bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-12 is an effective adjuvant to recombinant vaccinia virus-based tumor vaccines: enhancement by simultaneous B7-1 expression.
Authors: Rao J B , Chamberlain R S , Bronte V , Carroll M W , Irvine K R , Moss B , Rosenberg S A , Restifo N P
Published: December 2022

A number of cytokines and costimulatory molecules involved in immune activation have recently been identified including IL-12, a heterodimeric cytokine that supports the development of cell-mediated immunity, and B7-1, a costimulatory molecule involved in the activation of T lymphocytes. We explored the use of these immunomodulants as molecularly defined adjuvants in the function of recombinant anticancer vaccines using a murine model adenocarcinoma, CT26, transduced with a model Ag, beta-galactosidase (beta-gal). Although IL-12 given alone to mice bearing tumors established for 3 days did not have consistent antitumor activity, a profound therapeutic effect was observed when IL-12 administration was combined with a recombinant vaccinia virus (rVV) encoding beta-gal called VJS6. On the basis of the reported synergistic effects of IL-12 and the costimulatory molecule B7-1 (CD80) in vitro, we immunized mice with a double recombinant vaccinia encoding both the model tumor Ag and the costimulatory molecule B7-1, designated B7-1 beta-gal rVV. The adjuvant administration of IL-12 after immunization with this virus significantly enhanced survival in tumor-bearing animals. T cell subset depletions demonstrated that the in vivo activity of IL-12 was largely independent of CD4+ T lymphocytes, whereas the in vivo activity of a B7-1 rVV required both CD4+ and CD8+ T cells to elicit maximal therapeutic effect. To our knowledge, this is the first description of B7-1 and IL-12 cooperation in vivo and represents a novel strategy to enhance the efficacy of recombinant anticancer vaccines.
Tyrosine-phosphorylated Cbl binds to Crk after T cell activation.
Authors: Sawasdikosol S , Chang J H , Pratt J C , Wolf G , Shoelson S E , Burakoff S J
Published: December 2022

Crk is a Src homology 2 (SH2)/Src homology 3 (SH3)-containing adapter protein that has been implicated in intracellular signaling in fibroblasts and PC12 pheochromocytoma cells. Crk has been shown to bind to a tyrosine-phosphorylated protein of 116 kDa after TCR-mediated T cell activation. Here we demonstrate that the Crk-associated p116 phosphoprotein is not the Crk-associated substrate (Cas) but, rather, is a protein product of the c-cbl proto-oncogene. Whereas Cas was not tyrosine-phosphorylated after T cell activation, Cbl became highly phosphorylated. Crk immunoprecipitates from activated T cell lysates contain tyrosine-phosphorylated Cbl. This association is mediated by the SH2 domain of Crk, as evidenced by the interaction between Cbl and the fusion protein product of a glutathione S-transferase (GST) expression construct encoding the Crk-SH2 domain in vitro. Furthermore, phosphopeptide-binding studies revealed that the GST-Crk SH2 domain binds to a tyrosine-phosphorylated peptide corresponding to amino acids 770-781 of Cbl with high affinity. Cbl is a protein tyrosine kinase (PTK) substrate that becomes phosphorylated after engagement of numerous cell surface receptors including the TCR. Data revealed by genetic studies in the nematode, Caenorhabditis elegans, implicates a Cbl-like molecule, Sli-1, as a negative regulator of the Let-23-signaling pathway. Because the signal from the Let-23 pathway affects the activation status of the Let-60 (Ras homologue in C. elegans) pathway, the activation-dependent association between Crk and Cbl may represent another TCR-generated signal leading to Ras-related pathways.
Systemic administration of cellular IL-10 induces an effective, specific, and long-lived immune response against established tumors in mice.
Authors: Berman R M , Suzuki T , Tahara H , Robbins P D , Narula S K , Lotze M T
Published: December 2022

Cellular IL-10 (cIL-10), the collective term for human and murine IL-10, has both stimulatory and inhibitory effects on diverse cell types, including costimulation of T cell proliferation, chemoattraction of CD8+ T cells, and stimulation of lymphokine-activated killer cell activity. Human IL-10 (hIL-10) differs from its EBV homolog viral IL-10 (vIL-10) by only 16% at the amino acid level; however, vIL-10 shares with cIL-10 predominantly inhibitory effects, such as macrophage deactivation. We administered cIL-10 systemically to mice bearing established (day 7) sarcomas, melanomas, or colorectal carcinomas. At high doses (20 to 60 micrograms/day x 7 days), cIL-10 induced rejection of tumors, delaying tumor outgrowth or resulting in complete cure. Sublethal irradiation (500 rad) of mice prior to tumor inoculation abrogated the IL-10 effect. Cured mice were immune to subsequent rechallenge with 10-fold higher inoculation with the same, but not a different, tumor. IL-12 also has potent antitumor activity and interacts with IL-10 in both complementary and antagonistic ways; co-administration of both cytokines resulted in additive antitumor activity. To compare cIL-10 vs vIL-10 effects in vivo, we engineered CL8-1 melanoma transfectants bearing the vIL-10 or the murine IL-10 (mIL-10) gene. Local secretion of mIL-10 induced rejection of tumors, while vIL-10 resulted in accelerated outgrowth. Subsequent systemic administration of cIL-10 to mice bearing vIL-10-transduced tumors completely reversed the local suppressive effects, leading to rejection, suggesting distinct pathways for cIL-10 and vIL-10 effects. That cIL-10 can stimulate the acquisition of an effective, specific, and long-lived antitumor immune response in murine models and can reverse the local immunosuppressive effects of vIL-10 indicates a potential role for cIL-10 administration in the biologic therapy of cancer and suggests a broader interpretation of IL-10 biology.
Prevention of experimental autoimmune myasthenia gravis by a monoclonal antibody to a complementary peptide for the main immunogenic region of the acetylcholine receptors.
Authors: Araga S , Galin F S , Kishimoto M , Adachi A , Blalock J B
Published: December 2022

We have previously reported that a complementary peptide (denoted RhCA 67-16), encoded by RNA complementary to that of the Torpedo acetylcholine receptor (AChR) main immunogenic region (MIR), AChR residues alpha 61-76, induces polyclonal and monoclonal Ab reactive with Ig against the AChR MIR. RhCA 67-16 vaccination also protected against the development of experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. In the present report, we found that a mAb (denoted TCM 240, IgG1 kappa) against RhCA 67-16 recognized three different idiotypic Ab (mAb 6, mAb 35, and mAb 198), which were previously reported by others to recognize the AChR MIR and to cause EAMG. Based on these results, TCM 240 was tested for prophylactic effects in EAMG. EAMG induced passively by mAb 35 was inhibited by simultaneous injection with TCM 240. The disease severity was inversely paralleled by the ratio of mAb 35 to TCM 240. EAMG induced by immunization with purified native Torpedo AChR was also inhibited by TCM 240, but not a control mAb. The inhibitory effect of TCM 240 on actively induced EAMG occurred without significantly lowering the overall AChR Ab levels, which indicates a limited repertoire of disease-causing Ab in EAMG and perhaps MG. Such findings suggest the existence of an EAMG-associated Id and also support the concept of an MIR. In a more general sense, these results demonstrate that prophylactic and perhaps diagnostic mAb for autoimmune diseases can be produced by immunization with complementary peptides for disease-associated epitopes.
Antigen-receptor engagement in B cells induces nuclear expression of STAT5 and STAT6 proteins that bind and transactivate an IFN-gamma activation site.
Authors: Karras J G , Wang Z , Coniglio S J , Frank D A , Rothstein T L
Published: December 2022

The signal transducer and activator of transcription (STAT) family of transcription factors is triggered by cytokine and growth factor receptors in a number of cell types, and binds to a consensus sequence defined in part by the IFN-gamma activation site (GAS). It is not known whether these transcription factors respond to other kinds of growth stimuli, and, with particular relevance to lymphocytes, it is not known whether STAT proteins participate in Ag-specific responses. To determine the role of STAT proteins, coupling between Ag-receptor cross-linking and nuclear expression of DNA-binding protein complexes that recognize GAS sequences was evaluated. Ag-receptor triggering in primary B lymphocytes stimulated nuclear expression of a complex that specifically binds the IFN response factor-1 (IRF-1) GAS sequence, and is distinguished by electrophoretic mobility and GAS preference from IRF-1 GAS-binding complexes induced by IFN-gamma. Activation of nuclear IRF-1 GAS-binding activity by sIg was inhibited by the tyrosine kinase inhibitor, herbimycin A, and binding activity was eliminated by tyrosine phosphatase treatment. Activation of IRF-1 GAS-binding activity was blocked by depletion of protein kinase C. The IRF-1 GAS-binding activity induced by sIg engagement in B cells was transcriptionally active, and was found to consist of immunoreactive STAT5 and STAT6 proteins. This work demonstrates that the STAT signaling pathway previously associated with cytokine signaling is triggered in B lymphocytes through Ag-receptor engagement in a protein kinase C-dependent fashion. This heretofore described cytokine signaling pathway may play a role in bringing about Ag-specific proliferative and differentiative responses.
Mucosal immunity to HIV-1: systemic and vaginal antibody responses after intranasal immunization with the HIV-1 C4/V3 peptide T1SP10 MN(A).
Authors: Staats H F , Nichols W G , Palker T J
Published: December 2022

To optimize mucosal immune responses to the HIV-1 peptide vaccine candidate T1SP10 MN(A), we intranasally immunized BALB/c and C57BL/6 mice with C4/V3 HIV-1 peptide together with the mucosal adjuvant cholera toxin (CT). Four doses over a 4-wk period resulted in peak serum anti-peptide IgG titers of > 1:160,000 in BALB/c mice and > 1:520,000 in C57BL/6 mice, and significant levels (>1:30,000) persisted in both strains of mice for longer than 6 mo. Furthermore, intranasal immunization with peptide and CT induced serum IgG reactivity to HIV-1 gp120 and HIV-1(MN) neutralizing responses. The primary anti-peptide IgG subclass was IgG1, suggesting a predominant Th2-type response. In addition to elevated serum anti-peptide A responses, intranasal immunization with T1SP10 MN(A) and CT induced both vaginal anti-peptide IgG and IgA responses, which persisted for 91 days in both strains of mice. Vaginal anti-HIV IgA was frequently associated with secretory component, suggesting transepithelial transport of IgA into vaginal secretions. Cervical lymph nodes contained the highest relative concentration of anti-T1SP10 MN(A) IgG-producing cells, while the spleen was the next major site of anti-T1SP10 MN(A) IgG-producing cells. Ag-specific proliferative responses were also detected in cervical lymph node and spleen cell populations after intranasal immunization with T1SP10 MN(A) and CT. In addition, intranasal immunization with T1SP10 MN(A) and CT was able to induce anti-HIV cell-mediated immunity in vivo as indicated by the induction of delayed-type hypersensitivity. Therefore, intranasal immunization with hybrid HIV peptides provides a noninvasive route of immunization that induces both long-lived systemic and mucosal Ab responses as well as cell-mediated immunity to HIV.
Fc receptor off-signal in the B cell involves apoptosis.
Authors: Ashman R F , Peckham D , Stunz L L
Published: December 2022

By linking surface Ig to the FcR Fc gamma RII on the mouse B lymphocyte surface, whole anti-Ig has been shown to block cell cycle entry and subsequent Ab production, a phenomenon called the "Fc receptor off-signal." IL-4 or blocking Ab to Fc gamma RII, present with whole anti-Ig, restores cell cycle progress to the levels observed with F(ab')2 anti-Ig. The current study demonstrates that under "off-signal" conditions with whole anti-Ig, the early entry of B cells into apoptosis was accelerated relative to medium alone or equimolar F(ab')2 anti-Ig. All reagents tested which opposed the whole-anti-Ig-induced blockade of B cell cycle entry also protected B cells from apoptosis (IL-4, PMA, dextran sulfate, and the monoclonal anti-Fc gamma RII 2.4G2). This protective effect was most evident at 4 to 12 h, waning at later times. Low dose cycloheximide partially protected B cells from whole anti-Ig-induced apoptosis, but acted as a survival factor, failing to advance B cells from G0 phase or stimulate thymidine incorporation. Additive early apoptosis-associated membrane changes were transiently seen when whole anti-Ig was combined with other apoptosis-accelerating agents (trifluoperazine, staurosporine, dexamethasone, ionomycin, high-dose cycloheximide), but hypodiploid nuclei did not show this effect. B cells from bcl-2 transgenic mice showed less apoptosis when cultured with whole anti-Ig, or with any of the other agents tested. At 4 h the bcl-2-associated reduction in hypodiploid nuclei was greater than the reduction in membrane unpacking, but by 16 h this difference was much less. These results suggest that acceleration of apoptosis contributes to the inhibition of proliferation induced by whole anti-Ig.
Tenidap and other anion transport inhibitors disrupt cytolytic T lymphocyte-mediated IL-1 beta post-translational processing.
Authors: Perregaux D G , Svensson L , Gabel C A
Published: December 2022

LPS-activated murine peritoneal macrophages produce IL-1 beta but externalize little mature cytokine in the absence of a secondary stimulus, and CTLs previously were reported to serve this capacity. The release of 17-kDa IL-1 beta from LPS-activated BALB/c macrophages occurred rapidly after the addition of C57/B1-derived allogeneic CTLs; within 30 min of coculture, mature IL-1 beta was observed in the medium, and maximum release was achieved within 4 h. CTL-induced post-translational processing was efficient, and >80% of newly synthesized pro-IL-1 beta was released into the medium as the 17-kDa species. Externalization of IL-1 beta required active recognition of the macrophage target by the CTL preparation; C57/B1 CTLs promoted the release of mature IL-1 beta from allogeneic BALB/c macrophages, but not from syngeneic C57/B1 macrophages. In contrast, extracellular ATP promoted mature IL-1 beta release from both macrophage populations. CTL-induced cytokine post-translational processing was blocked by anion transport inhibitors, including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, UK5099, and the anti-inflammatory agent tenidap. An analogue of tenidap, CP-100,829, was more effective as an inhibitor of both IL-1 beta post-translational processing and anion transport. In contrast, the close structural analogue CP-236,492 inhibited neither process. Tenidap's activity was reversible and was not mimicked by cyclooxygenase inhibitors or by cycloheximide. Therefore, tenidap disrupted CTL-induced IL-1 beta post-translational processing by a mechanism dependent on anion transport inhibition. Multiple stimuli are likely to operate in vivo to promote IL-1 beta post-translational processing, and anion transport inhibitors such as tenidap that suppress cytokine processing independently of the initiating stimulus thus represent attractive candidates as therapeutic regulators of IL-1 production.
Impaired signaling in alloantigen-specific CD8+ T cells tolerized in vivo: employing a model of Ld-specific TCR transgenic mice transplanted with allogenic hearts under the cover of a short-term rapamycin treatment.
Authors: Chen H , Luo H , Xu D , Loh D Y , Daloze P M , Veillette A , Qi S , Wu J
Published: December 2022

Peripheral tolerance of T cells is necessary because thymic deletion is not complete, and tissue-specific Ags exist outside the thymus. We have reported that persistent Ag is required to maintain peripheral tolerance in vivo. We suspect that the TCR signaling pathway in in vivo tolerized cells is compromised due to continuous exposure to the Ags. In this study, the TCR signaling events in these cells were investigated using TCR transgenic mice (2C mice) whose T cells are predominantly Ld alloantigen-specific CD8 cells. The 2C mice were thymectomized and then rendered tolerant to Ld Ag by allogenic heart transplantation plus short-term treatment with rapamycin. We found that 1) the in vivo tolerized CD8 cells have compromised intracellular Ca2+ flux upon mitogen stimulation; and 2) their cellular tyrosine proteins fail to be phosphorylated properly upon TCR cross-linking. These results indicate that the signaling pathway in the in vivo tolerized CD8 cells is indeed defective. We also found that 1) the tolerized CD8 cells have no characteristic surface markers; and 2) the allograft is probably the place where the rejection response is initiated according to the appearance of an early activation marker of T cells on graft-infiltrating cells.
Brain-derived gangliosides regulate the cytokine production and proliferation of activated T cells.
Authors: Irani D N , Lin K I , Griffin D E
Published: December 2022

Gangliosides may regulate the activity of the immune system in vivo, particularly within tissues such as neoplasms or the central nervous system, where they are most abundant. However, the specific mechanisms by which gangliosides modulate immune function remain incompletely understood. We have characterized the effects that brain-derived gangliosides have on specific steps of the T cell activation process in vitro. Gangliosides inhibit T cell proliferation downstream from the early activation events that are bypassed pharmacologically using the combination of a phorbol ester plus a calcium ionophore. These lipids block IL-2 and IFN-gamma gene transcription without inhibiting the production of IL-4 and IL-10 mRNA. This may be accounted for by the ability of gangliosides to prevent the activation of NF-kappaB in mitogen-stimulated T cells. Despite inhibiting IL-2 production, the antiproliferative effects of gangliosides are not reversed by adding supplemental IL-2 to the culture media. This defect persists because gangliosides also block the entry of activated T cells into the cell cycle. In this setting, phosphorylation of the retinoblastoma gene product, a protein whose phosphorylation state is an important regulator of normal cell cycle progression, is prevented. These studies help to define how gangliosides modulate T cell effector function in vitro. They also highlight the fact that certain T cell responses, namely the production of Th2-associated cytokines, are not inhibited by their actions.
Deletion in HCDR3 rescues T15 antibody mutants from a secretion defect caused by mutations in HCDR2.
Authors: Martin T M , Kowalczyk C , Stevens S , Wiens G D , Stenzel-Poore M P , Rittenberg M B
Published: December 2022

We recently described mutants of the murine anti-phosphocholine Ab T15, with changes in heavy chain complementarity determining region 2 (HCDR2) that caused loss of secretion. Surprisingly, the T15 HCDR2 mutations did not alter secretion when placed into the related anti-phosphocholine Ab D16, which differs from T15 only in HCDR3 and light (L) chain. Here, we exploit the differences between these two Abs to assess the basis of the secretion defect. The T15 L chain is not secreted in the absence of heavy (H) chain. In contrast, D16 L chain is secreted in the absence of H chain, as are most L chains. We co-expressed the T15 wild-type (wt) and mutant H chains with the D16 L chain, as well as with another secreted L chain, J558L. The mutant H chains were not secreted when expressed with either heterologous L chain. These results establish that the T15 L chain is not uniquely associated with the defect. The T15 and D16 Abs also differ in HCDR3 length in that D16 lacks four amino acid residues (Ser99, Ser100, Tyr100a, Trp100b) present in T15. We deleted these four residues from T15 wt and mutant H chains. Secretion of T15 wt was unaffected by the deletion, but shortening HCDR3 restored secretion in the HCDR2 mutants regardless of L chain association. Together these data demonstrate that both the HCDR2 and HCDR3 domains contain structural information that may affect the secretion competence of Abs.
CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage cells and autoimmunity.
Authors: Sato S , Ono N , Steeber D A , Pisetsky D S , Tedder T F
Published: December 2022

CD19 serves as a cell surface response regulator that establishes signaling thresholds critical for B lymphocyte development and activation. B lymphocytes from CD19-deficient mice are hyporesponsive to transmembrane signals, while B lymphocytes from mice that overexpress CD19 to even a small extent (25% increase) become hyperresponsive. The B-1 subpopulation of B lymphocytes is particularly sensitive to CD19 regulation, since their development is severely decreased in CD19-deficient mice. The effect of altered CD19 expression levels on the development of B cells was therefore examined using transgenic mice that express varying levels of cell surface CD19. In this study, immature B cells within the bone marrow of wild-type mice were found to specifically up-regulate CD19 expression levels by twofold as they mature, while CD5+ B cells within the spleen and peritoneum expressed even higher levels of CD19. The development of CD5+ B cells was severely decreased in CD19-deficient mice, while there was a linear correlation between increased CD19 expression levels and the increased frequency of CD5+ B cells within the peritoneum and spleen. In fact, CD5+ B cells became a major B lymphocyte population in mice that overexpressed CD19. Increased expression of CD19 also correlated with increased levels of endogenous anti-DNA Abs and rheumatoid factor. These results indicate that up-regulated expression of CD19 is functionally important for B cell development and that CD19 establishes signaling thresholds that regulate the generation of B-1 lymphocytes as well as the development of autoantibodies.
Somatic hypermutation of a lambda 2 transgene under the control of the lambda enhancer or the heavy chain intron enhancer.
Authors: Klotz E L , Storb U
Published: December 2022

Previous experiments have suggested an important role for the Ig enhancers and transcription in targeting somatic hypermutation. To determine whether the requirement of the enhancers is Ig chain specific, we analyzed two lambda 2 light chain transgenes under the control of different enhancers, either the lambda 2-4 enhancer or the heavy chain intron enhancer. The transgenes were amplified and cloned from B220+PNAhigh B cells from either Peyer's patches (PP) or SRBC-immunized spleen. The lambda 2 transgene under the control of the lambda 2-4 enhancer underwent mutation in both the PP and splenic B cell populations, but at a frequency lower than endogenous light chain genes. This requirement for the lambda 2-4 enhancer could be replaced by the heavy chain intron enhancer. Interestingly, the heavy chain intron enhancer-driven lambda 2 construct showed evidence of statistically significant mutation in the PP B cell population, but not the spleen-derived population. This difference in somatic hypermutation suggests that a lower mutation frequency can be more readily detected in B220+PNAhigh B cells isolated from the PP. The ability of the heavy chain intron enhancer to replace the lambda 2-4 enhancer shows that the requirement for the Ig enhancers in somatic hypermutation is not Ig locus specific. Given that these Ig enhancers have very few elements in common, our results further suggest that the Ig enhancers are primarily important in the timing of transcription in the mutating B cell population.
Mutational analysis of the ligand-binding domain of M-T2 protein, the tumor necrosis factor receptor homologue of myxoma virus.
Authors: Schreiber M , McFadden G
Published: December 2022

The myxoma virus-encoded M-T2 protein shares extensive sequence homology with the ligand-binding domains of the TNF receptors (TNFRs) and has been shown to bind and inhibit rabbit TNF-alpha with affinities similar to those of TNF-alpha with cellular receptors. Here we show that M-T2 protein is secreted from infected cells as an N-linked glycoprotein, with both complex and hybrid or high mannose oligosaccharide chains. Since amino acid homology between M-T2 and cellular TNF receptors is limited to the four N-terminal cysteine-rich domains (CRDs), various M-T2 C-terminal truncations were created in recombinant vaccinia virus vectors. C-terminal deletions that include truncations up to the middle of the fourth CRD effectively bound and inhibited rabbit TNF-alpha. In contrast, removal of any one of the first three CRDs resulted in a mutant M-T2 protein incapable of binding or inhibiting rabbit TNF-alpha. The C-terminal portion of M-T2, which is not homologous to the cellular TNFRs, appears to be important for efficient secretion of M-T2 from infected cells, since all the C-terminal truncations, including a truncation removing only the last 24 amino acids, were effectively retained as intracellular proteins that were still capable of binding and inhibiting rabbit TNF-alpha. We conclude that the first three CRDs of M-T2 fulfill the same ligand-binding function as the cellular TNFRs, and the nonhomologous C-terminal region participates in protein trafficking of M-T2 in virus-infected cells.
IFN-gamma-independent production of IL-12 during murine endotoxemia.
Authors: Heinzel F P , Rerko R M , Ahmed F , Hujer A M
Published: December 2022

We examined whether IFN-gamma deficiency alters the in vivo IL-12 response occurring in the mouse model of acute endotoxemia. C57BL/6 IFN-gamma knockout mice (IFN-gamma 0/0) produced as much circulating IL-12 p40 and IL-12 p70 as did IFN-gamma +/+ mice following injection with S. enteritidis LPS, despite sustaining 11-fold reductions in circulating TNF-alpha. Pretreatment of IFN-gamma 0/0 mice with recombinant mouse IFN-gamma (rIFN-gamma) enhanced circulating TNF-alpha by as much as sixfold, but serum IL-12 p40 and IL-12 p70 responses increased by only twofold or less. Compared with IFN-gamma +/+ mice, the spleens of endotoxemic IFN-gamma 0/0 mice generated two- to threefold fewer IL-12 p40-secreting cells following in vitro or in vivo exposure to endotoxin. The addition of rIFN-gamma to IFN-gamma 0/0 splenocyte culture restored normal levels of LPS-stimulated IL-12 p40 production. Removal of Mac-1+ or F4/80+ cells from endotoxin-stimulated spleen reduced both TNF-alpha and IL-12 p40 production, but 33D1+ dendritic cell removal only affected IL-12 synthesis. These data suggest that the aggregate IL-12 p40 and p70 response to endotoxemia in vivo is IFN-gamma-independent and distinct from IFN-gamma-dependent serum TNF-alpha and splenic IL-12 responses. The cellular and/or cytokine basis for the unexpected preservation of IL-12 production in IFN-gamma-deficient mice may be relevant to normal and pathologic immune responses.
Effects of site-directed mutagenesis of basic residues (Arg 94, Lys 95, Lys 99) of lipopolysaccharide (LPS)-binding protein on binding and transfer of LPS and subsequent immune cell activation.
Authors: Lamping N , Hoess A , Yu B , Park T C , Kirschning C J , Pfeil D , Reuter D , Wright S D , Herrmann F , Schumann R R
Published: December 2022

LPS-binding protein (LBP) is a 60-kDa acute phase glycoprotein capable of binding the LPS of Gram-negative bacteria and facilitating its diffusion. This process is thought to be of potential importance in inflammatory reactions and pathogenic states such as septic shock syndrome. Here, we report on the identification of a LPS binding domain within the LBP molecule and on the identification of single amino acids important for binding of LPS by LBP. Several synthetic LBP peptides inhibited LPS-LBP interaction, and amino acids Arg 94 and Lys 95 were centrally located in these inhibitory peptides. LBP mutants with amino acid exchanges within this region were expressed and tested in five different functional assays: binding to immobilized LPS; facilitation of binding of LPS aggregates to monocytes; transfer of LPS monomers from aggregates to soluble CD14; transfer of soluble CD14-bound LPS monomers to high density lipoprotein (HDL); and enhancement of LPS-induced cell activation. The double mutant Glu 94/Glu 95 was completely lacking LPS binding, transfer, and cell stimulatory activity, indicating that the integrity of amino acids 94 and 95 is required for LBP function. While mutations of amino acids Arg 94 or Lys 95 into alanine reduced the LPS binding activity of LBP dramatically, the ability to facilitate binding of LPS aggregates to membrane CD14 at the cell surface was retained. These findings emphasize the distinction between binding of LPS aggregates to cells, which is not associated with cell stimulation, and binding of LPS monomers to CD14, which leads to cell stimulation.
CD44 is a cytotoxic triggering molecule on human polymorphonuclear cells.
Authors: Pericle F , Sconocchia G , Titus J A , Segal D M
Published: December 2022

In this study, we present evidence that CD44 is a cytotoxic triggering molecule on freshly isolated polymorphonuclear cells (PMN). PMN constitutively express high levels of CD44 as determined by FACS analysis, and immunoprecipitation studies using PMN lysates and an anti-CD44 mAb show a band of 80 to 90 kDa that migrates slightly faster than CD44 from PBL. A bispecific Ab consisting of anti-CD44 Fab cross-linked to anti-DNP Fab (anti-CD44(Fab) x anti-DNP(Fab)) induces PMN to lyse DNP-coated tumor cells in an 18-h assay, and this lysis is specifically inhibited by a polyclonal anti-CD44 F(ab')2. A second bispecific Ab, anti-CD16(Fab) x anti-DNP(Fab), that binds to Fc(gamma)RIIIb on PMN does not induce lysis, indicating that the bridging of target cells to PMN per se is not sufficient for killing. Moreover, CD44-directed killing by PMN results in the lysis of bystander cells, suggesting that the mechanisms of tumor cytolysis by CD44-targeted PMN does not require cell-cell contact. Lastly, PMN lyse target cells coated with hyaluronic acid (HA), the principal ligand for CD44, and this cytolytic activity is specifically blocked by the polyclonal anti-CD44 F(ab')2 and by an anti-CD44 mAb. We suggest that the interaction of HA with CD44 on neutrophils might initiate cytotoxic or inflammatory responses in vivo when neutrophils encounter high amounts of HA, for example on tumor cells, or in the extracellular matrix.
Modulation of human platelet-activating factor receptor gene expression by protein kinase C activation.
Authors: Thivierge M , Parent J L , Stankova J , Rola-Pleszczynski M
Published: December 2022

The effect of PMA on early and late regulation of platelet-activating factor receptor (PAF-R) expression was examined in human monocytes. Treatment with 16 nM PMA for 5 min initiated a rapid reduction (50-75%) in [3H]WEB 2086 binding, which was maximal between 5 and 15 min. Scatchard analysis revealed that PMA treatment reduced the number of binding sites to 50% of control cells without an appreciable change in their affinity. In parallel cultures, flow cytometry analysis using anti-PAF-R Abs failed to reveal any significant decrease in the level of PAF-R expression until after 4 h of treatment with PMA. By 24 h, PAF-R expression had declined by 80 to 90%. This PMA-induced down-regulation of surface PAF-R expression was preceded by a rapid down-regulation of PAF-R mRNA expression. PMA-mediated down-regulation could be blocked by the protein kinase C (PKC) inhibitors H-7 and calphostin C. Activation of PKC by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol also resulted in down-regulation of PAF-R mRNA accumulation, whereas the inactive phorbol diester 4alpha-PMA was ineffective. The rapid disappearance of the PAF-R transcripts was associated with decreased stability of receptor mRNA and not with a change in the nuclear transcription rate of the PAF-R gene. These findings indicate that PAF-R gene expression in human leukocytes can be regulated through a PKC-dependent pathway and involves post-transcriptional destabilization of receptor mRNA.
Breaking of B cell tolerance toward a highly conserved self protein.
Authors: Dalum I , Jensen M R , Hindersson P , Elsner H I , Mouritsen S
Published: December 2022

Self proteins are processed and presented by APCs in the same way as foreign proteins. Presentation of fragments derived from self proteins does not, however, lead to Th cell stimulation because of T cell tolerance. In this study, a novel approach was used to investigate whether B cell tolerance toward a self Ag could be due to the absence of this Th cell recognition. The highly conserved nonimmunogenic protein ubiquitin was used as a model protein. Two modified ubiquitin molecules were constructed with ubiquitin segments exchanged either with the T cell epitope, OVA(325-336), which binds to the mouse A(d) MHC class II molecule, or with the T cell epitope, hen egg lysozyme(50-61), which binds to the A(k) molecule. Mice were immunized with the resulting proteins. Both modified proteins elicited strong autoantibody responses toward soluble native ubiquitin, demonstrating that insertion of a single foreign T cell epitope can overcome the B cell nonresponsiveness. The T cell regulatory role of one of the inserted foreign T cell epitopes in ubiquitin was studied, and at least two different Th cell specificities were found to operate in the response. The T cells were directed against: 1) the inserted epitope, and 2) a combination of the inserted epitope and parts of the neighboring ubiquitin regions. Therefore, the absence of T cell help seems to be an important reason for B cell tolerance toward self proteins.
Recipient CD8+ cells are responsible for the rapid elimination of allogeneic donor lymphoid cells.
Authors: Fast L D
Published: December 2022

Blood transfusion can result in a variety of immunologic responses, including alloimmunization, transfusion-associated graft-versus-host disease, and immunosuppression that results in increased postoperative infection rate, and can also result in increased survival of allografts. One of the many factors influencing the resulting immunologic responses from a blood transfusion is the persistence of the donor leukocytes. A recent study has shown that almost all (99.9%) allogeneic leukocytes are removed within 2 days following transfusion but did not characterize the mechanism responsible for the rapid removal of allogeneic donor cells. Using a murine model these studies show that it is recipient CD8+ T cells that are responsible for the rapid elimination of allogeneic lymphocytes in naive recipients. Effective elimination was dependent on the donor/recipient combination and required that the donor cells express at least one MHC class I disparity to be recognized by the recipient CD8+ cells and that the donor cells also be able to induce additional responses that were needed by the CD8+ cells to manifest full activity. The perforin pathway was the predominant pathway used by the recipient CD8+ cells to mediate this elimination.
Binding of human complement component C4b-binding protein (C4BP) to Streptococcus pyogenes involves the C4b-binding site.
Authors: Accardo P , Sánchez-Corral P , Criado O , García E , Rodríguez de Córdoba S
Published: December 2022

A key step in the elimination of invading pathogens from the body is the covalent binding of complement proteins C3b and C4b to their surface. However, many pathogens have evolved mechanisms to avoid the complement system of the host. Understanding how these mechanisms work may lead to more efficacious forms of therapy. Here we provide an insight into the molecular basis of how Streptococcus pyogenes binds human plasma C4b-binding protein (hC4BP), a complement regulatory molecule that may decrease C3b and C4b deposition on the streptococcal surface. We show that streptococcal surface molecules bind to a site on hC4BP that is indistinguishable from the C4b binding site. This site involves multiple binding surfaces that span short consensus repeats 1 to 3 of the alpha-chain of hC4BP. We propose that hC4BP is bound to the bacterial surface because the streptococcal surface molecules involved in the interaction mimic human C4b epitopes.
Molecular characterization of Bip 1, a monoclonal antibody that modulates IgE binding to birch pollen allergen, Bet v 1.
Authors: Laffer S , Vangelista L , Steinberger P , Kraft D , Pastore A , Valenta R
Published: December 2022

Bet v 1 and homologous proteins represent major cross-reactive allergens for more than 95% of tree pollen-, fruit-, and vegetable-allergic individuals. To study the interaction of Bet v 1 and the immune system, we characterized a Bet v 1-specific mAb, Bip 1. Soluble rBip 1 Fabs were expressed in Escherichia coli and purified by affinity chromatography using immobilized Bet v 1. Bip 1 Fabs displayed a cross-reactivity to homologous allergens comparable with that of IgE Abs from allergic patients. Preincubation of Bet v 1 with Bip 1 led to an up to fivefold increase of allergic patients' IgE binding to Bet v 1. This enhancement in IgE binding may be interpreted as stabilization of a Bet v 1 state, in which certain IgE epitopes are better applicable. It also shows that allergic patients possess IgE Abs directed against different Bet v 1 conformations. The modulation of Ab binding to a given Ag by other Abs was observed also for human Bet v 1-specific IgG Abs, and may represent a novel mechanism for the regulation of specific humoral immune responses in a complex network.
Humanization of an anti-lymphocyte function-associated antigen (LFA)-1 monoclonal antibody and reengineering of the humanized antibody for binding to rhesus LFA-1.
Authors: Werther W A , Gonzalez T N , O'Connor S J , McCabe S , Chan B , Hotaling T , Champe M , Fox J A , Jardieu P M , Berman P W , Presta L G
Published: December 2022

Lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is involved in leukocyte adhesion during cellular interactions essential for immunologic responses and inflammation. mAbs against LFA-1 have been shown to inhibit several T cell-dependent immune functions in vitro and prevent graft failure after bone marrow transplantation in vivo. A murine anti-human CD11a mAb, MHM24, has been humanized and shown to prevent adhesion of human T cells to human keratinocytes and the proliferation of T cells in response to nonautologous leukocytes in the mixed lymphocyte response assay. However, of the nonhuman primate CD11a that we tested, the murine and humanized mAbs cross-reacted only with chimpanzee CD11a. To have a mAb available for preclinical studies in rhesus monkeys, the humanized mAb was reengineered to bind to rhesus CD11a by changing four residues in one of the complementarity-determining regions, CDR-H2, in the variable heavy domain. Cloning and molecular modeling of rhesus CD11a I-domain suggested that the changes from Lys197 and/or Arg189 in human CD11a I-domain to Glu197 and Gln189 in rhesus CD11a I-domain may be the reason that rhesus CD11a does not bind to the murine and humanized mAbs.
Glycoinositolphospholipids purified from Trypanosoma cruzi stimulate Ig production in vitro.
Authors: Bento C A , Melo M B , Previato J O , Mendonça-Previato L , Peçanha L M
Published: December 2022

We have investigated the effects of glycoinositolphospholipid (GIPL) purified from Trypanosoma cruzi on murine B cell activation. The GIPL neither stimulated any proliferative response by itself, nor affected the blastogenesis induced by surface IgD cross-linking or LPS. On the other hand, the GIPL significantly stimulated both low and high density B cells to secrete IgM in vitro. The GIPL induced B cells to produce IgM when added in the presence of either the surface Ig cross-linker, anti-delta-dextran, or a combination of IL-4 and IL-5. The T. cruzi-derived GIPL also stimulated Ig class switch to IgG1 in cultures stimulated with GIPL, IL-4, and IL-5. The IgG1 secretion was comparable to that induced by LPS plus IL-4. Production of IgG3 was also detected and the GIPL also potentiated the IgG3 production induced by LPS. The stimulatory effect of the T. cruzi-derived GIPL was mediated mainly by its oligosaccharide moiety. This isolated fraction induced a potent IgM secretory response, compared with a much lower response induced by the isolated GIPL ceramide. Taken together, our data suggest that the stimulatory effect of the T. cruzi-derived GIPL on B cell activation could play a role on the conspicuous Ig production observed during infection of the host with T. cruzi.
Loss of either CD4+ or CD8+ T cells does not affect the magnitude of protective immunity to an intracellular pathogen, Francisella tularensis strain LVS.
Authors: Yee D , Rhinehart-Jones T R , Elkins K L
Published: December 2022

Normal mice readily survive a sublethal intradermal (i.d.) infection with Francisella tularensis live vaccine strain (LVS), a model intracellular bacterium, and are strongly protected against subsequent lethal challenge. However, athymic nu/nu mice, which lack mature alphabeta TCR+ T lymphocytes, succumb to i.d. infection within 30 days. Here we characterize the alphabeta T cell subpopulations necessary for both resolution of i.d. infection and generation of optimal protective immunity to LVS. BALB/cByJ mice treated with anti-CD4 or anti-CD8 Abs before i.d. infection survived and cleared bacteria, and anti-CD4- or anti-CD8-treated immune mice survived a very strong i.p. challenge of 10,000 LD50s. Among mutant mice with targeted gene disruptions (knockouts), CD4-, beta2-microglobulin-deficient (which are also CD8-), and gammadelta TCR- mice all resolved a large sublethal i.d. infection. All CD4- and beta2-microglobulin-deficient mice readily survived subsequent lethal i.p. challenge of 10,000 LD50s, even in the absence of specific IgG Abs, as did most (86%) gammadelta TCR- mice. In contrast, alphabeta TCR- mice or alphabeta + gammadelta TCR- mice died about 35 days after i.d. infection. Depletion of gammadelta+ T cells from alphabeta TCR- mice had no effect on mean time to death from i.d. LVS infection. Therefore alphabeta TCR+ cells are required for protection, but either CD4+ or CD8+ T cells are individually sufficient to resolve a large sublethal i.d. LVS infection and to protect against a maximal secondary lethal challenge. These results emphasize the remarkable plasticity of the alphabeta T cell response in protective immunity to intracellular bacteria.
Lethal synergism between influenza infection and staphylococcal enterotoxin B in mice.
Authors: Zhang W J , Sarawar S , Nguyen P , Daly K , Rehg J E , Doherty P C , Woodland D L , Blackman M A
Published: December 2022

Superantigen hyperactivation of the immune system has variable, sometimes lethal consequences for the host. Here we show that concurrent influenza infection enhanced the effects of the bacterial superantigen staphylococcal enterotoxin B (SEB) in mice. The effect was T cell-dependent, and maximal synergism was observed when SEB was administered 7 days after the virus, a timepoint during infection associated with high viral titers, a vigorous cytotoxic T cell response, and extensive lung pathology. The influenza infection enhanced the SEB-induced cytokine response in terms of higher absolute levels of cytokine, sustained secretion, and localization to the respiratory tract. In particular, TNF and IFN-gamma were implicated in the mechanism of death because their neutralization protected the mice from death, and recombinant IFN-gamma and TNFalpha mimicked the lethal effect of SEB in influenza-infected mice. This lethal synergism between concurrent influenza infection and superantigen exposure points to the danger of secondary bacterial involvement in viral pneumonia, and suggests mechanisms that may contribute to sudden and unexpected death from influenza infection. In addition, these data demonstrate that the in vivo effects of superantigen exposure can be strongly influenced by the immune activation status of the host.
Modulation of natural IgM binding and complement activation by natural IgG antibodies: a role for IgG anti-Gal alpha1-3Gal antibodies.
Authors: Yu P B , Holzknecht Z E , Bruno D , Parker W , Platt J L
Published: December 2022

The most abundant natural IgG Abs in human serum are thought to be Abs specific for Gal alpha1-3Gal, a carbohydrate expressed in lower mammals. IgG Abs specific for Gal alpha1-3Gal have been postulated to contribute to host defense and to participate in the rejection of interspecies organ grafts. Our previous studies indicated, however, that IgM and not IgG anti-Gal alpha1-3Gal Abs activate complement on foreign surfaces, and thus the physiologic role of IgG anti-Gal alpha1-3Gal remains uncertain. We tested whether the IgG anti-Gal alpha1-3Gal in a human serum might in fact compete with IgM for binding and thus modulate complement fixation by IgM. Several lines of evidence suggested such competition might occur. First, the functional avidity of IgG and IgM for Gal alpha1-3Gal on cell surfaces were nearly within the same order of magnitude, and in some sera the molar concentrations of IgG and IgM anti-Gal alpha1-3Gal were comparable. Second, binding of human IgM to Gal alpha1-3Gal on cell surfaces was inversely correlated with the concentration of IgG anti-Gal alpha1-3Gal in serum. Third, combination of IgG and IgM Abs specific for Gal alpha1-3Gal demonstrated direct competition for binding. The presence of IgG anti-Gal alpha1-3Gal, which was predominantly IgG2, attenuated by up to 80% the fixation of C1q mediated by IgM, presumably by competing for antigenic sites recognized by IgM Abs that fix complement. Thus, IgG Abs specific for Gal alpha1-3Gal modulate complement activation by IgM specific for that structure and might in this way modulate the consequences that ensue when human blood is brought into contact with foreign organisms or xenogenic cells.
Inhibition of protein kinase C-mediated CD4 down-regulation by oxidative stress in T lymphocytes.
Authors: Nakamura K , Sasada T , Sono H , Yodoi J
Published: December 2022

The CD4 molecule acts as a receptor for class II MHC molecules to stabilize Ag recognition by the TCR and as a high affinity receptor for HIV-1. In this study, we investigated the effect of oxidative stress on the level of CD4 expression on cultured peripheral blood T lymphocytes (PBL blasts). As previously reported, we observed that the surface CD4 was down-regulated by PMA. Unexpectedly, treatment of PBL blasts with hydrogen peroxide (H2O2) or a sulfhydryl oxidative reagent, diamide, almost completely inhibited PMA-induced CD4 down-regulation, although these oxidants per se did not affect the level of CD4 expression. We next assessed the serine phosphorylation of CD4, which is reported to be an indispensable process for PMA-induced CD4 endocytosis. PMA could induce the serine phosphorylation even in the presence of oxidants. We also found that these oxidants had an additive effect on PMA-induced dissociation between CD4 and p56(lck), which is known to be another necessary step for CD4 endocytosis. These results indicate that in T cells, oxidants inhibit protein kinase C-mediated CD4 down-regulation due to perturbing a signaling process other than the above two steps, implying that oxidative stress may tune the functions of CD4+ T cells and their susceptibility to HIV-1 through the control of CD4 expression.