 are distinct gene products.
Genetics of resistance to the African trypanosomes. VII. Trypanosome virulence is not linked to variable surface glycoprotein expression.
Authors: Inverso J A , De Gee A L , Mansfield J M
Published: December 2022

The question of linkage of virulence traits to variable surface glycoprotein (VSG) expression in African trypanosomiasis was addressed. Previously we demonstrated that daughter cells arising in mice infected with a genetically homogeneous trypanosome population of Trypanosoma brucei rhodesiense were more virulent than the infecting population (J. A. Inverso and J. M. Mansfield, J. Immunol. 130:412, 1983). These virulent trypanosomes expressed differences in surface phenotype compared with the infecting variant types, and we proposed that virulence may be "linked" to VSG expression. In the present study, however, we have shown that expression of virulence is independent of the VSG phenotype displayed by trypanosome populations. A VSG-identical but highly virulent subpopulation of T. b. rhodesiense LouTat 1 was derived by rapid subpassage and subcloning in immunosuppressed mice. The virulent LouTat 1A subclone derived in this manner killed B10.BR/SgSnJ mice in 3 to 4 days postinfection compared with approximately 60 days for the parent clone, LouTat 1. The virulent subclone LouTat 1A appears to express the same VSG as the less virulent LouTat 1 population, as determined by polyspecific and monoclonal antibody-binding assays, cross-protection tests, and amino acid sequence analyses of the N-terminal portion of the VSG molecules. When LouTat 1 and subclone LouTat 1A were injected into a heterologous host species, multiple variant antigenic types (VATs) arising from each inoculum were isolated and characterized. VATs derived from the virulent subclone were as uniformly virulent for B10.BR mice as LouTat 1A. In summary, these results demonstrate that trypanosome virulence, once expressed, is a stable phenotype that does not seem to be associated with a particular VSG phenotype, nor does virulence change with the expression of different VSG genes.
cDNA cloning of granzyme C, a granule-associated serine protease of cytolytic T lymphocytes.
Authors: Jenne D , Rey C , Masson D , Stanley K K , Herz J , Plaetinck G , Tschopp J
Published: December 2022

A cDNA clone corresponding to the complete amino acid sequence of a putative protease CCP2 of murine cytotoxic T lymphocytes was isolated and sequenced. The clone encodes a 248-residue long serine esterase. The deduced N-terminal amino acid sequence is identical over 40 residues to that of granzyme C, a protease of unknown function present in granules of cytotoxic lymphocytes. Analysis of the sequence of granzyme C/CCP2 reveals high homology to other granzyme proteases, i.e. granzyme A (40%) and granzyme B (67%) and to rat mast cell protease II (46%). The amino acids lining the specificity pocket are well conserved between granzyme B, C, and rat mast cell protease II, but not granzyme A, suggesting a similar general specificity of these three proteases.
Heavy chain V gene-specific elimination of B cells during the pre-B cell to B cell transition.
Authors: Decker D J , Kline G H , Hayden T A , Zaharevitz S N , Klinman N R
Published: December 2022

As developing B cells acquire their surface Ig (sIg) receptors, they become highly susceptible to sIg-mediated negative selection, a process best exemplified by tolerance induction. Recent studies with sIg transgenic mice have suggested that B cells may become inactivated by tolerogens only after a developmental stage wherein they express low levels of sIgM and during the course of up-regulating their expression of sIgM. To determine whether inactivation of B cells of conventional mice occurs at this or other maturational stages, we have analyzed the ratio of productive vs nonproductive rearrangements of VH81X gene segments in developmental subsets of adult bone marrow cells. Earlier studies had demonstrated that cells whose productively rearranged H chain V region contained a VH81X gene segment were selectively disfavored both during pre-B cell development and subsequent to sIg expression. Contrary to the expectations for elimination by tolerance, no decrease in the proportion of cells expressing productive rearrangements of VH81X was observed as cells matured from the sIgMlow to the sIgMhigh maturational stage. However, a significant decrease in the proportion of productively rearranged VH81X gene segments was observed following the transition from sIg- pre-B cells to sIgMlow immature B cells. Additionally, the proportion of productively rearranged VH81X gene segments was significantly higher in sIgMhigh bone marrow cells than in splenic B cells. These findings demonstrate that B cells are susceptible to H chain-specific elimination at two developmental stages other than that wherein B cells are generally assumed to be negatively selected by tolerance.
In CD8+ T cell-deficient lpr/lpr mice, CD4+B220+ and CD4+B220- T cells replace B220+ double-negative T cells as the predominant populations in enlarged lymph nodes.
Authors: Giese T , Davidson W F
Published: December 2022

Mice homozygous for lpr or gld develop autoimmunity and progressive lymphoproliferative disease characterized by the accumulation of two unusual populations of B220+ TCR-alpha beta+ T cells, a predominant CD4-CD8- double-negative (DN) subset and a minor CD4dull+ subset. B220+ DN T cells appear to be derived from negatively selected thymocytes, but their immediate precursors have not been identified conclusively, and their relationship to CD4+B220+ T cells is unclear. Our previous studies of lpr and gld mice treated chronically with anti-CD8 mAb provided evidence that the majority of B220+ DN T cells are unrelated to CD4+B220+ T cells and may be descended from peripheral thymus-derived CD8+ T cells. To investigate the contributions of MHC class I-selected thymus-derived T cells to the production of B220+ DN T cells and to the accumulation of CD4+ T cell subsets, we studied C3H-lpr and -gld mice rendered deficient in CD8+ T cells by the introduction of disrupted beta 2-microglobulin (beta 2-m) genes. These CD8+ T cell-deficient mice developed massively enlarged lymph nodes, in which CD4+B220+ T cells and CD4+ T cells replaced B220+ DN T cells as the dominant T cell subsets. As a population, the CD4+B220+ T cells were depleted of autoreactive populations specific for endogenous retroviral superantigens and were enriched for V beta 8.3+ T cells. The deficiency of CD8+ T cells in beta 2-m(-/-)-lpr mice had no effects on the accumulation of primed CD4+ T cells or autoreactive B cells. The selective reduction in B220+ DN T cells and corresponding accumulation of CD4+B220+ T cells in beta 2-m(-/-)-lpr mice provide strong evidence that 1) the majority of B220+ DN T cells are unrelated to CD4+ T cells and their development and/or accumulation is dependent on MHC class I expression; and 2) CD4+B220+ T cells are a remarkably similar, but separate, lineage of cells that develop independently of thymus-derived CD8+ T cells and class I MHC expression.
In vitro maturation of clonal CD4+CD8+ cell lines in response to TCR engagement.
Authors: Groves T , Katis P , Madden Z , Manickam K , Ramsden D , Wu G , Guidos C J
Published: December 2022

Engagement of the TCR on immature CD4+CD8+ (DP) thymocytes by an appropriate peptide/MHC ligand evokes a complex program of maturation known as positive selection. As a result, DP thymocytes are rescued from programmed cell death, become committed to the CD4 or CD8 lineage, extinguish expression of V(D)J recombinase activity, and undergo further maturation. We describe here a panel of DP thymic lymphoma cell lines that, in response to in vitro TCR engagement, undergo many of the TCR-beta-induced maturation events that have been reported to accompany positive selection of DP thymocytes in vivo. These events include increased expression of CD5, CD69, CD45, TCR-alpha, and MHC class I, and decreased expression of Thy-1 and heat-stable Ag. In addition, we observed TCR-induced expression of the bcl-2 gene, a well described inhibitor of programmed cell death. Finally, TCR engagement decreased expression of recombinase-activating genes and terminal deoxynucleotidal transferase genes, as well as V(D)J recombinase activity. However, TCR engagement did not elicit demonstrable CD4/CD8 lineage commitment. These observations suggest that engagement of the TCR on these DP cell lines elicits multiple maturation events that are part of the positive selection developmental program, but not CD4/CD8 lineage commitment. Thus, these DP cell lines provide the opportunity to elucidate molecular mechanisms of maturation and CD4/CD8 lineage commitment in vitro.
Glycolipid ligands for selectins support leukocyte tethering and rolling under physiologic flow conditions.
Authors: Alon R , Feizi T , Yuen C T , Fuhlbrigge R C , Springer T A
Published: December 2022

Selectin interactions with glycolipids have been examined previously under static conditions, whereas physiologic interactions mediated by selectins take place under flow. We find that under physiologic flow conditions, sialyl Lewis(x) (sLe(x)) glycolipid and sialyl Lewisa (sLe(a)) neoglycolipid support tethering and rolling adhesions of Chinese hamster ovary (CHO) cells expressing E-selectin and lymphoid and myeloid cells expressing L-selectin. These selectin-mediated adhesions persist at the highest shear stresses that occur in postcapillary venules in vivo and occur at lower site densities than found for sLe(x) on neutrophils. The interactions are Ca(2+)-dependent and can be specifically and completely blocked with anti-selectin mAbs. Asialo nonfucosylated glycolipids are inactive, and sulfatide supports weak tethering, but not rolling, of L-selectin-expressing cells. Rolling velocities and resistance to detachment are related to the glycolipid site density and fall within the range measured for neutrophil and myeloid cell rolling on substrates containing purified selectins. These observations are the first indication that glycolipids can interact with selectins in physiologic flow conditions, and can contribute to rolling adhesions.
Endogenous macrophage CSF production is associated with viral replication in HIV-1-infected human monocyte-derived macrophages.
Authors: Gruber M F , Weih K A , Boone E J , Smith P D , Clouse K A
Published: December 2022

Human monocyte-derived macrophages (MDM) cultured in medium containing macrophage (M) CSF are more susceptible to infection with HIV-1. M-CSF increases the frequency with which MDM become infected, the level of HIV mRNA expressed per infected cell, and the level of proviral DNA expressed per infected culture. Because of these effects of M-CSF on HIV-1 replication and the reported function of this factor as a survival and differentiation factor for human monocytes, we investigated whether HIV-1 could induce endogenous M-CSF production by MDM and the potential role of endogenous M-CSF on HIV-1 infection in these cells. MDM infected with HIV and maintained in the absence of exogenous M-CSF produced this cytokine endogenously at levels 5- to 24-fold higher than uninfected cells. In contrast, the proinflammatory cytokines IL-1, IL-6, and TNF-alpha and the growth factor granulocyte-macrophage CSF were not detected. The kinetics of M-CSF production following infection paralleled the kinetics of virus replication. Furthermore, enhanced production of M-CSF was dependent on viral entry and active replication of HIV-1. Thus, endogenous M-CSF production may contribute to the survival of HIV-infected MDM, enable them to function as a reservoir for HIV, and facilitate the spread of virus in vivo.
Myelin oligodendrocyte glycoprotein induces a demyelinating encephalomyelitis resembling multiple sclerosis.
Authors: Johns T G , Kerlero de Rosbo N , Menon K K , Abo S , Gonzales M F , Bernard C C
Published: December 2022

Chronic relapsing experimental autoimmune encephalomyelitis, a demyelinating disease induced by injection of central nervous system (CNS) tissue, is widely used as a model for multiple sclerosis. However, it is unclear which Ag or combination of Ags in the CNS induce the demyelinating immune response. We now show in Lewis rats that a single injection of myelin oligodendrocyte glycoprotein, a specific CNS myelin component, or an appropriately derived myelin oligodendrocyte glycoprotein peptide produces a relapsing remitting neurologic disease with extensive plaque-like demyelination. Igs from affected animals reacted specifically with myelin oligodendrocyte glycoprotein and stimulated a myelin protease activity, leading to myelin basic protein degradation. The demonstrated involvement of myelin oligodendrocyte glycoprotein as a new demyelinating neural Ag may provide a deeper insight into the pathogenesis of multiple sclerosis and its treatment.
Analysis of apoptosis in lymph nodes of HIV-infected persons. Intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden.
Authors: Muro-Cacho C A , Pantaleo G , Fauci A S
Published: December 2022

The occurrence of in vivo apoptosis was investigated in lymph node sections obtained from HIV-infected persons at different stages of disease. The degree of apoptosis in lymph nodes from HIV-infected individuals was compared with that observed in lymph nodes obtained from HIV-negative individuals. Apoptosis was readily detected in lymph nodes obtained from both HIV-negative and HIV-positive persons; however, the degree of apoptosis in lymph nodes obtained from HIV-positive persons was three to four times higher than that observed in the lymph nodes obtained from HIV-negative persons. In contrast to HIV-negative lymph nodes in which apoptosis was confined largely to germinal centers, in HIV-positive lymph nodes all functional compartments of the lymph node (i.e., cortex, paracortex, and sinuses) were extensively involved by this phenomenon. Furthermore, a significant correlation was observed between intensity of apoptosis and degree of activation of the lymphoid tissue associated with HIV infection. In contrast, intensity of apoptosis correlated neither with the clinical stage of HIV disease nor with the viral burden in the lymph node. Finally, apoptosis was not restricted only to CD4+ T cells; both B cells and CD8+ T cells were found to undergo apoptosis. Taken together, these results indicate that the increased intensity of the apoptotic phenomenon in HIV infection is caused by the general state of immune activation, and is independent of the progression of HIV disease and of the levels of viral load.
Analysis of Ig VH region genes encoding IgE antibodies in splenic B lymphocytes of a patient with asthma.
Authors: Snow R E , Chapman C J , Frew A J , Holgate S T , Stevenson F K
Published: December 2022

An atopic patient with hypersensitivity against house dust mite died as a result of an asthmatic attack. A portion of the spleen was obtained and was used to analyze the spectrum of Ig heavy chain V regions involved in encoding IgE Abs. A nested PCR technique generated 14 cloned VH sequences that had distinct CDR3 regions; 5 of 14 were derived from the minor VH5 family, and the remainder derived from the larger families, VH3 (6 of 14) and VH4 (3 of 14). One of the VH3-derived sequences was present as a repeated sequence in three clones. A control PCR with the same VH primers in combination with JH primers yielded only 1 of 13 sequences from VH5, indicating preferential VH5 usage only for IgE. Analysis of VH5-C epsilon sequences revealed usage of a single gene, DP73, with extensive mutations and several "hot spots" containing common replacement amino acids. However, there was no concentration of replacement mutations in the CDRs, which conventionally would indicate a role for Ag selection. The VH3 and VH4 genes in combination with C epsilon also harbored extensive somatic mutations. From these findings in splenic B lymphocytes, and those of a previous study of blood lymphocytes, it seems that preferential usage of VH5 genes and extensive somatic hypermutation are characteristic of B cells synthesizing IgE in patients with allergic disease.
CD28 costimulation up-regulates long-term IL-2R beta expression in human T cells through combined transcriptional and post-transcriptional regulation.
Authors: Cerdan C , Martin Y , Courcoul M , Mawas C , Birg F , Olive D
Published: December 2022

Costimulatory molecules such as CD28 are required for induction of T cell clonal expansion and for prevention of T cell unresponsiveness. In combination with either CD3 or CD2 triggering, CD28 was shown to enhance T cell proliferation, cytolytic activity, production of cytokines and especially of IL-2, and expression of the IL-2R alpha-chain (IL-2R alpha). We and others have demonstrated that the costimulatory effect of CD28 on both IL-2 and IL-2R alpha expression results from a coordinated transcriptional activation of their genes and transcript stabilization. We show here that the CD28 stimulation, together with CD2, leads to a prolonged up-regulation of the constitutive expression of the IL-2R beta-chain in human peripheral T cells. As for IL-2R alpha, the increase in IL-2R beta gene expression seems to result from both transcriptional activation and transcript stabilization. In addition, IL-2 differentially regulates its own receptors, as only expression of IL-2R alpha, but not of IL-2R beta, is largely inhibited, at both the mRNA and protein levels, by blocking IL-2R mAbs. We propose that the long lasting T cell proliferation mediated by the CD2 and CD28 costimulation is mainly the consequence of the high and prolonged expression of both the IL-2R alpha- and beta-chains.
Staphylococcal enterotoxin B activates purified NK cells to secrete IFN-gamma but requires T lymphocytes to augment NK cytotoxicity.
Authors: D'Orazio J A , Burke G W , Stein-Streilein J
Published: December 2022

Staphylococcal enterotoxin B (SEB) is known for its effects on T lymphocytes; the mechanism of this activation is well studied. Although it is anticipated that there would be effects on other cells in the milieu, the mechanisms of activation of non-T immune cells are not well understood. This report examines the effects of SEB on human NK cells. Incubation of PBMC with SEB augmented NK cytotoxicity against the NK-sensitive targets K562 and Molt-4 and induced activity against the NK-resistant targets, Mel-30 and Raji. The degree of superantigen-augmented killer (SAK) activity induced by the SEB treatment correlated with the concentration of the superantigen in the mixture. Specific neutralizing Abs for lymphokines or monokines added to cultures showed that IL-2, IFN-gamma, or IL-12 each participated in the phenomenon; addition of all Abs together blocked most, but not all, SAK activity. Highly purified T lymphocytes (but not monocytes) supported the development of SAK activity in enriched cultures of CD56+ cells. The SAK activity was blocked by specific Abs to IL-2 and IFN-gamma in cocultures of T cells and CD56+ cells. In addition, SEB induced IFN-gamma secretion from sorted CD56+ cells. These data show for the first time that superantigen directly initiated cytokine synthesis in NK cells, and that SAK activity was induced as a consequence of products secreted from activated T lymphocytes. Such a superantigen effect on NK cells would place the NK lymphocyte in a pivotal role in determining the outcome of infection or immunopathology caused by superantigen exposure.
IFN-gamma and lipopolysaccharide induce DNA binding of transcription factor PU.1 in murine tissue macrophages.
Authors: Shackelford R , Adams D O , Johnson S P
Published: December 2022

The transcription factor PU.1, the Spi-1 oncogene product, is found principally in B cells and macrophages. PU.1 binds to a purine-rich sequence of DNA with a core consensus sequence of 5'-GAGGAA-3'. By using an electrophoretic mobility shift assay, we have been able to identify PU.1 in nuclear extracts of murine tissue macrophages. When macrophages are stimulated with IFN-gamma, an increase in PU.1 binding is observed. This increase is gradual, peaking at 6 to 9 h and decaying to basal levels 24 h after stimulation. The increase in PU.1 binding is unaffected by inhibiting protein synthesis, but is attenuated by either an inhibitor of the NA+/H+ antiporter or elevations in cAMP. In addition, we have found that low doses (1 ng/ml) of bacterial LPS induce PU.1 binding.
V gamma 2V delta 2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer.
Authors: Bukowski J F , Morita C T , Tanaka Y , Bloom B R , Brenner M B , Band H
Published: December 2022

The predominant subpopulation of gamma delta T cells in human peripheral blood expresses TCR V region genes V gamma 2 paired with V delta 2. Previous studies have shown that these V gamma 2V delta 2+ T cells proliferate in response to Daudi Burkitt lymphoma cells, synthetic alkyl phosphate molecules including monoethylphosphate (MEP), and an Ag chemically similar to MEP purified from mycobacterial extracts of several species including Mycobacterium tuberculosis. This proliferation is polyclonal and determined by the TCR V gene. However, because these alkyl phosphate molecules are so distinct from conventional peptides and superantigens, we questioned whether these substances induce gamma delta T cell proliferation via TCR-dependent recognition. Here we report that transfection of TCR- Jurkat T cells with cDNA constructs encoding a V gamma 2V delta 2 TCR enabled the transfectants to produce IL-2 in response to Daudi cells, mycobacterial extract, and MEP. The responses were dose dependent and Ag specific. These results demonstrate an essential role for the gamma delta TCR in V gamma 2V delta 2 T cell-mediated recognition of non-peptide Ags by human T cells and suggest a structural similarity or cross-reactivity between cellular and microbial Ags recognized by these gamma delta T cells.
Functional characterization of novel IL-2 transcriptional inhibitors.
Authors: Baine Y , Stankunas B M , Miller P , Hobbs C , Tiberio L , Koch J , Yoon K , Sawutz D , Surowy C
Published: December 2022

IL-2-mediated T cell proliferation is a critical early event in the inflammatory process. Formation of the NFAT-1 transcriptional complex on the IL-2 promoter is essential for IL-2 transcription. Using a cell line that is stably transfected with a trimer of the NFAT-1 regulatory element linked to a lac-Z reporter gene, we screened for inhibitors of NFAT-1-mediated beta-galactosidase activity. WIN 61058 and WIN 53071 were identified as microM inhibitors. These compounds also inhibited beta-galactosidase mRNA levels. Similar inhibition of NFAT-1-mediated gene expression was observed in a second cell line, which is stably transfected with NFAT-1 regulatory elements linked to the reporter gene for sCD8. At 10 microM, both compounds inhibited IL-2 mRNA and protein levels in the NFAT-1-linked lac-Z transfectants, and in human lymphocytes. Both compounds inhibited the mixed lymphocyte reaction, and this inhibition was reversed by exogenous IL-2. WIN 53071 inhibited IL-2 production induced in the calcium-dependent PMA and ionomycin pathway. Conversely, calcium-independent anti-CD28 Ab and PMA-induced IL-2 production was resistant. Both compounds altered the NFAT-1 transcriptional complex, causing its retarded mobility on gels. By these functional criteria, we believe we have identified two structurally distinct, novel inhibitors of NFAT-1-mediated transcription.
Regulation of human T lymphocyte chemotaxis in vitro by T cell-derived cytokines IL-2, IFN-gamma, IL-4, IL-10, and IL-13.
Authors: Tan J , Deleuran B , Gesser B , Maare H , Deleuran M , Larsen C G , Thestrup-Pedersen K
Published: December 2022

There has been a number of conflicting reports regarding the T lymphocyte chemotactic activities of several cytokines. IL-2 and IFN-gamma are known to promote augmentation of immune inflammation, whereas IL-4, IL-10, and IL-13 display immunomodulatory effects on inflammatory cells including inhibition of cytokine production. Their effects on chemotaxis of inflammatory cells are unknown. We observed that IL-1 alpha could induce chemotaxis both in overnight cultured and anti-CD3 mAb-activated T lymphocytes and that overnight culture and anti-CD3 activation increase the number of IL-1R on T lymphocytes. In contrast, IL-8 selectively attracts freshly isolated T lymphocytes. Staurosporine inhibits freshly isolated T lymphocyte chemotaxis toward IL-8, whereas tyrphostin 23 inhibits chemotaxis of overnight cultured and anti-CD3-activated T lymphocytes toward IL-1 alpha. We have found that IL-2 and IL-13 inhibit the chemotactic migration of both CD4+ and CD8+ T lymphocytes toward IL-8, and RANTES. IL-4 inhibits only CD8+ T lymphocyte chemotaxis toward RANTES, IL-8 and IL-10. IL-10 inhibits only CD4+ T lymphocytes in their chemotactic response toward RANTES and IL-8. IFN-gamma does on the other hand augment the sensitivity of human T lymphocytes to chemotactic stimuli. Thus, our results demonstrate that different proinflammatory cytokines will induce chemotactic migration of T lymphocytes under different circumstances acting through different signaling pathways. The T cell-derived cytokines IL-2, IL-4, IL-10, and IL-13 are able to block further T lymphocyte chemotaxis, thus leading to a focusing of T lymphocytes in an area of T lymphocyte activation. These mechanisms seem relevant in our understanding of the specific and continuous localization of T lymphocytes in allergic and autoimmune disorders.
Isolation of a kidney-specific peptide recognized by alloreactive HLA-A3-restricted human CTL.
Authors: Poindexter N J , Naziruddin B , McCourt D W , Mohanakumar T
Published: December 2022

The molecular nature of tissue-specific Ags involved in MHC-restricted CTL responses is as yet undefined. To determine the specificity of these peptides, their function, and their possible relationship to allograft rejection, we have utilized human kidney-specific CD8+ CTL clones to screen reversed-phase HPLC (RP-HPLC)-separated self peptides presented by allo-class I molecules. One of these clones is HLA-A3-restricted and the other HLA-B62-restricted, lysing human kidney cell lines but not MHC identical B lymphoblastoid cells which express the appropriate HLA molecules. We have identified a biologically active RP-HPLC fraction containing self peptides eluted from affinity-purified MHC molecules from HLA-A3+ kidney. This peptide is not expressed in HLA-A3+ spleen. Similarly, a HLA-B62-associated peptide fraction was identified in kidney but not in spleen using the HLA-B62-restricted CTL clone. Sequence analysis of the biologically active fraction from HLA-A3 kidney revealed multiple peptides. Because of the ambiguity of the peptide sequence, a mixed peptide library corresponding to this sequence was synthesized that included the HLA-A3 binding motif. The biologically active peptide library was RP-HPLC fractionated and the fraction containing HLA-A3-restricted CTL activity was sequenced. The resulting sequence of the alloreactive HLA-A3-restricted peptide epitope is GPPGVTIVK. By using this unique strategy, we describe the successful isolation and sequencing of an antigenic peptide that is recognized by a human alloreactive kidney-specific CTL clone.
Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.
Authors: Kawakami Y , Eliyahu S , Jennings C , Sakaguchi K , Kang X , Southwood S , Robbins P F , Sette A , Appella E , Rosenberg S A
Published: December 2022

Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-gamma release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.
Phospholipase A2-activating protein induces the synthesis of IL-1 and TNF in human monocytes.
Authors: Bomalaski J S , Ford T , Hudson A P , Clark M A
Published: December 2022

Phospholipase A2-activating protein (PLAP) is an important mediator of eicosanoid generation. PLAP can also be found in high concentrations in synovial fluid from patients with rheumatoid arthritis, and injection of PLAP into animal joints results in an inflammatory, rheumatoid-like lesion. We have demonstrated previously that TNF-alpha and IL-1 beta stimulate formation of PLAP before phospholipase A2 (PLA2) enzyme activation and production of eicosanoids. To further explore the mechanisms found in the inflammatory response, we examined the ability of PLAP to stimulate release of TNF and IL-1 from human peripheral blood monocytes. TNF and IL-1 protein levels were measured by ELISA, and IL-1 and TNF mRNA were determined by Northern blotting. PLAP, PLAP peptide, and melittin, a bee venom PLA2 activator with homology with PLAP, all increased IL-1 and TNF production in a time- and dose-dependent manner. Heat-denatured PLAP and actin (an irrelevant protein) failed to exert this effect. PLAP stimulation of TNF and IL-1 could be enhanced with co-treatment of cells with free fatty acids, such as arachidonic or linoleic acid, but it was not blocked completely by PLA2 inhibitors. These results demonstrate not only that synthesis of PLAP can be stimulated by cytokines, but also that PLAP may regulate cytokine synthesis and thus perpetuate an immune or inflammatory response.
Neutralization of endogenous IL-6 suppresses induction of IL-1 receptor antagonist.
Authors: Jordan M , Otterness I G , Ng R , Gessner A , Röllinghoff M , Beuscher H U
Published: December 2022

IL-1 is a potent cytokine that promotes host defense and inflammation. These processes may be modulated by an IL-1 receptor antagonist (IL-1Ra) that binds to and blocks IL-1 receptors. The objective of this study was to define the cellular origin and regulation of IL-1Ra production during bacterial infection. Oral infection of mice with Yersinia enterocolitica resulted in expression of IL-1Ra mRNA and synthesis of IL-1Ra in Peyer's patches (PP), the local site of infection, as well as in noninfected organs such as spleens. By immunostaining, recruited circulating neutrophils were identified to be the primary source of IL-1Ra in tissues. Only approximately 20% of the IL-1Ra-staining cells were accounted for by inflammatory macrophages. Strikingly, neutralization of IL-6 by anti-IL-6 antiserum caused a suppression of both IL-1Ra mRNA in PP and synthesis of IL-1Ra in circulating neutrophils. Confirmatory evidence that IL-6 participates in the generation of IL-1Ra was obtained when rIL-6 induced, and anti-IL-6 antiserum blocked, IL-1Ra expression in cultures of macrophage and polymorphonuclear leukocytes (PMN). These findings suggest that IL-6 induced induction of IL-1Ra may provide a negative feedback loop, facilitating resolution of the inflammatory response locally and presumably at remote sites of infection.
Glycosylphosphatidylinositol-modified murine B7-1 and B7-2 retain costimulator function.
Authors: Brunschwig E B , Levine E , Trefzer U , Tykocinski M L
Published: December 2022

Glycosylphosphatidylinositol (GPI)-modified variants of murine B7-1 and B7-2 cell surface costimulators were produced via chimerization with alternative GPI-modification signal sequences from decay-accelerating factor (DAF). GPI anchorage was verified by demonstrating phosphatidylinositol-specific phospholipase C (PI-PLC) sensitivity of the chimeric polypeptides in both immunofluorescence/flow-cytometric and immunoprecipitation analyses. The various GPI-modified chimeric B7-1:DAF and B7-2:DAF polypeptides were shown to retain costimulator function, in both an in vitro proliferation assay and an in vivo triggering of cytotoxicity assay. The findings indicate that costimulator function for both B7-1 and B7-2 is not dependent upon native hydrophobic transmembrane anchorage. Moreover, the functionality of the GPI-modified variants in enhancing the immunogenicity of the murine T lymphoma line EL-4 suggests a novel route for generating APC-centered immunotherapeutics, including cellular cancer vaccines, that is based upon protein transfer of GPI-modified costimulators.