bluuebunny/crossref_metadata_2025 huggingface dataset:
A role for TNF-alpha and mucosal T helper-1 cytokines in the pathogenesis of Crohn's disease.
Authors: Plevy S E , Landers C J , Prehn J , Carramanzana N M , Deem R L , Shealy D , Targan S R
Published: January 2023

Recent clinical studies of Crohn's disease patients demonstrated dramatic clinical responses following one i.v. infusion of a chimeric mAb to TNF-alpha (cA2). To assess the role of TNF-alpha in mucosal cytokine regulation, the effects of TNF-alpha on lamina propria mononuclear cell (LPMC) Th1 production were determined. Increased IFN-gamma production was demonstrated in anti-CD2-stimulated LPMC cultured in TNF-alpha. To determine the effects of cA2 on cytokine production, TNF-alpha- and IFN-gamma-producing cells were quantitated in LPMC from five Crohn's disease patients treated with cA2. In all four patients who demonstrated clinical and endoscopic improvement, decreased numbers of LPMC producing IFN-gamma and TNF-alpha following CD2/CD28 activation paralleled improvement in disease activity over 8 wk. In one patient who did not improve, increased numbers of TNF-alpha- and IFN-gamma-secreting LPMC were observed. In three of four responding patients, CD2/CD28-activated PBMC demonstrated increased IFN-gamma production over 8 wk. These observations suggest that TNF-alpha may be a cofactor for mucosal Th1 responses, and improvement in clinical parameters and intestinal inflammation induced by cA2 in Crohn's disease may be mediated by down-regulation of mucosal Th1 cytokines.
TNF-alpha down-regulates type 1 cytokines and prolongs survival of syngeneic islet grafts in nonobese diabetic mice.
Authors: Rabinovitch A , Suarez-Pinzon W L , Sorensen O , Rajotte R V , Power R F
Published: January 2023

Administration of TNF-alpha to autoimmune diabetes-prone nonobese diabetic mice and biobreeding rats inhibits diabetes development; however, the mechanism(s) of diabetes prevention by TNF-alpha has not been established. We used the model of syngeneic islet transplantation into diabetic nonobese diabetic mice to study the effects of TNF-alpha administration on the types of mononuclear cells and cytokines expressed in the islet grafts and on autoimmune diabetes recurrence. Twice daily i.p. injections of TNF-alpha (20 microg/day) from day 1 to day 30 after islet transplantation significantly prolonged islet graft survival; thus, 70% (16 of 23) of mice treated with TNF-alpha were normoglycemic at 30 days after islet transplantation compared with none (0 of 14) of vehicle-treated control mice. Islet grafts and spleens from TNF-alpha-treated mice at 10 days after islet transplantation contained significantly fewer CD4+ and CD8+ T cells, and significantly decreased mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and TNF-beta) than islet grafts and spleens from control mice. Regarding type 2 cytokines, IL-4 mRNA levels were not changed significantly in islet grafts or spleens of TNF-alpha-treated mice, whereas IL-10 mRNA levels were decreased significantly in islet grafts of TNF-alpha-treated mice and not significantly changed in spleens. TGF-beta mRNA levels in islet grafts and spleens were similar in TNF-alpha-treated and control mice. These results suggest that TNF-alpha partially protects beta cells in syngeneic islet grafts from recurrent autoimmune destruction by reducing CD4+ and CD8+ T cells and down-regulating type 1 cytokines, both systemically and locally in the islet graft.
IL-4 expression by grafts from transgenic mice fails to prevent allograft rejection.
Authors: Mueller R , Davies J D , Krahl T , Sarvetnick N
Published: January 2023

Cell-mediated tissue destruction, such as that occurring in allograft rejection, is thought to be mediated by Th1 cells and cytokines. We have recently shown that transgenic expression of the Th2 cytokine IL-4 by pancreatic beta cells completely protects nonobese diabetic (NOD) mice from autoimmune diabetes by inducing functional tolerance among autoreactive T cells. To investigate whether local IL-4 production could also induce functional tolerance among alloreactive T cells and thus prevent allograft rejection, we transplanted pancreata from transgenic neonatal mice and their nontransgenic littermates into allogeneic hosts. Within 2 wk, recipient mice had rejected their grafts regardless of the transgene's presence or absence. Considering that the vigorous immune response induced might have prevented any effect by IL-4, we injected recipient mice with anti-CD4 and anti-CD8 mAbs, thereby depleting them of T cells and thus providing the islets with an opportunity to mature and grow. This approach indeed delayed rejection of neonatal pancreata from nontransgenic mice by >1 wk. By that time, however, pancreata from transgenic mice had also been rejected. Our results indicate that the allograft rejection response under these conditions, in contrast to the autoimmune response in NOD mice, cannot be regulated by local IL-4 production, regardless of the cytokine's impact on Th1 cells.
Weak peptide agonists reveal functional differences in B7-1 and B7-2 costimulation of human T cell clones.
Authors: Anderson D E , Ausubel L J , Krieger J , Höllsberg P , Freeman G J , Hafler D A
Published: January 2023

The influence of costimulation on the T cell response to altered peptide ligands that act as either partial or weak agonists for human CD4+ T cell clones was examined. Using stable Chinese hamster ovary (CHO) cell transfectants expressing DR2 (DRB1*1501) and human B7-1 or B7-2 as APC, presentation of native myelin basic protein (MBP) p85-99 peptide Ag or a partial agonist of MBP p85-99 induced equivalent T cell activation as measured by [3H]TdR incorporation and cytokine secretion. In marked contrast, presentation of cross-reactive peptides of MBP p85-99 that act as weak agonists with B7-1, but not B7-2, costimulation resulted in significant T cell activation as measured by [3H]TdR incorporation and cytokine secretion. These data suggest that decreasing the strength of the signal provided to the TCR allows differences in B7-1 and B7-2 signaling to be observed. Thus, the costimulatory environment during T cell activation may be a mechanism of regulating T cell cross-reactivity in the periphery.
The avian chB6 (Bu-1) alloantigen can mediate rapid cell death.
Authors: Funk P E , Tregaskes C A , Young J R , Thompson C B
Published: January 2023

The control of cell death is critical in the immune system. T and B lymphocytes must be censored during their development to remove nonfunctional or self-reactive lymphocytes. However, the molecules controlling cell deletion during lymphopoiesis have not been defined. B cells removed from the avian bursa of Fabricius rapidly undergo cell death in culture. We screened bursal B cells with a panel of Abs and lectins to identify molecules affecting their viability. Abs to the chB6 alloantigen caused a rapid loss of cell viability as measured by staining with propidium iodide. ChB6 Abs also cause adhesion between B cells. Transfection of cDNA encoding chB6 reconstituted the allele-specific cell death and adhesion effects in avian cell lines. These effects can be separated by binding cells onto Ab-coated plastic dishes. In these experiments, cells were killed in the absence of cell:cell contact. The ability of chB6 cross-linking to evoke cell aggregation and cell death is also observed when chB6 is expressed in growth factor-dependent mammalian cells. In these cells growth factor can almost completely prevent cell death but not cell aggregation. This suggests that known cell survival stimuli can suppress the cell death brought about by chB6 cross-linking. These results show that chB6 may have an important role in controlling cell survival and/or adhesion during avian B cell development.
CD28-B7 T cell costimulatory blockade by CTLA4Ig in sensitized rat recipients: induction of transplantation tolerance in association with depressed cell-mediated and humoral immune responses.
Authors: Onodera K , Chandraker A , Schaub M , Stadlbauer T H , Korom S , Peach R , Linsley P S , Sayegh M H , Kupiec-Weglinski J W
Published: January 2023

We tested the effects of blocking CD28-B7 T cell costimulation by using CTLA4Ig in an established transplantation model in which LBNF1 cardiac allografts are rejected in an accelerated manner (<36 h) by LEW rats presensitized with Brown-Norway skin grafts. Treatment with CTLA4Ig with or without donor alloantigen in the sensitization phase (between skin and cardiac engraftment) minimally delayed accelerated rejection. However, adjunctive infusion of CTLA4Ig and donor alloantigen in the effector phase (after cardiac engraftment) resulted in long term graft survival and donor-specific tolerance in 30 to 50% of the recipients. The mutant form of CTLA4Ig, which blocks B7-1 but not B7-2, was ineffective. The tolerant state was accompanied by reduction of cell-mediated (MLR/CTL) responses and depression of humoral (circulating IgM/IgG allo-Abs) alloreactivity in vivo. Hence, the binding of CD28 on T cells to both CD80 and CD86 ligands represents a crucial initial costimulatory step leading to accelerated graft rejection. CTLA4Ig-mediated early blockade of the CD28 signaling pathway combined with transfusion of donor cells in the perioperative period interrupts sensitization and may produce transplantation tolerance. This regimen inhibits T cell costimulation and activation to provide help to CD8+ cytotoxic T and B cells, perhaps, via CTLA4Ig-induced clonal anergy or deletion.
T cell antigen receptor-induced IL-2 production and apoptosis have different requirements for Lck activities.
Authors: Chung C D , Lewis L A , Miceli M C
Published: January 2023

The T cell hybridoma BI-141 has been previously used to dissect the roles of Lck in Ag-induced IL-2 production. Here we demonstrate that BI-141 undergoes apoptosis in response to TCR stimulation using Ag or anti-TCR Abs. Using a panel of BI-141 transfectants expressing constitutively activated Lck (F505) or phosphotyrosine-binding (K154F505 and C156F505) or kinase-impaired (R273F505) mutants, we assess the relative requirements for Lck in TCR-mediated IL-2 production and apoptosis. While BI-141 transfectants expressing F505 are dramatically enhanced in their ability to produce IL-2 in response to Ag relative to K154F505-, C156F505-, or R273F505-expressing transfectants, no differences between these transfectants are observed in their ability to undergo TCR-induced apoptosis. TCR-induced Fas ligand (FasL) expression is demonstrated to be dependent on Lck SH2 and kinase activities, although FasL expression cannot be correlated with apoptosis. Low levels of Fas are constitutively expressed at equal levels on transfectants and are not increased in response to TCR ligation. Together, these data indicate that TCR-induced apoptosis in BI-141 is regulated through a mechanism(s) distinct from the Lck-induced expression of Fas, FasL, or IL-2 production. This TCR signal may be independent of Lck kinase and SH2 activities, or may require lower threshold activity. The identification of differential requirements for particular T cell functions is crucial to understanding how TCR engagement affects downstream T cell functions and may aid in the rational design of therapeutics aimed at specifically modulating particular T cell responses.
Induction of germinal center B cell markers in vitro by activated CD4+ T lymphocytes: the role of CD40 ligand, soluble factors, and B cell antigen receptor cross-linking.
Authors: Lahvis G P , Cerny J
Published: January 2023

Following primary immunization, B cells differentiate to memory cells with help from T cells. The specialized path to B cell memory takes place in lymphoid germinal centers (GC), where mouse B cells up-regulate peanut agglutinin receptor (PNA-R), B7-2 (CD86), and MHC class II expression. Using an in vitro culture system, we have studied how different stimuli can enhance the expression of these markers. We show that PNA-R is up-regulated when splenic B cells are cocultured with anti-CD3-stimulated CD4+, but not CD8+, T cells and that this process requires CD40-CD40 ligand engagement. Increased expression of PNA-R is also inducible with supernatants of activated CD4+, but not CD8+, T cells in combination with mitogenic signals, such as anti-Ig, anti-CD40, or LPS, but not by either supernatants or mitogenic signals alone. Unlike with PNA-R, increased expression of B7-2 and I-A occurs in response to activated T cells of either CD4+ and CD8+ subsets or their supernatants, does not require CD40 costimulation, and is readily induced with mitogenic signals alone. Taken together, these results indicate that PNA-R up-regulation has more restricted signaling requirements than B7-2 or I-A, and that it can be induced/maintained by Ag receptor cross-linking or CD40 engagement, as long as there is an appropriate cytokine milieu.
Fc epsilonRI-deficient mice infected with Schistosoma mansoni mount normal Th2-type responses while displaying enhanced liver pathology.
Authors: Jankovic D , Kullberg M C , Dombrowicz D , Barbieri S , Caspar P , Wynn T A , Paul W E , Cheever A W , Kinet J P , Sher A
Published: January 2023

The IgE/Fc epsilonRI interaction is postulated to play an important role in resistance to helminths both at the level of anti-parasitic effector cell function and in the initiation of Th2 responses through IL-4 produced by Fc epsilonRI+ non-B, non-T (NBNT) cells. To formally evaluate the role of IgE/Fc epsilonRI signaling in the host response to helminths we studied Schistosoma mansoni infection in Fc epsilonRI knockout (KO) mice. Infected wild-type (wt) and KO animals showed comparable adult worm and tissue egg burdens, arguing against a role for Fc epsilonRI interactions in host resistance. Significantly, NBNT cells from infected KO, in contrast to wt animals, did not secrete IL-4 when stimulated with anti-IgE Ab or soluble parasite Ag. Nevertheless, serum IgE levels and Th2 cytokine production profiles were comparable in both strains of mice, demonstrating that the Ag-dependent stimulation of IL-4 secretion by NBNT cells is not essential for helminth-induced Th2 differentiation. However, when stimulated with low Ag doses, splenocytes from infected Fc epsilonRI-deficient mice produced less IL-4 in vitro than similar cultures from infected wt animals, an effect attributable to their defective NBNT cell function. Moreover, infected KO mice showed enhanced egg granuloma formation and hepatic fibrosis, revealing that the IgE/Fc epsilonRI interaction, while not essential for Th2 response development or resistance to primary infection, plays a significant role in down-regulating host pathology.
DNA vaccines against lymphoma: promotion of anti-idiotypic antibody responses induced by single chain Fv genes by fusion to tetanus toxin fragment C.
Authors: Spellerberg M B , Zhu D , Thompsett A , King C A , Hamblin T J , Stevenson F K
Published: January 2023

Idiotypic determinants can act as tumor-associated Ags for B cell lymphoma. Vaccination with idiotypic protein and adjuvant is known to induce specific protection against lymphoma challenge in mice, largely mediated by anti-idiotypic Ab. For facilitating the approach for patients, the V(H) and V(L) genes used to encode the individual idiotypic determinants of each tumor can be obtained by PCR and assembled as single chain Fv (scFv). DNA vaccines containing scFv sequences alone induce low and poorly reproducible levels of anti-idiotypic Ab, likely to be insufficient to suppress tumor in patients. In addition, it may be necessary to break tolerance to Id in tumor bearers. By fusing the gene for fragment C of tetanus toxin to the C terminus of human scFv, we have promoted the anti-scFv Ab response in mice by >50-fold in three of three cases. The induced Abs are mainly against idiotypic determinants, and react specifically with patients' tumor cells, indicating optimal folding of the scFv molecule in the fusion protein. For both antigenic components of the DNA vaccine, the IgG subclass distribution showed a relative increase in IgG2a as compared with vaccination with IgM protein in adjuvant. In patients, the fusion gene should both promote anti-idiotypic Ab and induce Abs against fragment C of tetanus toxin. The latter response would provide a potentially useful comparative measure of the ability of patients to respond to conventional Ag delivered via DNA.
Cutting Edge: The B Cell Surface Protein CD72 Recruits the Tyrosine Phosphatase SHP-1 upon Tyrosine Phosphorylation
Authors: Adachi Takahiro , Flaswinkel Heinrich , Yakura Hidetaka , Reth Michael , Tsubata Takeshi
Published: January 2023

Activation signals of lymphocytes are negatively regulated by the membrane molecules carrying the immunoreceptor tyrosine-based inhibition motif (ITIM). Upon tyrosine phosphorylation, ITIMs recruit SH2-containing phosphatases such as SHP-1, resulting in down-modulation of cell activation. We showed that the cytoplasmic domain of the CD72 molecule carries an ITIM and is associated in vitro with SHP-1 upon tyrosine phosphorylation. Moreover, cross-linking of B cell Ag receptor (BCR) enhances both tyrosine phosphorylation of CD72 and association of CD72 with SHP-1 in B cell line WEHI-231. These results indicate that CD72 recruits SHP-1 upon tyrosine phosphorylation induced by BCR signaling, suggesting that CD72 is a negative regulator of BCR signaling.
A Critical Role for IL-18 in the Proliferation and Activation of NK1.1+CD3− Cells
Authors: Tomura Michio , Zhou Xu-Yu , Maruo Seiji , Ahn Hyun-Jong , Hamaoka Toshiyuki , Okamura Haruki , Nakanishi Kenji , Tanimoto Tadao , Kurimoto Masashi , Fujiwara Hiromi
Published: January 2023

Like IL-12, IFN-γ-inducing factor/IL-18 has been shown to stimulate T cells for IFN-γ production and growth promotion. Considering the NK-stimulatory capacity of IL-12, we investigated the effect of IL-18 on NK lineage cells. A CD4−CD8−surface Ig−Ia− fraction of freshly prepared C57BL/6 spleen cells proliferated strikingly in response to combinations of IL-12 + IL-18 or IL-2 + IL-18, but not to the individual cytokines or IL-2 + IL-12. Cells proliferating in response to IL-2 + IL-18 were NK1.1+CD3−, whereas IL-12 + IL-18-responsive cells were NK1.1−CD3−. Restimulation of the former cells with IL-12 + IL-18 or the latter cells with IL-2 + IL-18 resulted in the generation of NK1.1−CD3− or NK1.1+CD3− cells, respectively. Moreover, a NK1.1+CD3−CD4−CD8−surface Ig−Ia− population isolated from spleen cells was found to form NK1.1+CD3− or NK1.1−CD3− blasts by stimulation with IL-2 + IL-18 or IL-12 + IL-18, respectively, and the NK1.1 positivity on these blasts was again reversed after restimulation with an alternative combined stimulus. Both types of blasts produced enormously large amounts of IFN-γ in response to IL-12 + IL-18 and exhibited strikingly high levels of NK activity. These results indicate that IL-18 plays an obligatory role in inducing proliferation and activation of NK1.1+CD3−CD4−CD8− cells and that the expression of the NK1.1 marker is reversible, depending on the cytokine used for stimulation in combination with IL-18.
Pairing of Variable Heavy and Variable κ Chains in Individual Naive and Memory B Cells
Authors: Brezinschek Hans-Peter , Foster Sandra J. , Dörner Thomas , Brezinschek Ruth I. , Lipsky Peter E.
Published: January 2023

A functional Ig consists of two heterodimers each of which is composed of a heavy and a light chain. Although there is increasing knowledge about the events that govern the rearrangement of the genes encoding each individual chain, only very limited information is available about the mechanisms governing the pairing of variable heavy (VH) and variable light (VL) chains. Using a single cell PCR, we were able to obtain VH and Vκ chains from 144 individual human CD19+/IgM+ B cells. Pairing of specific VH or Vκ families was not observed, nor was the length or the amino acid composition of the CDR3s of VH and Vκ chains in individual B cells similar. Comparison of VH and Vκ genes in B cells in which one or both contained evidence of somatic hypermutation with those with no mutations revealed a significant decrease in the mean length of the VH CDR3. Moreover, there was a significant correlation between the frequencies of mutations in VH and Vκ gene pairs in individual B cells. These results indicate that Ag-mediated selection as opposed to VHDJH recombination or subsequent Ig chain pairing tended to approximate the CDR3 lengths and the frequency of mutations of VH and Vκ in individual B cells.
Phosphorylation Regulates the Delivery of MHC Class II Invariant Chain Complexes to Antigen Processing Compartments
Authors: Anderson Howard A. , Roche Paul A.
Published: January 2023

Transport of newly synthesized MHC class II glycoproteins to endosomal Ag processing compartments is mediated by their association with the invariant chain (Ii). Targeting to these compartments is dependent upon recognition of leucine-based endosomal/lysosomal targeting motifs in the Ii cytosolic domain. Ii, like many molecules that contain leucine-based endosomal targeting motifs, is phosphorylated in vivo. In this report we demonstrate that the cytosolic domain of the p35 Ii isoform is phosphorylated in class II Ii complexes isolated from human B lymphoblastoid cell lines or freshly obtained PBMC. Mutation of serine residue 6 or 8 prevents phosphorylation of Ii-p35 expressed in HeLa cells. Treatment of B lymphoblastoid cell lines with the serine/threonine kinase inhibitor staurosporine prevented Ii phosphorylation and significantly delayed trafficking of newly synthesized class II Ii complexes to endosomal Ag processing compartments. By contrast, staurosporine had no effect on the rate of transport of class I or class II glycoproteins through the Golgi apparatus and did not inhibit the delivery of the chimeric molecule Tac-DMβ to endocytic compartments, suggesting that staurosporine does not nonspecifically inhibit protein transport to the endocytic pathway. These results demonstrate that phosphorylation regulates the efficient targeting of MHC class II Ii complexes to Ag processing compartments and strongly suggest that this effect is mediated by phosphorylation of the MHC class II-associated Ii chain.
Phosphorylation of FADD/MORT1 and Fas by Kinases That Associate with the Membrane-Proximal Cytoplasmic Domain of Fas
Authors: Kennedy Norman J. , Budd Ralph C.
Published: January 2023

Fas (Apo-1, CD95), a member of the TNFR family, is expressed on a variety of cell types and transduces an apoptotic signal. Since Fas does not possess known enzymatic activities, proteins that interact with the cytoplasmic domain of Fas regulate the death signal. Several proteins have been identified, primarily using the yeast two-hybrid system, that associate with the death domain of Fas. One of these proteins, FADD/MORT1, can be phosphorylated, although the kinase that is responsible has not been identified. Furthermore, direct signaling connections between Fas and its known activation of sphingomyelinase or NF-κB have not been made, suggesting that other proteins may associate with Fas. In this study, a series of glutathione S-transferase fusion proteins was constructed that contained the cytoplasmic domain of murine Fas. These proteins were used to search for additional proteins that associate with Fas. Novel proteins, including kinases, were identified that associated specifically with the membrane-proximal, cytoplasmic tail of Fas but not with the death domain. One of these kinases phosphorylates FADD/MORT1. Moreover, the membrane-proximal region of Fas itself was phosphorylated by one of the associating kinases. These findings suggest that, similar to the Fas-related p55 TNFR, the membrane-proximal region of Fas likely participates in signaling.
Polymorphism in the Iα1 Germ-Line Transcript Regulatory Region and IgA Productivity in Patients with IgA Nephropathy
Authors: Yano Naohiro , Asakura Kumiko , Endoh Masayuki , Abe Yoshifumi , Nomoto Yasuo , Sakai Hideto , Kurokawa Kiyoshi , Tsukamoto Hideo
Published: January 2023

Enhanced in vivo and in vitro production of IgA has been reported in patients with IgA nephropathy (IgAN) and their family members. It is generally considered that IgA1 is a prominent subclass of IgA in IgAN. Although genetic mechanisms of IgA class switch recombination in IgAN have been studied enthusiastically, the critical factors that induce IgA1-specific class switching in IgAN have yet to be elucidated. A large body of data indicates that the germ-line transcript of Ig constant region (CH) genes that precedes actual class switching has regulatory effects on class switch recombination. To analyze structural abnormalities in the Iα1 germ-line transcript regulatory gene, a region about 1000 bp long located upstream of Iα1 exons was surveyed by the PCR-single strand conformation polymorphism method, and the polymorphism detected was confirmed by subsequent DNA sequencing. Three hot spots for point mutation were detected upstream of the promoter region of the Iα1 germ-line transcript, and the mutations were observed more frequently in patients than in controls. Patients with the mutations showed higher levels of serum IgA and higher in vitro IgA synthesis. In the luciferase assay, the regulatory gene with the mutations showed a potent effect for induction of the Iα1 germ-line transcript. The polymorphism in the Iα1 regulatory region possibly causes enhanced IgA production in some patients with IgAN.
HLA-E Surface Expression Depends on Binding of TAP-Dependent Peptides Derived from Certain HLA Class I Signal Sequences
Authors: Lee Ni , Goodlett David R. , Ishitani Akiko , Marquardt Hans , Geraghty Daniel E.
Published: January 2023

Previous studies showed that HLA-E was expressed in lymphoblastoid cell line (LCL) 721.221 cells, but surface expression was lacking. To determine the signals controlling surface expression, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner, a hybrid of HLA-E was identified, designated AEH, which differed from HLA-E by having the HLA-A2 signal sequence substituting for the HLA-E leader peptide. Transfection of LCL 721.221 cells with AEH induced HLA-E surface expression. Analysis of peptides bound to HLA-E revealed that a nonamer peptide derived from the A2 signal sequence was the predominant peptide bound. LCL 721.221 cells transfected with certain class I genes, including HLA-G, were also sufficient to promote peptide binding and HLA-E surface expression without increasing the level of HLA-E heavy chain synthesis. Peptides bound to HLA-E consisted of nine amino acids, with methionine at position 2 and leucine in the carboxyl-terminal position, and were nearly identical to the leader sequence-derived peptide previously shown to be a predominant peptide bound to the murine Qa-1 Ag. Signal peptides derived from certain HLA-B proteins with threonine in position 2 only marginally up-regulated HLA-E surface expression in .221 cells. An examination of HLA-E peptide binding in the TAP negative cell line .134 indicated that peptide binding to HLA-E was dependent on a functional TAP heterodimer regardless of whether peptide was available in cis, as in the AEH construct, or in trans, as in the class I transfectants of .221 cells.
TGF-β1 as an Endogenous Defender Against Macrophage-Triggered Stromelysin Gene Expression in the Glomerulus
Authors: Kitamura Masanori
Published: January 2023

Recent investigation has indicated that TGF-β1, the macrophage (Mφ) deactivator, may attenuate Mφ-mediated acute glomerular injury. Using stromelysin as an indicator, this study investigated whether and how endogenous TGF-β1 modulates the glomerular cell activation triggered by Mφ. Rat mesangial cells were stably transfected with a cDNA encoding the active form of TGF-β1 and a cDNA coding for a dominant-negative mutant of the TGF-βR type II. Compared with mock-transfected cells, TGF-β1 transfectants exhibited blunted expression of stromelysin in response to the Mφ-derived, inflammatory cytokine IL-1β. In contrast, mesangial cells expressing the dominant-interfering TGF-βR showed enhanced expression of stromelysin in response to IL-1β, suggesting that endogenous TGF-β functions as an autocrine inhibitor of the IL-1 response. In isolated, normal rat glomeruli, externally added TGF-β1 suppressed the induction of stromelysin by mediators that were elaborated by activated Mφ. Similarly, when isolated, nephritic glomeruli producing the active form of TGF-β1 were stimulated by IL-1β or Mφ-conditioned medium, the induction of stromelysin was dramatically suppressed as compared with normal glomeruli. To investigate whether endogenous TGF-β1 affects the glomerular cell activation triggered by Mφ, a technique for adoptive Mφ transfer was used. LPS-stimulated reporter Mφ were transferred into either normal rat glomeruli or nephritic glomeruli expressing active TGF-β1. In the normal glomeruli, stromelysin expression was markedly induced in resident cells after the transfer of activated Mφ. This induction was substantially repressed in those glomeruli producing active TGF-β1. These results reinforce the idea that TGF-β1 is an endogenous defender that attenuates certain actions of infiltrating Mφ in the glomerulus.
In Vivo Effects of a Bacterial Superantigen on Macaque TCR Repertoires
Authors: Kou Zhong-Chen , Halloran Matilda , Lee-Parritz David , Shen Ling , Simon Meredith , Sehgal Prabhat K. , Shen Yun , Chen Zheng W.
Published: January 2023

A macaque model was employed to explore staphylococcal enterotoxin B (SEB) superantigen-driven T lymphocyte responses. The SEB-reactive Vβ+ cell subpopulations demonstrated a striking tri-phase response in rhesus monkeys following an SEB challenge in vivo. The hyperacute down-regulation, seen as early as 2 h through 2 days after SEB injection, was characterized by a disappearance of the reactive Vβ-restricted PBL subpopulations from the circulation and decreased expression of these cell subpopulations in lymphoid tissues. Following this, a dominant expansion of reactive Vβ-expressing CD4+ cell subpopulations occurred in lymph nodes and spleens, whereas in the peripheral blood a preferential expansion of reactive Vβ-expressing CD8+ cell subpopulations was seen. An exhaustion of this response was then seen, with a prolonged decrease in the number of the reactive Vβ+ CD4+ lymphocyte subpopulations. Interestingly, monoclonal or oligoclonal dominance was seen in the reactive Vβ+ cell subpopulations in the period of the transition from the polyclonal cellular expansion to the exhaustion of the response, suggesting that some Vβ+ cell clones may be more resistant than others to superantigen-mediated depletion. These results indicate that in vivo SEB superantigen-mediated effect on lymphocyte subpopulations in macaques is complex, suggesting that profound dynamics in the TCR repertoires may in part account for the susceptibility of higher primates to SEB-induced diseases.
Cytokine Gene Therapy in Experimental Allergic Encephalomyelitis by Injection of Plasmid DNA-Cationic Liposome Complex into the Central Nervous System
Authors: Croxford J. Ludovic , Triantaphyllopoulos Kostas , Podhajcer Osvaldo L. , Feldmann Marc , Baker David , Chernajovsky Yuti
Published: January 2023

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system with many similarities to multiple sclerosis. The main effector cells involved are CD4+ T cells, recognizing encephalitogenic epitopes within the central nervous system, and macrophages, both of which secrete proinflammatory cytokines, such as IFN-γ and TNF. Studies have shown that immunomodulation of this inflammatory response by anti-inflammatory cytokines (IL-4, IL-10, IFN-β, and TGF-β) can reduce clinical severity in EAE. The importance of TNF in EAE has been demonstrated by using soluble TNF-receptor molecules to inhibit EAE. However, the limitation of this type of therapy is the necessity for frequent administration of cytokine proteins due to their short biologic half-life. This study demonstrates that EAE can be inhibited by a single injection of therapeutic cytokine (IL-4, IFN-β, and TGF-β) DNA-cationic liposome complex directly into the central nervous system. DNA coding for a novel, dimeric form of human p75 TNF receptor also ameliorated clinical EAE. Local administration of DNA-cationic liposome complex has identified gene targets that may be more efficiently exploited using vectors producing more stable expression for effective treatment of neuroimmunologic disease.
Short Peptide-Based Tolerogens Without Self-Antigenic or Pathogenic Activity Reverse Autoimmune Disease
Authors: Karin Nathan , Binah Ofer , Grabie Nir , Mitchell Dennis J. , Felzen Bella , Solomon Matthew D. , Conlon Paul , Gaur Amitabh , Ling Nicholas , Steinman Lawrence
Published: January 2023

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87–99), is a major target of T cells in brain lesions of multiple sclerosis (MS), and this peptide can trigger experimental autoimmune encephalomyelitis (EAE). We designed truncated peptides based on this pathogenic 13-mer that are not antigenic. These short peptides reduced production of IFN-γ and TNF-α in vivo. Moreover, paraplegic rats given the 7-mer FKNIVTP in soluble form showed total reversal of paralysis in 24 h. Truncated peptides that are too small to stimulate antigenic responses to pathogenic regions of myelin basic protein are nevertheless effective tolerogens and are able to anergize autoreactive T cells. Short peptide-based tolerogens, devoid of immunogenic and pathogenic potential, may be attractive for therapy of autoimmune diseases.
Treponema pallidumandBorrelia burgdorferiLipoproteins and Synthetic Lipopeptides Activate Monocytic Cells via a CD14-Dependent Pathway Distinct from That Used by Lipopolysaccharide
Authors: Sellati Timothy J. , Bouis Deborah A. , Kitchens Richard L. , Darveau Richard P. , Pugin Jerome , Ulevitch Richard J. , Gangloff Sophie C. , Goyert Sanna M. , Norgard Michael V. , Radolf Justin D.
Published: January 2023

Lipoproteins of Treponema pallidum and Borrelia burgdorferi possess potent proinflammatory properties and, thus, have been implicated as major proinflammatory agonists in syphilis and Lyme disease. Here we used purified B. burgdorferi outer surface protein A (OspA) and synthetic lipopeptides corresponding to the N-termini of OspA and the 47-kDa major lipoprotein immunogen of T. pallidum to clarify the contribution of CD14 to monocytic cell activation by spirochetal lipoproteins and lipopeptides. As with LPS, mouse anti-human CD14 Abs blocked the activation of 1,25-dihydroxyvitamin D3-matured human myelomonocytic THP-1 cells by OspA and the two lipopeptides. The existence of a CD14-dependent pathway was corroborated by using undifferentiated THP-1 cells transfected with CD14 and peritoneal macrophages from CD14-deficient BALB/c mice. Unlike LPS, cell activation by lipoproteins and lipopeptides was serum independent and was not augmented by exogenous LPS-binding protein. Two observations constituted evidence that LPS and lipoprotein/lipopeptide signaling proceed via distinct transducing elements downstream of CD14: 1) CHO cells transfected with CD14 were exquisitely sensitive to LPS but were lipoprotein/lipopeptide nonresponsive; and 2) substoichiometric amounts of deacylated LPS that block LPS signaling at a site distal to CD14 failed to antagonize activation by lipoproteins and lipopeptides. The combined results demonstrate that spirochetal lipoproteins and lipopeptides use a CD14-dependent pathway that differs in at least two fundamental respects from the well-characterized LPS recognition pathway.
Control ofLeishmania majorInfection in Mice Lacking TNF Receptors
Authors: Nashleanas Michelle , Kanaly Suzanne , Scott Phillip
Published: January 2023

TNF participates in the induction of nitric oxide (NO) production and macrophage activation, leading to the elimination of intracellular pathogens. We previously found that TNF receptor p55-deficient mice (TNFRp55−/−) control replication of Leishmania major in vivo but fail to resolve their lesions. Here we report that mice lacking the p75 receptor (TNFRp75−/−) or both receptors (TNFRp55p75−/−), also control parasite replication, albeit mice lacking the p55 receptor (either TNFRp55−/− or TNFRp55p75−/−) are delayed in their elimination of L. major compared with controls. All TNF receptor-deficient mice developed a Th1-type immune response and up-regulated inducible NO synthase (iNOS) mRNA gene expression in lesions during infection. Thus, neither TNF receptor appears to be absolutely required for NO production or elimination of L. major in vivo. In vitro, however, while macrophages from naive TNFRp75−/− mice could be activated to produce NO and kill L. major, we observed a defect in NO production and parasite killing by resident peritoneal macrophages from naive TNFRp55−/− or TNFRp55p75−/− mice. However, when macrophages were elicited with leishmanial Ag from 4-wk-infected TNFRp55−/−or TNFRp55p75−/− mice, they produced NO and were leishmanicidal. These data suggest that the TNFRp75 plays no essential role in L. major infection in mice and that the p55 receptor may be required for optimal macrophage activation. However, the results also show that a mechanism exists by which macrophages can be primed in vivo during L. major infection to produce NO and kill L. major in the absence of signaling through either of the TNF receptors.
Nonopsonic and Opsonic Association ofMycobacterium tuberculosiswith Resident Alveolar Macrophages Is Inefficient
Authors: Stokes Richard W. , Thorson Lisa M. , Speert David P.
Published: January 2023

The association of Mycobacterium tuberculosis with alveolar macrophages (Mφ) in a serum-free environment is a crucial first step in the pathogenesis of this facultative intracellular pathogen. We present data demonstrating that freshly explanted alveolar Mφ do not efficiently bind M. tuberculosis in a serum-free system, although a small subpopulation of these Mφ (10–15%) can bind mycobacteria. In contrast, almost 100% of a peritoneal Mφ population bind mycobacteria under the same conditions. The poor binding of mycobacteria by alveolar Mφ does not reflect a general inability to associate with particles; binding and ingestion of latex beads and zymosan particles were comparable with that seen with peritoneal Mφ. Resident alveolar Mφ did not efficiently bind mycobacteria in the presence of serum and expressed poorly several Mφ surface receptors, including CR3. Furthermore, we demonstrate that bovine surfactant protein A does not enhance the association of M. tuberculosis with alveolar Mφ. Differentiation of alveolar Mφ in vitro resulted in increased expression of Mφ surface receptors and an increased capacity to bind mycobacteria in the presence and absence of serum. Evidence is presented that opsonic binding of M. tuberculosis by differentiated alveolar Mφ is mediated by complement and CR3, and that the poor binding by resident alveolar Mφ is due to their poor expression of CR3. The receptor mediating nonopsonic binding of M. tuberculosis to differentiated alveolar Mφ was not unequivocally identified in this study, but could also be CR3.
The Fas Counterattack In Vivo: Apoptotic Depletion of Tumor-Infiltrating Lymphocytes Associated with Fas Ligand Expression by Human Esophageal Carcinoma
Authors: Bennett Michael W. , O’Connell Joe , O’Sullivan Gerald C. , Brady Ciaran , Roche Desmond , Collins J. Kevin , Shanahan Fergus
Published: January 2023

Various cancer cell lines express Fas ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression has been demonstrated in several human malignancies in vivo. We sought to determine whether human esophageal carcinomas express FasL, and whether FasL expression is associated with increased apoptosis of tumor-infiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Using in situ hybridization and immunohistochemistry, respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n = 6; adenocarcinoma, n = 2). The Extent of FasL expression was variable, with both FasL-positive and FasL-negative neoplastic regions occurring within tumors. TIL were detected by immunohistochemical staining for the leukocyte common Ag, CD45. FasL expression was associated with a mean fourfold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6- to 12-fold, n = 6, p < 0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean twofold increase in detectable cell death among TIL in FasL-positive areas compared with FasL-negative areas (range 1.6- to 2.4-fold, n = 6, p < 0.05). In conclusion, we demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest Fas-mediated apoptotic depletion of TIL in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer.
IL-3 Enhances Both Presentation of Exogenous Particulate Antigen in Association with Class I Major Histocompatibility Antigen and Generation of Primary Tumor-Specific Cytolytic T Lymphocytes
Authors: Yeh Kun-Yun , McAdam Alexander J. , Pulaski Beth A. , Shastri Nilabh , Frelinger John G. , Lord Edith M.
Published: January 2023

Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.14 (B3Z) restricted to Ova/Kb, was used as an indicator for presentation of particulate Ag with class I MHC. When activated, this hybridoma expresses lacZ, allowing a simple colorimetric measurement of Ag-specific T cell stimulation. We demonstrated that bone marrow cells stimulated by IL-3 in vivo and in vitro exhibited significantly increased presentation of exogenous OVA linked to beads. Lysate from OVA-transfected line 1 murine lung adenocarcinoma cells (line 1/OVA) was also presented by IL-3-stimulated bone marrow cells, suggesting that these APC can process tumor fragments or debris. Studies using TAP1/2-deficient mice and Ag presentation inhibitors indicate that this exogenous Ag presentation is mediated via the conventional class I MHC pathway. Adoptive transfer of IL-3-stimulated bone marrow cells pulsed with lysate from line 1/OVA tumor cells into naive recipient mice led to the generation of a potent CTL response. These observations indicate that use of such cells may provide a new avenue for development of tumor vaccines.
Relationships Among TCR Ligand Potency, Thresholds for Effector Function Elicitation, and the Quality of Early Signaling Events in Human T Cells
Authors: Hemmer Bernhard , Stefanova Irena , Vergelli Marco , Germain Ronald N. , Martin Roland
Published: January 2023

Determining how receptor ligand quality and quantity together control the biologic responses of T cells is central to understanding normal and pathologic T cell immunity. Here we have carefully examined how variations in antigenic peptide structure and dose affect multiple functional responses of human T cell clones and have correlated these observations with proximal TCR signaling events induced by the same set of related ligands. As the Ag concentration increases, effector functions are elicited according to a clone-specific hierarchy. The absolute amount of each peptide required to stimulate the entire set of effector functions (potency) differs markedly among ligands for a single TCR, correlating with the efficiency of TCR down-modulation and the extent of ZAP-70 activation. However, distinct patterns of TCR ζ-chain phosphorylation were observed, with the ratios of TCRζ isoforms relating to ligand agonist potency. The appearance of partially phosphorylated TCRζ isoforms was paralleled by relative changes in certain response thresholds within the hierarchy. Thus, a combination of density, potency, and quality of signaling all contribute to the distinct effects of agonist ligands on T cell immunity.
Expression and Characterization of Recombinant Soluble Peptide: I-A Complexes Associated with Murine Experimental Autoimmune Diseases
Authors: Radu Caius G. , Ober Bertram T. , Colantonio Lucia , Qadri Ayub , Ward E. Sally
Published: January 2023

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to β-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.
Structural and Functional Analysis of J Chain-Deficient IgM
Authors: Wiersma Erik J. , Collins Cathy , Fazel Shafie , Shulman Marc J.
Published: January 2023

Previous studies have discerned two forms of polymeric mouse IgM: moderately cytolytic (complement-activating) pentamer, which contains J chain, and highly cytolytic hexamer, which lacks J chain. To investigate the relationships among polymeric structure, J chain content, and cytolytic activity, we produced IgM in J chain-deficient and J chain-proficient mouse hybridoma cell lines. Both hexamer and pentamer were produced in the absence as well as the presence of J chain. Hexameric IgM activated (guinea pig) complement approximately 100-fold more efficiently than did J chain-deficient pentamer, which, in turn, was more active than J chain-containing pentamer. These results are consistent with the hypothesis that J chain-containing pentamer cannot activate complement. We also analyzed the structure of IgM-S337, in which the μ-chain bears the C337S substitution. Like normal IgM, IgM-S337 was formed as a hexamer and as both J chain deficient- and J chain-containing pentamers. Unlike normal IgM, IgM-S337 dissociated in SDS into various subunits. For IgM-S337 pentamer, the predominant subunits migrated as μ2κ2 and μ4κ4, and the subunit distribution was unaltered by J chain. However, J chain was found only in the μ2κ2 species, suggesting that some arrangement of inter-μ bonds directs incorporation of J chain. IgM-S337 hexamer also dissociated to μ2κ2 and μ4κ4, but also yielded several species migrating much more slowly in SDS-PAGE than wild-type μ12κ12. To account for these forms, we propose that each μ-chain can interact with three other μ-chains and that some hexameric molecules contain two catenated μ6κ6 circles.