bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of membrane Ig receptor density and affinity on B cell signaling by antigen. Implications for affinity maturation.
Authors: George J , Penner S J , Weber J , Berry J , Claflin J L
Published: January 2023

We have initiated a series of experiments to explore the effect of changes in density of the surface Ig receptor (mIg) on Ag:mIg interactions. We transfected into the surface Ig-negative B cell line M12.4 H and L chain constructs known to effect a 10-fold change in antibody binding affinity for the naturally occurring hapten phosphocholine (PC). Two sets of stable transfectants were generated and those expressing levels of mIg comparable to the range normally seen on splenic B cells were studied. One set expressed an unmutated VH and an unmutated VL. The second set expressed the same pair of V regions except for a single somatic change in CDR3 of VH; this substitution increases the affinity of antibody for PC from 3 x 10(4) M-1 to 3 x 10(5) M-1. Ag:mIg interactions were assessed in the transfected cell lines by measuring calcium mobilization induced by stimulation with soluble PC Ag. As expected, the mutation that increased affinity for PC increased the sensitivity of transfectants to PC Ag. Relatively small changes in receptor number had a dramatic effect in the quantity and quality of a calcium response. Significantly, we found that Ag-specific signaling could occur with only a few thousand receptors per cell. Signaling differences were most noticeable with PC protein Ag (T-dependent form) compared with PC polysaccharide Ag (T-independent form). These results suggest that the down-regulation of mIg that follows B cell activation may have evolved to assist in the selection of B cell clones with higher affinity for Ag. Furthermore, the results also provide an explanation for why selection of higher affinity clones can occur with protein Ag but only poorly so with polymeric Ag.
A cDNA clone encoding the mouse Qa-1a histocompatibility antigen and proposed structure of the putative peptide binding site.
Authors: Connolly D J , Cotterill L A , Hederer R A , Thorpe C J , Travers P J , McVey J H , Dyson J , Robinson P J
Published: January 2023

We have isolated a cDNA clone which encodes the Qa-1a histocompatibility Ag from a library prepared from Con A-activated B10.BR mouse spleen cells. The clone encodes a protein of 322 amino acids with three potential N-glycosylation sites. The coding sequence shows strongest similarity with that of the T23d gene of DBA/2 mice which encodes the Qa-1b molecule. Molecular modeling of the putative peptide-combining site indicates most of the differences between Qa-1a and Qa-1b are located peripheral to the binding cleft, with only two amino acid substitutions, at positions 9 and 24, which might affect peptide binding. Many features of the Qa-1 binding cleft are also conserved in the rat RTBM.1 and in human HLA-E molecules. This suggests that all of these molecules may associate with structurally similar peptides.
Beta 2M-/- knockout mice contain low levels of CD8+ cytotoxic T lymphocyte that mediate specific tumor rejection.
Authors: Lamousé-Smith E , Clements V K , Ostrand-Rosenberg S
Published: January 2023

C57BL/6 mice with a disrupted beta 2M gene (beta 2M-/- mice) express very low levels of MHC class I molecules and are deficient for CD8+ T lymphocytes. Because CD8+ T cells are thought to be a principle effector cell in tumor rejection, we have assessed the ability of beta 2M-/- mice to respond to tumors. beta 2M-/- knockout mice were challenged with seven independent MHC allogeneic and syngeneic tumors. The beta 2M-/- mice responded very similarly to their CD8+ beta 2M+/- littermates in that they rejected high dose challenges of 4/5 allogeneic tumors and were susceptible to 3/3 syngeneic tumors. In vivo depletion of CD4+ or CD8+ cells from the beta 2M-/- mice resulted in susceptibility to allogeneic tumor. The apparent requirement for CD8+ cells for tumor immunity was corroborated by in vitro assays in which depletion of CD8+ but not CD4+ T cells eliminated tumor-specific CTL activity. mAb blocking studies in which target tumor cells were incubated with MHC class I-specific mAb demonstrated that the tumor-specific CD8+ activity was MHC class I restricted. beta 2M-/- mice therefore contain very small quantities of potent, CD8+ T cells that are capable of rejecting large challenges of allogeneic tumor cells.
Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. Comparison with IL-4 and modulation by IFN-gamma or IL-10.
Authors: de Waal Malefyt R , Figdor C G , Huijbens R , Mohan-Peterson S , Bennett B , Culpepper J , Dang W , Zurawski G , de Vries J E
Published: January 2023

Recently, we described the cloning and expression of a human cDNA which is the homologue to P600, a gene transcribed by mouse Th2 clones. Based on its activities on human monocytes and B cells this gene was designated IL-13. In the present study we investigated the effects of IL-13 alone or in combination with IL-4, IFN-gamma, or IL-10 on human monocytes. IL-13 induced significant changes in the phenotype of monocytes. Like IL-4, it enhanced the expression of CD11b, CD11c, CD18, CD29, CD49e (VLA-5), class II MHC, CD13, and CD23, whereas it decreased the expression of CD64, CD32, CD16, and CD14 in a dose-dependent manner. IL-13 induced up-regulation of class II MHC Ag and its down-regulatory effects on CD64, CD32, and CD16 expression were prevented by IL-10. IFN-gamma could also partially prevent the IL-13-induced down-regulation of CD64, but not that of CD32 and CD16. However, IL-13 strongly inhibited spontaneous and IL-10- or IFN-gamma-induced ADCC activity of human monocytes toward anti-D coated Rh+ erythrocytes, indicating that the cytotoxic activity of monocytes was inhibited. Furthermore, IL-13 inhibited production of IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, macrophage inflammatory protein-1 alpha, granulocyte/macrophage-CSF, granulocyte-CSF, IFN-alpha, and TNF alpha by monocytes activated with LPS. In contrast, IL-13 enhanced the production of IL-1 ra by these cells. Similar results on cytokine production were observed or have been obtained with IL-4. Thus IL-13 shares most of its activities on human monocytes with IL-4, but no additive or synergistic effects of IL-4 and IL-13 on human monocytes were observed, suggesting that these cytokines may share common receptor components. Taken together, these results indicate that IL-13 has anti-inflammatory and important immunoregulatory activities.
Role of gamma delta T cells in murine collagen-induced arthritis.
Authors: Peterman G M , Spencer C , Sperling A I , Bluestone J A
Published: January 2023

Murine collagen-induced arthritis (CIA) is a T cell-mediated disease which is induced by injection of type II collagen. Previous studies have shown that CD4+ cells which express particular V beta TCR genes are involved in the induction of arthritis in this model. In the present report we demonstrate that CD4-, CD8-, TCR gamma delta cells are present in arthritic joints, expanded in peripheral lymphoid tissue of DBA/1 lac J mice with CIA, and respond in vitro to the anti-TCR gamma delta mAb UC7-13D5 (13D5). In order to directly investigate the role of the gamma delta TCR in murine CIA, DBA/1 lac J mice were injected with 13D5 before or 40 days after injection of type II collagen. Our results demonstrate that i.p. injections of 13D5 initiated 1 day before injection of type II collagen significantly delays both the onset and severity of CIA compared with treatment with type II collagen alone. In contrast, anti-TCR gamma delta mAb injection of arthritic mice 40 days after collagen injection resulted in the rapid onset of severe arthritis which was accompanied by increased bone erosion and cell infiltration into inflamed joints compared with arthritic mice injected with either control hamster IgG or F(ab')2 fragments of 13D5. Arthritic mice injected with intact 13D5 rapidly lost weight, suggesting that 13D5 may induce a cytokine-mediated syndrome similar to that observed in mice and humans after the injection of anti-CD3. Flow cytometry analysis of joint cells isolated after collagenase digestion from arthritic mice demonstrated that 13D5 injection induces the accumulation of CD4-, CD8-, PgP-1 (CD44)+ cells within arthritic joints, whereas arthritic joints from mice injected with control hamster IgG contained cells with a CD4+, CD8- phenotype. CD3+ T cell lines which express the gamma delta TCR from inflamed joints of arthritic mice were established and examined for V gamma usage by the polymerase chain reaction. V gamma 2 rearrangements were predominant in both T cell lines established from inflamed synovium as well as freshly isolated synovial cells from arthritic mice, whereas synovial cells from nonarthritic mice did not demonstrate V gamma 2 rearrangements. Taken together, the results described in this report suggest a direct role for gamma delta TCR T cells in the pathogenesis of CIA in DBA/1 lac J mice.
Influence of a recombinant human soluble tumor necrosis factor receptor FC fusion protein on type II collagen-induced arthritis in mice.
Authors: Wooley P H , Dutcher J , Widmer M B , Gillis S
Published: January 2023

A recombinant human TNF receptor Fc fusion protein (rhuTNFR:Fc) was assessed for antiarthritic activity using murine type II collagen-induced arthritis in mice. DBA/1 mice were immunized with bovine type II collagen and treated with rhuTNFR:Fc either from day 21 to day 28 (preventative protocol), or after disease onset for fourteen days (therapeutic protocol). Control mice received either sterile saline or human serum albumin injections. rhuT-NFR:Fc treatment significantly reduced both the incidence and the severity of collagen-induced arthritis in the preventative protocol. Mice receiving rhuTNFR:Fc therapeutically progressed to less severe disease than did control animals, and the arthritis index in rhuTNFR:Fc treated mice was significantly lower than the index in control mice from 7.5 weeks after treatment. The antibody response to collagen was significantly reduced by treatment with rhuTNFR:Fc in both the preventative and therapeutic protocols. No difference was observed in the proliferative response to type II collagen or Con A, but the response to LPS was significantly lower in rhuTNFR:Fc treated mice at the conclusion of both the preventative and therapeutic trials. The results suggest that rhuTNFR:Fc may have both immunosuppressive and antiarthritic properties in this experimental model, and may represent a useful approach to the treatment of autoimmune arthritis.
Induction of transforming growth factor-beta during cardiac allograft rejection.
Authors: Waltenberger J , Wanders A , Fellström B , Miyazono K , Heldin C H , Funa K
Published: January 2023

The polypeptides of the transforming growth factor-beta (TGF-beta) family are potent endogenous immuno-regulators. Using a rat cardiac allograft transplant model, we investigated the expression of the precursor forms of TGF-beta 1, TGF-beta 2, and TGF-beta 3 and the latent TGF-beta binding protein (LTBP) by immunohistochemistry. The activity of TGF-beta in the extracts from transplanted as well as normal hearts was measured using a bioassay, and Northern blot analysis was performed on RNA extracts. The transplanted hearts were analyzed both during acute rejection up to 6 days and during chronic rejection up to 6 mo after transplantation and compared with normal controls. The animals of the chronic rejection group received cyclosporin A for immunosuppression. The TGF-beta bioactivity dramatically increased in the transplanted allografts during the chronic rejection process compared to the normal hearts, and so did the immunostaining as well as the mRNA levels for TGF-beta 1 and, to a lesser extent, the immunostaining for TGF-beta 2. TGF-beta 3 expression remained unchanged and was only found in the myocardium in trace amounts. During the acute rejection process up to 6 days after transplantation, TGF-beta immunoreactivity increased only slightly, whereas the TGF-beta mRNA was severalfold increased. Control animals treated with cyclosporin A showed a similar pattern at day 6 with regard to TGF-beta expression. LTBP was induced simultaneously with TGF-beta 1 and occurred within interstitial spaces of the myocardium. The TGF-beta was produced by macrophage-like infiltrating lymphocytes. In conclusion, highly elevated levels of TGF-beta and LTBP were found during chronic rejection of cardiac allografts in rats. The induction of TGF-beta may counteract the rejection process and could be useful for new therapeutic approaches in the prevention of allograft rejection.
Identification of a transitional preapoptotic population of thymocytes.
Authors: Cohen G M , Sun X M , Snowden R T , Ormerod M G , Dinsdale D
Published: January 2023

Apoptosis, a major form of cell death in the immune system, is characterized by cell shrinkage, chromatin condensation, and cleavage into nucleosomal fragments. Apoptosis may be the mechanism for the elimination of autoreactive and unselected CD4+ CD8+ thymocytes in the thymus. A large number of diverse agents are capable of inducing apoptosis in immature thymocytes. Rat thymocytes were treated with etoposide, a DNA topoisomerase II reactive agent, or dexamethasone, a glucocorticoid, and separated on discontinuous Percoll gradients. We have identified and isolated a transitional preapoptotic population of thymocytes that exhibited early morphologic and biochemical changes associated with apoptosis. These preapoptotic cells were intermediate in size and density between normal and apoptotic thymocytes and exhibited a decreased surface expression of both CD4 and CD8 molecules compared to control thymocytes. On ultrastructural examination, they were shown to possess sharply defined clumps of condensed chromatin abutting onto the nuclear membrane. These morphologic changes, the first detectable signs of apoptosis, occurred prior to the internucleosomal cleavage of DNA, often regarded as the biochemical hallmark of apoptosis. Nucleosomal fragments of 180 to 200 base pairs or multiples thereof were, however, detected following subsequent dramatic changes in the nuclear structure of these preapoptotic cells that resulted in morphology typical of apoptosis. These results suggest that early critical events in apoptosis precede internucleosomal cleavage of DNA.
Analysis of T-cell receptor alpha beta chains of CD8+ suppressor T cells induced by tolerogenic conjugates of antigen and monomethoxypolyethylene glycol. Involvement of TCR alpha-CDR3 domain in immunosuppression.
Authors: Mohapatra S , Chen Y , Takata M , Mohapatra S S , Sehon A H
Published: January 2023

A number of investigators have demonstrated that there exists a relationship between Ag receptors of Ts cells (TCR) and their soluble suppressor factors. With a view to elucidating this relationship, the primary structures of receptors of Ts cells, induced in mice by tolerogenic conjugates of Ag and monomethoxypolyethylene glycol, were characterized in this study. The cDNA encoding the alpha and beta chains of TCR of cloned Ts cells specific for (i) OVA and (ii) human monoclonal (myeloma) IgG (HIgG) were produced by polymerase chain reaction. From the analysis of the V alpha genes of TCR of Ts cells it was deduced that these receptors utilized a new member of the V alpha 15 gene family, which was productively joined to the J alpha genes that differed for each of the Ts cells of the two distinct specificities. Similarly, sequence analysis of the beta chain cDNA of the two Ts cell clones revealed that both clones utilized the V beta 8.2 gene, and that their J beta gene differed from each other. It is inferred that the Ts cells generated in response to the different tolerogenic Ag(mPEG)n conjugates belonged to a subset of T cells utilizing similar TCR alpha beta chains and differed only in their J alpha/J beta regions. Most importantly, pretreatment of mice with a mixture of pentadecapeptides comprising the TCR alpha chain of the OVA-Ts cells, down-regulated the immune response specific to OVA, but not to HIgG. Moreover, injection of mice with a pentadecapeptide corresponding to the CDR3 region of the TCR alpha chain of either OVA-Ts or HIgG-Ts suppressed specifically the Ab response to OVA or HIgG, respectively. On the basis of all these results, it is concluded that the CDR3 of the TCR-alpha chain of Ts cells plays a pivotal role in the Ts network underlying the specific down-regulation of the immune responses induced by tolerogenic Ag(mPEG)n conjugates.
Differential tyrosine-specific protein phosphorylation in mouse T lymphocyte subsets. Effect of age.
Authors: Shi J , Miller R A
Published: January 2023

We have previously reported that activation of normal murine T lymphocytes with either anti-CD3 or Con A leads to increased phosphorylation of three phosphotyrosine-containing proteins with molecular masses of 120, 80, and 40 kDa, that antibody to the alpha beta TCR heterodimer induces phosphorylation of the 120- and 80-kDa species, and that aging impairs phosphorylation of all three substrates. In our study we determine if phosphorylation of these substrates differs among T cell subsets in old and young mice. In young mice, we found that the induced levels of all three phosphorylated substrates, 10 min after addition of the mitogenic stimulus, were significantly higher in CD8 than in CD4 cells for anti-CD3 and Con A stimulation; responses to anti-TCR were also higher in CD8 than in CD4 cells for the 80 kDa, although not the 120-kDa substrate. Aging led to significant declines in the ability to phosphorylate all three substrates in the CD8 population, and also of the 80-kDa substrate in CD4 cells. Comparison of unfractionated CD4 to purified CD4 memory T cells showed that the age-dependent shift from naive to memory T cells could explain the loss with age in p80 phosphorylation, in that memory cells were significantly less able to phosphorylate p80 than were the unseparated CD4 preparations. Phosphorylation of p40 also was significantly lower in CD4 memory cells, as was phosphorylation of p120 in responses to anti-CD3. We conclude that subsets of mouse splenic T cells display different patterns of tyrosine-specific protein phosphorylation in the first 10 min after activation, and that the accumulation of memory T cells with age can account for at least some of the age-dependent decline in tyrosine protein kinase pathways.
The human VH3b gene subfamily is highly polymorphic.
Authors: Adderson E E , Azmi F H , Wilson P M , Shackelford P G , Carroll W L
Published: January 2023

We have previously shown that human antibody (Ab) directed against the capsular polysaccharide of the important bacterial pathogen, Haemophilus influenzae type b (Hib) is encoded by a small group of VH3 gene family members. The majority of anti-Hib PS Ab use members of the smaller VH3b subfamily. To examine directly the available human VH3 repertoire, we have used PCR to amplify and clone candidate germ-line VH3b H chain V region genes from two unrelated subjects from whom anti-Hib polysaccharide mAb had been previously obtained. A single functional VH3b germ-line gene was obtained from one subject. This gene is identical throughout the coding region to the previously identified gene 9.1. Twelve distinct VH3b germ-line sequences, 87.6-99.8% homologous to one another, were obtained from the second subject. One of these genes, LSG1.1, is also identical to the 9.1 germ-line gene, and a second, LSG6.1, is identical to a previously reported cDNA, M85. These germ-line VH3b genes are 82.7-94.1% homologous to rearranged anti-Hib PS VH3b segments obtained from these subjects. Our findings further demonstrate that considerable polymorphism of VH segments exists in the human population. Despite the presence of very highly homologous VH elements in the germ line, particular genes are highly conserved within the outbred human population.
Functional identification of transcription control sequences of the mouse Crry gene.
Authors: Martin B K , Weis J H
Published: January 2023

The mouse C receptor-related gene Crry is expressed by a wide variety of cells. Those sequences required for the transcriptional control of this gene were identified by deletion analysis of regions 5' of the initiating ATG. Fusion of Crry promoter sequences to the reporter gene CAT identified a region approximately 1,500 bp upstream of the transcriptional start site that enhanced transcription of this gene construct. Gel shift and methylation interference assays were performed, and a specific protein-DNA complex was identified within this region. These assays defined a 16-bp sequence 1,642 bp 5' of the initiating ATG that bound a protein in nuclear extracts prepared from all murine cell lines and tissues examined. The methylation interference assay indicated that the core region of the DNA sequence recognized by the protein was GGAA, the common core binding site for the ets family of proto-oncogenes. Oligonucleotides prepared from this sequence with the GGAA sequence did inhibit the DNA/protein complex formation, whereas those with a mutant GGAA sites did not. The minimal site identified by methylation interference was able to up-regulate transcription when placed downstream of a heterologous promoter, whereas the same sequence with an altered GGAA site could not. Thus, this site functions as an enhancer.
Peripheral B cell maturation. II. Heat-stable antigen(hi) splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells.
Authors: Allman D M , Ferguson S E , Lentz V M , Cancro M P
Published: January 2023

HSAhi B cells comprise 5 to 10% of adult mouse splenic B cells and are phenotypically and functionally immature. To assess their origin and relationship to mature, heat-stable Ag (HSA)lo B cells, we determined HSA and surface IgD phenotype among splenic B cells throughout development, as well as during reconstitution of lethally irradiated adults given adult B-depleted bone marrow. In each case, HSAhi splenic B cells predominate during the earliest stages of B cell genesis. Furthermore, 5-bromo-2'-deoxyuridine labeling experiments indicate rapid turnover within both the marrow and peripheral HSAhi pools, and adoptive transfer studies show that peripheral HSAhi splenic B cells differentiate to HSAlo within 4 days. Finally, splenic HSAhi B cells reconstitute both primary and memory humoral responses. Together, these data indicate that splenic B cells in the HSAhi subset are an intermediate maturational stage in the adult periphery.
Lipopolysaccharide and tumor necrosis factor-alpha induce lipopolysaccharide receptor expression on bone marrow cells by different mechanisms.
Authors: Chaby R , Pedron T , Stütz P L , Girard R
Published: January 2023

Two cell types, monocytes/macrophages and neutrophilic granulocytes, play a prominent role in the pathogenic effects of endotoxins during Gram-negative infections. We previously established that nanomolar concentrations of LPS induce the expression of new specific LPS-binding sites (LpsR) in bone marrow granulocytes of LPS-responsive mice. To examine this induction process further, we asked whether it, like other LPS activities, can be mediated by TNF-alpha. We report that exogenous rTNF-alpha can induce LpsR expression in bone marrow cells (BMC) from both LPS-responsive (C3H/HeOU) and LPS-hyporesponsive (C3H/HeJ) mice. In BMC, LPS elicited a down-regulation of the TNF-alpha receptors (TNF-R) without direct binding to TNF-R. On the other hand, taxol, a microtubule stabilizer that has shown LPS mimetic activity in macrophages, was unable to elicit LpsR expression or induce TNF-R down-regulation in BMC. Thus, unlike the LPS-signaling receptor of macrophages, that of BMC is apparently not functionally associated with microtubules. The LPS-induced expression of LpsR was inhibited only partially with an anti-TNF-alpha serum, and with dexamethasone, suggesting that an autocrine activity of endogenous TNF-alpha cannot alone account for the LPS effect. Comparative analyses also indicated that dexamethasone inhibited the LPS-induced increase of LpsR, but enhanced the number of TNF-induced LpsR+ BMC. Furthermore, the synthetic lipid PPDm2 (a 1,4-bisphosphorylated and N,N-diacylated derivative of 2,3-diamino-2,3-dideoxy-D-glucose) inhibited LPS-induced, but not TNF-induced, expression of LpsR. These data show that in BMC, LPS and TNF-alpha induce LpsR expression by different mechanisms.
B cell antigen receptor cross-linking triggers rapid protein kinase C independent activation of p21ras1.
Authors: Harwood A E , Cambier J C
Published: January 2023

Membrane Ig (mIg) functions in binding and internalization of Ag for subsequent processing and presentation to T cells, as well as in transmembrane transduction of signals that lead to cell activation, proliferation, and differentiation. Tyrosine kinase activation and subsequent phosphatidylinositol hydrolysis and Ca2+ mobilization are clearly important intermediary events in receptor-mediated B cell activation. However, many details of the cellular signal transduction pathways utilized by this receptor are not resolved. Recent studies that demonstrated co-capping of mIg and the proto-oncoprotein p21ras suggested that this low m.w. GTP-binding protein may function in mIg-mediated signal transduction. p21ras has been implicated in some but not all protein tyrosine kinase/phospholipase C involving signaling pathways. To explore the potential role of p21ras in B cell Ag receptor-mediated signaling, we assessed the effect of Ag receptor ligation on the proportion of p21ras in the active GTP-bound state. We present evidence that p21ras is activated by mIgM and mIgG cross-linking by anti-receptor antibodies as well as by Ag. Depending upon the stimulus employed, this response is detectable within 1 min and occurs with similar kinetics as inductive protein tyrosine phosphorylation and Ca2+ mobilization. Ag dose dependence of this response is similar to that of inductive protein tyrosine phosphorylation. In these cells p21ras is also activated by PMA suggesting that p21ras activation after receptor cross-linking may be mediated by an effector molecule that functions downstream from protein kinase C (PKC). However, the kinetics of p21ras activation after mIg cross-linking are inconsistent with the possibility that PKC functions as the sole mediator of p21ras activation in this system. Finally, under conditions in which the PKC inhibitor calphostin C blocks PMA-induced p21ras activation, it does not inhibit Ag-induced p21ras activation. These data suggest that PKC effector mechanisms play a negligible role in p21ras activation during mIg-mediated signaling.
Human IL-10 is a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL-8-induced CD4+ T lymphocyte migration.
Authors: Jinquan T , Larsen C G , Gesser B , Matsushima K , Thestrup-Pedersen K
Published: January 2023

Human IL-10 (hIL-10) is a newly described cytokine that was originally identified as a cytokine synthesis inhibitory factor regulating the production of several pro-inflammatory cytokines such as IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, and IFN-gamma. Additionally, hIL-10 also inhibits the macrophage-dependent proliferative response of CD4+ T lymphocytes to Ag stimulation, and it down-regulates the constitutive class II MHC expression on human monocytes. We report hIL-10 to be a potent and specific chemotaxin for human T lymphocytes with optimum activity in the range between 10 and 100 U/ml. Checkerboard analysis shows the activity to be chemotactic and not chemokinetic. The chemotactic activity is directed toward CD8+ T lymphocytes and not towards CD(4+)-enriched cells. Also, hIL-10 lacks chemotactic activity toward human monocytes or neutrophil granulocytes. Further, we found that hIL-10 inhibits the chemotactic response of CD4+, but not CD8+, T cells toward IL-8. Because hIL-10 can be produced by several cells including CD4+ T cells of the Th2 type, our results suggest that hIL-10 participates in a complex regulatory circuit between CD4+ and CD8+ T cells with implications for the control of lymphocyte-mediated inflammatory responses.
Involvement of TCR-V beta 8.3+ cells in the cure of mice bearing a large MOPC-315 tumor by low dose melphalan.
Authors: Mokyr M B , Rubin M , Newell K A , Prokhorova A , Bluestone J A
Published: January 2023

We have previously shown that the curative efficacy of low dose melphalan (L-phenylalanine mustard; L-PAM) for mice bearing a large s.c. MOPC-315 tumor requires the participation of CD8+ (but not CD4+) T cell-dependent antitumor immunity. Here we show that CD8+ T cells obtained from regressing tumors on day 4 or 5 after low dose L-PAM therapy of MOPC-315 tumor bearers (L-PAM TuB mice) display a preferential enhancement in the utilization of the TCR-V beta 8.3 gene segment as compared to CD8+ T cells from normal lymph nodes. Treatment of L-PAM TuB mice with mAb F23.1, which leads to the depletion of V beta 8.3+ cells, as well as V beta 8.1 and 8.2+ cells, led to a significant reduction in the ability of their tumor-infiltrating lymphocytes as well as their spleen cells to lyse MOPC-315 tumor cells in vitro in a short term assay. In addition, the mAb F23.1 treatment almost completely abrogated the lytic activity of the tumor-infiltrating lymphocytes against another syngeneic, antigenically related plasmacytoma (the MOPC-104E). Moreover, the mAb F23.1 treatment significantly reduced the curative effectiveness of low dose L-PAM for mice bearing a large MOPC-315 tumor. In contrast, mAb KJ16 treatment, which leads to the depletion of V beta 8.1 and 8.2+ cells (but not V beta 8.3+ cells), did not reduce significantly the curative effectiveness of low dose L-PAM for such MOPC-315 tumor bearers. Thus, V beta 8.3+ T cells are important for the curative effectiveness of low dose L-PAM therapy for MOPC-315 tumor bearers, and it is conceivable that the V beta 8.3+ cells mediate their effect (at least in part) by contributing to the acquisition of CTL activity against plasmacytoma-shared Ag.
Cross-linking of the carcinoembryonic antigen-like glycoproteins CD66 and CD67 induces neutrophil aggregation.
Authors: Kuijpers T W , van der Schoot C E , Hoogerwerf M , Roos D
Published: January 2023

The carcinoembryonic Ag (CEA)-like glycoproteins present on human granulocytes have been designated non-specific cross-reacting Ag (NCA). We have recently demonstrated that granulocyte-specific CD66 and CD67 mAb recognize the three originally described NCA. CD66 binds to NCA-160 and NCA-90, whereas CD67 only recognizes NCA-95. As we have shown previously, NCA-160 and NCA-90 present sialylated Lewis-X oligosaccharide Ag (SLex) in a functional way, i.e., these Ag function as (one of many possible) molecules involved in neutrophil binding to the adhesion molecule E-selectin expressed on activated endothelial cells. In this study, we found that a polyclonal anti-CEA antiserum, either as intact Ig or as F(ab')2 fragments, induced neutrophil aggregation. This aggregation response was blocked by CD18 mAb. Neutrophils from a patient severely affected by paroxysmal nocturnal hemoglobinuria completely lacked expression of NCA-95 and NCA-90. The patient's neutrophils repeatedly showed no aggregation on addition of the anti-CEA antiserum. Thus, the presence of the phosphatidylinositol-linked NCA-95/90 seems to be essential for NCA-induced neutrophil activation. Our data indicate that NCA molecules on neutrophils may function via binding to E-selection or an as yet unknown ligand (as mimicked here by an antiserum) and subsequently induce CD18-dependent adhesive properties.
Synthetic peptide in mineral oil adjuvant elicits simian immunodeficiency virus-specific CD8+ cytotoxic T lymphocytes in rhesus monkeys.
Authors: Yasutomi Y , Palker T J , Gardner M B , Haynes B F , Letvin N L
Published: January 2023

In view of the importance of cell-associated virus in AIDS virus transmission, an HIV vaccine should be able to induce a virus-specific CTL response. Traditional subunit vaccines have not elicited virus-specific CD8+ MHC class I-restricted CTL. We have used the simian immunodeficiency virus of macaques (SIVmac)/rhesus monkey model to explore the use of CTL epitope peptide-helper peptide conjugates for the vaccine elicitation of AIDS virus-specific CTL. We found that both the CTL epitope peptide-helper peptide conjugate and the CTL epitope peptide alone, when delivered in an emulsion with IFA, induced CTL epitope-specific CD8+ MHC class I-restricted CTL. These effector cells recognized processed viral protein and were readily cloned from PBL of the immunized monkeys. Moreover, the cloned effector cells inhibited SIVmac replication in PBL. Immunization with the CTL epitope peptide used in this study also elicited a CD4+ PBL proliferative response, suggesting that the peptide also contained a helper epitope. These studies provide further evidence for the potential usefulness of peptide-based AIDS virus vaccines.
Characteristics of heterologous beta 2-m exchange into H-2Db at the cell surface.
Authors: Luscher M A , Newton B L , Barber B H
Published: January 2023

W6/32 is a monomorphic anti-HLA class I Ab that cross-reacts with the product of the exchange of murine beta 2-microglobulin (beta 2-m) with human or bovine beta 2-m on H-2 Db. Using W6/32 we have developed a simple and rapid flow cytometric method to measure the beta 2-m exchange kinetics for Db at the surface of intact H-2b cell lines. We find that 10 to 25% of the Db heavy chains exchange bound beta 2-m for soluble beta 2-m with a t1/2 of 10 to 15 min. The kinetics of the exchange are consistent with a mechanism that includes a free heavy chain intermediate on the reaction coordinate. The remainder of Db molecules appear to be refractory to beta 2-m exchange even after long-term culture with an exogenous source of beta 2-m. The exchange process proceeds at a similar rate on wild-type cells that bear a diverse complement of autologous peptides on their class I molecules, and on mutant RMA-S cells having class I molecules primarily occupied with a single, defined, high affinity synthetic peptide Ag. The t1/2 for dissociation of a radiolabeled analogue of a high affinity naturally presented peptide Ag is 10 h or more, implying that the free heavy chain intermediate in the beta 2-m exchange mechanism retains bound peptides. Further, we find that synthetic peptide Ags added to non-mutant cells bind both to class I molecules that subsequently exchange bound beta 2-m, and to molecules that retain endogenous beta 2-m.
Inhibition by insoluble immune complexes of both capacitative Ca2+ entry and Ca2+ mobilization by chemotactic agonists in human neutrophils.
Authors: Montero M , Steil A A , Sanchez Crespo M , Garcia-Sancho J , Alvarez J
Published: January 2023

Insoluble immune complexes (IIC) stimulate human neutrophils by binding to their FcR. It is known that they are able to release Ca2+ from intracellular stores but they induce little Ca2+ entry from the extracellular medium, a dissociation that cannot be explained within the framework of the capacitative model for Ca2+ entry, which is well established for these cells. We show here that IIC induce a strong and long-lasting inhibition of the Ca2+ pathway activated by emptying the Ca2+ stores (capacitative Ca2+ entry), which develops simultaneously with the activation of Ca2+ release from intracellular stores. This inhibition strongly resembles that previously described effected by FMLP and by phorbol dibutyrate, which seems to be mediated by phosphorylation. Inhibition by IIC, however, differs from that induced by FMLP and phorboldibutyrate in its lack of sensitivity to cytosolic-free calcium concentration and in its different sensitivity to the protein kinase inhibitors staurosporin and chelerythrine. It was also insensitive to the protein tyrosine kinase inhibitors genistein and herbimycin A. We also show that IIC inhibit Ca2+ mobilization induced by other agonists and that this inhibition is potentiated by the protein phosphatase inhibitor calyculin A. Our results therefore suggest that binding of IIC to the FcR activates a protein phosphorylation mechanism leading to a long-lasting inhibition of both capacitative Ca2+ entry and Ca2+ mobilization induced by other agonists such as FMLP, platelet-activating factor, or leukotriene B4.
IL-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis.
Authors: Romani L , Mencacci A , Tonnetti L , Spaccapelo R , Cenci E , Puccetti P , Wolf S F , Bistoni F
Published: January 2023

In murine models of systemic candidiasis, healing and nonhealing patterns of disease are associated with preferential expansion of Th1 and Th2 cells, respectively. As previous studies have shown IL-12 to be expressed transcriptionally in healer mice and to be required for Th1 development in vitro, this cytokine might play a role in Candida-driven Th1 cell differentiation in vivo. In the present study, IL-12-neutralizing Abs or recombinant IL-12 were administered to mice with healing or progressive candidiasis, respectively, and the animals were monitored for mortality, resistance to reinfection, serum levels of specific Abs, and IFN-gamma, IL-4, and IL-10 message/protein expression by CD4+ cells. In self-limiting infection by a yeast vaccine strain, neutralization of IL-12 ablated development of acquired anticandidal resistance and led to appearance of Candida-specific IgE and IL-4-producing cells. In mice with progressive systemic disease as well as in a mucosal infection model, administration of IL-12 did not result in therapeutic activity under conditions of yeast infection that would instead be resolved by serologic ablation of IL-4 or IL-10. Yet, in systemically infected mice cured by anti-IL-4 or anti-IL-10 therapy, the emergence of a Th1 response correlated with the detection of high levels of circulating IL-12 and splenic IL-12 transcripts. Although exogenous IL-12 may not be sufficient for Th conversion in the presence of an overwhelming IL-4/IL-10 response, endogenous production of IL-12 may be both required and prognostic for Th1 differentiation in vivo.
Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities.
Authors: Stepkowski S M , Tu Y , Condon T P , Bennett C F
Published: January 2023

Intercellular adhesion molecule-1 (ICAM-1) binds circulating leukocytes through interactions with beta 2 integrins, LFA-1, and macrophage Ag-1. The phosphorothioate antisense oligodeoxynucleotide, IP-3082, specific for ICAM-1 mRNA inhibited ICAM-1, but not vascular cell adhesion molecule-1, mRNA induction and expression of ICAM-1 molecules by mouse endothelioma cells. Scrambled control oligonucleotides were ineffective. Untreated C3H (H-2k) mice rejected C57BL/10 (H-2b) heart allografts with a mean survival time of 7.7 +/- 1.4 days. Administration i.v. of IP-3082 by a 7-day osmotic pump prolonged the survival of heart allografts in a dose-dependent fashion: 1.25 mg/kg, to 11 +/- 0 days; 2.5 mg/kg, to 12 +/- 2.7 days; 5 mg/kg, to 14.1 +/- 2.7 days; and 10 mg/kg, to 15.3 +/- 5.8 days (all p < 0.01). Control IP-1082 (10 mg/kg) was ineffective (7 +/- 0.8 days). Although 7-day anti-LFA-1 mAb (50 micrograms/day; i.p.) prolonged allograft survival to 14.1 +/- 2.7 days, the addition of IP-3082 (5.0 mg/kg x 7 days) induced donor-specific transplantation tolerance (> 150 days). Furthermore, IP-3082 (5 mg/kg x 7 days) acted synergistically with antilymphocyte serum, rapamycin, and brequinar, but not cyclosporin A: a single antilymphocyte serum (0.2 ml) i.p. injection alone prolonged graft survival to 10 +/- 0.5 days (p < 0.01) and in combination with IP-3082 (5 mg/kg), to 32.2 +/- 8.3 days (p < 0.001); rapamycin (0.1 mg/kg x 7 days; i.v.) alone prolonged survival to 13 +/- 7.5 days (p < 0.01), and with IP-3082, to 32.4 +/- 8.9 days (p < 0.03); brequinar (0.5 mg/kg x 7 days; oral gavage) alone to 12 +/- 2.4 days (p < 0.05), and with IP-3082 (5 mg/kg), to 38.8 +/- 30.2 days (p < 0.01); in contrast, cyclosporin A (5 mg/kg x 7 days; i.v.) alone produced graft survival of 9.8 +/- 1.3 days (p < 0.1 and in combination with IP-3082 (5 mg/kg), produced survival of 7.8 +/- 3.5 days (NS). Thus, antisense oligonucleotides may proffer a selective gene-targeted immunosuppressive therapy for organ transplantation.
Poly ADP-ribosylation of a 90-kDa protein is involved in TNF-alpha-mediated cytotoxicity.
Authors: Agarwal S , Piesco N P
Published: January 2023

Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ribosylation plays a crucial role in this reaction. We have studied the role of ADP-ribosylation during TNF-alpha action in TNF-alpha-sensitive and TNF-alpha-resistant cells. Treatment of 3T3 cells with TNF-alpha, in the presence of [adenylate-32P]NAD followed by SDS-PAGE, revealed the involvement of specific ADP-ribosylation of a 90-kDa protein in TNF-alpha-mediated cytotoxicity. The stability of the ADP-ribosyl linkage on the 90 kDa protein in 100 mM 2-(N-cyclohexylamino)ethanesulfonic acid at pH 9.0 confirmed that ADP-ribosylation of the 90 kDa protein was mediated by an enzymatic reaction. Analysis of ADP-ribose residues by phosphodiesterase hydrolysis showed that the 90-kDa protein was modified by poly ADP-ribosylation. Poly ADP-ribosylation of the 90-kDa protein concomitant with cytotoxicity was observed in all TNF-alpha-sensitive but not TNF-alpha-resistant cells. Inhibition of ADP-ribosylation of the 90-kDa protein by benzamide but not by benzoic acid abrogated cytotoxicity, which further suggested that the poly-ADP-ribosylation of the 90-kDa protein is causally related to TNF-alpha-induced cell death. Our results demonstrate that TNF-alpha modifies a specific protein by poly-ADP-ribosylation during its action. Furthermore, ADP-ribosylation of specific proteins may be yet another mechanism regulating protein function during cellular metabolism.
In vivo T cell activation by anti-CD3 monoclonal antibody induces soluble TNF receptor release in mice. Effects of pentoxifylline, methylprednisolone, anti-TNF, and anti-IFN-gamma antibodies.
Authors: Bemelmans M H , Abramowicz D , Gouma D J , Goldman M , Buurman W A
Published: January 2023

Injection of anti-CD3 is accompanied by an increase in systemic TNF, a mediator of the physiologic changes induced by anti-CD3 injection. Various mechanisms have been reported to be responsible for TNF inactivation and clearance. Recently, it has become evident that circulating soluble TNFRs (P55 and P75), which are known to increase in response to TNF inducers such as LPS, represent a natural protection mechanism against circulating TNF. Here we show that triggering the TCR by anti-CD3 injection results in a strong induction of both systemic TNF and soluble TNFR release. Maximal levels of TNF were reached after 2 h (10 ng/ml). Maximum levels of P55 (450 pg/ml) were reached between 0.5 and 8 h, whereas the highest levels of P75 were reached after 2 h (28 ng/ml). Because TNF and IFN-gamma are supposed to be involved in the pathophysiology of the anti-CD3 response, we investigated the influence of in vivo neutralization of TNF and IFN-gamma. Injection of mAb to TNF and IFN-gamma significantly reduced systemic TNF levels and both soluble TNFR levels. Two inhibitors of anti-CD3 induced TNF release; steroids and pentoxifylline both reduced TNF levels and P75 levels without affecting P55 levels. The results show that T cell activation induces both systemic TNF release and release of both soluble TNFRs. Although TNF and IFN-gamma are involved in this mechanism, their role does not seem to be crucial.
IL-10 inhibits transcription of cytokine genes in human peripheral blood mononuclear cells.
Authors: Wang P , Wu P , Siegel M I , Egan R W , Billah M M
Published: January 2023

Upon addition of LPS to human PBMCs, IL-1 beta, IL-6, and TNF-alpha were released in the culture media in a time-dependent manner. Cytokine release began 2 h after LPS addition and maximal release of all three cytokines was observed between 6 and 8 h. Northern analysis revealed that, for each cytokine, mRNA accumulation preceded protein release. When added either 2 h before or at the same time as LPS, IL-10 inhibited both the cytokine release and mRNA accumulation almost completely. Similar inhibition was observed when IL-10 was added 2 h after LPS, a time point that coincided with the onset of the rapid burst of cytokine mRNA accumulation. The inhibitory activity of IL-10 was abrogated by cycloheximide, suggesting an involvement of newly synthesized proteins in IL-10 action. By using nuclear run-on transcription assays, we have found that IL-10 inhibited transcription of all three cytokine genes in LPS-stimulated PBMCs. In contrast, IL-10 moderately enhanced degradation of IL-6 mRNA, but not of mRNAs for IL-1 beta and TNF-alpha. Thus, the present study provides the first evidence that, in human PBMCs, IL-10 inhibits cytokine synthesis by acting mainly at the level of cytokine gene transcription.
The role of self peptides in the allogeneic cross-reactivity of CTLs.
Authors: Kuzushima K , Sun R , van Bleek G M , Vegh Z , Nathenson S G
Published: January 2023

This study presents data relevant to understanding the molecular and structural basis for the cross-reactivity of many CTLs on multiple MHC targets. Five anti-H-2Kb alloreactive CTL clones derived from B6.C-H-2bm1 (bm1), B6.C-H-2bm8 (bm8), and B6.C-H-2bm11 (bm11) mice and a Sendai virus-specific H-2Kb-restricted CTL clone were studied. Self peptides extracted from Kb molecules were fractionated by HPLC and tested for their ability to be recognized on RMA/s (H-2b) cells by those clones. For each alloreactive clone, a single dominant peptide peak was found to sensitize target cells. In addition to recognizing peptides presented by the Kb molecule, the five alloreactive clones and the one Sendai virus-specific clone all showed cross-reactivities on a panel of Kbm mutant cells in a peptide-dependent manner. Two CTL clones, one alloreactive and one virus specific, cross-recognized Kbm targets by each responding to a unique self peptide in the context of the mutant MHC molecules. Our data underscore the prevalent idea that TCR-alpha beta have an inherent structural capability to react with several peptide/MHC structural patterns other than the original peptide/MHC pattern that might have been used to select that TCR. The high incidence of cross-reactivity seems to reflect a feature of the mechanism of positive selection in the thymus and the need for T cells in the repertoire to have an expanded capability for responding to a wide variety of foreign Ags.
Regulation of cyclooxygenase-2 expression in normal human articular chondrocytes.
Authors: Geng Y , Blanco F J , Cornelisson M , Lotz M
Published: January 2023

This study analyzes cyclooxygenase II (COX-2) gene expression, protein synthesis, and PGE2 release in normal human articular chondrocytes. Stimulation of chondrocytes in primary culture resulted in a dose-dependent induction of COX-2 mRNA in response to IL-1 with an ED50 between 0.1 and 1 ng/ml. COX-2 mRNA was detectable after 2 h, reached high levels at 6 h, and showed a remarkably long duration of expression for at least 72 h. Analysis of other extracellular stimuli showed that COX-2 mRNA was inducible by other cytokines including TNF-alpha, IL-6, and LIF and by bacterial LPS. Dexamethasone completely inhibited IL-1-induced COX-2 mRNA expression. Analysis of signaling pathways showed that PMA and calcium ionophore A23187, but not dibutyryl cAMP, induced COX-2 mRNA. The combination of IL-1 and A23187 resulted in synergistic increases. IL-1 effects were not reduced by the protein kinase C inhibitor staurosporine or by the protein kinase A inhibitor H89 but blocked by the protein tyrosine kinase inhibitor herbimycin A. COX-2 protein was detected at 71 kDa by Western blotting in IL-1-stimulated, and to almost similar levels in A23187-treated, cells. Flow cytometric analysis showed that after IL-1 stimulation 78% of the chondrocytes expressed COX-2 protein. The patterns of COX-2 protein expression and the levels of PGE2 release correlated with the effects of the different stimuli and inhibitors on mRNA expression.