bluuebunny/crossref_metadata_2025 huggingface dataset:
ICAM-1 expression on CD8+ T cells negatively regulates IFN-γ production. (50.28)
Authors: Zumwalde Nicholas , Shimizu Yoji
Published: January 2023

The factors that regulate the production of effector cytokines following antigen stimulation of naïve CD8+ T-cells are incompletely characterized. We utilized ICAM-1-/- mice to determine if the expression of ICAM-1 on naïve CD8+ T-cells modulates CD8+ T-cell activation and differentiation. Stimulation of purified wild-type OT-I CD8+ T-cells with purified MHC/Antigen, B7-1, and the inflammatory cytokine IL-12 results in robust proliferation and differentiation into CD8+ effector T-cells producing IFN-γ. This activation is associated with prominent T-cell:T-cell clusters and increased expression of ICAM-1. The presence of IL-12 sustains elevated levels of ICAM-1 on activated CD8+ T-cells and enhances T-cell:T-cell clusters. Activation of ICAM-1-/- OT-I T-cells results in comparable levels of proliferation as wild-type OT-I T-cells. However, ICAM-1-/- CD8+ T-cells fail to form significant homotypic clusters upon activation in this system. In addition, when compared to wild-type OT-I T-cells, antigen-specific activation of ICAM-1-/- OT-I T-cells results in a higher percentage of effector T-cells producing IFN-γ. These results suggest that inflammatory cytokines such as IL-12 may attenuate the generation of IFN-γ+ CD8+ effector T-cells via modulation of ICAM-1 expression following antigen stimulation of naïve CD8+ T-cells.
Inflammatory responses elicited by ocular herpes simplex virus type 1 infection are inhibited by upregulation of programmed cell death ligand 1 on trigeminal ganglionic neurons (50.29)
Authors: Cherpes Thomas , Jeon Sohyun , Vicetti Miguel Rodolfo , Phillips Jaclyn , Hendricks Robert
Published: January 2023

T cell effector responses are required for clearance of viral infection. In locations such as the central and peripheral nervous systems, where cell types lack regenerative ability, it is also critical that protective T cell responses produce minimal collateral damage. Using a murine model of ocular herpes simplex virus type 1 (HSV-1) infection, we report that programmed cell death ligand 1 (PD-L1) expression by trigeminal ganglia (TG) neurons is dramatically upregulated during acute infection, and that this increased PD-L1 expression is regulated by local expression of gamma-interferon. We further observed that treatment with anti-PD-L1 antibody significantly increased the overall number of CD4+ and CD8+ T cells as well as the number of HSV-specific CD8+ T cells present in the TG during acute infection. Anti-PD-L1 antibody treatment also affected T cell activation status as increased granzyme B expression was detected in all three of these T cell populations. Although control of viral replication in the TG appeared to be unaffected by treatment, during acute infection TG neurons from mice treated with anti-PD-L1 antibody became susceptible to perforin-mediated killing. Together, our data demonstrate a novel neuroprotective role for PD-L1 during acute viral infection, and may also suggest avenues for development of therapies capable of inhibiting the neuronal damage associated with many inflammatory conditions.
The Wiskott-Aldrich Syndrome protein (WASp) controls iNKT cell effector function. (50.39)
Authors: Locci Michela , Catucci Marco , Draghici Elena , Bosticardo Marita , Roncarolo Maria Grazia , Villa Anna
Published: January 2023

The Wiskott-Aldrich Syndrome protein (WASp) is a regulator of actin remodeling in hematopoietic cells. In T cells, WASp is required for a long-lived Immunological Synapse (IS) and TCR-mediated cytokine production. We recently published that WASp is fundamental in the biology of iNKT cells, a peculiar T cell subset activated by glycosphingolipid antigens presented by APC. We showed that murine was-/- iNKT cells were defective in IL-4 and IFN-γ production upon in vivo activation. It remains an open issue whether this functional impairment is caused by a cell autonomous functional defect of was-/- iNKT cells or by an impaired crosstalk between iNKT cells and APC. To address this issue we analyzed cytokine production upon in vitro activation of wt and was-/- iNKT cells by wt and was-/- DC. The data obtained indicate the defective IL-4 production due to a was-/- iNKT cell autonomous defect and the lower IFN-γ production caused by an inefficient crosstalk between was-/- iNKT cells and was-/- DC. However was-/- iNKT cells produced sustained levels of cytokines upon in vitro stimulation with TPA and Ionomycin, suggesting a TCR-mediated functional impairment of these cells. Supporting this hypothesis was-/- iNKT cells presented a decreased TCR avidity. Altogether these results demonstrate the relevance of WASp in TCR-mediated effector function of iNKT cells. Ongoing studies are aimed at evaluating the ability to properly form the IS by was-/- iNKT cell and APC.
IL-2/ IL-2 mAb treatment generates prolonged expansion and increased cytokine production by NKT cells (50.42)
Authors: Webster Kylie , Kyparissoudis Konstantinos , Godfrey Dale , Sprent Jonathan
Published: January 2023

Regulatory T cells can be dramatically boosted in vivo by stimulation with a complex of IL-2 and an IL-2 mAb (JES6-1). The swift 15-fold expansion creates a tolerogenic environment that significantly perturbs autoimmunity and allograft rejection. We recently observed that IL-2/mAb treatment also expands Natural Killer T (NKT) cells, a glycolipid-specific, CD1d-restricted T cell lineage implicated in both regulatory and immunogenic responses. Interestingly, the phenotype of the IL-2/mAb-expanded NKT cells differed from those expanded with the prototypic NKT ligand, alpha-galactosylceramide (α−GalCer). The IL-2/mAb-expanded NKT cells were relatively stable and contracted slowly. They were hyper-functional, with the capacity to rapidly produce cytokines for several days after stimulation. In comparison, α−GalCer stimulated NKT cells characteristically produce a quick burst of Th1 and Th2 cytokines, an ability that then dramatically declines along with their number in the following days. Interestingly, the range of cytokines produced by IL-2/mAb-NKT cells was broad and included not only IFNγ and IL-4, but also significantly high amounts of IL-10 and IL-13. IL-17, however, was notably reduced. These variations in cytokine production appeared to alter the phenotype of dendritic cells in their vicinity. These data suggest an alternate approach to the expansion of NKT cells, specifically within the context of a regulatory milieu that may be appropriate for tolerance induction.
Distinct sites in ADAP control IKKα/β phosphorylation, IKKγ ubiquitination and cell cycle progression in T cells. (50.43)
Authors: Srivastava Rupa , Burbach Brandon , Shimizu Yoji
Published: January 2023

NF-κB activation following TCR engagement involves PKCθ-dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IκB-kinase (IKK). We previously identified a novel role for the adhesion and degranulation-promoting adapter protein (ADAP) in regulating the assembly of the CBM complex via an interaction of ADAP with CARMA1. The ADAP interacts with another adaptor protein SKAP55 which is critical for for T cell integrin activation and T:APC conjugate formation. In this study, we identify a novel site in ADAP that is critical for association with the TGFβ activated kinase (TAK1). ADAP is critical for recruitment of TAK1 and the CBM complex, but not IKK, to PKCθ. While both the TAK1 and CARMA1 binding sites in ADAP are essential for IκB phosphorylation and degradation, only the TAK1 binding site in ADAP is necessary for IKK phosphorylation. In contrast, only the CARMA1 binding site in ADAP is required for ubiquitination of IKKγ. Loss of ADAP in ADAP-/- primary T cells led to a block in G1-S transition. Expression of ADAP mutants lacking TAK1 and CARMA1 binding sites do not rescue the block in cell cycle progression while ADAP mutant lacking the SKAP55 binding site rescued cell cycle progression. Thus, distinct sites within ADAP control two key activation responses that are required for NF-κB activation and provide unique signals for cell cycle cycle progression and T cell poliferation.
RIAM and RapL regulate distinct signaling events and functional outcomes upon TCR-mediated activation (50.8)
Authors: Patsoukis Nikolaos , Lafuente Esther , Boussiotis Vassiliki
Published: January 2023

Rap1, a small GTPase of the Ras superfamily, regulates inside-out activation of integrins and cell adhesion. Two new Rap1 effectors, RapL and RIAM, have been implicated in this function. Significant differences in their structure indicate that these molecules may mediate distinct signaling events. RIAM has a coiled-coil region, RA and pleckstrin homology (PH) domains, and proline-rich regions with specific motifs capable of interacting with the EVH1 domains of Ena/VASP proteins, with profilin and with SH3 domain containing proteins. RapL has an RA domain and a C-terminal coiled-coil region. In the present study we investigated the role of RIAM and RapL in regulating signaling and functional events activated via the TCR. Using shRNA we determined that activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by elimination of RIAM, whereas activation of these kinases was unaffected by elimination of RapL. Activation of p38 was unaltered in RIAM-KD but was abrogated in RapL-KD cells. Moreover, knockdown of RIAM but not RapL resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. Strikingly, RIAM-KD resulted in impaired IL-2 production, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon TCR/CD3-plus-CD28 crosslinking. Thus, RIAM and RapL have distinct roles in regulating signaling and functional outcomes of T cell responses.
Regulation of IL-13 expression in lymphocytes by the Let-7 miRNA family (51.11)
Authors: Gamper Christopher , Lee Youl-Nam , Brandal Stephanie , Mendell Joshua , Takemoto Clifford , McDevitt Michael , Powell Jonathan
Published: January 2023

IL-13 is a Th2 cytokine that plays a critical role in antibody production, parasite immunity, allergic disorders, and as a negative regulator of anti-tumor immunity. Curiously, microarray screens of activated Th1 clones demonstrated high levels of IL-13 RNA in spite of essentially no IL-13 protein production. Such findings were subsequently confirmed in primary cells cultured in Th1 conditions. We hypothesized that these observations might be due to regulation by miRNAs. Indeed, a highly conserved Let-7 site is predicted in the 3’ UTR of IL-13. Furthermore, several Let-7 family members are more highly expressed in activated Th1 cells when compared with activated Th2 cells. To test our hypothesis, we generated luciferase reporter constructs containing the native 3’ UTR of IL-13 (WT) or a 3’ UTR with the Let-7 site mutated (Mut). Overexpression of Let-7 mimetics inhibited the IL-13 WT but not the IL-13 Mut construct. In addition, the transfection of the mimetics or infection with Let-7g expressing retrovirus led to the inhibition of native IL-13 protein production. Alternatively, inhibitors of Let-7 family members or infection with a retrovirus encoding the Let-7 antagonist LIN28B led to enhanced IL-13 production. Overall our data suggest Let-7 miRNA plays a critical role in the regulation of IL-13 and that such molecules might be exploited to inhibit IL-13 in allergic diseases and to enhance anti-tumor immunity.
The Crohn’s Disease protective SNP rs11209026 mediates alternative splicing in human IL23R. (51.14)
Authors: Gallagher Grant , Brazaitis Jonothan , Yu Raymond
Published: January 2023

SNP rs11209026 in the human IL23R gene has been associated with susceptibility in human autoimmune diseases. Notably, the rarer “A” allele confers protection against Inflammatory bowel diseases. IL-23 and IL-23R signalling are key to the development of the pro-inflammatory T-cell subset, Th17 cells, where they stabilise the functionally-mature Th17 phenotype. Despite causing an Arg>Gln change at residue 381, inside the cell membrane, no differences in IL-23 signalling resulting from this change have been reported. Multiple splice-forms of the human IL23R transcript exist and one of these, lacking exon-9 (“Δ9”), encodes a potent IL-23 inhibitor comprising a truncated, soluble HuIL23Rα chain. Here, we show that the disease-protective “A” allele destroys the affinity of this site for the splice-factor Sf2 and so favours transcription of HuIL23RΔ9. Using both BAC and mini-gene approaches, we used anti-sense oligonucleotides to target the Sf2 binding site and cause dose-responsive, specific induction of Δ9. We applied this strategy to primary human CD4+ lymphocytes, again inducing Δ9 mRNA. In experiments designed to differentiate human Th17 cells in vitro this caused a reduction in wild-type IL-23R expression on the cells’ surface and reduced the expression and secretion of the signature Th17 cytokines, IL-17A and IL-17F. Thus, the presence of allele "A" predicts the elevated expression of Δ9 and a consequent lower ability to respond to IL-23 or to maintain Th17 activity.
Regulation of IL-27 Production by Antigen Presenting Cells in Mice (51.15)
Authors: Qin Fenghua , Li Sky , Dong Lingpu , Lin Xiao , Yang Xifeng
Published: January 2023

Interleukin-27 (IL-27) is a novel IL-6/IL-12 cytokine family member that consists of EBV-induced gene-3 (EBI3) and p28 subunits. IL-27 triggers expansion of antigen specific naïve CD4+ T cells, promotes polarization of Th1 cells, and inhibits the development of Th2 and Th17 cells. It is known that IL-27 is an early product of activated antigen-presenting cells. But the regulation of IL-27 production has not been well documented. Here, we studied the effects of IFN-γ and LPS on IL-27 production by mouse macrophages. Thioglycollate-elicited peritoneal cells from Balb/c mice were stimulated with IFN-γ and LPS with various combinations and different time periods. IL-27 expression was examined by flow cytometric analyses of intracellular staining with fluorochrome-conjugated anti-mouse IL-27 mAb (MM27.7B1) and bead-based analyses of the cultured supernatants. The data showed that LPS alone-stimulated macrophages yielded less than 10% of IL-27 positive cells on 24 hrs after stimulation. However, if the cells were primed with IFN-γ for 2 hours and then stimulated with LPS, higher frequency of IL-27 producing cells was observed (25% to 45%). Kinetic studies demonstrated that a significant level of IL-27 secretion was detected as early as 2 hours and the peak production was observed around 22 hours post stimulation.
Retinoic acid modulates IL-4 receptor alpha chain expression and signaling pathways in intestinal epithelial cells and macrophages (51.3)
Authors: Dawson Harry , Chen Celine , Fendlay Devin , Chitko-Mckown Carol , Wagner Bettina , Boyd Patricia , Lunney Joan , Urban Jr. Joseph
Published: January 2023

We have shown that all-trans retinoic acid (ATRA) increased mRNA expression of several IL-4-induced chemokines in explanted porcine alveolar macrophages. We now describe that ATRA modulated IL-4-induced signaling pathways in primary macrophages, and several macrophage and epithelial cell lines. ATRA increased epithelial cell mRNA and protein for the IL-4 receptor alpha chain (IL4RA), and IL-4 induced phosphorylation of STAT6 and expression of CLCA1 mRNA in vitro. In primary macrophages and several macrophage cell lines, ATRA-induced IL4RA mRNA and enhanced IL-4-induced expression of DHSR3, MRC1, PLAT, PTPN1, TGM2, and WARS mRNA and facilitated IL-4-inhibited expression of TLR2 and CYBB mRNA. However, ATRA blocked IL-4-induced expression of DHSR9, MAF, SEPP1 and STAT5A mRNA and counteracted IL-4 inhibited expression of BCL2L1, CCL2, NCF4, and PDGFB mRNA. These activities were largely shared by the RAR-a selective retinoid, Am580. ATRA variably affected the expression of a large number of other IL-4-induced mRNAs. These data indicated that ATRA and related retinoids can modulate Th2-related responses through mechanisms that utilize IL-4 and IL-4 receptor signaling pathways.
A naturally antagonist of human IL-23 inhibits the development and function of human Th17 cells (51.4)
Authors: Gallagher Grant , Yu Raymond
Published: January 2023

The Th17 subset of CD4 cells is critical to inflammation. Secretion of IL-17 by Th17 cells drives inflammation in human diseases and their mouse models, including IBD, psoriasis and arthritis. The differentiation of mature Th17 cells depends upon stimulation in the presence of IL-6, TGF-β and IL-21 and the induction of RORγt, but their phenotype is stabilised by IL-23, which is essential to Th17 stability and function. Initial induction of Th17 cells can be antagonised by IL-4 or IFN-γ, but mechanisms through which terminal differentiation can be inhibited have not been identified. We found that HuIL23Rα chain mRNA transcripts exist that lack exon-9 (“Δ9”); these are translated to a truncated receptor form containing the entire external domain of the wild-type protein. Here, we show that this soluble variant of the HuIL23Rα chain antagonises IL-23 signalling and Th17 maturation. The variant is secreted and present at low levels in the blood. This soluble form represents 10% of HuIL23Rα chain mRNA. It binds IL-23 in solution and inhibits the phosphorylation of STAT3 caused by IL-23. In experiments designed to differentiate human Th17 cells in vitro, the Δ9 form inhibits the production of Th17-associated cytokines, including IL-17A and IL-17F. Δ9 does not bind IL-12; thus it is a specific inhibitor of IL-23 and a modulator of Th17 cells. Our results indicate that this soluble form of HuIL23Rα likely functions to regulate Th17 activity.
Effects of cell-type specific hIL-10 expression on disease susceptibility (51.5)
Authors: Ranatunga Dilini , Hedrich Christian , Wang Fengying , McVicar Daniel , Norwak Nathan , Joshi Trupti , Feigenbaum Lionel , Grant Lindsay , Stager Simona , Bream Jay
Published: January 2023

IL-10 is an immunoregulatory cytokine produced by many cell types. Studies in mice suggest the cellular source of IL-10 plays a critical role in determining disease outcome. Yet the effects of tissue specific human IL-10 (hIL-10) expression are not well understood. To address this issue, we generated a hIL-10 transgenic mouse using a bacterial artificial chromosome (hIL10BAC). The hIL10BAC contains most if not all the regulatory sequences that govern hIL-10 production thus allowing tissue specific expression. As hIL-10 is biologically active in the mouse, reconstituting Il10-/- mice with the hIL10BAC allowed us to evaluate the effects of hIL-10 on known IL-10 dependant phenotypes. Expression of IL-10 from myeloid sources in WT mice protects against lethal endotoxic shock. Faithful expression of the hIL10BAC in myeloid cells rescued Il10-/- mice from this fate. In contrast, T-cell-derived IL-10 leads to high parasite burdens in the livers and spleens of WT mice infected with Leishmania Donovani. However both Il10-/- and Il10-/-/hIL10BAC mice cleared the parasites. The failure to reproduce the susceptibility phenotype is attributed to weak hIL-10 expression in CD4+ T cells. Similarly, inflammatory bowel disease (IBD) is also IL-10-dependant and resistance is thought to involve T-cell-derived IL-10. Surprisingly, we found that the hIL10BAC rescues Il10-/- mice from IBD, suggesting differential regulation of hIL-10 in CD4+ T cells found in the gut versus the spleen.
Generation of fully human monoclonal antibodies following anthrax vaccine absorbed (AVA) vaccination that provide protection from lethal toxin challenge. (52.15)
Authors: Crowe Sherry , Vineyard Amanda , Smith Kenneth , Carla Guthridge , Morris Jennifer , Wilson Patrick , James Judith
Published: January 2023

The release of Bacillus anthracis spores can cause significant morbidity and mortality and effective vaccination strategies and immunotherapeutics are needed. This study seeks to identify and characterize the major humoral targets that provide protection. To this end, we isolated antibody secreting cells from AVA vaccinated individuals and generated a panel of fully human monoclonal antibodies. Ten of these antibodies were highly specific for the protective antigen (PA), with affinities ranging from 3.7x10-7 to 1.4x10-10. Antibodies p6C01 and p6F01 were able to neutralize lethal toxin activity in vitro even at low concentrations (EC50 = 0.1µg/ml). Passive transfer of either human monoclonal antibody to A/J mice prior to lethal toxin challenge conferred 80-90% protection. Using a combination of western blotting and subunit ELISAs, we have shown that the p6C01 antibody binds to domain 3 of PA, which is critical for heptamer formation. Experiments are ongoing to further characterize the mechanism(s) of protection by these antibodies. These fully human antibodies can be used as a tool to identify protective humoral epitopes, as well as provide directed immunotherapeutics.
Immunization with Adenoviral-expressed salivary gland proteins (SALPs) decreases spirochete load in a murine model of Lyme borreliosis (52.2)
Authors: Ullmann Amy , Dolan Marc , Fikrig Erol , Piesman Joseph , Zeidner Nordin
Published: January 2023

It is known that tick saliva contains a vast array of proteins that function as anti-haemostatic, anti-inflammatory, and immunomodulatory factors when delivered to the vertebrate host. Previous studies have also demonstrated that prior exposure to tick saliva induced specific immunity to tick infestation, as well as affording protection against tick-transmitted Borrelia burgdorferi infection in the mammalian host. In our current studies we explored a vaccination technique to modulate tick feeding, using an adenovirus (Ad) expression system to deliver four salivary proteins (previously shown to modulate host immunity) derived from Ixodes scapularis; Salp15, Salp25A, Salp25D, and ISAC. C3H/HeJ mice were immunized individually with Ad-Salp15 and Ad-ISAC constructs, as well as all four constructs simultaneously. Immunized mice were then challenged with B. burgdorferi-infected I. scapularis ticks. Utilizing quantitative PCR, spirochete burden in target organs like heart and bladder was reduced by 55% compared to animals immunized with an empty Ad-cassette. The generation of anti-salivary protein antibodies in a murine model was also confirmed. Our results indicate that adenovirus expressed tick salivary proteins can stimulate dendritic cell expression of tick salivary proteins, as well as tick-specific humoral and cellular immune responses in mice, affording at least partial protection against subsequent tick-transmitted B. burgdorferi infection.
Multiple antigenic peptide vaccine targeting the loop neutralizing determinant of protective antigen from Bacillus anthracis completely protects rabbits from aerosol spore challenge (52.24)
Authors: Oscherwitz Jon , Yu Fen , Cease Kemp
Published: January 2023

The loop neutralizing determinant (LND) from domain 2 of Bacillus anthracis protective antigen (PA) has been shown to represent a potentially important target for an epitope-specific vaccine for anthrax. This region in the 2β2-2β3 loop of PA is immunologically inert, since rabbits immunized with PA or otherwise immune to PA do not contain antibody specific for this region. Herein we evaluated the immunogenicity in rabbits of a MAP peptide comprising the T* helper T cell epitope from Plasmodium falciparum colinearly synthesized at either the N- or C-terminus with a.a. 304-319 from PA. In initial studies, the MAP peptides formulated in Freund’s adjuvant were found to be highly immunogenic in rabbits, rapidly eliciting high levels of neutralizing antibody. To more fully evaluate this peptide as an anthrax vaccine, we immunized separate groups of rabbits (n=7) five times at two-week intervals with either of the two LND MAP peptides or with soluble PA in Freund’s adjuvant and then subjected all rabbits to a 200 LD50 aerosol spore challenge with B. anthracis Ames strain. 100% of rabbits immunized with the LND MAP in the B-T orientation and with soluble PA, and 89% of rabbits immunized with the LND MAP in the T-B orientation, survived the spore challenge, while all naïve rabbits died. Analysis of post-challenge serum suggested little or no spore germination in challenged rabbits. We conclude that the LND MAP peptide is a promising epitope-specific prototype vaccine for anthrax.
Recombinant human antibodies to IFN-α from the immune repertoires of MG/thymoma donors (83.13)
Authors: Hammond Philip , Boozer Christy , Branum Mark , Fox Raymond , Lilly Rebecca , Mosley Bruce , Ward Courtney , Whitman Darbie , Frey Steve , Cicirelli Mike , Olsen Ole , Moyle Matthew
Published: January 2023

A serum titer of autoantibodies to IFN-α has been reported in several autoimmune conditions and in many cases has been shown to neutralize IFN-α. The IFN-α antibody repertoire from 10 patients with autoimmune myasthenia gravis and associated thymoma (MG/T) was characterized. Surface IgG positive memory B-cells from peripheral blood mononuclear cells (PBMC) were cultured in 384-well plates to produce assayable quantities of antibody. Sensitive antibody microarray and AlphaScreen™ binding assays were used to identify antibodies reactive with IFN-α(2a). A range of IFN-α subtype specificity profiles was observed in competition assays for binding to the 12 subtypes of IFN-α. A subset of these antibodies was expressed recombinantly after recovery of heavy and light chain variable regions by RT-PCR and these recombinant antibodies recapitulated the activities demonstrated in the B-cell culture supernatants. These antibodies fell into 2 groups, blocking binding of IFN-α(2a) to either the IFNAR2 or IFNAR1 subunit of the Type 1 interferon receptor. The ability of these antibodies to neutralize the activity of IFN-α(2a) in a Daudi cell proliferation assay was confirmed. The antibody repertoires of MG/T patients include high affinity antibodies with both broad and narrow specificities for the different subtypes of IFN-α. These function-blocking human antibodies may have therapeutic utility in IFN-α dependent diseases such as SLE.
Activated IL-1R1 Signaling Pathway Induces Th17 Cell Differentiation in Patients with Relapsing Remitting Multiple Sclerosis (83.18)
Authors: Sha Yonggang , Troiani Luigi , Chopra Manisha , Markovic-Plese Silva
Published: January 2023

BACKGROUND: IL-1β plays a crucial role in the development of human Th17 cells. However, its mechanisms of action are still not completely elucidated. Recent has study discovered that Th17 cells are characterized by IL-1R1 expression. RESULTS: Our study has revealed that IL-1R1 and IL-1R2 gene expression is significantly increased in both naïve and memory CD4+ T-cells derived from patients with relapsing-remitting multiple sclerosis (RRMS) in comparison to matched healthy controls. IL-1R1 expression is up-regulated in the in vitro differentiated Th17 cells in comparison to the Th1 and Th2 cell subsets. IL-17A production was induced by IL-1β. This induction was blocked by IL-1R antagonist in the naïve CD4+cells cultured in the presence of Th17 polarizing cytokines. When IL-1R1 gene expression was silenced using siRNA, human naïve CD4+ T-cells cultured in the presence of Th17 polarizing cytokines had a significantly decreased expression of IL-21, IL-22, IL-23R, IRF4, RORC, IL-17A and IL-17F genes, confirming that IL-1R1 signaling induces Th17 cell differentiation. IRF4 gene expression silencing, similarly inhibited Th17 cell differentiation in the presence of IL-1b, indicating that IL-1R1 signals upstream of IRF4. CONCLUSION: Our study has identified that IL-1R1-mediated signaling pathway may be constitutively activated, leading to an increased Th17 cell expansion and autoimmune response in patients with RR MS.
CD44act as a marker for T cell regulatory activity in inflammatory bowel disease (83.21)
Authors: Estess Pila , Shah Rajesh , Siegelman Mark
Published: January 2023

Inflammatory bowel disease (IBD) is an autoimmune condition that results from attack of intestinal tissue by effector T cells. While CD4+CD25+ naturally occurring regulatory T cells (Treg) have great potential for treatment of IBD and other autoimmune conditions, therapeutic use is hindered by the inability to identify those most specific for controlling disease and difficulty in distinguishing them from T effector cells that induce disease. We have investigated the isolation of highly competent Treg from patients with Crohn’s Disease and Ulcerative Colitis using the conformationally activated hyaluronan-binding form of CD44 (CD44act), previously shown to be associated with markedly improved Treg function and traffic to sites of inflammation. Using a CD44act-dependent primary adhesion assay, we have demonstrated that this marker can be used to reliably identify and isolate functional regulatory T cells from PBMC of affected individuals. Among IBD patients, greater numbers of circulating T lymphocytes bearing CD44act are found in peripheral blood during disease flare or during resolution of disease flare, consistent with a regulatory role. Isolated CD44act T cells from patient blood prevent the activation of Teffector cells from the same individual. After isolation and in vitro expansion, these cells retain regulatory behavior and express cytokines consistent with their being regulatory T cells
Resting B cells as Inducers of Antigen-Specific T Regulatory Cells (83.22)
Authors: Zhai Yougang , Zhou Lan , Zhong Zhenyu , Qiao Liang
Published: January 2023

We have previously shown that resting B cells are able to expand T regulatory cells through TGF-beta3 whereas activated B cells induce increased apoptosis of T regulatory cells. Thus, we hypothesize that resting B cells can be used to induce antigen-specific T regulatory cells. To test our hypothesis, we isolate resting B cells from mice and load the cells with Ova, and then adoptively transfer the B cells to Ova-specific TCR transgenic mice. We determine numbers of OVA-specific T effector cells and T regulatory cells in the adoptively transferred mice and in control mice that receive untreated resting B cells alone by flow cytometry (Foxp3). We further determine if these T regulatory cells mediate immunosuppressive activities by using proliferation assay in vitro and by in vivo prevention of immune response against challenge with OVA + adjuvant. This study will provide basis to develop approaches to induce antigen-specific T regulatory cells to treat inflammatory diseases such as allergy and autoimmune diseases.
Induction of antigen specific T regulatory cells to suppress inflammatory responses by tolerogenic vaccination (83.6)
Authors: Wang Bin , Jin Jin
Published: January 2023

Inflammatory disorders, such as asthma, could be detrimental. Dust mites may cause 50-80% of asthma cases. No effective treatments, particularly those targeted to antigen-specific inhibition, are still lacking. In our previous study demonstrated that co-immunization of DNA and protein vaccines together as tolerogenic vaccination into animal could inhibit inflammatory responses in a antigen-specific matter. Using dust mite induced asthma as model, we assess the immunotelarogenic effect of co-immunization. Mice immunized with a Derp1 tolerogenic vaccine were examined their ability to prevent this experimental asthma by assessment of infiltrations of eosinophils and lymphocytes into lungs. The immunization with the tolerogenic antigens not only induced significant decrease of inflammatory responses in the lungs, but also to inhibit Th2 cytokines and production of IgE. The induced tolerogenic responses are associated tightly with the generation of CD4+CD25-Foxp3+ iTregs. We observe that these iTregs exhibit phenotypic markers including IL10+, CTLA4low, GITRlow, PD-1- and ICOSlow, which differ from those documented natural CD4+CD25+ Tregs and iTreg. Blockade of TGF-β1 could demolish the induction of this iTregs and suppressive responses. This tolerogenic vaccination induces the iTreg via TGF-β1 and present a novel therapeutic strategy to treat or prevent inflammatory disorder related diseases.
Computational modeling of B cell signaling shows B cell affinity discrimination requires kinetic proofreading (84.2)
Authors: Tsourkas Philippos , Raychaudhuri Subhadip
Published: January 2023

B cells signaling in response to antigen is proportional to antigen affinity, a process known as affinity discrimination. B cells can acquire antigen in membrane-bound form on the surface of antigen-presenting cells (APCs), with signaling being initiated within a few seconds of contact. During the early stages of contact, B cell receptors (BCRs) on protrusions of the B cell surface bind to antigen on the APC surface and form micro-clusters of 10-100 BCR-antigen complexes. We use a Monte Carlo computational model to show that B cell affinity discrimination at the level of micro-clusters requires a threshold antigen binding time, similar to kinetic proofreading. We find that if BCR molecules become signaling-capable immediately upon binding antigen, there is a loss in serial engagement as BCR-antigen affinity increases, due to the increased bond lifetime of high-affinity bonds. This results in an unnatural decrease in signaling strength with increasing affinity. A threshold antigen binding time favors high affinity BCR-antigen bonds, as these long-lived bonds can better fulfill the threshold time requirement than low-affinity bonds. A threshold antigen binding time of ~10 seconds results in monotonically increasing signaling with affinity, as seen in B cell activation experiments. This time matches well with the experimentally observed time (~20 seconds) of lipid raft-mediated conformational changes in BCR signaling domains that lead to association with Syk.
BCR mediated c-Rel induction facilitates NF-κB2 and RelB expression (84.22)
Authors: Wright Jacqueline , Castro Iris , Damdinsuren Bazarragchaa , Hoek Kristen , Carlesso Gianluca , Shinners Nicholas , Gerstein Rachel , Woodland Robert , Sen Ranjan , Khan Wasif
Published: January 2023

B cell receptor (BCR) and B cell activating factor receptor (BAFF-R) signaling are critical for the generation of mature splenic B cells. BCR expressing Transitional type 1 (T1) cells migrate from the bone marrow to the spleen where they must successfully pass BCR mediated checkpoints to become Transitional type 2 (T2) cells and subsequently mature follicular B (FoB) cells. T1 and T2 cells are fundamentally different in their response to BCR or BAFF-R stimulation; T1 cells do not survive whereas T2 cells do when triggered through BCR or BAFF-R. Our results demonstrate that Bruton’s tyrosine kinase (Btk) plays a critical role in both BCR and BAFF-R mediated T2 and mature B cell survival. These results suggest that a potential mechanism that provides T2 cells with a survival advantage is their ability to sustain c-Rel expression in response to BCR engagement via mechanisms involving Btk. The increased c-Rel expression coincides precisely with the up-regulation of anti-apoptotic members of the Bcl-2 family as well as BAFF-R and its substrate p100 (NF-κB2). Thus, BCR indirectly enhances the survival function of BAFF-R in part through c-Rel. Consistent with a supporting role for BCR/c-Rel signaling in the activation of alternative NF-κB pathway, c-Rel-deficient B cells are impaired for BCR-induced expression of p100 as well as RelB.
CD40 signaling regulates both adaptive and innate immune receptors through TRAF degradation (84.3)
Authors: Graham John , Bishop Gail
Published: January 2023

CD40, a tumor necrosis family member, is a costimulatory molecule central to adaptive immune responses. CD40 activates many of the same pathways as innate immune receptors; however, the outcome of CD40 signaling is much different. Whereas innate immune receptors rapidly initiate early protective immune responses, CD40 engagement and signaling results in a fundamental change toward a regulated, adaptive immune response. The specific regulatory signals that restrain the CD40 response have remained elusive. Here we show that upon signaling, CD40 specifically negatively regulates its own signals as well as those delivered by other receptors that rely on TRAF2 and TRAF3 for signaling. This negative regulation of CD40 and TLR signals is caused by CD40’s unique ability to induce both TRAF2 and TRAF3 degradation upon signaling. Hence CD40, as a molecule central to immune responses, is not only able to regulate its own signals, it is able to transition an immune response from an early inflammatory stage to a regulated adaptive stage.
Fc receptor-like 5 has dominant inhibitory function in B cells that is mediated via Lyn and SHP-1 (84.5)
Authors: Zhu Zilu , Davis Randall
Published: January 2023

An extended family of Fc receptor-like (FCRL) molecules has tyrosine-based signaling properties and is preferentially expressed by B lineage cells. In mice, FCRL5 is distinguished by its discrete expression among innate B lineage lymphocytes including splenic marginal zone B cells and peritoneal cavity-derived B1 B cells. FCRL5 has five Ig-like extracellular domains and a cytoplasmic tail containing an ITAM-like sequence and a consensus ITIM. To evaluate its signaling functionality, chimeric receptors comprised of the extracellular and transmembrane portions of FcγRIIb in frame with cytoplasmic Y>F variants of FCRL5 were generated in the mouse A20-IIA1.6 B cell line. Pervanadate treatment of chimeric transductants revealed that both tyrosine-based motifs can be phosphorylated. Coligation of the unmodified FcγRIIb/FCRL5 chimeric receptor with the B cell receptor (BCR) inhibited calcium mobilization, whole cell tyrosine phosphorylation, and Erk kinase activation via the ITIM-dependent (Y566) recruitment of the SHP-1 phosphatase. Conversely, coligation of a chimeric mutant bearing the intact ITAM-like (Y543/Y556) sequence with the BCR resulted in subtle but consistent enhancement of calcium mobilization. This activation effect correlated with the recruitment of the Src-family kinase, Lyn. The inhibitory influence of FCRL5 was similarly observed in primary splenic marginal zone B cells. These data indicate that FCRL5 serves as a dominant inhibitory co-receptor in B cells.
Pax5 and E47 regulate AID and class switch recombination in young and aged BALB/c mice (84.6)
Authors: Landin Ana Marie , Frasca Daniela , Harrison Patrick , Scallan Martina , Hagman James , Blomberg Bonnie
Published: January 2023

Our laboratory has shown that stimulated B cells from senescent mice (19 to 24 months) have an intrinsic defect in class switch recombination (CSR) that is coincident with reduced E47. In aging, the decrease in E47 correlates proportionally with levels of activation induced cytidine deaminase (Aicda/AID) transcripts; but, other transcription factors may be involved. Pax5 has been shown to play a role in CSR. The purpose of the present study was to determine whether 1) Pax5 is decreased with age, 2) the decrease in Pax5 correlates with decreased CSR in aged B cells, 3) increased E47 and/or Pax5 can rescue the decreased CSR observed in these cells and 4) depletion of Pax5 down-regulates E47 and/or AID expression. Here, we demonstrate that E47 and Pax5 are both down-regulated in mature splenic B cells of aged mice. Endogenous E47, Pax5 and IgG1 are increased in old as well as young primary stimulated B cells in response to exogenously expressed E47. We successfully rescued levels of both E47 and Pax5 mRNA and protein in transduced splenic B cells of aged mice when compared to young untransduced controls. Restoration of Pax5 expression increased E47, AID and CSR in B cells from aged mice. Conversely, silencing Pax5 by means of siRNA transfection decreased E47 and AID expression significantly. These results suggest a possible feedback mechanism between E47 and Pax5. Our results show that E47 and Pax5 cooperate to regulate AID and CSR in both young and old B lymphocytes.
MS4a4B, a CD20 Homologue in T cells, inhibits T cell propagation by modulation of cell cycle (85.1)
Authors: Xu Hui , Yan Yaping , Williams Mark , Carey Gregory , Yang Jingxian , Li Hongmei , Zhang Guang-Xian , Rostami Abdolmohamad
Published: January 2023

MS4a4B, a homologue of CD20 in T cells, is a novel member of the MS4A gene family (Membrane-spanning 4-domain family, subfamily A). Recent data showed that MS4a4B may play functional roles in regulation of Th1 and regulatory T cells. We have demonstrated previously that MS4a4B negatively regulates T cell proliferation. Over-expression of MS4a4B in primary T cells and EL4 thymoma cells by viral vector inhibits cell proliferation. In this study, we determined whether MS4a4B inhibited T cell proliferation by modulating cell cycle progression. We transfected EL4 cells with either MS4a4B or mock lentiviral vectors and isolated stably transfected cells by flow cytometry. Transfected EL4 cells were synchronized by serum starvation and thymidine treatment, and then were stimulated with complete medium containing 15% fetal calf serum. We found that overexpression of MS4a4B in EL4 cells interfered with cell cycle progression from G0/G1 to S-G2/M phase. To further confirm the role of MS4a4B in cell cycle regulation, we knocked down expression of MS4a4B in T32 cells, a MS4a4B+ T cell line, by shMS4a4B-lentiviral vector. The results showed that shMS4a4B-mediated reduction of MS4a4B expression promoted cell cycle progression. Our data suggest that MS4a4B inhibits T cell proliferation by suppression of cell cycle progression. Supported by American Heart Association grant AHA-0830072N (to H.X)
Localization of Fas/CD95 to lipid raft microdomains sensitizes human CD4+ effector memory T cells to Fas-induced apoptosis (85.13)
Authors: Cruz Anthony , Ramaswamy Madhu , Siegel Richard
Published: January 2023

Fas-FasL interactions play a critical role in TCR-induced apoptosis of activated CD4+ T cells. However, naïve T cells activated through acute antigen stimulation expand and contract independently of the presence of the death receptor Fas. Fas deficiency prompts the accumulation of CD44+ memory phenotype T cells and autoimmunity in mice, suggesting Fas may play a critical role in memory and/or autoreactive T cell apoptosis. We have previously shown a correlation between lipid raft-associated Fas and sensitivity to apoptosis. Whether or not lipid raft microdomains influence Fas signaling in primary T cells, and whether lipid raft association of Fas is regulated in different primary cell types is unknown. Therefore, we have compared the sensitivity of purified naïve versus memory human CD4+ T cells to undergo Fas-mediated apoptosis. Effector memory phenotype cells lacking both the chemokine receptor CCR7 and the TNFSFR CD27 were the most sensitive to both anti-Fas antibody as well as FasL-induced cell death. Memory T cells have increased lipid raft-associated Fas receptor versus naïve cells, which could explain the increased Fas sensitivity in the memory pool. Using a competitive inhibitor to palmitoylation, we found sensitivity of effector memory T cells to Fas-induced apoptosis depended on Fas palmitoylation. These results suggest that effector memory cells are intrinsically more sensitive to Fas-induced apoptosis, possibly due to lipid raft localization of Fas.
Bim regulates the entry of poorly functional and/or low avidity Th1 effector cells into the memory pool (85.15)
Authors: Williams Matthew , Jay David
Published: January 2023

Following the clearance of acute infection, most pathogen-specific effector T cells die, with a small minority (5-10%) surviving the contraction phase and populating the memory T cell compartment. The pro-apoptotic Bcl-2 family member Bim is central in eliminating end-stage effector T cells during contraction. We recently found that not all clones that populate the effector Th1 pool are represented in the memory phase, and failure to survive corresponded to low TCR structural and functional avidity as well as increased Bim expression. We hypothesized that Bim was required to remove unwanted low avidity Th1 responders during the transition from effector to memory. We found that Bim-deficiency rescued the survival of end-stage Th1 effector clones and allowed their entry into the memory pool. However, these cells displayed decreased primary effector function and poor recall capacity, demonstrating that while Bim played a T cell intrinsic role in the death of Th1 effector cells, its absence did not permit the rescue of effector or memory Th1 cell function. Additionally, Bim-deficient CD4+ memory T cells declined at a rate similar to wildtype memory cells, suggesting that their survival is regulated by Bim-independent factors. We propose that Bim plays a crucial role in shaping optimally functional CD4+ memory T cell repertoires by removing low avidity and/or poorly functional Th1 clones during contraction.
Two distinct functions for the BTB domain of the CD4-differentiating factor Thpok (85.20)
Authors: Zamisch Monica , Wang Lie , Yockey Laura , Vinson Charles , Bosselut Remy
Published: January 2023

Thpok is a zinc-finger (ZF) transcription factor essential for CD4 T cell development. Thpok promotes CD4 T helper lineage commitment, in part through the repression of genes necessary for CD8 differentiation. A member of the BTB-ZF subclass of ZF transcription factors, Thpok contains two conserved motifs: a carboxy-terminal ZF DNA binding domain, and an amino-terminal BTB motif. Although the BTB domain is required for Thpok to promote CD4 lineage differentiation, its contribution to Thpok function is not clear. In vitro analyses of the BTB domain of the prototype family member, Bcl6 (B cell lymphoma 6) revealed a role of the BTB domain in oligomerization and transcriptional repression. In the present study, we examined whether the Thpok BTB domain simply serves to promote dimerization of Thpok molecules, or whether it also serves to recruit other transcriptional regulators. We generated chimeric Thpok proteins by replacing the BTB domain of Thpok with that of other family members, or with a heterologous dimerization domain. All chimeric molecules containing the BTB domains tested blocked CD8 lineage specific gene expression; in contrast, the chimeric molecule carrying a heterologous dimerization domain was only partially functional. These results indicate that the BTB domain of Thpok not only promotes dimerization but it also serves to control transcription, presumably through interactions with transcriptional corepressors.
WOX1 and MEK1 interactions determine T cell sensitivity to PMA-mediated apoptosis (85.4)
Authors: Chang Nan-Shan , Lin Hsin-Ping , Hsu Li-Jin
Published: January 2023

Phorbol myristate acetate (PMA) mimicks the function of diacylglycerol in binding to PKC and activating downstream Ras/Raf/MEK/ERK pathway. Here, we determined that PMA, at μM levels, rapidly induced ERK phosphorylation and WOX1 expression in less than 30 min, and subsequent apoptosis in 24 hr in Jurkat T cells (determined by DNA fragmentation and cell cycle analysis). WOX1, also named WWOX or FOR, is a tumor suppressor and a proapoptotic protein. In resting cells, WOX1 is present in the cytoplasm. When these cells were pretreated with a MEK inhibitor U0126 or overexpressed with dominant-negative WOX1, PMA-induced cell death was blocked. In stark contrast, less differentiated Molt-4 T cells were refractory to PMA-induced apoptosis. Most intriguingly, U0126 dramatically reduced the resistance and rendered apoptosis in Molt-4 T cells. Induction of Molt-4 T cell differentiation by nanomolar PMA restored its sensitivity to micromolar PMA-induced apoptosis. By co-immunoprecipitation and FRET, WOX1 was shown to physically interact with MEK1 in Jurkat T cells. PMA rapidly induced the dissociation of the WOX1/MEK1 complexes and U0126 blocked the dissociation, thereby supporting cell survival. Together, we described a novel PMA signaling to induce dissociation of the cytosolic WOX1/MEK1 complexes, thereby allowing WOX1 nuclear relocation for inducing apoptosis.