bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression cloning of complementary DNA encoding three distinct isoforms of guinea pig Fc receptor for IgG1 and IgG2.
Authors: Yamashita T , Shinohara K , Yamashita Y
Published: December 2022

Three cDNA clones encoding the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2 R) have been isolated by an expression cloning strategy using mAb directed against the receptor. When transfected into COS-7 cells, these cDNA induced cell surface expression of the receptor that bound IgG1 and IgG2 antibodies complexed with the Ag. The ligand-binding affinities of these receptors were indistinguishable. Nucleotide sequencing has indicated that one of these clones, Fc gamma 1/gamma 2R-B1, is identical to the previously isolated cDNA clone homologous to the b2 isoform of human Fc gamma RIIB and that of murine Fc gamma RII, encoding a transmembrane glycoprotein containing two Ig-like extracellular domains. Two other clones, Fc gamma 1/gamma 2R-B2 and -B3, are identical to Fc gamma 1/gamma 2R-B1 except for an inframe insertion in the cytoplasmic region. The 48-nucleotide insertion found in Fc gamma 1/gamma 2R-B2 is identical to the first 48 nucleotides of the B3 insert that comprises 132 bp. Based on the size and homology of the inserted sequence, Fc gamma 1/gamma 2R-B2 and -B3 are identified as the homologues of the b1 isoform of human Fc gamma RIIB and that of murine Fc gamma RII, respectively. Reverse transcription and polymerase chain reaction of RNA revealed that macrophages and polymorphonuclear leukocytes expressed preferentially Fc gamma 1/gamma 2R-B1. On the other hand, B lymphocytes expressed all three forms, among which Fc gamma 1/gamma 2R-B2 and -B3 were selectively expressed in LPS-activated B lymphocytes that showed a dramatic increase in the levels of cell surface expression of Fc gamma 1/gamma 2R. These results suggest that the inserted sequences of Fc gamma 1/gamma 2R-B2 and -B3 are important to generate responses specific for B lymphocytes, which may include regulation of cell activation.
Responses of mesangial cells of autoimmune mice to cytokines.
Authors: Liu Y J , Ooi B S
Published: December 2022

Glomerulonephritis is one of the most important complications of SLE. The genesis of the renal lesions in this disorder is determined by a cascade of events that leads to mesangial cell proliferation as one of the hallmarks of the kidney disease. The current study examines the postulate that there may be intrinsic abnormalities in the mesangial cells of the autoimmune host which predisposes it to develop nephritis. It investigates the possibility that growth responses of mesangial cells of the autoimmune MRL/MpJ-lpr/lpr mice are different from those of their congenic normal MRL/MpJ-(+)/+ (MRL-(++)) counterparts when cultured with various cytokines. To test this possibility, growth responses of mesangial cells as assessed by [3H]TdR uptake were measured when these cells were cultured with epidermal growth factor, platelet-derived growth factor, transforming growth factor-beta, and 1,25-dihydroxy vitamin D3. Epidermal growth factor stimulated the growth of mesangial cells of both strains of mice equally, but platelet-derived growth factor appeared to elicit a more pronounced proliferative response from mesangial cells of the autoimmune mice. In regard to inhibitory substances tested, mesangial cells of autoimmune MRL-lpr mice appeared to be relatively insensitive to the suppressive effects of transforming growth factor-beta and 1,25-dihydroxy vitamin D3. Collectively, the response profile of mesangial cells of the MRL-lpr mice suggests that these cells have a tendency to proliferate when they are acted on by cytokines, so that they may be more susceptible to develop the proliferative lesions seen in the nephritis of SLE.
T cell epitopes of house dust mite major allergen Der p II.
Authors: Joost van Neerven R , van t'Hof W , Ringrose J H , Jansen H M , Aalberse R C , Wierenga E A , Kapsenberg M L
Published: December 2022

Recent work has indicated the significance of IL-4- and IL-5-secreting allergen-specific human Th2 lymphocytes in the control of immune responses to allergens in atopic individuals. The precise allergenic epitopes that activate these allergen-specific Th2 cells are, however, hardly known. We analyzed the epitope-specificity of T lymphocytes specific for Der p II, one of the major allergens of house dust mite Dermatophagoides pteronyssinus. Using a panel of overlapping synthetic peptides that span the entire Der p II molecule, we could demonstrate that polyclonal Der p II-specific T cell lines prepared from the peripheral blood of five atopic patients can react with at least 10 different epitopes of the molecule. Each donor showed a different pattern of reactivity with the synthetic peptides, suggesting that Der p II contains multiple T cell epitopes that may differ from individual to individual. We studied the specificity of the T cell response to Der p II in more detail in one atopic patient using a short term polyclonal T cell line that strongly reacted to one single peptide (116-129) of the allergen. From this patient we established a panel of 11 Der p II-specific TLC. Ten TLC were of the CD3+ CD4+ phenotype and showed a high IL-4/IFN-gamma production ratio, whereas another TLC expressed CD3 and CD8 and failed to secrete substantial IL-4 and IFN-gamma. The use of at least four different TCR V beta gene segments was shown within this panel TLC. All TLC tested recognized the allergen in an HLA-DR1-restricted manner. Although this patient reacted to only one peptide on the polyclonal level, two T cell epitopes were identified on the clonal level by using synthetic peptides and autologous APC to stimulate the TLC. Combining data of CD4/CD8 expression, TCR V beta usage, and epitope specificity, at least six different types of Der p II-specific TLC could be identified within this patient. Binding of IgE to all synthetic peptides of Der p II is low and of low affinity, which may be of particular importance with respect to possible desensitization protocols using such peptides.
Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes.
Authors: Mehrotra P T , Wu D , Crim J A , Mostowski H S , Siegel J P
Published: December 2022

We have studied the effects of human rIL-12 on the proliferation and generation of cytotoxic activity in human CTL precursors. Purified human blood CD8+ T lymphocytes were stimulated overnight with immobilized alpha-CD3 and cultured 3 to 4 additional days under various conditions. The addition of IL-12 resulted in a marked (10- to 20-fold), dose-dependent, augmentation of cytotoxicity per cell with a smaller (2-fold) increase in cell number. IL-12 augmentation of proliferation and cytotoxicity of CD8+ T cells was not inhibited by a mAb to the p55 subunit of the IL-2 receptor (alpha-Tac) at a concentration sufficient to block the activity of exogenously added IL-2, indicating that the activity of IL-12 did not require IL-2. Addition of IL-12 at the time of alpha-CD3 activation or 1 day later was highly effective at augmenting cytotoxicity, whereas delayed addition of IL-12 (day 2 or 3) resulted in a smaller increase in CTL activity with no increase in cell number. IL-12 at all doses tested synergized with low dose IL-2 in inducing the proliferation and differentiation of CD8+ T cells. The synergistic effect was not blocked by adding neutralizing serum to IFN-gamma. In contrast to this synergistic effect, IL-12 significantly inhibited the proliferation observed in the presence of higher concentrations of IL-2 (4,500 and 13,500 pg/ml). An inhibitory effect of IL-12 was also observed when IL-12 was added to CD8+ T lymphocytes 3 days subsequent to activation with alpha-CD3 and IL-2. This broad set of potent effects of IL-12 on CD8+ T cell responses suggests that IL-12 may play an important immunoregulatory role on CTL development in vivo and may be a useful tool for manipulating this process in vivo for investigational and immunotherapeutic purposes.
Prominent usage of V beta 8.3 T cells in the H-2Db-restricted response to an influenza A virus nucleoprotein epitope.
Authors: Deckhut A M , Allan W , McMickle A , Eichelberger M , Blackman M A , Doherty P C , Woodland D L
Published: December 2022

The spectrum of TCR usage has been analyzed for virus-specific CD8+ T cells isolated from the regional mediastinal lymph modes and from the lung by bronchoalveolar lavage (BAL) of C57BL/6 (B6) mice with influenza pneumonia. Lymphocytes were recovered during the acute phase of the primary response in mice infected with an H3N2 (A/HKx31) virus, or in immune animals that were secondarily challenged with an H1N1 virus (A/PR8). Cells taken directly from the BAL of infected mice exhibited an increase in the frequency of V beta 8.3+/CD8+ T cells. In addition, 20 to 50% of proliferating CD8+ T cells in the mediastinal lymph nodes and BAL populations stimulated in vitro with A/HKx31 were V beta 8.3 TCR+. These observations indicated that the V beta 8.3+/CD8+ T cells were specifically involved in the inflammatory process during influenza infection. However, in vivo depletion of V beta 8+ T cells in CD4-depleted mice did not adversely affect viral clearance, suggesting that other CD8+ T cells can compensate for the absence of these cells. The spectrum of TCR usage was also analyzed for influenza-specific T cell hybridomas derived from freshly isolated BAL of mice with pneumonia. Many of these T cell hybridomas were V beta 8.3+, although other TCR V beta elements were used. All of the V beta 8.3+ hybridomas recognized the H-2Db-restricted NP epitope, 365-380. Although the V beta 8.3 TCR contain similar TCR D beta and J beta elements, V alpha usage was surprisingly variable. Therefore, recognition of this particular epitope was dominated by the beta-chain of the TCR. We conclude that the murine CD8+ response to influenza A virus infection of B6 mice is limited in terms of the diversity of the responding T cells. However, there is significant plasticity in the CD8+ response, which readily compensates for the absence of the dominant T cell population.
IL-2 up-regulates but IFN-gamma suppresses IL-8 expression in human monocytes.
Authors: Gusella G L , Musso T , Bosco M C , Espinoza-Delgado I , Matsushima K , Varesio L
Published: December 2022

IL-2 has pleiotropic properties and is a potent activator of monocytic functions. Since monocytes are an important source of the chemoattractant cytokine IL-8, we studied the effects of IL-2 on the expression of IL-8 in human monocytes. IL-8 mRNA expression was detectable in resting human monocytes. Treatment of monocytes with IL-2 increased IL-8 mRNA expression by a protein synthesis-independent process. The augmentation of IL-8 mRNA by IL-2 was associated with an increase in IL-8 secretion. The expression of IL-8 mRNA was not a nonspecific response to any stimulus of monocyte activation. In fact, IFN-gamma, which is also a potent monocyte activator, not only failed to induce IL-8 expression but inhibited the stimulation of IL-8 by IL-2. Nuclear run-on experiments demonstrated that both the enhancement of IL-8 mRNA expression and its down-regulation by IFN-gamma occurred at the transcriptional level. These results show for the first time that in fresh human monocytes, IL-8 expression is differentially regulated by IL-2 and IFN-gamma and suggest that the interactions among IL-2, IL-8, and IFN-gamma may be important for the development and control of the inflammatory response.
Particle opsonization and lung macrophage cytokine response. In vitro and in vivo analysis.
Authors: Kobzik L , Huang S , Paulauskis J D , Godleski J J
Published: December 2022

Inhaled inert particles and organisms cause a spectrum of pulmonary responses, ranging from minimal changes to marked acute inflammation. During ingestion and clearance of such particles, alveolar macrophages (AM) can initiate pulmonary inflammation by production of TNF and neutrophil chemoattractant cytokines. We tested the role of opsonization in determining the AM response to ingestion of inert particles in vitro and in vivo. As measures of AM activation after phagocytosis in vitro, we measured release of TNF and mRNA expression for the platelet-factor 4 family neutrophil chemoattractants, KC and macrophage-inflammatory protein-2 (MIP-2). Using albumin-coated fluorescent latex particle as phagocytic targets, we found a marked release of TNF by AM ingesting particles opsonized with antialbumin IgG, although uptake of similar numbers of unopsonized particles caused little or no release (e.g., 4648 +/- 1147 pg/ml, opsonized beads vs 576 +/- 205 unopsonized, 10:1 particle:cell ratio, n = 4, mean +/- SD). Flow cytometry confirmed equal uptake of the two particle types. Northern analysis of AM mRNA showed marked induction of KC and MIP-2 mRNA after ingestion of opsonized particles only. Instillation of opsonized particles into hamster lungs caused a marked neutrophil influx, although unopsonized particles did not. TNF was elevated in lavage fluid after instillation of opsonized particles, but not after unopsonized beads (92.7 +/- 136 pg/ml opsonized, n = 7 vs 1.3 +/- 3.6 unopsonized, n = 6). KC and MIP-2 mRNA were induced in lavaged cells after instillation of opsonized but not after unopsonized particles or vehicle control. The nature of particle interaction with the AM surface during phagocytosis determines the subsequent AM response. Although many unopsonized inert particles are ingested with minimal AM activation, specific opsonization of pathogens or non-specific adsorption of Ig onto other particles may activate AM and lead to pulmonary inflammation.
Rescue, expression, and analysis of a neutralizing human anti-hepatitis A virus monoclonal antibody.
Authors: Lewis A P , Lemon S M , Barber K A , Murphy P , Parry N R , Peakman T C , Sims M J , Worden J , Crowe J S
Published: December 2022

A human anti-hepatitis A virus mAb was rescued from a hybridoma cell line by conventional cDNA cloning, and expressed in CHO cells. The full nucleotide sequences of the mAb H and L chains were determined, revealing a VHI/V lambda II V region combination. Comparisons with germline V genes suggest that the V regions had undergone somatic mutations characteristic of an Ag-driven immune response. A comparison of the binding to hepatitis A virus between mAb derived from the CHO cells and the original hybridoma cell line using ELISA, radioimmunoprecipitation, and solid-phase competition RIA, indicated that the CHO cell-derived mAb fully retained the specificity of the mAb produced by hybridoma cells. Analysis of viral neutralization using a radioimmunofocus inhibition assay demonstrated the retention of antibody functionality after expression in CHO cells, demonstrating the use of this technique in the rescue and high level expression of unstable efficacious human mAb.
Induction of antibodies to a kappa V region by gene immunization.
Authors: Watanabe A , Raz E , Kohsaka H , Tighe H , Baird S M , Kipps T J , Carson D A
Published: December 2022

Direct gene transfer into muscle can lead to sustained gene expression at the injected sites. Here we tested the ability of in vivo gene transfection to immunize mice against an isolated human IgV region. The Humkv325 germline kappa L chain V gene encodes the kappa L chain in V regions of several human IgM autoantibodies and is used frequently in chronic lymphocytic leukemia. This gene was inserted into a mammalian expression vector, pREP7, to produce pREVk3. Mice injected i.m. with pREVk3 produced antibodies against the V region of Glo, a human monoclonal IgM paraprotein whose kappa L chain is encoded by Humkv325. Co-injection of an expression vector encoding IL-2 enhanced anti-Glo antibody production fivefold and induced a localized delayed hypersensitivity reaction. Antibody production also was induced when vectors encoding Humkv325 and IL-2 were injected s.c. These experiments demonstrate that gene immunization vectors can stimulate immune responses to antibody V region determinants.
Clustered CD3/TCR complexes do not transduce activation signals after bispecific monoclonal antibody-triggered lysis by cytotoxic T lymphocytes via CD3.
Authors: Blank-Voorthuis C J , Braakman E , Ronteltap C P , Tilly B C , Sturm E , Warnaar S O , Bolhuis R L
Published: December 2022

Bispecfic mAb (bsmAb) directed against the CD3/TCR complex and a tumor-associated Ag (TAA) induces CTL-mediated lysis of TAA+ target cells. We have investigated whether bsmAb-pretargeted CTL can enter multiple lytic cycles. BsmAb-pretargeted CTL retained bsmAb-targeted lytic capacity for at least 24 h when exposed to medium without TAA+ target cells. Exposure of bsmAb-pretargeted CTL to TAA+ target cells resulted in a rapid loss of bsmAb-targeted cytotoxicity of TAA+ or Fc gamma R+ target cells, although the CTL retained surface bsmAb. Moreover, addition of rabbit anti-mouse lg to these CTL did not induce calcium mobilization. These CTL still showed Ag-specific cytotoxicity and cytolysis of anti-CD3 mAb-expressing hybridoma cells. Readdition of bsmAb to CTL that had lost bsmAb-targeted cytotoxicity instantly restored the bsmAb-targeted lytic activity of the CTL. Hence, as in Ag-specific cytolysis, bsmAb-pretargeted CTL can enter multiple bsmAb-targeted cytolytic cycles. Surprisingly, exposure of bsmAb-pretargeted CTL to Fc gamma R+ cells did not result in loss of bsmAb-targeted cytolysis of TAA+ cells. Fluorescence microscopic analysis revealed that bsmAb-mediated interaction with TAA+ cells, but not with Fc gamma R+ cells, resulted in clustering of bsmAb-pretargeted CD3/TCR complexes on the CTL surface. On the basis of the observed correlation between clustered bsmAb-pretargeted CD3/TCR complexes and loss of bsmAb-targeted cytotoxicity, we hypothesize that clustered CD3/TCR complexes can no longer transduce signals.
Development of T cell receptor-gamma delta cells. Phenotypic and functional correlations of T cell receptor-gamma delta thymocyte maturation.
Authors: Tatsumi Y , Pena J C , Matis L , Deluca D , Bluestone J A
Published: December 2022

The development of TCR-gamma delta cells during thymic ontogeny has been studied using fetal thymic organ cultures of normal and transgenic (Tg) mice. The expression of the cell-surface markers--heat stable Ag (HSA), MEL-14, CD5, CD25 (IL-2R), and CD44 (Pgp-1)--correlated with TCR-gamma delta maturation. As the fetal thymus developed, there was an increase in HSA-, CD5dull, and CD44+ cells for each TCR-gamma delta cell subset. Moreover, the expression of recombination activating genes-1 and -2 (RAG-1 and RAG-2) also correlated with TCR-gamma delta maturation as only HSA+ TCR-gamma delta cells transcribed these genes. Cyclosporin A inhibited the development of the TCR-gamma delta thymocytes if it was introduced early during thymic ontogeny by arresting the differentiation of TCR-gamma delta thymocytes at the HSA+ stage. Immature HSA+ TCR-gamma delta thymocytes isolated from both TCR-gamma delta Tg and normal mice did not respond to nominal Ag or anti-TCR mAb unless exogenous IL-2 was added to the cultures. In contrast, HSA- TCR-gamma delta cells from Tg and normal mice responded to TCR/ligand interactions in the absence of additional IL-2. Finally, the development of functionally mature TCR-gamma delta cells could be induced in vitro. Interaction of the HSA+ Tg+ TCR-gamma delta cells with anti-TCR-gamma delta mAb or Ag-bearing thymic stromal cells resulted in RAG-1 and RAG-2 down-regulation. These data strongly suggest that TCR-gamma delta HSA+, RAG+ thymocytes differentiate into a more mature stage under the pressure of positive selection and that TCR-gamma delta cell development is regulated in a manner similar to TCR-alpha beta cells. In addition, the ability of Cyclosporin A to inhibit TCR-gamma delta cell development combined with the findings that Ag-bearing stromal cells can induce Tg TCR-gamma delta cell development suggests that maturation and selection of TCR-gamma delta cells depends on receptor-mediated physiologic stimuli delivered during thymic development.
Hen egg-white lysozyme-specific T cells elicited in hen egg-white lysozyme-transgenic mice retain an imprint of self-tolerance.
Authors: Yule T D , Basten A , Allen P M
Published: December 2022

The characteristics of T cell self-tolerance were examined in hen egg-white lysozyme (HEL)-transgenic (Tg) mice that were tolerant to a dose of HEL that was immunogenic in non-Tg littermates. HEL-specific T cells were identified in the periphery of the Tg mice after immunization with 100-times more HEL than was required to achieve a response in normal littermates. The Tg T cells were functional in vivo as they were capable of providing help to generate a HEL-specific antibody response. Selective deletion of T cells specific for the dominant T cell determinant of the native protein was not the primary mechanism of T cell tolerance in the HEL-Tg mice because, similar to non-Tg littermates, the majority of lymph node (LN) and T cell clones from HEL-Tg mice were specific for the dominant T cell determinant of HEL. Rather, our findings support the idea that the HEL-reactive T cells were anergic in vivo, but could be partially activated with a strong stimulus to the immune system (i.e., 20 nmol HEL and CFA). This conclusion is based on three observations: 1) proliferation in vitro to HEL by Tg LN T cells was subnormal (25% of control) and required 2 log more Ag to proliferate when compared with proliferation of LN from non-Tg littermates; 2) T cell clones isolated from HEL-Tg mice also proliferated poorly upon stimulation with HEL and Con A, although lymphokine production from the same stimuli was similar to that obtained from non-Tg clones; 3) invariably, upon repeated antigenic stimulation in vitro, the Tg T cell clones acquired full proliferative capacity to Ag and mitogens.
Activated platelets induce superoxide anion release by monocytes and neutrophils through P-selectin (CD62).
Authors: Nagata K , Tsuji T , Todoroki N , Katagiri Y , Tanoue K , Yamazaki H , Hanai N , Irimura T
Published: December 2022

Activated platelets expressing P-selectin in their surfaces are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes are functionally modified by their adhesion to activated platelets. We used human peripheral blood monocytes and neutrophils and measured superoxide anion generation by these cells cultured with platelets. The levels of superoxide anion production were found to be markedly elevated when thrombin-activated platelets were used. The extent of this enhancement was much smaller when leukocytes were cultured with resting platelets than activated platelets. The increase depended on incubation time and platelet concentration. The membranes prepared from activated platelets also induced superoxide anion production, but the culture supernatant of activated platelets did not. The enhanced superoxide anion production was inhibited by anti-P-selectin antibody, anti-sialyl-Lewis X antibody, or a soluble recombinant P-selectin fusion protein. These results indicate that the adhesion of activated platelets to the leukocytes through P-selectin was a crucial step for the activation of leukocyte function, and support the idea that activated platelets are actively involved in inflammation processes.
Isolation of erythrocyte membrane inhibitor of reactive lysis type II. Identification as glycophorin A.
Authors: Tomita A , Radike E L , Parker C J
Published: December 2022

When incubated with activated CoF and normal human serum (as the C source), a portion of the E from most patients with PNH are hemolyzed. In contrast, normal E are completely resistant to this form of C-mediated cytolysis called reactive lysis. This observation implies that normal E express membrane proteins that inhibit reactive lysis. In previous studies, we have shown that when E proteins are subjected to anion exchange chromatography, two peaks of inhibitory activity are observed. We isolated the inhibitor from the first peak and identified it as an 18-kDa protein that we call MIRL (CD59). The purpose of the studies presented herein was to isolate the inhibitory factor (MIRL type II) from the second peak. After anion exchange and gel filtration chromatography, aliquots of the fractions containing MIRL II activity were subjected to preparative SDS-PAGE (nonreducing conditions), and protein was eluted from gel slices electrophoretically. Inhibitory activity was found primarily in two adjacent slices, corresponding to an M(r) range of 84-51 kDa. When MIRL II was analyzed by SDS-PAGE and silver staining, two prominent bands representing proteins with M(r) of 77 and 39 kDa were observed under both reducing and nonreducing conditions. This behavior in SDS-PAGE is characteristic of GP-A, which migrates primarily as a nondisulfide-linked homodimer in equilibrium with a monomeric form. Immunoblotting studies confirmed that MIRL II is GP-A. The protein caused a concentration-dependent inhibition of reactive lysis, with approximately 100 ng producing 50% inhibition. GP-A inhibited reactive lysis by blocking the formation or binding of C5b-7, and this inhibitory activity was immunoprecipitated by monoclonal anti-GP-A. Furthermore, GP-A that was isolated by affinity chromatography also inhibited reactive lysis. These studies demonstrate that the major E sialoglycoprotein functions as an inhibitor of the membrane attack complex of C.
In vitro T cell immune responses to the preS2 antigen of the hepatitis B virus envelope protein in preS2 + S vaccine recipients. Absence of cross-reactivity of subtypes at a major T cell recognition site.
Authors: Cupps T R , Tibbles J , Hurni W M , Miller W J , Ellis R W , Milich D , Wetter N
Published: December 2022

The immune responses to hepatitis B virus envelope antigen were investigated in 16 vaccine recipients after immunization with a recombinant yeast-derived preS2 + S (adw) vaccine for hepatitis B virus. After the completion of the three-slot immunization series, all vaccine recipients developed antibody to the S domain and anti-preS2 antibody. In vitro proliferative responses to preS2 (120-174) peptide were demonstrated in 10 of 16 vaccine recipients. Although reactivity could be demonstrated through the length of the preS2 peptide, the principal site of proliferative activity was contained within the preS2 (146-165) region of the peptide. The principal T cell reactive site coincides with a region of significant amino acid variability of the different hepatitis B virus serotypes. Cross-reactivity with a serotype (ayw) not present in the preS2 + S vaccine could not be demonstrated at this widely recognized T cell epitope. The low level of cross-reactivity demonstrated in a limited subset of the vaccine recipients was mediated through nondominant T cell reactive sites contained in the relatively conserved preS2 (120-146) region of the molecule. The identification of widely recognized but serotype-specific T cell epitopes in the preS2 region of the hepatitis B virus envelope antigen may be an important consideration in future vaccine development.
Cytotoxic T cells in HIV2 seropositive Gambians. Identification of a virus-specific MHC-restricted peptide epitope.
Authors: Gotch F , McAdam S N , Allsopp C E , Gallimore A , Elvin J , Kieny M P , Hill A V , McMichael A J , Whittle H C
Published: December 2022

A preliminary study of gag-specific, MHC-restricted CD8+ CTL has been performed in nine Gambian patients infected with HIV2. Such CTL were present in at least 55% of patients in fresh peripheral blood mononuclear cells without the requirement for in vitro restimulation. We have identified a nonamer peptide from HIV2 gag that is recognized by CD8+ HLA-B53 CTL using an amino acid sequence motif predicted from analysis of endogenous peptides eluted from HLA-B53 molecules. This peptide, from an HIV2/SIV conserved sequence, has previously been reported to be recognized by CTL from non-human primates vaccinated with recombinant vaccinia virus expressing the gag protein of SIV or infected with SIV virus. HLA-B53-restricted, HIV2 gag-specific CTL did not recognize target cells expressing HIV1 gag proteins, indicating that no cellular cross protection to HIV1 could be expected in this case.
Regulation of apoptosis in vitro in mature murine spleen T cells.
Authors: Perandones C E , Illera V A , Peckham D , Stunz L L , Ashman R F
Published: December 2022

Previously it has been shown that thymocytes undergo apoptosis, a form of programmed cell death, in response to glucocorticoids. This classic form of apoptosis is prevented by inhibition of protein synthesis. The current paper demonstrates that mature T cells also undergo apoptosis, but that the regulation of apoptosis in spleen T cells differs from that of thymocytes. Mature mouse spleen T cells were shown to die by apoptosis, not necrosis, when cultured without an added stimulus. Assays for apoptosis included internucleosomal DNA cleavage by gel electrophoresis, percent fragmentation of DNA by the diphenylamine method, and percent of cells with hypodiploid DNA by flow cytometry. The percent of apoptotic cells was 2% in fresh spleen T cells, and increased at least until 16 h, when 21% were apoptotic. Dexamethasone caused apoptosis in both thymus and spleen T cells, but only thymocytes showed a requirement for protein synthesis in dexamethasone-induced death. Cycloheximide increased apoptosis in spleen T cells, indicating that apoptosis was controlled by newly synthesized protective proteins. Spontaneous apoptosis was decreased in spleen T cells by protein kinase C activation, and was increased by H7 and staurosporine, which inhibits protein kinases, in contrast with the behavior of thymocytes. The protein kinase A/G inhibitor HA1004 also decreased spleen T cell apoptosis. The contrasting effects of cycloheximide on thymocytes and spleen T cells occurred over the same concentration range, and the same was true for PMA. The dexamethasone dose-response curves were similar, except that a greater proportion of spleen T cells were dexamethasone-resistant. These data support the hypothesis that the apoptosis program in T cells undergoes a transition during their maturation, such that apoptosis in mature T cells is regulated more like that of mature B cells than that of thymocytes.
Apoptosis of activated CD8+/CD57+ T cells is induced by some combinations of anti-CD2 mAb.
Authors: Rouleau M , Bernard A , Lantz O , Vernant J P , Charpentier B , Senik A
Published: December 2022

Peripheral blood CD8+/CD57+ T cells display poor proliferative responses when stimulated with CD3 or CD2 in vitro, but can be induced to proliferate in the presence of exogenous IL-2. Although GT2+T11(1), a mitogenic anti-CD2 mAb pair, could synergize with IL-2 to induce sustained cell divisions in this population (as did immobilized OKT3), D66+T11(1), another anti-CD2 mAb pair, could only induce a small abortive proliferative response. All these antibodies were in contrast strongly mitogenic for CD8+/CD57- T cells. Assuming that D66+T11(1) were exerting inhibitory effects on CD8+/CD57+ T cells, we indeed found that such antibodies profoundly suppressed the anti-CD3 and IL-2-induced proliferation of those cells, but not that of CD8+/CD57- cells. CD2-mediated growth arrest was correlated with rapid cell death occurring within 2 h, once the cells had been submitted to D66+T11(1), and cells susceptible to the death signal were large cells committed in the cell cycle. D66+T11(1)-treated cells had the well-known ultrastructural form of apoptosis, and the DNA extracted from these cells showed the typical ladder pattern of DNA fragmentation accompanying this process. For apoptosis to occur, two anti-CD2 mAb had to be applied to the cells, one of them being D66, suggesting that the corresponding anti-CD2 mAb pairs were imposing on the CD2 molecule a particular conformational change appropriate to transduce a death signal. Notably, CD8+/CD57- T cells were largely resistant to apoptosis in the conditions just described. When exposed to anti-CD3 and IL-2 in primary and secondary cultures, CD8+/CD57+ T cells retained high viability, whereas in contrast, when exposed to D66+T11(1), important cell loss occurred, concomitant with apoptosis, illustrating the specificity of the CD2-derived death signal. Our results suggest that the expansion of CD8+/CD57+ T cells is critically dependent on the CD2 pathway which, according to the conformational change of the CD2 molecule and the activation state of the cells, will direct them either towards proliferation or towards cell death.
Activation of human neutrophils induces an interaction between the integrin beta 2-subunit (CD18) and the actin binding protein alpha-actinin.
Authors: Pavalko F M , LaRoche S M
Published: December 2022

Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily of adhesion molecules, rapidly change from a low avidity to a high avidity state on activation of neutrophils by various agonists. The control of CD18 integrin-dependent neutrophil adhesion and the mechanisms that regulate integrin avidity are poorly understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integrins are necessary for proper integrin function and suggest that interactions with intracellular proteins are involved. We have focused on identifying cytoskeletal proteins that interact with the cytoplasmic domain of the beta-subunit (beta 2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein alpha-actinin associates in vitro with a peptide corresponding to a portion of the CD18 cytoplasmic domain in solid phase binding assays and affinity chromatography experiments. The peptide sequence within the CD18 cytoplasmic domain that binds alpha-actinin is homologous with a region in the cytoplasmic domain of the integrin beta 1-subunit, which also binds alpha-actinin. We demonstrate that the association of alpha-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with alpha-actinin from stimulated human neutrophils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to coprecipitate with alpha-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-alpha. Very little CD18 coprecipitates with alpha-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with alpha-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between alpha-actinin and CD18 was transient, peaking 5-10 min after activation and decreasing to near resting levels by 20 min. CD18 did not coimmunoprecipitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an alpha-actinin-mediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, alpha-actinin has a major function as an actin membrane linker molecule, and integrin avidity may be affected by an association with the actin cytoskeleton involving alpha-actinin.
Clonal deletion of V beta 5+ T cells by transgenic I-E restricted to thymic medullary epithelium.
Authors: Burkly L C , Degermann S , Longley J , Hagman J , Brinster R L , Lo D , Flavell R A
Published: December 2022

A variety of cell types expressing MHC class II molecules is known to function as APC in vitro. We employed the Ig kappa gene enhancer and promoter to target the class II E alpha gene, and thereby I-E, exclusively to B cells to address their APC function in vivo. Although transgenic I-E was expressed on B lymphocytes, we unexpectedly obtained I-E on thymic medullary epithelium but not macrophages and at low frequency on dendritic cells. Using these transgenic mice, we constructed bone marrow irradiation chimeras with I-E expressed only on medullary epithelium, in order to determine the role of this cell type in tolerance by clonal deletion in the thymus. Although it is accepted that bm-derived cells play a primary role in deletion, and thymic epithelium can delete clones to a lesser degree, the role of cortical vs medullary thymic epithelium has not been directly dissected. We demonstrate that medullary epithelium alone can tolerize by partial deletion of I-E-reactive V beta 5+ T cells. These results indicate a role for medullary epithelium in deletion during the later stages of thymic development, and support the notion that positive and negative selection of developing T cells can occur in distinct temporal and anatomic compartments.
Production and characterization of soluble and transmembrane murine CD2. Demonstration that CD48 is a ligand for CD2 and that CD48 adhesion is regulated by CD2.
Authors: Kaplan A J , Chavin K D , Yagita H , Sandrin M S , Qin L H , Lin J , Lindenmayer G , Bromberg J S
Published: December 2022

The baculovirus expression vector was used to produce full length, two amino-terminal Ig-like extracellular domains, and one amino-terminal Ig-like extracellular domain soluble murine CD2 products. The products were monomeric, glycosylated, and of the correct predicted m.w. Sf9 insect cells infected with recombinant baculovirus encoding the full length construct display cell surface CD2 by flow cytometry and rosette with murine cell lines that express the ligand for CD2. Uninfected Sf9, wild-type baculovirus-infected Sf9, and Sf9 expressing truncated products do not display cell surface CD2 nor do these latter Sf9 bind to murine cell lines. Cell binding is inhibited by anti-CD2 mAb. All CD2 products possess ligand binding activity since purified preparations of these block cell adhesion. All CD2 antigenic epitopes are close to the ligand binding site because all mAb tested can inhibit cell adhesion. The ligand for CD2 is shown to be CD48. Only CD48+ cell lines can bind CD2+ Sf9 and this is inhibited by anti-CD48 mAb. Antibodies against the closely related cell surface Ag Ly-6A.2 and Ly-9.2 do not inhibit binding. Purified, soluble CD2 also inhibits the binding of anti-CD48 mAb to the cell surface. Unexpectedly, additional mAb blocking studies show that CD2 on the surface of CD48+ cell lines influences adhesion to CD2+ binding partners. The use of cells expressing CD2 and/or CD48 provides evidence for a cis CD2-CD48 interaction on the cell surface in which CD2 negatively regulates CD48 adhesion properties.
Distinct requirements of positive and negative selection for selecting cell type and CD8 interaction.
Authors: Schönrich G , Strauss G , Müller K P , Dustin L , Loh D Y , Auphan N , Schmitt-Verhulst A M , Arnold B , Hämmerling G J
Published: December 2022

We investigated the requirements of positive and negative selection in the thymus for CD8 interaction and the selecting cell type. Thymic epithelial cells are known to mediate positive selection, whereas thymocytes fail to do so. The reason for this failure could be either the low amount of MHC class I molecules on thymocytes or the lack of other properties required for positive selection. To address this question a CD2.Kb transgenic mouse was prepared in which the expression of the Kb gene is under control of the CD2 promoter. In these mice the thymocytes exhibited very high levels of Kb. The mice were crossed with two TCR transgenic mice expressing either the Des.TCR (anti-Kb), which is positively selected on Kk and negatively on Kb, and the 2C.TCR (anti-Ld) with positive selection on Kb and negative on Ld. Despite the high Kb expression on thymocytes in CD2.Kb x 2C.TCR mice no positive selection was observed, whereas efficient negative selection was found in CD2.Kb x Des.TCR F1 mice. Thus, although thymocytes can negatively select, even a strong increase of MHC class I expression cannot convert them into positively selecting cells. This is consistent with the notion that thymic epithelium is specialized for positive selection, but the respective difference between thymocytes and thymic epithelium is not clear. The influence of CD8 was investigated using transgenic mice expressing a Kk/A2 or a Kb/A2 hybrid gene in which the promoter, the alpha 1, alpha 2 domains were of mouse origin and the alpha 3 domain of human origin. Because the murine CD8 molecule does not efficiently bind to human HLA class I, in these mice the CD8 interaction was impaired. In Kb/A2 x 2C.TCR and Kk/A2 x Des.TCR mice no positive selection of the respective TCR was found, whereas in Kb/A2 x Des.TCR mice negative selection was still functional. Altogether, the results indicate that positive selection depends more strictly on CD8 interaction and cell type than negative selection.
Rapid activation of C-Raf-1 after stimulation of the T-cell receptor or the muscarinic receptor type 1 in resting T cells.
Authors: Siegel J N , June C H , Yamada H , Rapp U R , Samelson L E
Published: December 2022

The c-Raf-1 serine/threonine kinase is an important component of signal transduction pathways mediating the effects of a variety of growth factors. In activated T cells, IL-2 has been shown to induce activation of c-Raf-1, but c-Raf-1 has not previously been shown to be activated through the T-cell receptor (TCR) in resting G0 T cells. Using a sensitive immune complex kinase reaction, we show that cross-linking of the stimulatory and costimulatory receptors CD3, CD4, or CD28 induces c-Raf-1 activation in highly purified resting peripheral blood human T cells. In contrast, cross-linking the nonstimulatory receptor CD45 did not induce c-Raf-1. Surprisingly, although earlier studies had shown delayed kinetics in response to Thy-1 stimulation in murine cells, c-Raf-1 activation in response to CD3 cross-linking was one of the earliest measurable events. In spite of its early kinetics, c-Raf-1 activation was found to be downstream of several other early signal transduction events, including activation of a tyrosine kinase and a tyrosine phosphatase. Several lines of evidence suggest that activation of c-Raf-1 in response to TCR stimulation may be PKC-dependent: first, phorbol esters are extremely potent activators of c-Raf-1 in human T cells; second, the kinetics of accumulation of products of phosphatidylinositol hydrolysis coincides with the kinetics of c-Raf-1 activation; and third, physiologic activation of the PLC/PKC pathway through a transfected, G-protein-coupled receptor HM1 induced similar levels of c-Raf-1 activation with a similar time course. We conclude that c-Raf-1 activation is tightly coupled to TCR stimulation and may participate in signal transduction pathways in resting, G0 T cells. The observation that the HM1 receptor can also activate c-Raf-1 suggests that T cells have the capability to utilize both tyrosine kinase-dependent and tyrosine kinase-independent mechanisms of c-Raf-1 activation.
Xid-associated resistance to experimental Chagas' disease is IFN-gamma dependent.
Authors: Minoprio P , el Cheikh M C , Murphy E , Hontebeyrie-Joskowicz M , Coffman R , Coutinho A , O'Garra A
Published: December 2022

In contrast to normal Balb/c, Balb.Xid immunodeficient mice are naturally resistant to Trypanosoma cruzi infection. Thus, Balb.Xid mice control parasitemia, do not show the characteristic wasting in the acute infection and develop no tissue pathology in the skeletal or cardiac muscles in the chronic phase of disease. By in situ hybridization and semiquantitative polymerase chain reaction, the expression of IL genes in spleen cells from Balb/c and Balb.Xid mice were compared after T. cruzi infection. The results showed that Balb.Xid mice produce considerably higher levels of IFN-gamma, IL-2, and IL-4, but lower levels of IL-10, from as early as 4 days after parasite injection. By day 12 of the infection, although IFN-gamma, IL-2, and IL-4 expression was now comparable in both groups, IL-10 levels continue to be lower in Balb.Xid than in control Balb/c animals. The central role of IFN-gamma in the resistance to T. cruzi was confirmed by treatment of Balb.Xid mice with anti-IFN-gamma antibodies that reestablished susceptibility and lead to increased parasitemia and mortality.
Tumor-specific lysis of human renal cell carcinomas by tumor-infiltrating lymphocytes. I. HLA-A2-restricted recognition of autologous and allogeneic tumor lines.
Authors: Schendel D J , Gansbacher B , Oberneder R , Kriegmair M , Hofstetter A , Riethmüller G , Segurado O G
Published: December 2022

Metastatic renal cell carcinoma (RCC), like melanoma, belongs to the small group of human tumors in which partial or complete remission has been observed in some patients after treatment with various forms of immunotherapy. In contrast to melanoma, CTL showing MHC-restricted lysis of RCC have not been easily found among tumor-infiltrating lymphocytes (TIL). This has led to the suggestion by some that responses to immunotherapy are mediated predominantly by non-MHC-restricted effector cells. We have characterized an MHC-restricted, CD8+ CTL line obtained from an uncloned TIL population of a primary RCC using a low concentration of rIL-2; in fact, these CTL represented a majority of the short-term cultured TIL population. The CTL lysed autologous tumor cells but not normal kidney cells or target cells sensitive to non-MHC-restricted effector cells. In contrast, lymphokine-activated killer (LAK) cells grown in a high concentration of rIL-2 from the patient's PBL lysed autologous tumor and normal kidney cells in addition to several allogeneic tumors. The TIL could be expanded optimally using an autologous tumor line retrovirally transduced with the human cDNA encoding IL-2. TIL were 20-fold more potent than LAK cells in eliminating the IL-2 expressing tumor cells in vitro. The cultured TIL utilized a restricted number of V alpha gene families, suggesting that they may recognize only a limited number of MHC-peptide complexes presented by autologous tumor cells. HLA-A2 was identified as an MHC restriction molecule for presentation of one tumor-derived peptide to these CTL. Only some allogeneic HLA-A2 RCC tumors were lysed. Sequencing of the second and third exons of the HLA-A2 alleles of these cells revealed that both heterogeneity in MHC and peptide availability influenced CTL recognition. These studies demonstrate that some RCC express common antigenic determinants that can be recognized by MHC-restricted CTL and open the possibility of defining the nature of RCC-derived peptides, which, combined with HLA-A2, can generate specific immune responses.
Suppression of monocyte function and differential regulation of IL-1 and IL-1ra by IL-4 contribute to resolution of experimental arthritis.
Authors: Allen J B , Wong H L , Costa G L , Bienkowski M J , Wahl S M
Published: December 2022

IL-4 has diverse effects on hematopoietic cells, including the ability to suppress certain mononuclear cell functions. To evaluate the effect of IL-4 on the evolution of acute and chronic arthritis, murine recombinant IL-4 was administered systemically to animals receiving an arthropathic dose of group A streptococcal cell wall fragments. Daily treatment with IL-4 had a minimal effect on the acute phase, but significantly suppressed the chronic, destructive phase. By 4 wk after initiation of disease, the articular index of IL-4-treated animals was reduced > 60% (articular index = 4 +/- 0.9) compared with the untreated rats (11.5 +/- 0.48, p < 0.001). A substantial decrease in the influx of inflammatory cells and virtual elimination of pannus and erosions occurred after IL-4 therapy. Associated with the reduced accumulation of mononuclear leukocytes was a decrease in their proinflammatory functions including cytokine production and reactive oxygen intermediate metabolism. These observations are consistent with the selective effects of IL-4 on phagocytic cell function demonstrated in vitro. Furthermore, IL-4 induced gene expression for IL-1ra, a protein that antagonizes the action of IL-1 by binding to the IL-1 receptor without agonist activity. Through an expanding spectrum of effects on monocyte-macrophage phenotypic and functional parameters, IL-4 is emerging as an important inhibitor of cell-mediated immune responses and pathogenic processes.