bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflationary expansion and maintenance of an MHC class Ib-restricted response to polyomavirus infection differ in their dependence on CD4 T cell help and CD28 costimulation. (105.45)
Authors: Hofstetter Amelia , Ford Mandy , Hadley Annette , Mlaver David , Pack Christopher , Tompkins S. , Lukacher Aron
Published: January 2023

We recently identified a protective MHC class Ib-restricted CD8 T cell response to infection by mouse polyomavirus (PA Swanson et al. 2008. JEM 205). These CD8 T cells recognize a peptide corresponding to amino acids 139-147 of the VP2 capsid protein presented by the nonpolymorphic Q9 molecule. This Q9:VP2.139-specific CD8 T cell response exhibits inflationary kinetics in MHC class Ia-deficient mice - a gradual expansion in the first three months post-infection with minimal contraction thereafter. We previously demonstrated that Q9:VP2.139-specific CD8 T cells are dependent on antigen for their expansion, but not for their long-term maintenance (PA Swanson et al. 2009. JI 182). Here, we tested the hypothesis that the expansion and maintenance phases of the Q9:VP2.139-specific T cell response are also differentially dependent on CD4 T cell help and CD28 and CD40 ligand costimulation. Either in vivo CD4 cell depletion or CD28/CD40L costimulation blockade inhibited expansion of Q9:VP2.139-specific CD8 T cells. We further found that CD28 blockade alone was sufficient to impair this expansion. In contrast, CD4 cell depletion, but not CD28/CD40L blockade, during the maintenance phase resulted in a decline in frequency of Q9:VP2.139-specific CD8 T cells. These results indicate that the Q9:VP2.139-specific CD8 T cell response depends on CD4 T cell help and CD28 costimulation for its inflationary expansion, but only on CD4 T cell help for its maintenance.
Reverse correlation between CD39+CD25+ Treg and activated CD8+ T cells / NK cells in HIV-1- patients with low CD4 cell counts (105.48)
Authors: Schuler Patrick , Buchanan William , Riddler Sharon , Whiteside Theresa , Bernard Macatangay , Rinaldo Charles
Published: January 2023

The role of regulatory T cells (Treg) in HIV has been studied intensely but remains unclear regarding their effect on cellular immune activation. Frequencies of CD4 and CD8 T cells, CD39+CD25+ Treg, Th17 cells, MDSC, B cells, NK cells and the activation status of T cells (CD38+HLADR+) were determined by flow cytometry in 10 healthy controls and 32 HIV-1+ patients. Immunosuppressive functions of single-cell sorted Treg were determined in the CFSE proliferation assay. Cytokine production (IL1b, 2, 4, 5, 6, 8, 10, INFg, TNFa, GM-CSF), C-reactive protein and soluble CD14 were measured in patients’ plasma by Luminex and ELISA. The absolute number of CD39+CD25+ Treg was significantly decreased in HIV-patients compared to normal controls (p=0.001). However, the frequency of Treg (p=0.04) and HLADR+CD38+CD8+ T cells (p=0.05) was increased only in patients with anti-retroviral therapy (ART). HIV+ patients with ART and low CD4+ cell count had the highest level of immune activation (p=0.04), and in this group we observed a significant negative correlation between CD39+CD25+ Treg and activated T cells (r2=0.5) as well as increased numbers of NK-cells. In ART-treated HIV+ patients with CD4+ T cell recovery, the frequency of Treg, NK cells and CD8+ T cell activation normalized. ART restores the frequency of Treg and activated T cells to normal levels. However, in patients whose CD4+ T cell count did not recover during ART, Treg frequency negatively correlated with T cell immune activation.
The clinical use of cord blood-derived virus-specific cytotoxic T lymphocytes reactive against Cytomegalovirus, Adenovirus, and Epstein-Barr Virus (105.50)
Authors: Hanley Patrick , Martinez Caridad , Leung Kathryn , Cruz Conrad Russell , Savoldo Barbara , Rodgers John , Leen Ann , Dotti Gianpietro , Gee Adrian , Rooney Cliona , Heslop Helen , Krance Robert , Shpall Elizabeth , Bollard Catherine
Published: January 2023

CMV, EBV and Ad-specific CTL(mCTL) have been used successfully to treat these viruses after HSCT. However, the development of virus-protective T cell therapy for patients undergoing CB Transplantation (CBT) requires the priming and extensive expansion of naïve T cells rather than the more limited and simple direct expansion of pre-existing virus-specific memory T cell populations from virus-experienced donors. We have developed a novel protocol utilizing an initial round of stimulation with CB-derived dendritic cells transduced with a recombinant Ad5f35-CMVpp65 vector in the presence of IL12, IL15 and IL7, followed by weekly stimulations with Ad5f35-CMVpp65-transduced EBV-LCL. CB-derived mCTL generated for clinical use from only 20-40 million CB mononuclear cells showed specificity for all 3 viruses and contained a mean of 48% CD8+ and 42% CD4+ cells with various stages of memory T cells. Two patients received mCTL on day 63 post-CBT. One patient remains negative for all 3 viruses. In the other patient, elevated CMV DNA levels were observed in the peripheral blood (PB) 4 weeks post CTL. The CMV cleared within 2 weeks of additonal CTL therapy corresponding to a 31-fold rise in CMV-CTL in the PB. The patient also developed Ad+ diarrhea which resolved without additonal therapy. In summary, we are the first to deliver antigen-specific CTL from CB: infusion of these cells increased virus-specific T cells in the PB dramatically earlier than observed immune reconstitution post CBT.
Viral persistence and fatal CD8 T cell-associated pathology are consequences of NK cell-mediated killing of activated CD4 T cells (105.6)
Authors: Waggoner Stephen , Selin Liisa , Welsh Raymond
Published: January 2023

Natural killer (NK) cells directly control virus replication during some infections and are postulated to indirectly regulate antiviral immunity during other infections through modulation of antiviral T cell responses, but the consequences and mechanisms of this regulation remain largely unexplored. Using the relatively NK cell-resistant lymphocytic choriomeningitis virus (LCMV), we identified a crucial role for NK cells in repression of antiviral T cell responses to a degree that alters the balance between virus clearance and immune pathology. Depletion of NK cells prior to infection of mice with a LCMV clone 13 dose (2 x 105 PFU) normally associated with fatal T cell-mediated immunopathology completely prevented virus-associated morbidity and promoted rapid viral clearance. In contrast, mice depleted of NK cells succumbed to a higher viral dose (2 x 106 PFU) normally associated with non-lethal persistent infection and clonal exhaustion of T cells. During the first three days of infection, NK cells eliminated activated CD4 T cells in a perforin-dependent manner. This early NK cell-mediated decrease in CD4 T cells resulted in lower numbers and diminished cytokine-producing capacity of LCMV-specific CD8 T cells. Thus, NK cells can dramatically alter viral clearance and infection-associated immunopathology by reducing the ability of CD4 T cells to support antiviral CD8 T cell responses in the context of high viral loads and disseminated infection.
Late interleukin-6 promotes T follicular helper cell response and resolution of chronic viral infection (105.8)
Authors: Harker James , Lewis Gavin , Mack Lauren , Zuniga Elina
Published: January 2023

Multiple inhibitory molecules create a profoundly immunuosuppressive environment during chronic viral infections in humans and mice, making generation of effective immunity in this context a challenge. Using murine chronic lymphocytic choriomeningitis virus (LCMV) infection we found that non-hematopoietic cells produced interleukin 6 (IL-6) in a unique biphasic manner, with later IL-6 playing an essential role for viral control. The underlying mechanism involved IL-6 upregulation of the transcription factor BCL6 in virus specific CD4 T cells during chronic (but not acute) infection. This resulted in escalation of T follicular helper cell, germinal center and antibody responses at late stages of chronic infection. Notably, while CD4 T cells showed intact IL6 signaling both early and late after chronic infection only late IL6 stimulation resulted in significant BCL6 up-regulation. Finally ablation of the common IL-6 cytokine family receptor gp130 not only curtailed BCL6 upregulation in CD4 T cells but also reduced their numbers and IL-21 expression in a cell-intrinsic fashion. Our results uncover a novel antiviral strategy that overcomes the immunosuppressive environment to promote CD4 helper functions and safely resolves a persistent infection in vivo. These findings could illuminate new therapies to control viral persistence in humans.
Domain-specific variation in hemagglutinin-specific T-cell responses elicited by licensed seasonal influenza vaccines (106.11)
Authors: Basha Saleem , Hazenfeld Staci , Brady Rebecca , Subbramanian Ramu
Published: January 2023

Persistent antigenic drift in influenza hemagglutinin (HA) results in the emergence of serologically novel influenza viruses that result in seasonal epidemics globally. While the globular domain of the HA antigen is the primary repository of drift mutations, the viral-fusion domain is highly conserved. T cells are being increasingly recognized as a significant component of influenza immunity in humans and may target variant regions of HA that are not effectively targeted by type-specific antibodies. While a trivalent inactivated influenza vaccine (TIV) and a live-attenuated influenza vaccine (LAIV) are now licensed for use in humans, their comparative ability to elicit T cell responses against influenza HA is not well understood: specifically, the relative magnitude of T cell responses elicited by TIV and LAIV and their domain-specificity toward HA regions is unknown. In this study, we systematically compare immune responses elicited by TIV and LAIV in a cohort of healthy adults (18 to 49 years old) against the rapidly evolving H3N2 HA antigen. Data suggests TIV elicits higher geometric mean antibody titers than LAIV; whereas, LAIV elicits superior T cell responses. Importantly, the two vaccines vary significantly in their ability to elicit T cell responses targeting the variant and conserved regions of HA. These results have important implications for the deployment of influenza vaccines in years of antigenic mismatch and shift.
The B-subunit of ricin toxin is involved in eliciting protective antibodies (106.18)
Authors: Yermakova Anastasiya , Mantis Nicholas
Published: January 2023

The ricin toxin B subunit (RTB) is a galactose-specific lectin that promotes attachment and entry of the ricin enzymatic subunit (RTA) into host cells. RTB is an appealing alternative to RTA-based vaccines, because it is non-toxic and inherently antigenic. However, the degree to which RTB elicits protective immunity against ricin remains unresolved. Here we report that parenteral immunization of mice with RTB elicited high ricin-specific serum antibody titers, yet conferred only partial protection against systemic ricin challenge. Analysis of ~2000 RTB-specific B cell hybridomas identified only a handful of neutralizing monoclonal antibodies (mAbs). Two neutralizing mAbs, 24B11 and SylH3, were shown by passive immunization to be sufficient to protect mice against a 5xLD50 dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to primary macrophages, suggesting that they function by steric hindrance and recognize epitopes located on RTB’s galactose binding sub-domains, 1α or 2γ. By contrast, analysis of four non-neutralizing mAbs revealed that they recognized epitopes situated within sub-domains of RTB not involved in galactose recognition (i.e., 1β, 2α). Based on these data, we propose using specific RTB sub-domains as antigens, rather than RTB itself, as a means to more efficiently elicit neutralizing antibodies and protective immunity against ricin.
Passive oral rice-based antibody prophylaxis and therapy against rotavirus infection (106.19)
Authors: Tokuhara Daisuke , Gonzalez Beatriz , Mejima Mio , Takahashi Yuko , Kurokawa Shiho , Hiroiwa Tomoko , Kuroda Masaharu , Oyama Masaaki , Kozuka-Hata Hiroko , Nochi Tomonori , Aladin Farah , Marcotte Harold , Frenken Leon , Iturriza Miren , Kiyono Hiroshi , Hammarstrom Lennart , Yuki Yoshikazu
Published: January 2023

Rotavirus-induced diarrhea is a life-threatening disease in children in developing countries. We have developed a novel system for prophylaxis and therapy against rotavirus using transgenic rice expressing the neutralizing variable domain of llama heavy-chain antibody fragments against rhesus-monkey rotavirus serotype G3P (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. MucoRice-ARP1 was water-soluble and expressed in protein body II as a rice storage organelle and cytoplasm between protein bodies of in rice. We confirmed the full sequence of 123 amino acids of ARP1 by mass spectrometry. MucoRice-ARP1 neutralized human RV strains (ST-3 G4P[6], 69M G8P[10], F45 G9P[8], P G3P[8] and Va70 G4P[8]) in vitro. When administered orally, MucoRice-ARP1 markedly decreased the prevalence of diarrhea and viral load in a murine pup model. Long-term storage (>1yr) at room temperature of MucoRice-ARP1 did not impair its in vivo neutralizing activity. Oral MucoRice-ARP1 thus offer a novel cost-effective approach to prevent and treat rotavirus-induced diarrhea not only in children but also in adults who would not be expected to respond to the live attenuated vaccine.
Design of T cell epitope rich OmpC peptide and its conjugation with Vi-polysaccharide: a candidate vaccine for typhoid fever (106.26)
Authors: Haque Shabirul , Jailkhani Bansilal , Sengupta Sanjukta , Mukhopadhyay Asok , Bhan Maharaj kishan , Kumar Ramesh
Published: January 2023

Here, we conjugated Vi polysaccharide with outer membrane protein C (OmpC) peptide-39 amino acid residues of S.Typhi and its immunogenicity was evaluated in mice. T cell epitopes in the OmpC were predicted using program T sites. Predominantly two regions with high content of T-cell epitopes, loop # 3a & 7 were chosen and synthesized commercially. Carbodiimide-mediated conjugation of Vi polysaccharide with synthetic OmpC peptide was achieved using ADH as a linker molecule. In various batches; obtained polysaccharide/peptide ratio was between 0.43-0.65. Total and IgG antibodies against Vi polysaccharide and OmpC peptide were measured by ELISA. No Vi antibody was detected in OmpCp and saline control group. Vi polysaccharide alone elicited very low levels of Vi antibody without any booster effect while Vi-OmpCp conjugate evoked 20 fold more efficient immune response than Vi polysaccharide. The Vi-OmpCp conjugate elicited strong booster response which persisted for longer duration. Further, adequate level of IgG antibodies were induced only by the Vi-OmpCp conjugate suggesting that T-helper cells had been induced. Although no immune response was generated against OmpCp. Therefore, selected OmpC short peptide used as carrier with Vi is assumed to be a promising candidate vaccine against typhoid fever.
Antibody-blockade of a Type I interferon receptor decoy cures a viral disease by restoring liver interferon signaling (106.27)
Authors: Xu Renhuan , Sigal Luis
Published: January 2023

Type 1 interferons (T1-IFNs) play a major role in antiviral defense. Orthopoxviruses, including those that produce smallpox and mousepox encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protects locally in the liver rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infectious foci in the liver to impair their ability to signal interferon, thus facilitating virus spread. Remarkably, this process can be reverted late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Our findings provide insights on how T1-IFNs function in vivo during viral infection, the first evidence on how an immune evasion gene operates in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.
T cell responses in rheumatoid arthritis patients treated with TNF antagonists (107.7)
Authors: Roumanes David , Kobie James , Cavenaugh James , Quataert Sally , Mosmann Tim
Published: January 2023

Rheumatoid Arthritis (RA) is a chronic inflammatory disease characterized by the overproduction of pro-inflammatory cytokines such as TNFα and IL1. An effective RA treatment is to block the action of TNFα with inhibitors. Due to the central role of TNFα in the inflammatory phase of the immune response, we investigated whether diminishing the TNFα level could lead to an impaired T cell response in RA patients. We first measured a panel of 71 proteins in the serum of healthy donors, early/new RA patients without any treatment and RA patients under anti-TNF therapy. We detected higher levels of several chemokines and cytokines in RA patients. Most levels returned to near-normal values after TNF antagonist therapy, but some chemokines showed no alterations in levels either in RA subjects or after therapy. We then evaluate T cell cytokine secretion by 16-color flow cytometry. A previous in vitro study showed that proliferative responses to recall antigens were reduced in RA patients, but these responses were restored after TNFα antagonist treatment. In contrast, ex vivo analysis of antigen-induced cytokine secretion (after 10 hours) in our studies showed that normal, RA and TNF antagonist-treated patients all showed recall responses against influenza antigens.
Inducible expression of kallikrein in kidney tubular cells protected mice from spontaneous lupus nephritis (107.8)
Authors: Li Quanzhen , Shao Xinli , Yang Ru , Li Yajuan , Xie Shangkui , Mohan Chandra , Igarashi Peter
Published: January 2023

Inducible gene modification using the Cre/loxP system provides a valuable tool for analysis of gene function in specific tissues of live animals. To investigate the therapeutic role of kallikreins (klk) against spontaneous lupus nephritis, we created a transgenic mouse model, KSP-CreERT2/CAG-LacZ(flox)-mKlk1-GFP/Sle1Sle3. This model is based on the expression of a tamoxifen-inducible Cre recombinase under the control of kidney specific promoter (KSP), and its activation will cause the deletion of loxP-flanked LacZ gene and turned on the mKlk1 expression under the control of CAG promoter. In the transgenic mice, the expression of CreERT2 was restricted to KSP+ kidney tubular cells. The transgenic mice were injected with either tamoxifen or vehicle (controls) at 3-mo age and followed up for 6 months for the klk expression and nephritis phenotype. The elevated expression of mKlk1 in the kidney and urine was detected in tamoxifen induced mice but not in controls. At 9-mo age, more than 80% of vehicle injected mice developed severe lupus nephritis as evidenced with increased level of serum BUN (72 ±23mg/dL), proteinuria (21±7mg/24hr) and renal pathology. However, the tamoxifen injected mice showed significantly reduced level of serum BUN(17±8mg/dL), proteinuria (7±3mg/24hr) and mild or no renal pathology change (p<0.05). Tamoxifen induced upregulation of mKlk1 in mouse kidney increased the NO production and suppressed the apoptosis of kidney tubular cells in B6.Sle1.Sle3 mice.
Prostaglandin I2 regulates IL-17 production by invariant natural killer T cells (108.7)
Authors: Simons Bryan , Ferrini Maria , Jaffar Zeina , Roberts Kevan
Published: January 2023

PGI2 is an immunoregulatory prostanoid that is known to limit allergic inflammation. Our previous studies have shown that the PGI2 receptor, IP, also plays an important role in the development of IL-17-producing γδ T cells. Using mice lacking the PGI2 receptor (IPKO), we have investigated whether PGI2 is required for programming invariant natural killer T cells (iNKT) to produce IL-17. The GalCer response of spleen cells from the IPKO mouse was markedly reduced compared to wild type (WT) control animals. Paradoxically, the IL-17 production elicited by iNKT cells isolated from the lungs of IPKO mice stimulated with GalCer was 1.7-5.7 fold elevated over WT control animals. The elevated GalCer response in IPKO lungs was not associated with an increase in CD1d expression or the number of CD4+NK1.1+ or CD4+DX5+ iNKT cells in the lung. The exposure of IPKO animals to 1.5 ppm O3 resulted in a further increase in IL-17 production by lung mononuclear cells (LMC) in response to GalCer. These observations are consistent with iNKT responses being limited by a suppressive mechanism operative in the WT, but not IPKO lung tissue. The regulatory cells were not γδ T cells since the IL-17 produced by LMC in mice lacking the δ TCR chain remained low. Collectively, these results demonstrate that the GalCer response in normal lungs is depressed by a PGI2 dependent mechanism. The role for PGI2 in promoting the regulation of iNKT cells by regulatory cells, including CD4+ nTreg will be discussed.
Regulation of CD40 Ligand (CD40L) mRNA stability by Protein Kinase C (109.15)
Authors: Castro Moraima , Matus-Nicodemos Rodrigo , Covey Lori
Published: January 2023

The expression of the CD40 ligand (CD40L) is controlled at the posttranscriptional level by an activation-induced program of regulated mRNA stability whereby at early times of activation the mRNA is highly unstable and at later times (24-48 hr) the transcript becomes significantly stabilized. Regulated stability is dependent on the formation of a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex (Complex I) that binds to the 3’UTR of the CD40L mRNA. Here we report that the broad-spectrum PKC inhibitor H7 destabilizes the CD40L mRNA in both a Jurkat subclone (Jurkat/D1.1) that recapitulates the “stability” phase of the CD40L regulated decay program and in primary human CD4+ T cells activated with PMA and ionomycin. In both cell populations, treatment with H7 also resulted in a corresponding decrease in surface CD40L expression. Analysis of RNA binding complexes by electrophoretic mobility shift assays (R-EMSA) following H7 treatment of Jurkat/D1.1 cells corresponded to an increase in Complex I as well as the formation of a novel PTB-independent complex that bound to the CD40L stability element. These results demonstrate the relationship between PKC-regulated pathways and the control of CD40L expression through mRNA stability. Future work is underway to identify the protein component(s) of this new complex, as well as to identify PKC isoforms that are involved in regulating PTB-mediated decay.
Increased kon of TCR-pMHC interaction influences activation and development of CD4+ T cells (109.18)
Authors: Lynch Jennifer , Donermeyer David , Weber K. Scott , Allen Paul
Published: January 2023

While T cell activation has been correlated with half-life of the TCR-pMHC interaction, the importance of on-rate is only now being realized. In contrast to other studies, our lab has generated a system that separates the kon component from koff by comparing the response of two T cells, 3L2 and M2. 3L2 responds specifically to Hb(64-76) peptide presented on I-Ek. M2 differs from 3L2 by two point mutations in the CDR1α loop, resulting in a 4-fold higher affinity for Hb(64-76) due entirely to a faster kon. We generated an M2 TCR transgenic mouse to compare the affect of kon on T cell responses in vivo and in vitro to 3L2 T cells. M2 expressing T cells are positively selected and detected with a clonotypic antibody in the periphery though at a reduced number in comparison to 3L2. This could be due to decreased positive selection and/or increased negative selection of M2 T cells by alteration of the kinetics of M2 interactions with endogenous pMHC. M2 responds to 12 Hb(64-76) altered peptides (APLs) that are null peptides for 3L2 and has increased reactivity to APLs that are agonists for 3L2. M2 cells have a greater proliferative response at day 3 than 3L2 when exposed to Hb(64-76). While both M2 and 3L2 proliferate by day 3, a larger number of M2 cells than 3L2 cells have divided. This suggests that a fast kon increases early T cell responses. By selectively altering kon, we have revealed the importance of kon for TCR binding pMHC in T cell development and activation.
T cell triggering thresholds are modulated by the number of antigen within individual T cell receptor clusters (109.20)
Authors: Manz Boryana , Jackson Bryan , Petit Rebecca , Dustin Michael , Groves Jay
Published: January 2023

T cells are known to react to extremely small numbers of activating agonist peptides. Spatial organization of T cell receptors (TCR) and their peptide-MHC (pMHC) ligands into microclusters is correlated with T cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Experiments in which the overall agonist density and the number of agonist per TCR cluster are independently varied in primary T cells indicate that single pMHCs are unlikely to be able to trigger calcium signals. Analysis of activating pMHC distribution among clusters suggests the most probable minimal triggering unit for calcium signaling is at least 4 pMHC per cluster in this system. This is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.
Regulation of T-cell receptor signaling by activation-induced zinc influx (109.21)
Authors: Lee Won-Woo , Yu Mingcan , Tomar Deepak , Li Guangjin , Czesnikiewicz-Guzik Marta , Pryshchep Sergey , Lamar David , Singh Karnail , Weyand Cornelia , Goronzy Jörg
Published: January 2023

Zinc is a trace element that is essential for innate and adaptive immune responses. In addition to being a structural element of many proteins, zinc also functions as a neurotransmitter and an intracellular messenger. Although cytoplasmic zinc concentrations are strictly controlled, temporal or spatial changes in bioavailable zinc may influence the activity of several enzymes, including kinases and phosphatases. In this study, we provide evidence that zinc functions as an ionic signaling molecule after T-cell activation. Cytoplasmic zinc concentrations increased within one minute after TCR triggering by super antigen presented by myeloid dendritic cells. In contrast to activation-induced calcium influx, increased zinc concentrations were compartmentalized in the subsynaptic area. The zinc increase was dependent on the extracellular zinc concentrations and was inhibited by silencing the plasma membrane zinc transporter Zip6 consistent with influx from extracellular compartments. Increased zinc availability augmented ZAP70 phosphorylation, inhibited SHP-1 recruitment to the T cell signaling complex, slowed down Lck rephosphorylation at position 505 and sustained calcium influx. As a result, increased extracellular zinc bioavailability facilitated the induction of T cell proliferative responses to suboptimal stimuli. We propose that zinc calibrates TCR activation thresholds by inhibiting negative feedback mechanisms in TCR signaling.
Drak2 and PKD coordinate early T cell activation events to maintain survival (109.24)
Authors: Newton Ryan , Walsh Craig
Published: January 2023

T cells deficient in Drak2 hyper-respond to suboptimal stimulation through the antigen receptor, exhibiting enhanced calcium mobilization and high Il-2 production. Despite this lack of negative regulation, mice deficient in Drak2 are resistant to models of induced organ-specific autoimmune disease. This has been attributed in part to a defect in survival following activation, and can be rescued in vitro with exogenous co-stimulation and in vivo through expression of a Bcl-xL transgene. Drak2 kinase activity is crucial to maintain normal calcium responses required for productive activation of T cells, and has recently been shown to require the kinase activity of protein kinase D (PKD). PKD and Drak2 associate at mitochondria, and downstream events are dependent on calcium-induced mitochondrial reactive oxygen generation. PKD and Drak2 are thus part of a critical negative feedback loop whereby calcium influx is both required and targeted by Drak2 activity. Recent work suggests that specific ion channels may be directly targeted by Drak2, with consequences on both the activity of these channels and downstream metabolic events that contribute to proliferative capacity. This implicates both PKD and Drak2 as part of a formerly unappreciated signaling complex that coordinates immediate T cell activation events to limit unproductive activation of T cells and maintain survival.
Decoupling age from cytomegalovirus-associated changes in the immune system (109.28)
Authors: Furman David , Shen-Orr Shai , Kidd Brian , Maecker Holden , Dekker Cornelia , Davis Mark
Published: January 2023

CMV infection has been associated with premature immunosenescence and shorter lifespan. To date, no systems-wide analysis for decoupling age from CMV associated changes have been conducted. To address the differences between an immune system in naïve versus CMV infected humans and to decouple the age-related from CMV-related changes, we studied a comprehensive set of assays comprising serum cytokines, cell subset frequencies, whole genome gene expression and various stimulations of immune cells in 30 young and 60 older, CMV seropositive or seronegative individuals. For each measurement we performed a multiple regression analysis on all samples by age, CMV serostatus and the interaction between both variables (age × cmv). We found dramatic differences in multiple measurements of the immune system by age - mostly affecting T cell signaling responses, inflammatory cytokines and many immune-related genes, by CMV - which seems to preferentially modulate the CD4 and B cell compartments at different levels - or when both factors are present with changes mostly restricted to B cell functions. We present, for the first time, a systems immunology view of CMV- and age-associated changes in the immune system.
LAT-independent triggering of granule-mediated cytotoxicity of CD8 T cells (109.32)
Authors: Ouyang Chihwen (Claudia) , Zhu Minghua , Li Qi-jing , Zhang Weiguo
Published: January 2023

LAT (Linker for activation of T cells) is a transmembrane adaptor protein that is integral to the TCR-mediated signaling. LAT has been shown to play an essential role in thymocyte development, T cell activation, and cytokine production by CD4 T cells. Conventional thinking assumed that LAT is also required for the effector function of CD8 T cell. In this study, we used LAT conditional knockout mice to study the function of LAT in the cytotoxicity of CD8 T cells. Surprisingly, although CTL cytotoxicity is reduced in the absence of LAT, it is not completely abrogated. This cytotoxicity appears to be mediated through a granule-dependent, Fas-independent pathway. We further dissected the effect of the LAT deletion within each step of granule exocytosis. LAT-deficiency led to the formation of unstable synapses, subsequently causing unstable T:APC conjugates. Moreover, MTOC polarization and granule reorientation were impaired by LAT-deficiency. Further investigation revealed that, even though TCR-mediated signaling was severely impaired, the residual calcium flux and PLC activity in LAT-deficient CTLs were sufficient to support cytotoxicity. Our study reveals a novel finding that, in contrast to helper T cells, CTL function is not entirely dependent on LAT and, perhaps is not governed by the overall TCR signaling strength.
Discs Large Homolog 1 splice variants couple T cell receptor engagement to distinct T cell effector functions (109.34)
Authors: Silva Oscar , Humphries Lisa , Crocetti Jillian , Round June , Miceli M.
Published: January 2023

The recognition of antigen by the TCR triggers the polarized recruitment of receptors, signal transducers and cytoskeletal components to the T-cell:APC interface, providing a molecular platform for selectively instructing T effector transcription and polarized secretion. How TCR signals are specified to regulate distinct downstream signaling and functional outputs remains poorly understood. We report that T cells utilize distinct protein variants of Dlgh1, a scaffold that regulates synaptic polarity and signaling, to independently regulate pathways controlling TCR-induced transcriptional activation and secretion of effector molecules. T cells express two Dlgh1 protein variants, Dlgh1 i1A-i1B and Dlgh1 i1B, which differ by inclusion of the alternatively spliced i1A-domain, hypothesized to control binding of the TCR proximal kinase, Lck. Over-expression of Dlgh1 i1A-i1B in T cells selectively enhanced TCR-induced TNFα and NFATc1, but not IκBα or IL-2 mRNA expression, while over-expression of Dlgh1 i1B did not. In contrast, knockdown of the Dlgh1 i1B variant in CD8+ T cells attenuated TCR-induced granzyme B secretion, but did not alter TCR induced granzyme B mRNA upregulation. Knockdown of Dlgh1 i1A-i1B reduced TCR-induced TNFα and NFATc1 mRNA upregulation, but not granzyme B secretion. Therefore, alternative splicing specializes Dlgh1 variants to orchestrate TCR signal specificity, selectively coupling TCR engagement to transcriptional activation or secretion of effectors.
The emerging role of Mg2+ availability on B cell receptor signaling (109.4)
Authors: Towne Francina , Perraud Anne-Laure , Schmitz Carsten
Published: January 2023

Hypomagnesemia in patients, as well as in animal models, results in a weaker humoral immune response. The mechanism underlying this Mg2+ dependent modulation of the humoral response remains undefined. The master regulator of Mg2+ homeostasis, TRPM7, is a unique cation channel with a fused serine/threonine kinase domain. Interestingly, TRPM7 interacts with several phospholipase isoforms including PLCγ2, an essential component of B cell receptor (BCR) signaling. Here we address our hypothesis that in response to environmental availability of Mg2+, TRPM7 acts as a homeostatic sensor to adjust BCR signaling strength by serine/threonine phosphorylation of PLCγ2. We show in the DT40 avian B cell line that PLCγ2-dependent BCR-mediated Ca2+-response is reduced under low Mg2+ conditions. Using in vitro phosphorylation assays and a complementation approach in PLCγ2 deficient DT40 cells, we show that TRPM7 kinase is phosphorylating PLCγ2 resulting in the Mg2+ sensitive modulation of BCR-elicited Ca2+ responses. These findings suggest that TRPM7 actively responds to the availability of environmental Mg2+, in turn regulating downstream BCR signaling responses through serine/threonine phosphorylation of PLCγ2. This novel interaction, between an ion channel kinase and a BCR signaling molecule, could be a key piece of the puzzle in explaining why animal models of nutritional hypomagnesemia have weaker humoral immune responses.
Stim1, PKCδ and RasGRP proteins set a threshold for pro-apoptotic Erk signaling during B cell development (109.6)
Authors: Limnander Andre , Depeille Philippe , Freedman Tanya , Liou Jen , Leitges Michael , Kurosaki Tomohiro , Roose Jeroen , Weiss Arthur
Published: January 2023

Antigen encounter induces proliferation in mature B cells but triggers apoptosis in developing B cells. Thus, developing B cells that strongly recognize auto-antigens undergo apoptosis, one mechanism that contributes to prevent autoimmunity. Developing B cells display increased Ca2+ entry in response to antigen as compared to mature B cells, and Ca2+ is required for antigen-induced apoptosis. However, the Ca2+-driven signals involved are poorly defined. Here we identify and characterize a previously unrecognized Ca2+-driven Erk activation pathway, which is pro-apoptotic and biochemically distinct from diacylglycerol (DAG)-induced Erk activation. This pathway requires PKCδ and RasGRP proteins and depends on Stim1 levels, which control the magnitude of Ca2+ entry. We identify a novel phosphorylation site on RasGRP1 as a putative target of PKCδ that is absolutely required for Ca2+-dependent Erk activation but is dispensable for DAG-mediated Erk activation. Developmental regulation of these proteins is associated with selective activation of the pathway in bone marrow B cells prone to negative selection. This checkpoint is impaired in PKCδ-/- mice, which develop B cell autoimmunity. Conversely, Stim1 overexpression confers a competitive disadvantage to developing B cells, an effect that depends on PKCδ. Our findings demonstrate that DAG and Ca2+ can mediate the activation of functionally distinct Erk pathways to determine whether B cells proliferate or die upon antigen encounter.
Prohibitins and the cytoplasmic domain of CD86 are necessary to mediate signaling in B lymphocytes (109.8)
Authors: Lucas Christopher , Campbell Kerry , Bhatia Sumeena , Hodes Richard , Sanders Virginia
Published: January 2023

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that lead to an increase in Oct-2 and mature IgG1 mRNA and protein expression, without affecting class switch recombination. The most proximal signaling intermediates identified thus far are Lyn and phospholipase Cγ2α/β, which are usually activated after recruitment and binding to phospho-tyrosine residues on receptors, but the cytoplasmic domain of CD86 lacks tyrosines. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins, prohibitin-1 (Phb1) and Phb2, bind to CD86. The basal association is low in resting cells, but increased after priming with CD40L/IL-4. The level of total Phb1/2 expression increased primarily via a CD40-dependent mechanism. Phb1 protein expression and binding to CD86 were consistently greater than that for Phb2 in CD40L/IL-4-primed B cells. The CD86-induced increase in Oct-2 and IgG1 was reduced when either Phb1/2 expression was silenced by shRNA or when the cytoplasmic domain of CD86 was truncated or mutated at the serine/threonine protein kinase C-phosphorylation sites. Truncation of the CD86 cytoplasmic domain did not affect the binding of Phb1/2 to CD86, or the level of T cell activation and class switch recombination. These findings suggested that Phb1/2 and the CD86 cytoplasmic domain are each necessary to mediate the CD86-induced signaling in B cells.
Regulation of interleukin-27 production by Mycobacterium tuberculosis (110.2)
Authors: Liu Jianguo , Zhang Jidong , Qian Xuesong , Ning Huan , Hoft Daniel
Published: January 2023

Mycobacterium tuberculosis (Mtb) remains a major global challenge to human healthcare and the mechanisms of how Mtb evades host immune surveillance to favor its survival are still largely unknown. In this study, we found that BCG and Mtb could activate IL-27 expression in human and mouse macrophages by induction of p28 subunit transcription. However, in parallel with these effects, BCG and Mtb stimulation of macrophages induces activation of p38 MAPK signaling molecules MLK3/MKK3/MK2 to prevent maximal IL-27 production. AP-1/c-Fos was shown to bind directly to the p28 promoter and induce p28 expression after Mtb stimulation. Overexpression of p38α inhibited the binding of c-Fos to the p28 promoter but had no effect on c-Fos protein expression or phosphorylation in response to Mtb. Furthermore, blockade of p38 by SB203580 enhanced Mtb-induced AP-1 binding to the p28 promoter. Importantly, we show that adding exogenous IL-27 to increase the levels produced by PBMCs stimulated with live mycobacteria enhances the ability of BCG-expanded T cells to inhibit intracellular mycobacterial growth in human macrophages. Taken together, our data demonstrate that mycobacterial stimulation induces both IL-27 production and p38 MAPK activation. Strategies designed to tip the balance toward positive regulation of p28 induction by mycobacteria could lead to enhanced protective TB immunity.
Recognition and functional activation of human FcαRI by C-reactive protein (110.22)
Authors: Marjon Kristopher , Lu Jinghua , Marnell Lorraine , Wang Ruipeng , Mold Carolyn , Sun Peter , Du Clos Terry
Published: January 2023

C-Reactive Protein (CRP) is a member of the pentraxin family and an acute phase serum protein. It has been established that CRP and other pentraxins bind FcγRs and induce phagocytosis, activation of signaling, and cytokine synthesis. We report a novel structural and functional interaction between CRP and FcαRI with μM affinity and the binding site for CRP on FcαRI is distinct from that of IgA. Although FcγR and FcαRI bind their immunologlobulin ligands differently, the binding between CRP and FcαRI or FcγRII was inhibited by C1q indicating a similar binding orientation. However, the CRP mutant T173A, which binds poorly to FcγR, showed enhanced binding to FcαRI compared to wild type CRP. CRP crosslinking of FcαRI induced degranulation, ERK phosphorylation and IL-4 production in rat basophilic leukemia cells expressing FcαRI. CRP increased phagocytosis, surface expression of FcαRI, and the production of TNF-α in human neutrophils. The finding that CRP binds to an additional FcR on leukocytes has broad implications for the roles of CRP in host defense, autoimmunity and regulation of the immune response.
Heme oxygenase-1 induction stimulates host defense mechanism against infections (110.3)
Authors: Dhawan Subhash
Published: January 2023

Innate immune response is pivotal for host defense against pathogenic infections. It is, however, often ineffective to provide protection against infections and fails to efficiently respond to the existing conditions. Induction of host response or direct targeting of the invading pathogens then becomes desirable to reduce the disease progression. Activation of HO-1 by a natural substrate hemin effectively enhanced the ability of human macrophage to resist infections by several pathogens, including HIV-1, dengue virus, West Nile virus, poxvirus, and Leishmania donovani. HO-1 expression in a prostate cancer cell line susceptible to infection by XMRV resulted in substantial reduction in virus production by infected cells. Reduction of intracellular pathogens in HO-1-activated cells was attenuated by pretreatment of cells with SnPP IX, a specific inhibitor of HO-1 activity, indicating a key role of this endogenous enzyme in the host defense mechanism. Considering effective treatment of these infections is a continuous daunting challenge due to the emergence of drug resistant mutants, unlocking of HO-1-dependent regulatory genes may provide novel biomarkers and therapeutic targets for treating multiple infections and disease conditions.
Significant role of IL-1 signaling, but limited role of inflammasome activation in oviduct pathology during Chlamydia muridarum genital infection (110.6)
Authors: Nagarajan Uma , SIkes James , Yeruva Laxmi , Prantner Daniel
Published: January 2023

IL-1β has been implicated in the development of oviduct pathology during Chlamydia muridarum genital infection in the mouse model. The goal of this study was to characterize the role of IL-1 signaling and the inflammasome activation pathways during C. muridarum genital infection. Compared to control mice, IL-1 receptor KO (knockout) mice display delayed clearance and increased infection burden, but very few incidences of oviduct hydrosalpinx. Consistent with this observation, IL-1R antagonist KO mice display an opposite phenotype of rapid infection clearance. Flow cytometry analysis at day 10 and day 20 post infection reveal significantly more macrophages in the oviducts of IL-1R KO mice in comparison to control mice. Despite increased infection and recruited macrophages, IL-1R-KO mice have less tissue fibrosis and dilation, suggesting an anti-inflammatory role for these cells in the absence of IL-1 signaling. To investigate the role of inflammasome components during in vivo genital infection, mice lacking NLRP3, NLRC4 and ASC were tested. Although IL-1β secretion is dependent on caspase-1 during in vitro infection of primed macrophages and requires ASC, it is independent of NLRP3 and NLRC4 activation. Surprisingly, during in vivo genital infection, mice lacking NLRP3, NLRC4, as well as ASC display no reduction in oviduct pathology. These data suggest a major role for IL-1 signaling but a limited role for inflammasome pathway in development of oviduct pathology.
Activation of membrane TGFβ1 on alveolar macrophages by co-cultured alcohol primed alveolar epithelial cells (111.1)
Authors: Joshi Pratibha , Venado Aida , Curry-McCoy Tiana , Guidot David
Published: January 2023

Alcohol abuse impairs pulmonary innate immunity rendering individuals susceptible to pneumonia. We have previously shown that alcohol abuse causes oxidative stress in the lung and increases TGFβ1 expression. TGFβ1 downregulates GM-CSF receptors resulting in the alveolar macrophage and epithelial cell dysfunction. However, the exact mechanism of alcohol-mediated activation of TGFβ1 in vivo is still under investigation. We examined TGFβ1 expression on alveolar macrophages and epithelial cells from control- and alcohol-fed rats by real time PCR and flow cytometry. TGFβ1 gene expression was significantly higher in alcoholic macrophages and TGFβ1 protein was predominantly membrane bound in macrophages. In addition, as compared to control, alveolar epithelial cells from alcohol-fed rats had significantly higher expression of alpha5 integrin chain that is implicated in TGFβ1 activation. Therefore, we hypothesized that alcohol-induced increase in epithelial alpha5 would activate membrane TGFβ1 on macrophages. To examine this, macrophage (NR8383) and epithelial (L2) cell lines were co-cultured on transwell membranes to assess the changes in paracellular permeability. Only alcohol primed co-cultures showed a decrease in epithelial barrier function that was reversed by anti-TGFβ1 treatment. Taken together, these results suggest that cellular contact between alveolar macrophages and epithelial cells is essential for alcohol mediated activation of TGFβ1 in the lung.
SerpinB1 functions in generating neutrophil extracellular traps (111.11)
Authors: Farley Kalamo , Stolley James , Cooley Jessica , Remold-O'Donnell Eileen
Published: January 2023

SerpinB1 is a potent inhibitor of neutrophil serine proteases (NSPs), and mice deficient for serpinB1 showed reduced capacity to clear Pseudomonas aeruginosa lung infection, increase of proinflammatory cytokines and neutrophil death compared with wild type (WT) mice. Here we questioned whether this deficient anti-microbial protection might be caused by altered production of neutrophil extracellular traps (NETs). NETs, extruded chromatin associated with granule proteins including NSPs, capture and kill microbes, but excess NETs can cause tissue injury, e.g., impairment of lung function in patients with cystic fibrosis. On stimulation with phorbol myristate actetate (PMA) or platelet-activating factor (PAF), isolated bone marrow neutrophils of serpinB1-/- mice produced increased NETs compared with WT. Isolated lung neutrophils of LPS-treated serpinB1-/- mice also produced increased NETs compared with WT in response to PMA, and increased numbers of neutrophils with expanded nuclei. NET production was NADPH oxidase-dependent, and NADPH oxidase activity did not differ between the genotypes. Fluorescent microscopy showed that SerpinB1 translocated in PMA treated human neutrophils from the cytoplasm to the nucleus prior to nuclear membrane disintegration. SerpinB1 was also found as an extracellular component of NETs. These findings indicate that SerpinB1 contributes to regulation of NET formation and, by its presence in NETs, may protect against protease-induced tissue injury.