bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of the p27kip1 cyclin-dependent kinase inhibitor expression during IL-4-mediated human B cell activation.
Authors: Blanchard D A , Affredou M T , Vazquez A
Published: December 2022

IL-4 activates resting B cells and, in conjunction with cosignals such as anti-IgM (anti-mu) Ab or CD40 ligand, modulates progression of B cells through the cell cycle, leading to proliferation. In this study, we show that the mitogenic combination of IL-4 and anti-mu Ab triggered induction of cyclin D3 and up-regulated cyclin-dependent kinase (cdk) 6 expression, whereas such regulation was not observed in B cells activated by IL-4 or anti-mu Ab alone. Furthermore, cyclin D3 immunoprecipitated fron as associated with cdk6, and the cyclin D3/cdk6 complex was able to phosphorylate recombinant retinoblastoma protein in vitro. In addition, B cells activated with either IL-4 or 1L-13 alone expressed a higher amount of p27kip1 (p27) cdk inhibitor than nonstimulated cells. In contrast, p27 expression was decreased when cells were activated with mitogenic combinations of IL-4 and anti-mu Ab or anti-CD40 mAb. We also observed that the IL-4-mediated inhibition of the proliferation of anti-mu/IL-2- or anti-mu/phorbol 12,13-dibutyrate-activated human leukemic B cells was associated with the maintenance of large amounts of p27 in these cells. These data suggest that IL-4 controls B cell proliferation by action during at least two steps of the regulation of the cell cycle, cyclin D3/cdk6 complex regulation and p27 inhibitor expression.
Hemopoietic progenitors in the murine fetal liver capable of rapidly generating T cells.
Authors: Kawamoto H , Ohmura K , Hattori N , Katsura Y
Published: December 2022

We investigated the hemopoietic progenitors in fetal liver (FL) compared with those in adult bone marrow (BM) for their ability to differentiate into T, B, and myeloid cells. Lineage marker-negative (Lin-) c-kit+ cells isolated from FL and BM were used as the source of progenitors. No apparent difference was observed between FL and BM progenitors in their ability to generate B and myeloid cells when these progenitors were cultured on a monolayer of a BM-derived stromal cell line. In marked contrast, FL progenitors were able to generate T cells much more rapidly than BM progenitors in both in vivo intrathymic cell transfer and fetal thymus organ culture systems. The same type of FL progenitors were also found in the FL of congenitally athymic nu/nu mice, indicating that these progenitors arise in FL independently of the fetal thymus. The present study have provided circumstantial evidence suggesting the presence of T cell lineage-committed progenitors in FL.
Tyrosine and serine protein kinase activities associated with ligand-induced internalized TCR/CD3 complexes.
Authors: Luton F , Legendre V , Gorvel J P , Schmitt-Verhulst A M , Boyer C
Published: December 2022

Ligand engagement of the TCR/CD3 complex leads to its internalization and modulation from the cell surface. In the present study, we analyzed the intracellular fate of internalized TCR/CD3 complexes following activation of a CTL clone with an anti-clonotypic mAb (anti-TCR mAb). Confocal microscopy using fluorescent anti-TCR mAb showed that after 15 min the TCR/CD3 complex colocalized with the transferrin receptor within endosomes, whereas at later times (2 h) it migrated in late endocytic compartments devoid of transferrin receptor. Using a cell fractionation technique, CD3 components could be detected in early endosomes in the absence of ligand-induced internalization, but were detected in late endosomes only after 2-h anti-TCR-induced internalization. In late endosomes, the internalized TCR/CD3 complex was found to be associated with an active protein kinase, distinct from p56(lck) and p59(fyn), which were mainly present in early endosomes, and ZAP-70, which was only present in the postnuclear supernatant. Phosphoamino acid analysis following an in vitro kinase assay of CD3 immunoprecipitates from early and late endosome fractions showed that the CD3 zeta- and epsilon-chains were phosphorylated exclusively on tyrosine, whereas the CD3 gamma- and delta-chains were phosphorylated on serine and tyrosine, as were 40-kDa and 60-kDa associated proteins. Furthermore, the serine phosphorylation was increased in late endosomes compared with early endosomes. These results suggest that the TCR/CD3 may be associated with different kinase activities during its intracellular pathway following ligand triggering.
Degenerate MHC restriction reveals the contribution of class I MHC molecules in determining the fine specificity of CTL recognition of an immunodominant determinant of HIV-1 gp160 V3 loop.
Authors: Shirai M , Kozlowski S , Margulies D H , Berzofsky J A
Published: December 2022

The novel allogeneic presentation of an immunodominant determinant within the HIV-1 gp160 V3 loop by three different class I MHC molecules to the same CD8+ CTL is used to study the influence of the MHC molecule on the fine specificity of CTL recognition. We previously reported that four distinct class I molecules of H-2d,u,p,q presented the V3 decapeptide P18-I10 (RGPGRAFVTI) to CTL. Surprisingly, we found that H-2d,u,p cells mutually cross-present the P18-I10 peptide to allogeneic CTL clones of each of the other haplotypes, whereas none of these cross-presents to H-2q CTL, nor do H-2q targets present to CTL of the other haplotypes. Here, we explore the critical amino acid residues for the cross-presentation using 10 variant peptides with single amino acid substitutions. The fine specificity examined using these mutant peptides presented by the same MHC class I molecule showed striking similarity among the CTL of each haplotype, expressing either V beta 8.1 or V beta 14. In contrast, the fine specificity is different between the distinct MHC class I molecules even for the lysis by the same CTL, as shown by reciprocal effects of the same substitutions. Thus, peptide fine specificity of a single TCR is influenced by changes in the class I MHC molecules presenting the Ag.
MHC class I ligation of human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis.
Authors: Skov S , Bregenholt S , Claesson M H
Published: December 2022

Cross-linking of MHC class I (MHC-I) molecules on human T cells induces signal-transduction events, including activation of tyrosine kinases, tyrosine phosphorylation of phospholipase C-gamma 1, and elevation of the intracellular free calcium concentration. In this study, we demonstrate that the ZAP70 tyrosine kinase is tyrosine phosphorylated in Jurkat T cells and in purified peripheral T cells after MHC-I ligation. The tyrosine-phosphorylated ZAP70 kinase exhibits a particular phenotype with low affinities for proteins at 21, 40, 60, and 120 kDa, proteins normally co-precipitated with ZAP70 after TCR/CD3 stimulation. The phosphorylation of ZAP70 after MHC-I ligation was dependent on TCR/CD3 surface expression. One of the natural substrates for ZAP70 is the zeta-chain dimer of the TCR/CD3 complex. MHC-I cross-linking induces a phosphorylated zeta-protein that migrates as a dimer at 42 kDa in SDS-PAGE and differs from the 38-kDa phosphorylated zeta-protein dimer induced by TCR/CD3 cross-linking. Furthermore, we demonstrate that the p56lck tyrosine kinase is tyrosine phosphorylated following MHC-I ligation, and that a p56lck-negative Jurkat T cell mutant does not induce phosphorylation of the zeta-chain and the ZAP70 kinase following MHC-I ligation. Previous studies have demonstrated that lack or diminished activation of ZAP70 is involved in the induction of anergy or apoptosis in T cells. Likewise, MHC-I cross-linking of Jurkat T cells results in growth arrest and induction of apoptosis that is strongly inhibited by herbimycin A, suggesting an essential role of tyrosine kinase activity in the process leading to apoptosis.
Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gamma-dependent and -independent pathways.
Authors: Perry L L , Feilzer K , Caldwell H D
Published: December 2022

Mucosal immunity to Chlamydia trachomatis in a mouse model of female genital tract infection is mediated predominantly by Th1-type cells, as shown by in vivo neutralization of cytokines involved in the Th1 vs Th2 pathways. Neutralization of IL-12 was associated with an apparent decrease in the infiltration of CD4+ T cells into infected tissues, systemic reductions in the production of IFN-gamma, and prolonged shedding of high levels of bacteria. Neutralization of IL-4 had no detectable effect on host immunity or on bacterial clearance. To dissociate the protective role of IL-12 from that of IL-12-induced IFN-gamma, resistance to C. trachomatis was compared in IL-12-depleted and IFN-gamma-deficient animals. IL-12-depleted mice displayed minimal bacterial clearance for 1 mo post-infection but eventually resolved genital tract infections completely. IFN-gamma-deficient mice, on the other hand, cleared 99.9% of genital Chlamydia within the first 3 wk but then developed systemic disease associated with dissemination of bacteria to multiple organs. Animals surviving this stage often maintained low level persistent infections within the urogenital tract. These results indicate that the bulk of chlamydial clearance from the genital mucosa is mediated by an IL-12-dependent, IFN-gamma-independent mechanism, while prevention of disseminated disease requires the action of IFN-gamma.
Inhibition of human neutrophil apoptosis by platelets.
Authors: Andonegui G , Trevani A S , López D H , Raiden S , Giordano M , Geffner J R
Published: December 2022

In the absence of appropriate stimuli, polymorphonuclear neutrophils rapidly undergo characteristic changes indicative of programmed cell death or apoptosis. We report here that neutrophils cultured in the presence of platelets (neutrophil:platelet ratios of 1:50, 1:25, and 1:10) show a dramatic inhibition of apoptosis compared with neutrophils cultured alone. Similar degrees of apoptosis delay were induced by viable unstimulated platelets, fixed unstimulated platelets, or fixed activated (1 U/ml thrombin) platelets. Inhibition of apoptosis was associated with prolongation of the functional lifespan of the neutrophil, as indicated by the higher capacity of platelet-treated neutrophils to display chemiluminescence responses triggered by FMLP, immune complexes, and zymosan. The mechanism responsible for the inhibition of neutrophil apoptosis by platelets has not yet been defined. However, it seems that classical recognition systems such as those mediated by the interaction between platelet P-selectin (CD62) or glycoprotein IIb/IIIa complex and their counter-receptors expressed by neutrophils are not involved.
Evidence for the differential expression of the functional alpha-melanocyte-stimulating hormone receptor MC-1 on human monocytes.
Authors: Bhardwaj R , Becher E , Mahnke K , Hartmeyer M , Schwarz T , Scholzen T , Luger T A
Published: December 2022

alpha-Melanocyte-stimulating hormone (alpha-MSH) is released by immunocompetent cells as well as the pituitary gland and functions as a potent inhibitor of immune and inflammatory reactions. Therefore, it was investigated whether normal human monocytes express melanocortin (MC) receptors specific for alpha-MSH. Upon FACS analysis using biotin-labeled alpha-MSH, a low number of alpha-MSH binding sites was detectable on unstimulated monocytes. alpha-MSH receptor expression was up-regulated when monocytes were treated with endotoxin (LPS) or mitogen (PHA) for 3 to 5 days and was further augmented by the addition of cytokines such as IL-2, IFN-gamma, IL-4, and IL-10. Adrenocorticotropin, a precursor of alpha-MSH, but not the structurally unrelated beta-MSH, competitively inhibited alpha-MSH binding, suggesting that the receptor expressed on monocytes is specific for alpha-MSH. This was further confirmed by reverse transcription-PCR, which demonstrated that monocytes express mRNA specific for the MC receptor MC-1, which binds alpha-MSH and adrenocorticotropin, whereas mRNA specific for other known melanocortin receptors was not detectable. To investigate whether the immunosuppressing capacity of alpha-MSH is associated with the up-regulation of MC-1, its effect on the expression of costimulatory molecules (CD86 and CD80) on human monocytes was investigated. alpha-MSH significantly inhibited the expression of CD86 on LPS-treated monocytes, which exhibited a high density of MC-1, whereas CD80 expression was not altered. These findings indicate that human monocytes, depending on their activation and maturation state, are able to express MC-1, and up-regulation of MC-1 seems to be required to enable alpha-MSH to modulate immune responses in which costimulatory molecules play a decisive role.
Uromodulin and Tamm-Horsfall protein induce human monocytes to secrete TNF and express tissue factor.
Authors: Su S J , Chang K L , Lin T M , Huang Y H , Yeh T M
Published: December 2022

Effects of uromodulin (URO) and Tamm-Horsfall protein (THP), the most abundant proteins in the urine of pregnant and normal women, respectively, on the induction of TNF-alpha secretion and tissue factor (TF) expression of human monocytes were studied. THP, URO, and its fragments stimulated human mononuclear cells to proliferate and secrete TNF-alpha. The release of URO and THP-induced TNF-alpha in monocytes was dependent upon protein tyrosine kinase activation that results in tyrosine phosphorylation. URO and THP also induced TF expression of human monocytes and monocytic cell line U937 in a dose-dependent manner. TF expression was transient, reached its peak at 6 h and declined toward basal levels by 24 h. Reverse transcriptase-PCR and dot-blot analysis confirmed the induction of TF mRNA synthesis. URO and THP-induced TF expression were inhibited by actinomycin D and pentoxifylline further supporting the requirement of de novo TF mRNA synthesis. The possibility of LPS contamination of URO and THP was excluded because: 1) URO and THP-induced TF expression were inhibited by specific Ab; 2) URO was less capable of inducing TF in HUVEC as compared with LPS; 3) polymyxin B blocked the induction of Limulus clotting by LPS but not by URO and THP; 4) both LPS-sensitive (C3H/HeN) and -resistant (C3H/HeJ) mice produced little or no TNF-alpha after URO challenge. Therefore, our findings suggest that URO and THP play a significant role in the innate immunity of the urinary system and that the immunostimulatory activity of URO is potentially useful for immunotherapy.
Nuclear factor-kappa B potently up-regulates the promoter activity of RANTES, a chemokine that blocks HIV infection.
Authors: Moriuchi H , Moriuchi M , Fauci A S
Published: December 2022

The complex network of cytokines that are involved in inflammatory and immunoregulatory responses plays a critical role in the pathogenesis of HIV infection. RANTES (regulated upon activation, normal T cell expressed and secreted) is a cytokine that belongs to the beta-chemokine family; it is chemoattractant for CD4+/CD45RO T cells, it is produced by various cell types including CD8+ and CD4+ T cells as well as monocytes/macrophages, and has recently been shown to suppress replication of macrophage-tropic strains of HIV in CD4+ T cells. To investigate the molecular mechanisms of RANTES expression, the RANTES promoter region was analyzed by transient expression and gel-mobility shift assays. We demonstrate that: 1) RANTES promoter activity is up-regulated by PMA plus ionomycin, coexpression of the p65 subunit of nuclear factor (NF)-kappa B, the proinflammatory cytokines TNF-alpha and IL-1 beta, and the CD28 costimulatory pathway; 2) the RANTES promoter region contains four NF-kappa B binding sites at positions -30, -44, -213, and -579 relative to the transcription start site; 3) one site (-213) is an NF-AT (nuclear factor of activated T cells) binding site that also has weak affinity to NF-kappa B, and the most distal site (-579) also serves as a CD28-responsive element; and 4) mutation on any of those NF-kappa B sites or coexpression of I kappa B alpha (cytoplasmic inhibitor of NF-kappa B) markedly reduced the promoter activity. Thus, NF-kappa B, a potent transcriptional activator of HIV expression, is also involved in the expression of RANTES, a chemokine that blocks infection by macrophage-tropic strains of HIV.
Vaccine-specific antibody responses induced by HIV-1 envelope subunit vaccines.
Authors: Pincus S H , Messer K G , Cole R , Ireland R , VanCott T C , Pinter A , Schwartz D H , Graham B S , Gorse G J
Published: December 2022

The first generation of candidate vaccines to prevent HIV infection consisted of recombinant envelope proteins (Env, gp120, and gp160) derived from a single laboratory strain of HIV, designated IIIB/LAV, but produced with different expression systems. In this study we examined the fine specificity of the human Ab response to each vaccine and compared them to the responses of laboratory workers infected with the same strain of HIV. The best responders from each vaccine protocol were studied and compared. Detailed comparisons of the fine specificity of the Ab response were possible because all immunologic assays were performed using homologous recombinant proteins, peptides, and virus stocks. Although the total amounts of anti-Env Ab were comparable, the groups exhibited significant differences in epitope specificity, avidity, and functional capacity of the Ab response. The data demonstrate that the form of the immunogen (e.g., live virus or recombinant protein) is important in determining the quality of the Ab response. Conclusions are also drawn regarding characteristics of the anti-HIV-neutralizing Ab response. These studies represent one of the most detailed analyses of the human Ab response to any Ag and have implications for the development of vaccines for HIV as well as for other microbial pathogens.
Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice.
Authors: Christianson S W , Greiner D L , Hesselton R A , Leif J H , Wagar E J , Schweitzer I B , Rajan T V , Gott B , Roopenian D C , Shultz L D
Published: December 2022

Genetic crosses produced NOD/LtSz mice doubly homozygous for the severe combined immunodeficiency (scid) mutation and the beta2m (B2m) null allele. Both NOD/LtSz-scid/scid and NOD/LtSz-scid/scid B2m(null) mice lacked mature lymphocytes and serum Ig. However, homozygosity for the B2m(null) allele also resulted in the absence of MHC class I expression, loss of NK cell activity, accumulation of iron in the liver, and rapid clearance of human IgG1. NOD/LtSz-scid/scid B2m(null) mice supported markedly elevated levels of human T cell engraftment, compared with NOD/LtSz-scid/scid control animals, following injection with human PBMC. The increased engraftment was associated with a major increase in the number of human CD4+ T cells. Following injection with 20 million human PBMC, levels of human CD4+ T cells in the peripheral blood and spleen of NOD/ LtSz-scid/scid B2m(null) mice were 6- to 7-fold higher than those in NOD/LtSz-scid/scid mice and >50-fold higher than those in C.B-17-scid/scid mice. The resulting normalization of CD4+/CD8+ ratios in NOD/LtSz-scid/scid B2m(null) mice is in sharp contrast to that observed in NOD/LtSz-scid/scid mice or in C.B-17-scid/scid mice. Circulating human IgG was cleared 6-fold more rapidly in NOD/LtSz-scid/scid B2m(null) mice than in NOD/LtSz-scid/scid mice. This rapid IgG clearance suggested a failure of the engrafted human lymphoid cells to maintain high circulating levels of human IgG. The higher levels of human CD4+ T cells and the normalization of the CD4:CD8 ratio that are observed in human PBMC-engrafted NOD/LtSz-scid/scid B2m(null) mice suggest that this system may be an excellent model for studies of HIV pathogenesis.
A tumor-associated and self antigen peptide presented by dendritic cells may induce T cell anergy in vivo, but IL-12 can prevent or revert the anergic state.
Authors: Grohmann U , Bianchi R , Ayroldi E , Belladonna M L , Surace D , Fioretti M C , Puccetti P
Published: December 2022

Ag-specific CD8+ cell responses, including delayed-type hypersensitivity in vivo and IFN-gamma production in vitro, are initiated by host immunization with P815AB, a self peptide bearing CTL epitopes and expressed by murine mastocytoma cells. Using P815AB-pulsed dendritic cells (DC) and monitoring class I-restricted skin test reactivity in DC-primed mice, we have previously shown that the development of a Th1-like response to P815AB requires T helper effects, such as those mediated by coimmunization with class II-restricted (helper) peptides or by the use of rIL-12. The adjuvanticity of IL-12 was suggested to involve improved recognition of class II-restricted epitopes of P815AB. In the present study, we provide evidence for the occurrence of I-A(d)-restricted epitopes in the tumor peptide. We also show that in the absence of helper peptide or rIL-12, P81 5AB not only failed to initiate CD8+ cell responses in vivo and in vitro, but resulted in a transient state of functional unresponsiveness, characterized by a profound inability of CD4+ cells to produce IL-2 in vitro. Ag-specific T cell anergy was also observed after neutralization of endogenous IL-12 at the time of priming with P815AB plus helper peptide. All of these effects were reversed by rIL-12, which was added to DC cultures and administered to the DC-recipient mice. Anergy induction may thus contribute to P81 5AB unresponsiveness in vivo. IL-12 may act to prevent or revert anergy to this tumor-associated and self peptide.
Molecular characterization and role in T cell activation of staphylococcal enterotoxin A binding to the HLA-DR alpha-chain.
Authors: Thibodeau J , Dohlsten M , Cloutier I , Lavoie P M , Bjork P , Michel F , Léveillé C , Mourad W , Kalland T , Sékaly R P
Published: December 2022

Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific V beta regions of the TCR. Two distinct regions of staphylococcal enterotoxin A superantigen (SEA) have been shown to affect the binding to MHC class II molecules. Results presented here demonstrate for the first time that the SEA-DR interaction can be affected by mutations on the class II alpha-chain. Furthermore, we have precisely mapped the interaction of the SEA N-terminal domain with the alpha1 domain of HLA-DR. Scatchard analysis using DAP cells transfected with mutant class II molecules showed a role for residue DR alpha K39 in the binding of SEA. Also, complementation experiments using mutant SEA molecules revealed an interaction between SEA residue F47 and position alphaQ18 on an outer loop of HLA-DR. These interactions between SEAF47 and the DR alpha-chain are critical, as they allow the recognition by an otherwise nonreactive V beta1+ T cell hybridoma and induction of tyrosine phosphorylation through the TCR.
Complement-inhibiting peptides identified by proximity to indels in the C3/4/5 protein family.
Authors: Ogata R T , Low P J
Published: December 2022

To find protein-protein interactive sites in complement component C3, we examined regions of C3 that are proximal to sites of length polymorphism or indels in the C345 protein family. We reasoned that indels probably mark protein interactive sites because they usually involve residues at protein surfaces. To test for the involvement of individual indels, we examined the effects on complement function of synthetic peptides corresponding to indel-proximal segments of C3. We inferred that if such a segment made direct contact with a C3 binding protein, then the corresponding peptide might also bind to that protein and inhibit binding to C3. Twenty-one peptides were tested; four of these inhibited complement-mediated erythrocyte lysis at < or =100 microM and complement-mediated killing of Escherichia coli at about threefold higher levels. These results indicate that the four peptides act as specific inhibitors of complement. They also suggest that indels can be effective guides for locating interactive sites in C3 and in any protein that is a member of a protein family. Because only a linear sequence is required, a focus on indels may be particularly useful for identifying interactive sites in proteins for which a three-dimensional structure is unavailable.
Variable expression of WASP in B cell lines of Wiskott-Aldrich syndrome patients.
Authors: Remold-O'Donnell E , Cooley J , Shcherbina A , Hagemann T L , Kwan S P , Kenney D M , Rosen F S
Published: December 2022

The Wiskott-Aldrich syndrome (WAS) arises from defects of the X-chromosome gene WASP. Severe platelet defects, thrombocytopenia with small platelets, are a hallmark of the disease, but clinical immunodeficiency based in lymphocyte dysfunction varies from negligible to life threatening among WAS patients. To address the connection between WASP mutations and clinical outcomes, we generated and characterized a panel of patient B cell lines. Three cell lines from patients with exon 2 missense mutations and mild immune dysfunction were found to express substantial levels of WASP mRNA and protein. On the other hand, 8 of 10 cell lines from patients with moderate or severe immune dysfunction lack detectable WASP protein. The findings suggest that the clinical variability of the WAS can partially be explained by the level of WASP protein in the patient's cells.
Engagement of the signaling lymphocytic activation molecule (SLAM) on activated T cells results in IL-2-independent, cyclosporin A-sensitive T cell proliferation and IFN-gamma production.
Authors: Aversa G , Chang C C , Carballido J M , Cocks B G , de Vries J E
Published: December 2022

The expression and function of the novel signaling lymphocytic activation molecule (SLAM) are described. SLAM is present on immature thymocytes, CD45R0(high) memory T cells, T cell clones, CD56+ T cells, EBV-transformed B-cell lines and on a proportion of B cells. SLAM is rapidly induced on naive T cells, TCR gammadelta+ T cells, and B cells following activation. Engagement of SLAM by the agonistic anti-SLAM mAb A12 resulted in proliferation of T cell clones and Ag- or PHA-activated peripheral blood T cells. mAb A12-induced T cell proliferation is independent of IL-2 and sensitive to inhibition by cyclosporin A. The extent of the anti-SLAM-induced proliferation was related to the activation state of the T cells, since fully rested T cell clones failed to proliferate in response to mAb A12 stimulation, despite unchanged SLAM expression on their surface. Ligation of SLAM on activated peripheral T cells or T cell clones by mAb A12 induced IFN-gamma production in the absence of other exogenous stimuli, even in allergen-specific Th2 clones. These data indicate that stimulation via SLAM reverses the cytokine production profile of Th2 clones to a Th0 phenotype, whereas it further polarizes cytokine production by Th1 clones. Thus, SLAM is a novel receptor that mediates IL-2-independent expansion of activated T cells during immune responses, while concomitantly directing these proliferating cells to a Th0/Th1 pathway.
Synergistic induction of CTLA-4 expression by costimulation with TCR plus CD28 signals mediated by increased transcription and messenger ribonucleic acid stability.
Authors: Finn P W , He H , Wang Y , Wang Z , Guan G , Listman J , Perkins D L
Published: December 2022

T cell activation requires at least two signals transduced by the Ag-specific TCR plus a costimulatory receptor. The CD28 costimulatory molecule has been shown to promote T cell proliferation and cytokine production. CTLA-4, a cell surface molecule homologous to CD28, can function as a repressor of T cell activation. Thus, CTLA-4 and CD28 may have opposing functions during T cell activation. CTLA-4 is expressed at low levels on resting T cells and up-regulated after T cell activation. Regulation of CTLA-4 expression is critical to the normal regulation of immunity. For example, CTLA-4-deficient mice develop early onset lethal autoimmunity. We previously showed that CTLA-4 transcription is increased after T cell activation and that induction was controlled by 335 bp of CTLA-4 upstream sequence. In this work, we show that cell surface CTLA-4 expression is increased synergistically by TCR plus CD28 signals. Synergistic induction is mediated by two mechanisms: an enhanced rate of transcription and increased mRNA stability. In contrast to the regulation of IL-2 and IL-2R expression, which is inhibited by cyclosporin A-, but not rapamycin-dependent signal transduction pathways, CTLA-4 expression is inhibited by either cyclosporin A or rapamycin. Thus, synergistic induction of CTLA-4 expression requires both cyclosporin A- and rapamycin-dependent signals.
Differential CC chemokine-induced enhancement of T helper cell cytokine production.
Authors: Karpus W J , Lukacs N W , Kennedy K J , Smith W S , Hurst S D , Barrett T A
Published: December 2022

Chemokines are a family of small m.w. cytokines that induce chemotaxis and chemokinesis of leukocytes. These molecules are ligands for seven-transmembrane, Gi protein-linked receptors that induce a signaling cascade in human T cells and provide costimulation for T cell activation, in addition to participating in transendothelial migration of leukocytes. To address the role of chemokines in the regulation of Th cell cytokine production, we utilized an OVA-specific TCR transgenic (Tg+) model. Cells stimulated through the TCR and incubated in the presence of macrophage inflammatory protein-1alpha (MIP-1alpha) showed enhanced IFN-gamma production, whereas cells incubated in the presence of monocyte chemotactic protein-1 (MCP-1) showed enhanced IL-4 production. Similar results were obtained whether TCR Tg+ T cells were stimulated with anti-CD3 mAb or OVA peptide. Primary stimulation of T cells in the presence of chemokines, followed by secondary stimulation and tertiary stimulation with anti-TCR clonotype mAb alone (no exogenous chemokines), revealed an enhanced IFN-gamma production for MIP-1alpha stimulation and IL-4 production for MCP-1 stimulation. Naive Tg+ T cells, obtained from Tg+ mice crossed to RAG-1-deficient mice, showed enhanced IFN-gamma production when incubated with MIP-1alpha and enhanced IL-4 production when incubated with MCP-1. These results suggest CC chemokines play a role in regulating naive Th cell cytokine production, in addition to regulating leukocyte trafficking.
HIV-1 env DNA vaccine administered to rhesus monkeys elicits MHC class II-restricted CD4+ T helper cells that secrete IFN-gamma and TNF-alpha.
Authors: Lekutis C , Shiver J W , Liu M A , Letvin N L
Published: December 2022

The Th cell response elicited by an HIV-1 env plasmid DNA vaccine was assessed in rhesus monkeys by isolation of gp120-specific, MHC class II-restricted CD4+ T cell lines from PBL of vaccinated animals. The Env-specific CD4+ T cell lines recognized epitopes located in the second hypervariable region and in the carboxyl terminus of HIV-1 gp120. These cell lines proliferated in response to APC in the presence of recombinant gp120, as well as to APC expressing virally encoded Env. All of the CD4+ T cell lines responded to Env peptide by secreting IFN-gamma and TNF-alpha without appreciable IL-4 production. Recombinant gp120 stimulation of PBL from these vaccinated monkeys elicited the secretion of a similar profile of cytokines. Demonstration of a nucleotide vaccine eliciting a Th1-like immune response is consistent with the well documented ability of naked DNA vaccines to induce durable CD8+ CTL responses.
The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites.
Authors: Iwasaki A , Torres C A , Ohashi P S , Robinson H L , Barber B H
Published: December 2022

Although plasmid DNA immunization provides an effective means of inducing CTL responses to an expressed Ag, the mechanism by which CTL precursors are activated remains to be established. Insights could be gained by identifying the cells responsible for Ag presentation when DNA is introduced into different tissue sites. By immunizing parent into F1 bone marrow chimeric mice with an influenza nucleoprotein-expressing plasmid, we have demonstrated that the key cells in this presentation process for both gene gun-mediated epidermal injection and needle intramuscular injection of plasmid DNA are bone marrow derived. Furthermore, as assessed by intramuscular injection, coexpression of nucleoprotein with the costimulatory molecule B7-2, or the cytokines granulocyte-macrophage CSF and IL-12, did not convert nonhemopoietic cells into APCs. Thus, for two distinctly different modes of DNA immunization, in one case with or without coexpressed immunostimulatory factors, the APCs were consistently found to be of hemopoietic origin.
Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope.
Authors: Garrity R R , Rimmelzwaan G , Minassian A , Tsai W P , Lin G , de Jong J J , Goudsmit J , Nara P L
Published: December 2022

Immunodominant epitopes are known to suppress a primary immune response to other antigenic determinants by a number of mechanisms. Many pathogens have used this strategy to subvert the immune response and may be a mechanism responsible for limited vaccine efficacies. HIV-1 vaccine efficacy appears to be complicated similarly by a limited, immunodominant, isolate-restricted immune response generally directed toward determinants in the third variable domain (V3) of the major envelope glycoprotein, gp120. To overcome this problem, we have investigated an approach based on masking the V3 domain through addition of N-linked carbohydrate and reduction in net positive charge. N-linked modified gp120s were expressed by recombinant vaccinia virus and used to immunize guinea pigs by infection and protein boosting. This modification resulted in variable site-specific glycosylation and antigenic dampening, without loss of gp120/CD4 binding or virus neutralization. Most importantly, V3 epitope dampening shifted the dominant type-specific neutralizing Ab response away from V3 to an epitope in the first variable domain (V1) of gp120. Interestingly, in the presence of V3 dampening V1 changes from an immunodominant non-neutralizing epitope to a primary neutralizing epitope with broader neutralizing properties. In addition, Ab responses were also observed to conserved domains in C1 and C5. These results suggest that selective epitope dampening can lead to qualitative shifts in the immune response resulting in second order neutralizing responses that may prove useful in the fine manipulation of the immune response and in the development of more broadly protective vaccines and therapeutic strategies.
Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response.
Authors: Dalal M , Kim S , Voskuhl R R
Published: December 2022

Female SJL mice are more susceptible than male mice to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-specific T lymphocytes. In the present study, we examined mechanisms involved in this gender-related difference in disease susceptibility. MBP-specific T lymphocytes derived from spleens of males during the effector phase of adoptive EAE produced significantly higher levels of IL-10, an anti-inflammatory cytokine in EAE. A protective effect of testosterone was then shown. Females implanted with dihydrotestosterone pellets demonstrated a significantly less severe course of EAE as compared with females implanted with placebo pellets. Finally, MBP-specific T lymphocytes derived from dihydrotestosterone-implanted females produced significantly higher levels of IL-10 than those from placebo. Together these data indicate that testosterone exerts a protective effect in EAE that is mediated at least in part by enhanced production of IL-10 by autoantigen-specific T lymphocytes.
Macrophage inflammatory protein-1alpha (MIP-1alpha) is required for the efferent phase of pulmonary cell-mediated immunity to a Cryptococcus neoformans infection.
Authors: Huffnagle G B , Strieter R M , McNeil L K , McDonald R A , Burdick M D , Kunkel S L , Toews G B
Published: December 2022

Our objective was to define the role of the chemokine macrophage-inflammatory protein-1alpha (MIP-1alpha) in the efferent phase of pulmonary cell-mediated immunity (CMI) against Cryptococcus neoformans. Following intratracheal inoculation of C. neoformans (24067) into CBA/J mice, the development of CMI was required for leukocyte recruitment into the lungs at 2 wk postinfection. MIP-1alpha mRNA was expressed by day 6 postinfection, and MIP-1alpha protein in bronchoalveolar lavage fluid was detectable at day 6, but significantly elevated at days 19 and 33. Administration of neutralizing anti-MIP-1alpha Abs from days 7 to 13 blocked the increase in bronchoalveolar lavage fluid MIP-1alpha and resulted in a 37% decrease in total leukocytes in the lungs at day 16. There were 66% fewer macrophages/monocytes and 42% fewer neutrophils in the lungs of anti-MIP-1alpha-treated mice, and the pulmonary burden of C. neoformans was threefold higher. There was no significant difference in the number of eosinophils, CD4+, CD8+, or B220+ lymphocytes between the two groups of mice. Neutralization of MIP-1alpha did not significantly decrease the levels of monocyte chemotactic protein-1 (MCP-1); however, neutralization of MCP-1 significantly decreased MIP-1alpha levels, demonstrating that induction of MIP-1alpha was largely dependent on MCP-1 production. Neutralization of MIP-1alpha also blocked the cellular recruitment phase of a recall response to cryptococcal Ag in the lungs of immunized mice. Thus, in both the contexts of active cryptococcal infection or rechallenge with cryptococcal Ag, MIP-1alpha was required during the efferent phase of CMI for maximal leukocyte recruitment into the lungs, most notably the recruitment of phagocytic effector cells (neutrophils and macrophages).
Differential CD45 isoform expression accompanies reduced natural antibody binding in L5178Y-F9 tumor progression.
Authors: Zhang Z Y , Chow D A
Published: December 2022

Considerable evidence supports a role for polyclonal serum natural Ab (NAb) as a mediator of natural resistance against tumors. However, the molecular mechanisms of this NAb activity are not known. Flow cytometry selection of L5178Y-F9 murine T lymphoma cells for high NAb binding provided the variant LYNAb+, which exhibited an inversely corresponding reduction in tumorigenicity. Accompanying the increased NAb binding, LYNAb+ bound more monoclonal 14.8 anti-CD45RA and DNL-1.9 anti-CD45RC and less 13/2 anti-pan CD45, and the binding of MB23G2 anti-CD45RB was eliminated. However, neuraminidase treatment increased NAb binding and detection of pan CD45, CD45RA, and CD45RC but reduced CD45RB expression, suggesting that the epitopes recognized by the former Abs are masked by sialic acid, while the latter includes sialic acid. Growth of the LYNAb+ from a threshold s.c. inoculum in syngeneic DBA/2 mice yielded more tumorigenic cells which bound less NAb, anti-CD45RA, and anti-CD45RC; the same very low anti-CD45RB; and more anti-pan CD45. In accord with the mAb binding, the L5178Y-F9 and an in vivo passaged LYNAb+ variant expressed predominantly lower m.w. CD45 isoforms while the LYNAb+ expressed predominantly higher 200-kDa isoforms. The consistent correspondence between CD45RA and CD45RC determinant expression, CD45 isoform expression, tumorigenicity, and NAb binding exhibited by T lymphoma cells selected for high NAb binding in vitro or through tumor progression in vivo suggests that asialo high m.w. isoforms of the cell surface-signaling molecule CD45 are differentially expressed during tumor development and furthermore that they participate in NAb-mediated antitumor mechanisms.
A mechanism for selective recruitment of CD8 T cells into B7-1-transfected plasmacytoma: role of macrophage-inflammatory protein 1alpha.
Authors: Maric M , Chen L , Sherry B , Liu Y
Published: December 2022

An important aspect of immunity is the recruitment and accumulation of lymphocytes into target tissues where Ags are localized. Because the TCR recognizes antigenic peptides presented by MHC molecules, the subset of T cells that exert effector function is determined by the class of MHC molecules expressed on a given tissue. We and others have demonstrated that CD8 T cells are preferentially recruited into B7-1-transfected class II-negative plasmacytoma J558, and the virus-infected central nervous system. However, the mechanism for such specificity has not been addressed. Here we analyzed the mechanism for selective recruitment of CD8 T cells into B7-1-transfected plasmacytoma J558. We show that sustained accumulation of CD8 T cells in vivo requires local expression of B7-1. In addition, we have observed a striking correlation between expression of macrophage-inflammatory protein 1alpha (MIP1alpha) and selective accumulation of CD8 T cells in the tumors. The selective recruitment of CD8 T cells is blocked by in vivo administration of neutralizing anti-MIP1alpha antisera. Moreover, gene-transfer studies reveal that locally produced MIP1alpha is sufficient to induce selective recruitment of CD8 T cells. Taken together, our study reveals that costimulation by B7 leads to sustained local production of MIP1alpha, which selectively recruits CD8 T cells into tumors. These results have important implications for T cell recruitment in vivo and for tumor immunotherapy.
Induction of procoagulant function in porcine endothelial cells by human natural killer cells.
Authors: Malyguine A M , Saadi S , Holzknecht R A , Patte C P , Sud N , Platt J L , Dawson J R
Published: December 2022

NK cells may mediate effector functions other than target cell cytotoxicity. To explore such noncytotoxic effector mechanisms, we tested whether human PBL and purified NK (CD56+) cells might induce expression of tissue factor by cultured porcine aortic endothelial cells. Tissue factor is the major coagulation factor that binds to factor VIIa and initiates coagulation. The addition of freshly isolated NK cells but not T cells to endothelial cells resulted in the induction of tissue factor activity. NK-depleted (CD56-) effector cells did not induce tissue factor activity; however, the combination of CD56+ cells and NK-depleted cells induced tissue factor activity to the same extent as unseparated cells. PBL induced tissue factor mRNA in porcine endothelial cells and NK depletion resulted in a significant decrease of the induction. Induction of tissue factor activity in porcine endothelial cells by human NK cells required direct cell-to-cell contact, as transfer of supernatants from NK-endothelial cell cultures to secondary cultures did not induce tissue factor activity, and anti-LFA-1alpha Abs inhibited the induction of tissue factor activity. Induction of tissue factor activity in endothelial cells by NK cells may represent one of a variety of ways in which NK cells mediate noncytotoxic effects.
Natural processing sites for human cathepsin E and cathepsin D in tetanus toxin: implications for T cell epitope generation.
Authors: Hewitt E W , Treumann A , Morrice N , Tatnell P J , Kay J , Watts C
Published: December 2022

Cathepsin E is an aspartic proteinase that has been implicated frequently in Ag processing for presentation on class II MHC molecules, but no information exists on its cleavage specificity within Ags in relation to known T cell epitopes. We have analyzed the processing by cathepsin E of a large C-terminal domain of tetanus toxin (residues 872-1315), and we have compared the processing products with those liberated by cathepsin D, a related aspartic proteinase also thought to be involved in class II MHC-restricted Ag processing. Processing products were analyzed by N-terminal Edman degradation and mass spectrometry following reverse-phase HPLC separation of peptides. A total of 28 cleavage sites was identified, 11 of which were recognized by both cathepsins E and D. Most, although not all, sites were between pairs of hydrophobic residues and were located within the 200-amino-acid C terminal region known to contain several human T cell epitopes. Previously described T cell epitopes, for example, between residues 1273 and 1284, were flanked by cathepsin E and D cleavage sites. These data are consistent with an important role for cathepsins E and/or D in Ag processing in the human immune system.
Constitutive activation of JAKs and STATs in BCR-Abl-expressing cell lines and peripheral blood cells derived from leukemic patients.
Authors: Chai S K , Nichols G L , Rothman P
Published: December 2022

An important step in the oncogenic transformation of hemopoietic cells and the subsequent development of leukemia is the proliferation of tumor cells in the absence of exogenous growth factors. In most cases of chronic myelocytic leukemia and in some cases of acute myelocytic leukemia and acute lymphocytic leukemia, the bcr-abl oncogene is involved in this process. Although the BCR-Abl oncoprotein demonstrates enhanced tyrosine kinase activity in leukemic cells, the mechanism by which this leads to growth factor independence remains poorly defined. One proposed mechanism is the activation of cytokine signal transduction pathways, possibly by an autocrine loop involving IL-3 and/or granulocyte-macrophage CSF. Examination of several different cell lines expressing BCR-Abl demonstrates that some of these cells have constitutive activation of the JAK/STAT signaling pathway. We have found the constitutive activation of STAT5 in most, but not all, cell lines expressing BCR-Abl. This constitutive activation of STAT5 is variably associated with a corresponding activation of JAK kinases. Ab blocking studies show that the activation of STAT5 in these cell lines cannot be attributed to the activation of an IL-3/granulocyte-macrophage CSF-driven autocrine loop. Interestingly, samples of peripheral blood cells derived from patients with acute myelocytic leukemia and chronic myelocytic leukemia, which express BCR-Abl, demonstrate constitutive activation of STAT family members. These studies suggest that in a variety of leukemic states, BCR-Abl may use a bypass mechanism to activate cytokine signal transduction pathways.
Pivotal role of endogenous TNF-alpha in the induction of functional inactivation and apoptosis in NK cells.
Authors: Jewett A , Cavalcanti M , Bonavida B
Published: December 2022

The interaction of purified nonactivated human NK cells with target cells overnight results in functional anergy and apoptosis in NK cells and in a change of the NK phenotype from CD16+ CD56+ CD69- to CD16(dim/-) CD56(+/dim/-) CD69+. These studies suggested that signaling triggered by the FcR CD16 may be implicated in target cell-mediated anergy/apoptosis of NK cells. We hypothesized that triggering CD16 by anti-CD16 Ab should result in NK cells exhibiting functional and phenotypic properties similar to those obtained following triggering of NK cells with target cells. The findings demonstrate that the anti-CD16 mAb-treated NK cells acquired the CD16- CD56(+/dim) CD69+ Fas+ phenotype and lost their cytotoxic function, a significant number of the NK cells underwent apoptosis, and a selective induction of TNF-alpha synthesis and secretion was observed. The coaddition of IL-2 to anti-CD16 Ab-treated NK cells resulted in enhanced secretion of TNF-alpha and augmentation of the frequency of cell death. However, a minor subset of NK cells exhibited potent cytotoxic function and proliferated. The anti-CD16-induced effects in NK cells were largely abrogated by the addition of either anti-TNF-alpha Ab or TNF-binding protein in the cultures. There was an enhancement of NK cell killing following the addition of exogenous TNF-alpha into the culture. Cytochalasin B selectively triggered the secretion of TNF-alpha and significantly augmented the frequency of apoptosis of anti-CD16-treated NK cells. These findings demonstrate an important and pivotal role of endogenous TNF-alpha synthesis and secretion by NK cells in the induction of functional anergy and apoptosis in anti-CD16-treated NK cells.