bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloid differentiation treatment to diminish the presence of immune-suppressive CD34+ cells within human head and neck squamous cell carcinomas.
Authors: Young M R , Wright M A , Pandit R
Published: January 2023

Within human head and neck squamous cell carcinomas (HNSCC) that produce granulocyte-macrophage CSF are CD34+ cells that exhibit natural suppressive (NS) activity. The present study aimed to identify how these NS cells mediate suppression and how to diminish their presence. CD34+ cells that were immunomagnetically isolated from fresh surgical HNSCC specimens produced a soluble product that blocked normal T cell stimulation through the TCR/CD3 complex. This inhibitory activity could be neutralized with Abs to TGF-beta1. Since prior studies showed that the CD34+ NS cells within HNSCC cancers are myelomonocytic progenitor cells, the feasibility of using cytokines that can induce myeloid cell differentiation to diminish the presence of CD34+ NS cells was tested. Adding low doses of 100 U/ml IFN-gamma plus 10 U/ml TNF-alpha to bulk cultures of dissociated HNSCC cancers diminished the frequency of CD34+ cells. Studies with CD34+ cells that were isolated from the HNSCC cancers showed that this cytokine treatment induced differentiation of the CD34+ cells predominantly into monocytic cells. The consequence of treating CD34+ NS cells with the myeloid differentiation treatment was the loss of suppressive activity, a decline in TGF-beta production, and the production of TNF-alpha by the resulting monocytic cells. In HNSCC bulk cultures containing high levels of CD34+ NS activity, IFN-gamma/TNF-alpha not only reduced CD34+ cell levels, but also increased the capacity of the intratumoral T cells to express the p55 IL-2R. These studies show that IFN-gamma/TNF-alpha can induce differentiation of TGF-beta-secreting CD34+ NS cells into nonsuppressive monocytic cells that secrete TNF-alpha.
Ig Sgamma3 DNA-specifc double strand breaks are induced in mitogen-activated B cells and are implicated in switch recombination.
Authors: Wuerffel R A , Du J , Thompson R J , Kenter A L
Published: January 2023

The looping-out and deletion model of switch recombination suggests that double strand breaks (DSBs) may occur in switch regions and participate in the recombination transaction. A DSB assay based on the ligation-mediated PCR method has been devised for the Sgamma3 region. Mitogen-inducible DSBs have been detected in Sgamma3 DNA of normal splenic B cells but not in splenic T cells or thymocytes. The breaks are restricted to the Sgamma3 region and are sequence specific. Examination of the sequence surrounding the DSBs has led to the derivation of a consensus sequence for DSB formation. Induction of Sgamma3-specific DSBs is correlated with the onset of switch recombination. The DSBs flank clusters of Sgamma3 recombination breakpoints, suggesting that processing of the broken ends may occur during the recombination reaction.
The PU.1 binding site is a cis-element that regulates pro-B/pre-B specificity of Vkappa-Jkappa joining.
Authors: Hayashi R , Takemori T , Kodama M , Suzuki M , Tsuboi A , Nagawa F , Sakano H
Published: January 2023

We have previously shown that the PU.1 binding motif (GAG GAA) in the 3'-enhancer region in the Ig kappa gene is responsible for the negative regulation of tissue (B/T)-specific Vkappa-Jkappa joining. Here we report that the PU.1 binding site also regulates the stage (pro-B/pre-B) specificity of Vkappa-Jkappa joining. In the substrate with base substitutions in the PU.1 binding motif, recombination took place in both pro-B (B220dull/CD43+) and pre-B (B220dull/CD43-) cells. In the transcriptional regulation, the PU.1 motif acts in a positive manner cooperatively with the nuclear factor-EM5 (or PIP) motif (GAAAAC), which is located 2 bp downstream from the PU.1 motif. Interestingly, base substitutions in the nuclear factor EM5 (PIP) motif did not affect the pro-B/pre-B specificity of Vkappa-Jkappa joining. Thus, the PU.1 motif regulates both temporal and tissue-specific rearrangements, while nuclear factor-EM5 is not involved in the regulation of Ig kappa recombination.
Stromal cell modulation of negative regulatory signals that influence apoptosis and proliferation of B lineage lymphocytes.
Authors: Borghesi L A , Smithson G , Kincade P W
Published: January 2023

The bone marrow microenvironment influences whether a given B cell proliferates, differentiates, or undergoes apoptosis. In this report, we demonstrate that apoptosis of primary murine B lymphocyte precursors can be regulated either positively or negatively by stroma. Several stromal lines that support lymphocyte outgrowth suppressed the spontaneous apoptosis of pre-B cells by as much as 90%. Direct contact with stromal cells more effectively protected lymphocytes than did stromal cell-CM or a collection of recombinant cytokines. In contrast, one unique stromal cell clone actually induced lymphocyte apoptosis, and a second line appeared inert. A survey of adherent cell lines suggested that expression of life-sparing molecules is widespread but not ubiquitous. Experiments with neutralizing Abs to CD44, vascular cell adhesion molecule-1 (VCAM-1), CD9, intercellular adhesion molecule-1 (ICAM-1), or ICAM-2 suggested that these interaction molecules do not deliver short-term survival signals to B cell precursors. Of particular interest, direct interaction with lymphocyte-supportive stromal cells minimized the negative regulatory effects of IL-1alpha, and a glucocorticoid, but not IFN-beta or PGE2. These results demonstrate that the effect of negative regulators depends upon the context in which these signals are presented. As molecules that influence B lymphopoiesis are better defined, it will be important to consider the role of each in combination with other stimuli.
Differential effects of B7-1 blockade in the rat experimental autoimmune encephalomyelitis model.
Authors: Gallon L , Chandraker A , Issazadeh S , Peach R , Linsley P S , Turka L A , Sayegh M H , Khoury S J
Published: January 2023

Blocking the CD28-B7 T cell costimulatory activation pathway protects animals from developing experimental autoimmune encephalomyelitis (EAE). In the mouse EAE model, selective blockade of B7-1 by specific mAbs has been shown to protect animals from EAE. In the Lewis rat model, we have shown that CD28-B7 blockade by systemic administration of CTLA4Ig prevents actively induced EAE. Since CTLA4Ig binds to both B7-1 and B7-2, we used a mutant form of CTLA4Ig (CTLA4IgY100F) that binds only B7-1, to study the role of B7-1 blockade in this model. Such a reagent avoids the potential of signaling by mAbs. Systemic administration of CTLA4IgY100F in several dosing regimens did not protect from EAE, and in some protocols worsened disease, while CTLA4Ig was always protective. In contrast, systemic injection of APCs preincubated ex vivo with the encephalitogenic peptide of myelin basic protein and either CTLA4Ig or CTLA4IgY100F protected recipients from disease. In vitro studies confirmed the in vivo observations and showed that primed lymph node cells from protected animals had decreased proliferative responses to myelin basic protein as compared with controls, while lymphocytes from animals treated with systemic CTLA4gY100F did not. More importantly, systemic administration of CTLA4IgY100F abrogated the protective effect of ex vivo treated APCs. These data suggest an important regulatory role for B7-1, perhaps through binding to CTLA4, in this model of EAE. Understanding the role and mechanisms of selective blockade of costimulatory molecules has implications for therapy of autoimmune disease.
TCR-beta repertoire development in the mouse embryo.
Authors: Ema H , Cumano A , Kourilsky P
Published: January 2023

We studied the TCR beta-chain repertoire in thymocytes along embryonic development. At day 14 of gestation, complete V-D-J rearrangements of the TCR-beta locus were detected at the messenger RNA level, and a total of 56 different rearrangements were observed in one thymus. At this stage, thymocytes showed preferential usage of TCR-betaV1, TCR-betaV2, and TCR-betaV8 together with TCR-betaJ1S1, -J1S2, and -J2S7 segments. This frequent usage of V and J segments was similar to the usage seen in the adult thymus. Assuming that day 14 thymocytes were not subjected to specificity selection, we conclude that the expression of TCR-betaV and J segments is biased from the beginning of T cell repertoire creation. The first detectable transcripts of rearranged TCR-betas were different among individual fetuses and constituted a random representation of the ones seen at later stages of thymic ontogeny. The diversity of TCR-beta rearrangements increased with time and became indistinguishable from that of the adult by day 16. The first rearranged TCR-alphas were expressed at day 17 and their diversity increased thereafter. We conclude that the diversification of the beta-chain repertoire takes place in 3 days, before the alpha-chain rearrangement begins in transition from CD4/CD8 double negative to double positive cells.
CD40-mediated signals inhibit the binding of TNF receptor-associated factor 2 to the CD40 cytoplasmic domain.
Authors: Chaudhuri A , Orme S , Eilam S , Cherayil B J
Published: January 2023

TNF receptor-associated factor 2 (TRAF2) is a signal-transducing protein associated with the CD40 cytoplasmic domain. It has been hypothesized that during signal transduction, TRAF2 must be released from CD40 in order for it to interact with downstream signaling molecules. We found that CD40 and TRAF2 were constitutively associated with each other in a human B cell line. Following stimulation with an anti-CD40 Ab, a decrease in the amount of CD40-associated TRAF2 was observed that could not be explained by a change in total level of either of the proteins. These results, as well as similar findings obtained with 293 cells overexpressing CD40 and TRAF2, suggested that CD40-mediated signals inhibited the CD40-TRAF2 interaction. We then conducted binding studies using CD40 cytoplasmic domain fusion proteins and TRAF2 derived from either control or CD40-stimulated cell lines. These in vitro studies also indicated that the binding of TRAF2 to the CD40 cytoplasmic domain was inhibited by CD40 stimulation. The results of these experiments, as well as differences between the in vitro and in vivo findings, indicated that multiple mechanisms were involved in the inhibition of the CD40-TRAF2 interaction by CD40 signals. Possible mechanisms of inhibition are discussed based on mapping of the TRAF2 binding site on the CD40 cytoplasmic domain.
Specific resistance of T cells to CD95-induced apoptosis during S phase of the cell cycle.
Authors: Dao T , Huleatt J W , Hingorani R , Crispe I N
Published: January 2023

We have examined the susceptibility of mouse thymocytes and Con A-activated mature T cells to CD95 (Fas; APO-1)-induced apoptosis at different phases of the cell cycle. Signaling through CD95, induced by murine CD95 ligand expressed on fibroblasts, resulted in the preferential apoptosis of T cells in G0-G1 phase of the cell cycle. T cells in S phase were selectively protected. CD95-induced apoptosis was inhibited by exogenous IL-2, which increases the percentage of cells in S phase. The inverse relationship between DNA synthesis and apoptosis in CD95-ligated T cells was not observed during the spontaneous death of T cells in culture or during propriocidal apoptosis due to TCR cross-linking, to which cells in S phase were susceptible. These results show that in T cells there is no distinctively apoptosis-vulnerable phase of the cell cycle; instead, apoptosis can occur at different phases of the cycle depending on the apoptotic stimulus.
In vivo administration of flt3 ligand markedly stimulates generation of dendritic cell progenitors from mouse liver.
Authors: Drakes M L , Lu L , Subbotin V M , Thomson A W
Published: January 2023

The study of liver dendritic cells (DC) and their progenitors is restricted by the small numbers that can be isolated or propagated from normal hepatic tissue. We examined the ex vivo growth, phenotype, and function of these cells after the administration to mice of the recently cloned hemopoietic growth factor flt3 ligand (FL), which is highly effective in mobilizing stem/progenitor cells. FL treatment (10 microg/day for 10 days) resulted in a mean 14-fold increase in the absolute number of nonparenchymal cells recovered from collagenase-digested livers compared with the control value. Culture of these nonparenchymal cells in granulocyte-macrophage CSF (GM-CSF; 1000 U/ml) resulted in the early formation of proliferating cell clusters and maximal release (within 4-5 days) of markedly increased numbers of nonadherent, low buoyant density cells per liver. Maximal release of low buoyant density cells propagated from control livers was at the later time of 6 to 8 days. Cells from both sources were DEC-205+, CD11c+, MHC class II+, CD80(low) (i.e., low level of CD80), CD86(low) and CD40(low). This immature phenotype was linked to poor T cell allostimulatory activity, indicative of DC progenitors. Propagation of cells from livers of FL-treated mice in GM-CSF and IL-4 resulted in a more mature DC phenotype and function. Maturational changes were also observed following exposure of the GM-CSF-stimulated progenitors to type 1 collagen for 3 additional days. The ability of FL to boost production of large numbers of liver DC progenitors provides opportunities for the further study of these important APC in normal liver immunobiology and in immune-mediated hepatic disorders.
The cytoplasmic domain of CD5 mediates both TCR/CD3-dependent and -independent diacylglycerol production.
Authors: Simarro M , Pelassy C , Calvo J , Places L , Aussel C , Lozano F
Published: January 2023

CD5 is a 67-kDa surface glycoprotein found in association with the Ag receptor complex on both T and B lymphocytes. CD5 modulates Ag receptor-mediated immune responses, but the molecular mechanisms of its action remain unclear. In this respect, the assessment of the relative and unique contribution of CD5 in cell signaling events is a crucial point. We have used Jurkat variants and anti-CD5 mAbs to show that the CD5 signaling pathway is distinct from that used by the TCR/CD3 complex. We hereby identify two independent mechanisms of CD5-mediated diacylglycerol release by virtue of their different kinetics: 1) an early and transient diacylglycerol increase that results from the activation of a phosphatidylcholine-specific phospholipase C, and 2) a late and sustained increase that requires de novo phospholipid synthesis. Studies performed on a TCR/CD3-deficient Jurkat cell variant indicate that only the CD5-mediated phosphatidylcholine-specific phospholipase C activation is dependent on TCR/CD3 expression. Mutational analyses of CD5 demonstrate that both mechanisms are dependent on the integrity of the CD5 distal cytoplasmic region. Our results show that CD5 is a signaling molecule per se that uses mechanisms resembling those used by some cytokine receptors (such as IL-1 or TNF receptors) to modulate lymphocyte activation.
Mechanism for enhanced external transfer of dimeric IgA over pentameric IgM: studies of diffusion, binding to the human polymeric Ig receptor, and epithelial transcytosis.
Authors: Natvig I B , Johansen F E , Nordeng T W , Haraldsen G , Brandtzaeg P
Published: January 2023

Transport of polymeric Igs (pIgA and pIgM) across secretory epithelia is mediated by the polymeric Ig receptor (pIgR), also known as the transmembrane secretory component. Compared with local production, external transfer of pIgA is favored 6- to 12-fold over that of pIgM on a molar basis. This transfer may be modulated at several levels: diffusion through matrix and basement membranes, ligand affinity for pIgR, and efficiency of epithelial transcytosis. To investigate these possibilities, we compared the ability of Madin-Darby canine kidney epithelial cells transfected with human pIgR to transport pIgA vs pIgM from the basolateral to the apical face, and examined the inhibitory effect of various filter types used for mounting of the monolayer. Binding data showed that pIgR bound pIgA and pIgM with similar affinity. Internalization of both ligands was fast and took place at similar rates; transcytosis was also found to be equally efficient at the molar level. Thus, the overall rate of transport across the epithelial monolayer was comparable for pIgA and pIgM, and was not further enhanced by ligand stimulation over a 20-fold increased concentration level. Conversely, pIgA had a considerable advantage over pIgM in passive diffusion assays performed in vitro. Moreover, in situ immunofluorescence staining showed retention of IgM over IgA and IgG in mucosal basement membrane zones, in contrast to the preferential epithelial uptake of IgA and, less so, IgM. The biologic consequences of the highly efficient epithelial pIg transport, and the diffusion advantage of pIgA over pIgM, are discussed in relation to the evolution and function of secretory Abs.
Alteration of glycosylation renders HIV sensitive to inactivation by normal human serum.
Authors: Reed D J , Lin X , Thomas T D , Birks C W , Tang J , Rother R P
Published: January 2023

Retroviruses from various mammalian species, excluding humans, are effectively inactivated in normal human serum (NHS). Recent studies have shown that NHS inactivation of retroviruses occurs through natural Ab recognition of a terminal glycosidic moiety on the viral envelope that is acquired during replication in the host cell. This carbohydrate structure (the alpha-galactosyl epitope) is expressed on the cells of most mammals, with the exception of humans and other Old World primates. In this study, NHS sensitivity of HIV was assessed following viral propagation in human cells that were manipulated to express the alpha-galactosyl epitope. HUT-78 cells were transduced with an exogenous alpha1-3-galactosyl transferase gene, which codes for the terminal glycosyl transferase responsible for generation of the alpha-galactosyl epitope. The transduced HUT-78 cells expressed high levels of the alpha-galactosyl epitope on their membrane surface, rendering them sensitive to killing in NHS. Similarly, HIV passaged through these cells acquired the alpha-galactosyl epitope in association with the envelope glycoprotein gp120 and was also effectively inactivated in NHS. Viral inactivation was abolished by the addition of a synthetic disaccharide that contains the alpha-galactosyl epitope, indicating that virolysis is mediated by anti-alpha-galactosyl natural Ab. These results demonstrate that, like other retroviruses bearing the alpha-galactosyl epitope, HIV modified to express this epitope is inactivated in NHS. Furthermore, these data suggest that expression of the alpha-galactosyl epitope on the surface of viruses may have implications in the interspecies transmission of such viruses to humans.
Somatic hypermutation: mutations 3' of rabbit VDJ H-chain genes.
Authors: Lanning D K , Knight K L
Published: January 2023

VDJ genes of rabbit B cells are diversified by a somatic gene conversion-like mechanism in which V(H) gene segments 5' of the VDJ gene serve as donors. To assess whether somatic mutation also contributes to Ab diversification in the rabbit, we searched for mutations 3' of VDJ genes because mutations in this region presumably result from somatic mutation rather than gene conversion. We PCR-amplified and cloned the region extending 534 bp 3' of VDJ genes from splenic DNA of young and adult rabbits. Results of nucleotide sequence analysis of the clones revealed a high mutation frequency (2.4%) within the first 120 bp downstream of VDJ genes. This frequency decreased rapidly with increasing distance 3' of the VDJ genes. The mutations demonstrated both strand bias and dinucleotide preferences characteristic of somatic hypermutation. We conclude that Ab diversity in rabbit is generated not only by somatic gene conversion but also by somatic hypermutation.
B7-2 blockade enhances T cell responses to Leishmania donovani.
Authors: Murphy M L , Engwerda C R , Gorak P M , Kaye P M
Published: January 2023

Infection with Leishmania donovani has been reported to induce a dominant Th1-type response in all strains of mice examined, providing a model for examining the regulation of Th1 responses in the relative absence of Th2 cross-regulation. Here we demonstrate that blockade of the costimulatory molecule B7-2, but not B7-1, has significant effects on disease progression, measured as day 28 parasite burden in the liver. The effects of B7-2 blockade were associated with increased IFN-gamma production, as determined 1) following restimulation with specific Ag, 2) by enumeration of IFN-gamma-secreting cells using ELISPOT assays, and 3) by analysis of IFN-gamma mRNA by reverse transcription-PCR. Surprisingly, IL-4-producing cells were also readily detected in infected mice by ELISPOT analysis. The frequency of these IL-4-producing cells and of IL-4 mRNA levels was also enhanced in the liver of infected mice treated with anti-B7-2 mAb. Administration of anti-B7-2 from the day of infection or delaying its administration until day 3 after infection had similar effects. Parasite-specific IgG1 and IgG2a responses were either unaffected or marginally increased following anti-B7-2 administration, contrary to the inhibitory effect of this treatment on responses to the T-dependent Ag DNP-BSA. These data support a model of T cell activation whereby B7-2/CD28 interactions play a relatively redundant role in initial T cell activation, but in which interference with later B7-2/CTLA4 interaction potentiates established cytokine responses.
Differential roles of selectins and the alpha4-integrin in acute, subacute, and chronic leukocyte recruitment in vivo.
Authors: Johnston B , Walter U M , Issekutz A C , Issekutz T B , Anderson D C , Kubes P
Published: January 2023

Adhesion blocking mAbs specific for rat P-, E-, and L-selectin and the alpha4-integrin were used to characterize leukocyte recruitment mechanisms in models of LTC4 (acute), LPS (subacute), and adjuvant-induced (chronic) inflammation. Intravital microscopy was employed to measure leukocyte rolling and adhesion in rat mesenteric venules. Superfusing the mesentery with 20 nM LTC4 elicited an increase in leukocyte rolling (66.8 +/- 3.8 vs 18.2 +/- 3.2 cells/min control) that was completely eliminated by an anti-rat P-selectin mAb. Superfusion with 1 microg/ml LPS induced a significant increase in leukocyte rolling within 15 min (73 +/- 8 vs 33 +/- 6 cells/min control). Rolling increased further starting at 105 min and peaked by 150 min (141 +/- 23 cells/min). LPS-induced leukocyte rolling was eliminated during the first 90 min by the P-selectin mAb. The later increase in leukocyte rolling was not prevented by a second treatment with P-selectin mAb or a function-blocking mAb against rat E-selectin. This later phase of leukocyte rolling was blocked by treatments with mAbs against either the alpha4-integrin or L-selectin. Twelve days following Mycobacterium butyricum immunization, 300 to 500 rolling cells/min were observed. This could be reduced approximately 50 to 60% by mAb against either the alpha4-integrin or L-selectin. The combination of both mAbs eliminated approximately 90% of rolling. Neither the P- nor E-selectin mAbs reduced rolling in this chronic inflammatory model. This study highlights differences in leukocyte adhesive mechanisms elicited by different stimuli and at different time points within the same vascular bed.
The fate of pro-TNF-alpha following inhibition of metalloprotease-dependent processing to soluble TNF-alpha in human monocytes.
Authors: Solomon K A , Covington M B , DeCicco C P , Newton R C
Published: January 2023

Human monocytes rapidly produce TNF-alpha following activation by bacterial LPS. The message for TNF-alpha encodes a 26-kDa protein that is proteolytically processed to the secreted 17-kDa form. Sequencing of the N terminus of the protein secreted by monocytes shows processing of the 26-kDa pro-TNF-alpha to a mature form at the projected metalloprotease cleavage site to generate 17-kDa TNF-alpha with the N terminus VRSSSR-. The addition of hydroxamic acid-based metalloprotease inhibitors to the cell culture is capable of blocking >95% of the production of soluble TNF-alpha and leads to a transient, but reproducible, increase in cell surface TNF-alpha as measured by FACS analysis. The cell surface TNF-alpha was demonstrated to increase the cell's ability to kill L929 tumor targets and induce PG production from human gingival fibroblasts. The buildup of cell surface TNF-alpha is unable to account for the TNF-alpha that is not secreted when inhibitor is present. Pulse-chase analysis of the cells demonstrates rapid degradation of the pro-TNF-alpha that remains unprocessed in the monocytes. Through inhibition of processing and secretion by brefeldin A, processing was shown to occur at a postendoplasmic reticulum site and is closely associated with movement to the cell surface.
Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation.
Authors: Lamkhioued B , Renzi P M , Abi-Younes S , Garcia-Zepada E A , Allakhverdi Z , Ghaffar O , Rothenberg M D , Luster A D , Hamid Q
Published: January 2023

Presently, there is considerable evidence for the participation of eosinophils in the pathophysiology of human bronchial asthma. Although increased numbers of eosinophils are present in the airways and bronchoalveolar lavage (BAL) fluid of atopic asthmatics, the mechanisms responsible for their preferential accumulation are still largely unknown. Eotaxin is a chemokine that promotes the selective recruitment of eosinophils. We report that atopic asthmatic patients have high concentrations of eotaxin in BAL fluid and an increased expression of eotaxin mRNA and protein in the epithelium and submucosa of their airways when compared with normal controls. In the BAL cells from asthmatic patients, eotaxin immunoreactivity colocalized predominantly to macrophages (62.2%), with a lesser contribution from T cells (16.3%) and eosinophils (8.9%). BAL fluid from asthmatics contained chemotactic activity for eosinophils that was attributable in part to the presence of eotaxin. Moreover, eotaxin was more effective at inducing in vitro eosinophil chemotaxis when eosinophils were stimulated with IL-5 (a cytokine that enhances the effector capacity of mature eosinophils). These observations suggest that eotaxin contributes to the pathogenesis of asthma by the specific recruitment of eosinophils into the airways.
Myelin protein expression is increased in lymph nodes of mice with relapsing experimental autoimmune encephalomyelitis.
Authors: MacKenzie-Graham A J , Pribyl T M , Kim S , Porter V R , Campagnoni A T , Voskuhl R R
Published: January 2023

Myelin proteins had been thought to be sequestered behind the blood-brain barrier. Recently, however, myelin proteins have been found to be expressed in lymphoid tissues. The myelin basic protein (MBP) gene is embedded within a larger transcription unit called the golli-MBP gene. This larger gene encodes both the "classic" MBPs as well as the structurally related golli-MBPs. In this study, golli-MBP expression in lymph nodes was examined in four different models of relapsing experimental autoimmune encephalomyelitis (rEAE). Disease in these rEAE models was induced by the adoptive transfer of T lymphocytes specific for 18.5-kDa MBP, MBP peptide 83-102, or PLP peptide 139-151 in the SJL/J mouse and the adoptive transfer of T lymphocytes specific for MBP peptide Ac1-9 in the (SJL/J x PL/J)F1 mouse. In all four models, expression of golli-MBP BG21 mRNA was increased two- to fivefold in lymph nodes of mice 45 to 60 days post-transfer. Immunohistochemical analysis indicated that expression occurred principally in macrophages within lymph nodes. Endogenous golli-MBP epitopes within lymph node cells stimulated "classic" MBP 1-44-specific T lymphocytes, and this stimulatory ability resided within the adherent lymph node cell population. An increase in myelin protein expression within lymph nodes during rEAE has implications with regard to intra- and intermolecular epitope spreading. This is the first report describing an increase in target autoantigen expression within lymphoid tissue during an autoimmune disease.
Skin-homing, CLA+ memory T cells are activated in atopic dermatitis and regulate IgE by an IL-13-dominated cytokine pattern: IgG4 counter-regulation by CLA- memory T cells.
Authors: Akdis M , Akdis C A , Weigl L , Disch R , Blaser K
Published: January 2023

Cutaneous lymphocyte-associated Ag (CLA) is a skin-homing receptor displayed by memory/effector T cells recognizing skin-related allergens. Here we demonstrate that peripheral blood CLA+ CD45RO+ T cells in patients with atopic dermatitis (AD) are in vivo activated. They spontaneously proliferate and release an IL-13-dominated Th2 cytokine profile and are capable of inducing IgE in autologous B cells without further activation. The spontaneous cytokine release occurred within the first hour of culture and was not inhibited by cycloheximide or by other immunosuppressive drugs, indicating that cytokine transcription and translation had been completed in vivo. In contrast, the CLA- CD45RO+ T cells from the same patients and from nonatopic controls represented a resting memory T cell subset, secreted borderline quantities of cytokines, and induced IgG4. Polyclonal activation by the anti-CD2/anti-CD3/anti-CD28 mAb mixture generated distinct cytokine patterns in the two memory/effector T cell subsets. CLA+ T cells secreted Th2 cytokines with high IL-13 levels, and the CLA- subset mainly produced IFN-gamma. There was no difference in in vitro activated cytokine pattern between AD patients and nonatopic subjects. These results indicate that the CLA+ memory/effector T cells of AD patients are activated in vivo and play a pivotal role in allergic inflammation by production of IL-13 and induction of IgE Abs. In contrast, the CLA- resting memory T cell population may exert immunoprotective properties toward allergen by high IFN-gamma secretion and induction of IgG4.
CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice.
Authors: Balasa B , Krahl T , Patstone G , Lee J , Tisch R , McDevitt H O , Sarvetnick N
Published: January 2023

The nonobese diabetic (NOD) mouse spontaneously develops T cell-dependent autoimmune diabetes. Here, we investigate the role of CD40 ligand (CD40L)-CD40 costimulation in the initiation and progression of this disease. Anti-CD40L mAb treatment of 3- to 4-wk-old NOD females (the age at which insulitis typically begins) completely prevented the insulitis and diabetes. In contrast, treatment of such mice with anti-CD40L at >9 wk of age did not inhibit the disease process. These results suggest that a costimulatory signal by CD40L is required early but not in the effector phase of disease development. Anti-CD40L treatment affected the priming of islet Ag-specific T cell responses in vivo. Cytokine analysis revealed a dramatic decrease in IFN-gamma and IL-2 release without a concomitant increase in IL-4 production by T cells from anti-CD40L-treated mice. Thus, anti-CD40L impaired the islet Ag-specific Th1 cell response in vivo, and the prevention of diabetes by anti-CD40L was not associated with switching of the response from a Th1 to a Th2 profile. Cotransfer of splenocytes from anti-CD40L-treated mice with splenocytes from diabetic NOD mice into NOD/scid mice did not inhibit the transfer of disease, indicating that anti-CD40L does not prevent the disease by inducing regulatory cells. Since anti-CD40L clearly prevented the insulitis by inhibiting the development and further accumulation of pathogenic Th1 cells to islets of Langerhans, we conclude that CD40L-CD40 costimulation is required for early events in the development of spontaneous autoimmune diabetes.
Cutting Edge: CTLA-4 Ligation Delivers a Unique Signal to Resting Human CD4 T Cells That Inhibits Interleukin-2 Secretion but Allows Bcl-XL Induction
Authors: Blair Patrick J. , Riley James L. , Levine Bruce L. , Lee Kelvin P. , Craighead Nancy , Francomano Tara , Perfetto Steven J. , Gray Gary S. , Carreno Beatriz M. , June Carl H.
Published: January 2023

We have assessed the functional effects of a panel of CTLA-4 mAbs on resting human CD4+ T cells. Our results demonstrate that some CTLA-4 mAbs can inhibit proliferative responses of resting CD4+ cells and cell cycle transition from G0 to G1. The inhibitory effects of CTLA-4 were evident within 4 h, at a time when cell surface CTLA-4 expression remained undetectable. Other CTLA-4 mAbs had no detectable inhibitory effects, indicating that binding of Ab to CTLA-4 alone is not sufficient to mediate down-regulation of T cell responses. Interestingly, while IL-2 production was shut off, inhibitory anti-CTLA-4 mAbs permitted induction and expression of the cell survival gene bcl-XL. Consistent with this observation, cells remained viable and apoptosis was not detected after CTLA-4 ligation.
Reduced Cell Surface Expression of HLA-C Molecules Correlates with Restricted Peptide Binding and Stable TAP Interaction
Authors: Neisig Anne , Melief Cornelis J. M. , Neefjes Jacques
Published: January 2023

HLA-C molecules are poorly expressed at the cell surface compared with HLA-A and HLA-B locus products. The reason for the low surface expression and the underlying mechanism is unclear. We show that the HLA-C4 allele is expressed intracellularly in amounts similar to HLA-A and HLA-B alleles. However, the majority of the HLA-C4 molecules is not transported, but is retained in the endoplasmic reticulum by stable interaction with TAP. This pool does not appear to participate in the formation of HLA-C4/peptide complexes, but is degraded in the endoplasmic reticulum. HLA-C4 molecules can dissociate from TAP upon binding of specific peptide. However, they require a 10-fold higher concentration of a completely degenerated 9-mer peptide mixture for release from TAP than the HLA-A and HLA-B alleles. Our data show that the HLA-C molecules tested are more selective in their peptide binding than HLA-A and HLA-B molecules, resulting in prolonged association with TAP and a reduced formation of intracellular HLA-C/peptide complexes. The restricted peptide binding of certain HLA-C alleles provides one explanation for the reduced expression of HLA-C molecules at the cell surface. Other mechanisms will be discussed.
Cutting Edge: Identification of the Mouse IgG3 Receptor: Implications for Antibody Effector Function at the Interface Between Innate and Adaptive Immunity
Authors: Gavin Amanda L. , Barnes Nadine , Dijstelbloem Hilde M. , Hogarth P. Mark
Published: January 2023

Mouse IgG3 appears early in immune responses independently of T cell help and, as such, is an early effector molecule of the immune system. Yet, a specific IgG3 cellular receptor remains undefined. In transfection experiments, mouse FcγRI was clearly able to bind immune complexes of IgG3, whereas mouse FcγRII could not. Furthermore, macrophages from mice expressing FcγRII and FcγRIII but lacking FcγRI were unable to phagocytose IgG3 immune complexes, thus identifying mouse FcγRI as the sole receptor for IgG3 immune complexes. Competition studies demonstrated that monomeric mouse IgG3 could inhibit IgG2a binding to mouse FcγRI with an ID50 ≈10−7 M (fivefold lower than IgG2a). The identification of mouse FcγRI as the IgG3 receptor establishes FcγRI as a participant in events at the interface between innate and adaptive immunity, implying a greater role for this receptor in the development of normal and pathologic immune responses than previously recognized.
Monoclonal Anti-Cardiolipin Antibodies from New Zealand Black × New Zealand White F1 Mice React to Thrombomodulin
Authors: Haruta Kazuhiko , Kobayashi Shigeto , Hirose Sachiko , Horiai Aiko , Ohyanagi Mutsuko , Tanaka Mitsuhiko , Kawano Terunaga , Shirai Toshikazu , Takasaki Yoshinari , Hashimoto Hiroshi
Published: January 2023

The reactivity with and affinity for thrombomodulin (TM) of monoclonal anti-cardiolipin Abs (MoaCL), derived from a New Zealand Black × New Zealand White F1 (NZB/W F1) mouse, were studied to investigate the pathogenicity of anti-cardiolipin Abs (aCL). Four of eighteen MoaCL were found to react with rabbit TM when examined using ELISA. These four MoaCL also reacted with synthetic peptide that included the epidermal growth factor-like domain of human TM, a binding site for thrombin. The reaction with TM of these four MoaCL was inhibited by bovine thrombin. When the affinity for TM of the MoaCL was determined, the dissociation constants (Kd) ranged from 4.8 × 10−9 to 4.7 × 10−8 M. By contrast, examination of the affinity for cardiolipin (CL) gave values from 8.3 × 10−6 to 7.4 × 10−5 M. Thus, these MoaCL reacted to TM with a higher affinity than to CL. Moreover, these MoaCL also bound to TM on HUVEC and down-regulated the expression level of TM on the surface of HUVEC due to internalization of TM. The binding of thrombin to TM is known to initiate rapid protein C activation, and complexes of activated protein C and protein S show anticoagulatory activity. Thus, the present studies suggest that certain pathogenic aCL cross-react with TM and induce down-regulation of TM on endothelial cells, followed by induction of thrombosis.
VH Repertoire of a Marsupial (Monodelphis domestica)
Authors: Miller Robert D. , Grabe Hilary , Rosenberg George H.
Published: January 2023

When contrasted with information available for placental mammals, very little is known about the development of immunocompetence in marsupials. Marsupials, however, provide interesting immunology problems, since most appear to be born at a stage of development much less mature than that of placental mammals. To further understand the marsupial immune system, the Ig repertoire of the short-tailed opossum, Monodelphis domestica, was characterized. The majority of the VH clones were isolated in an unbiased manner by screening a spleen cDNA phage library, using C region probes, or anchored PCR, using C region-specific primers paired with vector specific primers. Analysis of 54 unique VH sequences from this marsupial revealed the presence of two VH families in the expressed Ig repertoire. The larger family, which contributed the majority of the clones identified, appears to be derived from 10 to 12 germline VH segments. The second family of clones is derived from a single germline VH. Both VH families are related to the group III sequences described in other vertebrates. Unusual codon bias differences between the two families may result in very different patterns of somatic mutation within the opossum Ig repertoire.
Molecular Analysis of the Major MHC Recombinational Hot Spot Located Within the G7c Gene of the Murine Class III Region That Is Involved in Disease Susceptibility
Authors: Snoek Margriet , Teuscher Cory , van Vugt Huub
Published: January 2023

Recombination within the MHC does not occur at random, but crossovers are clustered in hot spots. We previously described a recombinational hotspot within the 50-kb Hsp70.3–G7 interval in the class III region of the mouse MHC. The parental haplotypes of recombinants with crossovers in this region represent the majority of the laboratory haplotypes (a, b, d, dx, k, m, p, px, q, s, and u). Using microsatellite markers and sequence-based nucleotide polymorphisms, the breakpoint intervals of 30 recombinants were mapped to a 5-kb-long interval within the G7c gene adjacent to G7a. Recombination within the G7c hot spot does not appear to be restricted to certain haplotypes. Sequence motifs that had been suggested to be associated with site-restricted meiotic recombination were absent in the vicinity of the G7c hot spot, and hence, these sequence motifs are no prerequisite for meiotic recombination. The G7c hot spot resides in a region to which a number of disease susceptibility loci have been mapped, including susceptibility to cleft palate, experimental autoimmune allergic orchitis, and chemically induced alveolar lung tumors. The exact localization of crossovers in recombinants that have been used in functional studies is important for mapping susceptibility genes and limits the number of candidate genes.
Mechanism of Protective Immunity Against Influenza Virus Infection in Mice Without Antibodies
Authors: Epstein Suzanne L. , Lo Chia-Yun , Misplon Julia A. , Bennink Jack R.
Published: January 2023

There is considerable interest in developing viral vaccines intended to induce T cell immunity, especially cytotoxic CD8+ T lymphocytes, when Abs are not protective or are too narrow in viral strain specificity. We have studied protective immunity in doubly inactivated (DI) mice devoid of Abs and mature B cells. When infected with influenza B virus, these mice cleared the virus in a process dependent upon CD8+ T lymphocytes. Cytotoxic activity was detected in lung lymphocytes of DI mice after primary or secondary infection, and was abrogated by depletion of CD8+ cells in vivo. Challenge experiments showed that DI mice could be protected by immunization against reinfection 1 mo later, and protection was virus specific. Depletion of CD4+ or CD8+ T cells in vivo during the challenge period partially abrogated, and depletion of both subsets completely abrogated, the protection. This indicates that both CD4+ and CD8+ T cells are required effectors in the optimal control of virus replication. Thus, when Abs fail to protect against varying challenge viruses, as is the case with variant strains of influenza and HIV, there is hope that T cells might be able to act alone.
IL-1 and TNF Antagonists Inhibit the Inflammatory Response and Bone Loss in Experimental Periodontitis
Authors: Assuma R. , Oates T. , Cochran D. , Amar S. , Graves D. T.
Published: January 2023

Periodontal disease is the most frequent cause of tooth loss in humans and is the most prevalent disease associated with bone loss, including osteoporosis. Periodontal destruction is initiated by bacteria that colonize the tooth surface, leading to inflammation and bone resorption. To assess the roles of IL-1 and TNF in this process, studies were conducted in a Macaca fascicularis primate model of experimental periodontitis. Function-blocking soluble receptors to IL-1 and TNF were applied by local injection to sites with induced periodontal destruction and compared with similar sites injected with vehicle alone. The results indicate that injection of soluble receptors to IL-1 and TNF inhibited by approximately 80% the recruitment of inflammatory cells in close proximity to bone. The formation of osteoclasts was reduced by 67% at the experimental sites compared with that at the control sites, and the amount of bone loss was reduced by 60%. All results were statistically significant (p < 0.01). These findings indicate that a significant component of the pathologic process of periodontitis is due to IL-1/TNF activity, since inhibiting IL-1/TNF reduces both inflammatory cell recruitment and bone loss. The data also suggest that inflammation associated with gingivitis is actively protective, since blocking further up-regulation of the host response with IL-1/TNF inhibitors does not cause periodontal damage. Furthermore, these results coupled with recent evidence that IL-1 and TNF participate in endocrine-associated osteoporosis suggest that multiple pathologies involving excessive loss of bone may operate through a common mechanism involving IL-1 and/or TNF.
RANTES-Induced T Cell Activation Correlates with CD3 Expression
Authors: Dairaghi Daniel J. , Soo Kenneth S. , Oldham Elizabeth R. , Premack Brett A. , Kitamura Toshio , Bacon Kevin B. , Schall Thomas J.
Published: January 2023

The chemokine RANTES induces a unique biphasic cytoplasmic Ca2+ signal in T cells. The first phase of this signal, similar to that of other chemokines, is G-protein mediated and chemotaxis associated. The second phase of this signal, unique to RANTES and evident at concentrations greater than 100 nM, is tyrosine kinase linked and results in a spectrum of responses similar to those seen with antigenic stimulation of T cells. We show here that certain Jurkat T cells responded to RANTES solely through this latter pathway. A direct correlation between the RANTES-induced second phase response and CD3 expression was demonstrated in these cells. Sorting the Jurkat cells into CD3high and CD3low populations revealed that only the CD3high cells were responsive to RANTES. Furthermore, stimulation of these Jurkat cells with anti-CD3 mAb significantly depresses their subsequent response to RANTES. While a RANTES-specific chemokine receptor is expressed at a low level on these Jurkat cells, the RANTES-induced activation is dependent on the presence of the TCR. Thus, stimulation through TCR may partially account for RANTES’ unique pattern of signaling in T cells.
Regulation of Chemokine Gene Expression in Human Peripheral Blood Neutrophils Phagocytosing Microbial Pathogens
Authors: Hachicha Mohamed , Rathanaswami Palaniswami , Naccache Paul H. , McColl Shaun R.
Published: January 2023

Production of chemokines (chemotactic cytokines) by neutrophils is likely to be important in the regulation of inflammation and the control of infection. In this study we show that exposure of human neutrophils to various microbial pathogens leads to the production of both macrophage inflammatory protein 1α (MIP-1α) and IL-8. The bacterial microbes, Salmonella typhimurium and Pseudomonas aeruginosa, and Staphylococcus aureus all strongly induced both IL-8 and MIP-1α secretion, whereas Streptococcus pneumoniae, Staphylococcus epidermidis, and the opportunistic yeast Candida albicans were less potent. Saccharomyces cerevisiae and zymosan both induced IL-8 secretion but failed to stimulate that of MIP-1α. Coincubation of neutrophils with the proinflammatory cytokine TNF-α and the micro-organisms also led to differential expression of MIP-1α and IL-8. Significant enhancement of the induction of both MIP-1α and IL-8 by S. typhimurium, P. aeruginosa, and S. pneumoniae as well as by C. albicans was observed. In contrast, while IL-8 production in response to S. cerevisiae and zymosan was enhanced in the presence of TNF-α, no MIP-1α was produced. These combined results indicate that while neutrophils exposed to some micro-organisms alone or in the presence of inflammatory cytokines such as TNF-α will produce both MIP-1α and IL-8, resulting in generation of signals for the recruitment of mononuclear leukocytes and neutrophils, respectively, certain types of micro-organisms can skew this response toward synthesis of IL-8.