bluuebunny/crossref_metadata_2025 huggingface dataset:
A quantitative approach to T cell killing (TUM2P.1043)
Authors: Whitlock Ben , Altan-Bonnet Gregoire
Published: January 2023

Previous research has identified the pathways and proteins cytotoxic T lymphocytes (CTLs) use to kill antigen presenting cells (APCs); however, it is currently impossible to predict how many T cells are needed to kill a specific number of APCs in vivo. Current methods for examining killing rely on bulk measurements and effector to target ratios of 30:1, which are thought to be unphysiological. We have developed a single-cell killing assay that occurs within a 50x50μm well, which we image with time-lapse fluorescent microscopy. This has allowed us to make new observations that would have been impossible to discern using conventional bulk killing assays. Using this approach, we observe that T cells have a greater affinity for living cells than dead cells. We hypothesize that this phenomenon could be a way for T cells to avoid dead cells in vivo and are performing additional experiments to examine this.
Unmasking cancer to innate sensing by anti-CD47 antibody triggers adaptive immune-mediated tumor destruction (TUM3P.1045)
Authors: Xu Meng
Published: January 2023

Using xenograft and in vitro models, anti-CD47 antibody is thought to enhance "eat me" signaling to kill tumor cells by macrophage-mediated phagocytosis. However, the potential for adaptive immune response in anti-CD47 mediated tumor control has been ignored. Here we reveal a novel mechanism in which tumor regression after local anti-CD47 antibody treatment depends on cytotoxic T lymphocyte (CTLs) response. Mechanically, tumor recognition by DC following anti-CD47 enhances type I IFNs production and that subsequent IFNs signaling strengthens the cross-priming capability of DCs within the tumor microenvironment. Intriguing, anti-CD47 mediated antitumor immunity requires adaptor protein STING for the innate sensing of tumor cells. Additionally, we delineate a clinically relevant combinational strategy of chemotherapeutic drugs and anti-CD47 antibody to enhance the reduction of tumor burden without abrogating anti-CD47 initiated immunity.
Selective inhibition of the T cell p38 alternative activation pathway and pancreatic cancer (TUM3P.1046)
Authors: Alam Muhammad , Gaida Matthias , Bergmann Frank , Lasitschka Felix , Giese Thomas , Giese Nathalia , Hackert Thilo , Hinz Ulf , Hossain S. , Kozlov Serguei , Ashwell Jonathan
Published: January 2023

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm with a five year survival rate of ~5%, that is characterized by a marked fibro-inflammatory microenvironment, the presence of which can promote cancer induction and growth. Therefore, selective manipulation of local cytokines is an attractive if unrealized therapeutic approach. T cells possess a unique mechanism for activation of p38 MAPK downstream of T cell receptor (TCR) engagement in which it is phosphorylated on Tyr-323 (pY323), which is required for pro-inflammatory cytokine production. Analysis of 192 human pancreatic carcinomas revealed that having a high percentage of infiltrating pY323+ T cells was accompanied by large numbers of TNFa and IL-17-producing CD4+ TIL and very aggressive disease. Cancer progression was inhibited in two different murine pancreatic tumors models by genetic ablation of the alternative p38 activation pathway. Strikingly, a plasma membrane-permeable peptide derived from Gadd45α, the naturally-occurring inhibitor of p38 pY323+, reduced infiltrating CD4+ T cell production of TNFa, IL-17A, IL-10, and induction of secondary cytokines, slowed the growth of implanted tumor cells and inhibited progression of spontaneous K-ras-driven adenocarcinoma. Thus, TCR-mediated alternative p38 activation in tumor-infiltrating CD4+ T cells, which promotes the production of pro-tumorigenic inflammatory factors, can be targeted for therapeutic benefit.
Comparison of anti-OX40 to PD-1 and CTLA-4 blockade in T cell immunization/priming models (TUM3P.1047)
Authors: Vercellini Jonna , Tucker Christopher , Moran Amy , Polesso Fanny , Weinberg Andrew
Published: January 2023

OX40 agonists have shown to enhance anti-tumor immunity in several mouse tumor models with similar efficacy to PD-1 and CTLA-4 blockade. We wanted to compare these T cell immune enhancing strategies in subcutaneous vaccine models to understand the role that each of these Abs might have in immune priming. We used both the OVA-specific CD4 and CD8 TCR transgenic adoptive transfer models, D011.10 and OT1, to determine the effect these antibodies have upon vaccination with ovalbumin (OVA). Mice were immunized with whole OVA protein in the absence of adjuvant (soluble OVA). The Abs were delivered on the day of immunization and one day later. Peripheral blood and spleens were analyzed at several time points after immunization. We found that the OX40 agonist in both T cell models enhanced the % and numbers of Ag-specific T cells to a greater extent then either CTLA-4 or PD-1 blockade. This was true both early after activation (4 days) as well as survival of T cells 28 days following immunization in both models. Based on this evidence we hypothesize that OX40 agonists may be more important for immune cell priming to tumor Ags and that PD-1 and CTLA-4 blockade may play a role in decreasing T cell tolerance within the tumor microenvironment. Hence these immune activating pathways most likely have non-overlapping pathways to activate T cells and we now have compelling in vivo evidence that combining OX40 agonists with checkpoint inhibitors have synergistic effects in mouse tumor models.
PD-1 mediates paralysis of ovarian cancer-infiltrating dendritic cells through SHP-2 dependent and independent NFkB inactivation (TUM3P.1048)
Authors: Karyampudi Lavakumar , Lamichhane Purushottam , krempski James , Kalli Kimberly , Behrens Marshall , Vargas Doris , Hartmann Lynn , Janco Jo Marie , Hedin Karen , Dietz Allan , Goode Ellen , Knutson Keith
Published: January 2023

The PD-1:PD-L1 axis is a major immune regulatory pathway that blunts T cell activation in the tumor microenvironment. We recently identified that PD-1 is also upregulated on ovarian mouse tumor-infiltrating myeloid dendritic cells (TIDCs) that exhibit an immune suppressive phenotype. Extending those observations in this study, we now report that PD-1 is also expressed on human myeloid TIDCs of ovarian cancer patients, on which it suppresses the expression of co-stimulatory molecules (CD40 and CD80) as well as production of inflammatory cytokines. Mechanistic studies revealed that PD-1 on TIDCs is associated with Src homology domain-containing phosphatase-2 (SHP-2) and that PD-1-mediated blockade of NFkB-dependent cytokine release is SHP-2 dependent. PD-1 also inhibits NFkB-mediated antigen presentation which appears, however, to be independent of SHP-2. Thus, suppression of NFkB is critical in mediating PD-1-induced paralysis of ovarian tumor-associated immune TIDCs and is mediated through multiple signaling circuits that are both SHP-2 dependent and independent.
Follicular CD8+ T cells: a novel subset in immune protection and antibody responses. (VAC3P.1062)
Authors: Cross Eric , Edwards Lindsay , Kedl Ross
Published: January 2023

Endogenous antigen-specific CD8 T cells have been detected within B cell zones of the splenic white pulp during the primary response to multiple infections. The physiologic implications of CD8 T cell recruitment to B cell zones are unknown. However, such CD8 T cell localization may be beneficial for immunity in multiple ways, including, but not limited to: 1) direct CD8 T cell-mediated costimulation of B cells, 2) CD8 T cell-derived signals lowering the threshold of help required from CD4 follicular helper T cells (Tfh) for B cell maturation, and/or 3) direct CD8 T cell-mediated support of Tfh cells. We show here that after a combined TLR agonist and anti-CD40 antibody immunization (TLR/CD40) the follicular-homing CD8 T cell population is increased compared to infection, particularly at 2-3 days post-immunization. Concurrently, CD8 T cells upregulate Tfh cell-associated markers including CXCR5, PD-1, and ICOS. IL-21 is secreted robustly by CD8 T cells at this time point, which could help support B cell germinal center maintenance, class-switching, and Tfh cell differentiation. Indeed, in the context of TLR/CD40 immunization, the absence of CD8 T cells does not affect the IgM response, but reduces an isotype thought to be directed by IL-21, IgG2c, to levels seen in CD4 T cell-depleted animals. Our data indicates that CD8 T cells can assume a Tfh-like phenotype, we call follicular cytotoxic T cells (Tfc), early after challenge and direct antibody responses.
Toll-like receptor ligands differentially activate human innate immune system and distinctively enhance antigen-specific T cell response to vaccination in humanized mice (VAC4P.1105)
Authors: Cheng Liang , Zhang Zheng , Zurawski Sandra , Zurawski Gerard , Levy Yves , Su Lishan
Published: January 2023

TLR-Ls represent a new class of novel vaccine adjuvant. However, their immunologic effects in humans remain poorly defined in vivo. Here we investigated how TLR-Ls stimulated human immune system in vivo and their application as immune adjuvant using the humanized mouse model. To our surprise, we found that various TLR-Ls (CpG-A/CpG-B/CpG-C, R848, R837, MPLA and Poly I:C) stimulated human cytokines very differently in humanized mice in vivo compared to that in human PBMCs in vitro. The different in vivo response to TLR-Ls was not due to the improper development of human immune cells in vivo because splenocytes from humanized mice showed identical responses as human PBMC in vitro to various TLR-Ls. Importantly, the human innate immune response to specific TLR-Ls in humanized mice in vivo was different from that reported in C57/BL6 mice, but similar to that reported in nonhuman primates. Furthermore, we showed that distinct TLR-Ls differentially activated and mobilized human pDCs, mDCs and monocytes in different organs in vivo. Finally, we showed that Poly I:C and R848 were superior to CpG-B, for enhancing antigen specific CD4+ and CD8+ T cell responses to a CD40-targeting HIV candidate vaccine in humanized mice, which correlated with their ability to activate human CD141+ mDCs and IL-12 induction. Thus, we conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans.
A novel DNA vaccine against Mycobacterium tuberculosis infection (VAC4P.1109)
Authors: Tang Jiansong , Liang Jianguo , Cai Yi , zhou jingying , TAN ZHIWU , Tang Xian , Zhang Chi , Cheng Lin , Liu Wan , Yam Wing , Chen Xinchun , Chen Zhiwei
Published: January 2023

Background: Mycobacterium tuberculosis (M.tb.) pandemic continues to grow together with serious issues of increasing drug resistance, HIV co-infection and mortality. Since the existing BCG vaccine does not provide significant protection in adults, novel protective strategies should be explored with urgency. Methods: DNA vaccines that express TB antigens Ag85B, ESAT-6, Rv2660c and fusion BER, were investigated in BALB/c mice intramuscularly with electroporation (EP). Immunogenicity and efficacy assessments of these DNA vaccines were then determined before or after aerosol challenge of 100CFU M.tb H37Rv strain. Results: Compared with BCG and other DNA/EP vaccines, pBER/EP induced high frequency of Ag85B-specific CD8+ T cells (up to 41%) in tetramer assay. Moreover, pBER/EP also elicited significantly higher frequencies of antigen-specific CD4+ T cells by ELIspot and ICS assays. Surprisingly, these T cell responses were also significantly higher than those induced by a heterologous DNA/EP prime and vaccinia-85B boost regimen. Critically, pBER/EP conferred significantly better protection against M.tb H37Rv challenge when compared to other DNA/EP vaccines, with lower CFU count in the lungs (p<0.01) and the absence of lung pathology. Conclusions: pBER/EP is a promising DNA vaccine candidate that does not have major issues of safety and pre-existing anti-vector immunity (e.g. vaccinia vector). Our findings warrant further investigation of pBER/EP for TB prevention and immunotherapy.
Temporal regulation of adenovirus vector-elicited antibody responses by CD4 T cells (VAC4P.1111)
Authors: Provine Nicholas , Badamchi-Zadeh Alexander , Bricault Christine , Penaloza-MacMaster Pablo , Larocca Rafael , Parenteau Lily , Blackmore Stephen , Borducchi Erica , Barouch Dan
Published: January 2023

BACKGROUND: Recombinant adenovirus (Ad) vectors have recently garnered interest as vaccine platforms to elicit robust antibody (Ab) responses. In this study we seek to determine the timing of CD4 T cell help for the induction of these Ab responses. METHODS: B6 mice were immunized intramuscularly with 1010 vp of Ad serotype 26 expressing SIVmac239 Env. Anti-CD4 Ab (GK1.5) was used to deplete CD4 T cells. Serum Env-specific IgG was quantified by ELISA. Statistical analysis was performed by Mann-Whitney U test. RESULTS: Depletion of CD4 T cells at immunization completely abolished SIV Env-specific Ab responses at day 30 post-immunization. Surprisingly, by day 60 post-immunization Env-specific IgG responses spontaneously developed in these mice, and by day 90 these responses were of equal titer to those in control mice (Endpoint titer of 18225 for untreated vs. 12150 for anti-CD4 treated mice; P=0.9). Delayed Ab responses coincided temporally with the recovery of CD4 T cells following anti-CD4 Ab treatment. Recovery of CD4 T cells was necessary for this phenomenon, as repeated administration of anti-CD4 Ab blocked the development of Env-specific Ab responses long term. CONCLUSION: These data demonstrate that CD4 T cells actively promote the development of Ad vector-elicited Ab responses even when CD4 T cells are absent at the time of immunization. These data have potential implications for the utility of Ad vectors in immunocompromised individuals, such as AIDS patients.
Immunogenicity of TB vaccine H56 in non-human primates (VAC4P.1113)
Authors: Wong Eileen , Agger Else , Andersen Peter , Flynn JoAnne
Published: January 2023

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health problem. Several TB vaccine candidates are currently in various stages of development and clinical testing, including the protein subunit vaccine H56, from Statens Serum Institute (Copenhagen, Denmark). The cationic liposome adjuvant CAF09 was recently developed to enhance the CD4 and CD8 T cell response when administered with H56 vaccine. We vaccinated non-human primates (NHP) with H56 vaccine and CAF09 adjuvant by intranasal and subcutaneous routes and compared differences in immunogenicity. We measured the immune response to the vaccine in blood samples at multiple time points by IFN-γ ELISPOT and intracellular cytokine staining (IFN-γ, IL-2, TNF, IL-17, IL-6, IL-10) and in bronchoalveolar lavage fluid (BAL) by intracellular cytokine staining. NHPs were challenged with Mtb, and recall responses assessed. Frequencies of activated T cells producing cytokines transiently increased following vaccinations, but were generally low. CD4 responses were highest to the Ag85B vaccine component, while CD8 responses were highest to the Rv2660c vaccine component. There were no significant differences in cytokine production between the two vaccination routes in blood or BAL. This may lead to further H56/CAF09 vaccine trials in NHPs with Mtb challenge and future clinical trials, while strengthening understanding of the immune response necessary for protection from TB.
Direct involvement of neutrophils and Th17 cells in the protective immune response to tuberculosis after vaccination (VAC4P.1067)
Authors: Trentini Monalisa , de Oliveira Fábio , Kipnis André , Junqueira-Kipnis Ana Paula
Published: January 2023

Introduction: In 2013, 1.5 million died from TB, the BCG vaccine is the only TB vaccine currently available, but does not protect adults. The mc2CMX vaccine developed by our group was able to generate similar protection in mice, but induced higher levels of Th17 cells when compared with BCG. Objective: To study the role of Th17 cells in the protection induced by mc2CMX vaccine. Methodology: WT, IL22KO, and IL17KO mice were immunized twice with mc2CMX. Mice were challenged with Mycobacterium tuberculosis. Fifteen days after the last immunization, the lesions were collected and stained with HE, and the spleen cells were analyzed by flow cytometry before and after Mtb challenge. Results: Although WT mice induced vaccine lesions showing peripheral neutrophilia, central necrosis and few macrophages, both KO mice strains showed reduced injuries with lower neutrophils migration. Only IL-22KO and WT vaccinated mice presented vaccine specific Th1 and Th17 responses and reduced the lung and spleen bacterial load 30 days after challenging with Mtb. Adoptive transfer of splenocytes from vaccinated WT mice to naïve IL17KO and WT animals restored partially the protection. WT mice neutrophils depletion provoked reduction in lung specific Th1 and Th17 responses after mc2CMX vaccination. Conclusion: The results show for the first time, the protective role of vaccine induced Th17 specific cells, and also the assistance of neutrophils in the generation of such protection against TB.
Abrogation of CD59 function restores activities of neutralizing and non-neutralizing antibodies in triggering antibody-dependent complement-mediated lysis of HIV-1 virions and provirus-activated latently infected cells (VIR1P.1125)
Authors: Yang Kai , Lan Jie , Shepherd Nicole , Hu Ningjie , Byrd Daniel , Amet Tohti , Jewell Corlin , Gupta Samir , Yu Qigui
Published: January 2023

Abrogation of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env Abs or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs) including 2G12, 2F5, and 4E10 and non-nAbs including 2.2C, A32, N5-i5, and N12-i15 to investigate whether the ADCML enhancement by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to both laboratory-adapted R5, X4, and R5/X4 strains of HIV-1 and 6 primary isolates examined, as well as provirus-activated ACH-2 cells. In contrast, non-nAbs 2.2C and N12-i15 reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions of both laboratory strains and primary isolates and provirus-activated latently infected cells. Thus, abrogation of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells
The establishment of immune protection against a CNS viral challenge (VIR1P.1157)
Authors: Garcia Samantha , Hooper Douglas
Published: January 2023

The neuroinvasive rabies virus (RABV) requires CD4 T and B cell infiltration into CNS tissue and local production of virus neutralizing antibody (VNA) in order to be successfully cleared. This occurs during attenuated but not in wild-type dog RABV infection. Effective protection against a CNS challenge with dog RABV is only established by a vaccination regimen that delivers RABV-specific immune effectors to the CNS. Mice that recover from an attenuated RABV of the CNS are protected long term against subsequent CNS challenge by pathogenic dog RABV, whereas long term protection is not seen in mice that received attenuated RABV in the gastrocnemius. This difference is likely to be due to the increased formation of memory like cells which remain in the brain following a direct CNS vaccination, resulting in a more robust and effective response during CNS challenge. Since the blood-brain barrier (BBB) evidently remains intact during dog RABV infection, it is likely that immune memory cells capable of reactivating to produce VNA upon challenge must be resident in the CNS rather than enter from the periphery. In support of this concept, adoptive transfer of RABV immune cells fails to protect against a dog RABV infection of the CNS. A robust CNS immune response during initial vaccination appears to be the only effective way to establish long term protection against a pathogenic RABV infection that reaches the CNS.
Glycosylphosphatidylinositol deficiency attenuates the production of infectious HIV-1 and renders the virions sensitive to complement attack (VIR1P.1128)
Authors: Amet Tohti , Byrd Daniel , Lan Jie , Shepherd Nicole , Yang Kai
Published: January 2023

Human immunodeficiency virus type 1 (HIV-1) escapes complement-mediated destruction by incorporating regulators of complement activation (RCA) from the host cells. Abrogation of RCA function leads to complement-mediated virolysis. However, little is known about the mechanism by which HIV-1 incorporates RCA from the host cell. Here we used glycosylphosphatidylinositol (GPI)-deficient Jurkat (Jurkat-7) cells that only express intracellular CD59 (a key member of RCA), but not on the surface, to study how HIV-1 incorporates CD59 from an infected cell. Compared to wild type Jurkat cells, Jurkat-7 cells are less susceptible to HIV-1 infection, and produces lower amount of virions due to the impaired GPI biosynthesis. Interestingly, virions produced from Jurkat-7 cells did not contain CD59, while virions from wild-type Jurkat cells did contain CD59 with a certain amount of viral protein expression. This indicates that HIV-1 incorporates CD59 from the surface of host cell rather than from the intracellular compartments. Virions produced from Jurkat-7 cells are less infectious, but more susceptible to complement-mediated virolysis due to the failed CD59 incorporation into the virus surface. Furthermore, transient restoration of GPI anchor in Jurkat-7 cells resulted in recovered surface expression of CD59. These results reveal a critical role of GPI anchor and GPI-anchored proteins in HIV-1 life cycle and incorporation of CD59, probably other host cell proteins into the HIV-1 virions.
Triggering of organ-specific autoimmunity through synergy of defects in key tolerance checkpoints
Authors: Proekt Irina , Miller Corey , Jeanne Marion , Fasano Kayla , Lowell Clifford A , Gould Douglas , Anderson Mark S , DeFranco Anthony L
Published: January 2023

Studies of genetic factors associated with human autoimmunity suggest a multigenic origin of autoimmune susceptibility. To address how defects in different immune tolerance pathways cooperate to result in disease, we investigated whether genetic alterations in two such pathways might synergize to produce autoimmunity in mice. The intracellular protein tyrosine kinase Lyn is critical for inhibitory receptor function in innate immune cells and B cells. Lyn-deficient mice develop systemic but not organ-specific autoimmunity. In contrast, Autoimmune Regulator (Aire) promotes expression of tissue-specific antigens by thymic epithelial cells and T-cell negative selection. Aire-deficient mice develop multiorgan autoimmunity, including autoimmune uveitis, due to lack of thymic expression of the retina-specific interphotoreceptor retinoid binding protein (IRBP). AireGW/+ knockin mice express a dominant negative allele of Aire, have low but detectable thymic expression of IRBP and do not develop uveitis. To determine whether lack of Lyn inhibitory pathways could cooperate with a partial defect in T cell central tolerance, we generated AireGW/+ Lyn−/− double mutants. Remarkably, over 50% of these mice developed severe uveitis that was accompanied by a rise in anti-IRBP antibodies and an expansion of IRBP-specific CD4 T cells. To determine the contribution of different Lyn-expressing cell subsets to uveitis, we crossed AireGW/+ mice to a panel of Cre lines with an inducible Lyn knockout allele. Deletion of Lyn selectively in dendritic cells (DCs) was sufficient to drive disease, suggesting that Lyn-deficient DCs may have increased ability to prime IRBP-specific T cells that have escaped negative selection in the AireGW/+ thymus.
The effect of dietary phytoestrogen content on the development of systemic lupus erythematosus in the MRL/lpr mouse.
Authors: Edwards Michael Richard , Dai Rujuan , Heid Bettina , Cowan Catharine R , Ahmed S Ansar
Published: January 2023

Systemic lupus erythematosus is a prototypical autoimmune disease, characterized by the loss of immune cell tolerance to self-antigens, and the subsequent production of autoantibodies leading to multi-organ damage. SLE is an incurable, highly variable autoimmune disease that most often affects women of child-bearing age. We investigated the effects of three standard commercial diets with varying phytoestrogen content on the development of lupus in female MRL/lpr mice. The diets include Harlan 2018 which contains soybean meal, Teklad 7013 which contains both soy and alfalfa, and Research Diets Inc. D11112226 (RD) purified ingredients diet, containing no measurable phytoestrogens. Mice fed the 2018 diet developed severe glomerulonephritis with high levels of complement C3 protein deposition and moderate IgG immune-complex deposition, while mice fed the RD diet developed minimal evidence of renal damage and protein deposition. The mice fed the RD diet, however, had increased levels of multiple pro-inflammatory cytokines, while immune cell populations remained unchanged. Immunofluorescence revealed distinct infiltrating cellular phenotypes in the kidney, dependent on dietary source. Splenocyte miRNA-148a was markedly elevated in mice fed the 2018 and 7013 diets, while circulating miRNA values were not statistically different. Our results suggest that dietary isoflavones can worsen the development of glomerulonephritis due to enhanced immune complex protein deposition and altered cellular infiltration phenotypes. Our results also support that each organ system must be evaluated independently, and that what is measured in circulation does not necessarily reflect what is occurring in disease target organs.
The transcriptional regulator Bcl-3 impaires the function of regulatory T cells leading to the development of intestinal inflammation
Authors: Reissig Sonja , Nikolaev Alexei , Weigmann Benno , Gerlach Katharina , Glasmacher Elke , Hövelmeyer Nadine , Waisman Ari
Published: January 2023

The oncoprotein and transcription factor Bcl-3 (B cell leukemia-3) is a member of the IκB family of NF-κB inhibitors and was originally identified as a component of a chromosomal rearrangement in some B cell lymphomas. In contrast to other IκB proteins, Bcl-3 associates with p50 and p52 homodimers and is predominantly a nuclear protein involved in regulating nuclear NF-κB activity.

We could demonstrate that Bcl-3 plays a crucial role in the maintenance of Treg function as well as in the development of IBD. Data of patient cohorts illustrate that Bcl-3 expression levels correlate with activity of disease. Treatment of IBD patients with anti-TNFa therapy significantly reduced the expression of Bcl-3. To assess the role of Bcl-3 in IBD, we generated mice that overexpress Bcl-3 solely in T cells. Interestingly, these mice develop severe spontaneous colitis. This intestinal inflammation was not mediated by CD4+ T cells, which even display memory formation defects. Instead, we found that the development of disease is caused by an impaired function of Tregs and demonstrate that Bcl-3 regulates NF-κB activity intrinsically in Tregs. Our data emphasize a novel pathway in the maintenance of viability and suppressive function of Tregs in the gut that critically involves Bcl-3 as a negative regulator, qualifying this protein as an interesting pharmacological target for the treatment of IBD.
Suppression of acetylcholine receptor endocytosis in neuromuscular junction by carbonic anhydrase III in the pathogenesis of myasthenia gravis.
Authors: Huang shiqian , Zhao Xiaonan , Xu Congfeng
Published: January 2023

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifestated as fatigable muscle weakness. The disorder typically caused by pathogenic autoantibodies against the postsynaptic acetylcholine receptor in the endplate of skeletal muscle. Our previous studies have identified carbonic anhydrase III as a specific protein insufficient in skeletal muscle from myasthenia gravis patient. In this study, we determined to investigate the underlying mechanism how carbonic anhydrase III insufficiency contributes to myasthenia gravis. Based on experimental autoimmune myasthenia gravis animal model and myocyte C2C12 cells, we find that inhibition of carbonic anhydrase III promotes acetylcholine receptor, which is through lipid raft-mediated pathway, leading to accelerated degradation of surface acetylcholine receptor. While activation of carbonic anhydrase III negatively regulates acetylcholine receptor degradation, through suppressing the endocytosis of surface acetylcholine receptor, thus contributes to attenuation of myasthenia gravis. Further study demonstrates that carbonic anhydrase III regulates endocytosis of AChR through actin polymerization and Src phosphorylation. Collectively, our study illustrates that carbonic anhydrase III in skeletal muscle cells is critical for acetylcholine receptor homeostasis in neuromuscular junction, while carbonic anhydrase III deficiency induces accelerated degradation of acetylcholine receptor, leading to myasthenia gravis. This novel mechanism highlights the essential role of carbonic anhydrase III in regualtion acetylcholine receptor endocytosis, providing a potential therapeutic approaches for myasthenia gravis.
Leupaxin Regulates LFA-1 Related Function in Cytotoxic T Lymphocytes (CTL)
Authors: Yao Shugang , Ostergaard Hanne
Published: January 2023

Background

Leupaxin is an adaptor protein that functions downstream of integrin receptor engagement. It contains N-terminal LD domains and C-terminal LIM domains. Although leupaxin is primarily expressed in leukocytes, it is very little known about the localization and function in leukocytes.

Results

Degranulation follows the formation of the immunological synapse (IS) and recruitment of MTOC to the IS. Leupaxin co-localizes with MTOC and microtubules, and is recruited to the IS during CTL degranulation. Through deletion of leupaxin domains, it shows that both LD2 and LD4 domains are sufficient for leupaxin recruitment to the IS. Leupaxin fails to be recruited to the IS after deleting LD2–4 domains.

Leupaxin is a focal adhesion protein in adherent cells. Leukocytes do not form classical focal adhesions during migration. We have found that leupaxin is recruited to the contact zone during CTL migration on ICAM-1, and forms very dynamic focal adhesion (FA)-like structures. These structures are assembled in the leading edge, and disappear in the trailing edge. Leupaxin also co-localizes with vinculin and talin at the FA-like structures, which are both FA proteins. Different from the role of leupaxin at the IS, leupaxin C-terminal LIM domain contributes to the localization of leupaxin at the FA-like structures.

Conclusions

Leupaxin localization suggests that it contributes to LFA-1 related processes in CTL, including degranulation and migration, but with different domains. Leupaxin LD domains are necessary for the recruitment to the IS during degranulation, whereas LIM domains are necessary for the formation of FA-like structures during migration.
CD11c+ cells are required for lymphocyte trafficking into previously infiltrated pancreatic islets during type 1 diabetes.
Authors: Sandor Adam , Lindsay Robin S , Gebert Matthew , Bradley Brenda J , Haskins Kathryn , Jacobelli Jordan , Friedman Rachel Sharon
Published: January 2023

Type 1 diabetes (T1D) is a largely T cell mediated autoimmune disease that destroys the beta cells of the pancreatic islets. Initiation of T1D requires T cell activation in the pancreatic lymph node, and then activated T cells must enter the islets to destroy beta cells. The requirements for lymphocyte entry into islets are not fully understood. We show that lymphocyte entry into previously infiltrated islets is dependent on CD11c+ cells in the islets. T cells and B cells transferred prior to CD11c+ cell depletion were able to enter the islets; whereas, short-term CD11c+ cell depletion rapidly prevented further entry. CD11c+ cells are highly efficient antigen presenters; however, entry into the islets was not reliant on antigen for either activated or naïve T cells. An alternative role for CD11c+ cells in lymphocyte entry into the islets is the production of chemokines and cytokines, which can have direct chemotactic effects on lymphocytes or can activate vascular endothelium. CD11c+ cells in the islets express high levels of CXCL9, a chemoattractant for T and B lymphocytes. CXCR3, the receptor for CXCL9, is present on both T and B lymphocytes in the islets. We hypothesize that CD11c+ cells facilitate lymphocyte recruitment to the islets through a combination of chemokine production and activation of the islet vascular endothelium. Chemokine and cytokine production by CD11c+ cells may be targeted therapeutically for the treatment of T1D to prevent T cell entry into remaining or transplanted islets.
IL-15 stimulates core 2 O-glycan synthesis and regulates trafficking of memory CD8 T cells during viral skin infection
Authors: Mooster Jana L , Osborn Jossef , Khan Tahsin , Nolz Jeffrey
Published: January 2023

Following successful vaccination or pathogen clearance, recently activated CD8+ T cells that survive contraction differentiate into long-lived memory populations and provide host protection against re-infection. We have previously demonstrated that memory CD8+ T cells acquire the capacity to generate core 2 O-glycans in an antigen-independent manner, a post-translational modification required for binding to P- and E-selectin. In addition, we have shown that de novo synthesis of core 2 O-glycans can be regulated by IL-15 signaling and this modulates the ability for memory CD8+ T cells to traffic to inflamed tissues. Herein, we show that the trafficking of memory CD8+ T cells to Vaccinia Virus (VacV)-infected skin occurs in an antigen-independent manner, but that protective immunity requires antigen-specificity. Following epicutaneous infection with VacV, migratory dendritic cells express IL-15Rα, suggesting this cell type could trans-present IL-15 to T cells. In fact, nearly all memory CD8+ T cells in the draining lymph node during VacV infection synthesize core 2 O-glycans regardless of antigen specificity. IL-15 stimulates memory CD8+ T cells to specifically express ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (St3Gal4), an enzyme known to facilitate formation of sialyl Lewis X structures on core 2 O-glycans. Finally, we show that neutralizing IL-15 during VacV infection reduces E-selectin binding by circulating memory CD8+ T cells and results in decreased recruitment of these cells to the infected skin. Overall, these data identify IL-15 and core 2 O-glycan synthesis as critical regulators of memory CD8+ T cell trafficking to viral infection of the skin and perhaps other peripheral tissues.
CXCR4 mediates B-1 cell localization to the bone marrow and production of atheroprotective IgM antibody
Authors: Upadhye Aditi , McSkimming Chantel , Taylor Angela , McNamara Coleen
Published: January 2023

Existing evidence demonstrates that B1 cells exert protective effects in atherosclerosis through production of anti-inflammatory natural IgM antibodies that recognize oxidation-specific epitopes (OSE), such as MDA-LDL, present in diseased arteries. The bone marrow is a known niche for B1 antibody production. However, the mechanisms underlying B1 localization to the bone marrow and B1 antibody production are currently unclear. Our lab has correlated surface marker expression on peripheral blood mononuclear cells with clinical markers of atherosclerosis in a human cohort. Expression of the chemokine receptor CXCR4 on circulating B1 cells associates with decreased plaque burden in coronary arteries and increased plasma levels of anti-MDA-LDL IgM antibodies. To study the role of CXCR4 in modulating B1 cell function more directly, we generated mice with B cell-specific loss of CXCR4 (CXCR4BKO). 8-week-old CXCR4BKO mice demonstrate a significant reduction in B1 number in the bone marrow relative to littermate controls (CXCR4WT). No significant changes in B1 number were seen in the aorta, peritoneal cavity, or spleen. Using ELISA and ELISPOT, CXCR4BKO mice were demonstrated to have decreased circulating IgM levels and fewer IgM antibody-secreting cells (ASC) in the bone marrow, but not in spleen. CXCR4BKO and CXCR4WT mice were placed on 9 weeks Western diet (WD) or control chow diet and then analyzed for IgM ASC number. CXCR4WT WD-fed mice have increased IgM ASC’s in the bone marrow relative to CXCR4WTchow-fed controls. This increase did not occur in WD-fed CXCR4BKO mice. Our data demonstrate that CXCR4 mediates B1 localization to the bone marrow and IgM production in mice, and indicate an atheroprotective role for CXCR4 on B1 cells.
Multiplex Immunoassays for Simultaneous Quantification of Soluble Adhesion Biomarkers in Human and Mouse Samples
Authors: Sun Binggang , Yao Jingjing
Published: January 2023

Cells bind to each other or to the extracellular matrix via Cell Adhesion Molecules (CAMs). Most of the CAMs belong to four protein families, including the immunoglobulin superfamily (e.g., ICAM-1, VCAM-1 and ALCAM), the selectins (e.g., P-, E- and L-selectins), the cadherins (e.g., E-cadherin) and the integrins (e.g., LFA-1). CAMs control intercellular adhesion and mediate cell growth, migration, proliferation and apoptosis. As a result, they play important roles in a wide range of cellular and disease processes. The soluble forms of these CAMs have increasingly become popular biomarkers in various diseases including inflammatory disorders, cardiovascular diseases, wound repair and cancers. Therefore, accurate measurement of these biomarkers is critical for understanding disease progression and immune responses.

We have developed both human and mouse adhesion panels for simultaneous quantification of nine adhesion biomarkers, including ALCAM, ICAM-1, PECAM-1, VCAM-1, E-selectin, L-selectin, P-selectn, E-cadherin and ESM-1 (Endocan). Our multiplex panels are bead-based sandwich immunoassays using fluorescence-encoded beads that can be separated by a variety of flow cytometers. Both panels have been validated for use with cell culture supernatant, serum and plasma samples. Each antibody pair has been carefully selected for assay specificity, accuracy and reproducibility. Our assays exhibit low-pg/mL sensitivity, broad dynamic range over 4 orders of magnitude, and high precision with <15% inter CV. No or negligible cross-reactivity was found for the analytes within each panel. In summary, our adhesion multiplex panels offer a reliable and efficient tool to help researchers advance our knowledge of these soluble CAMs.
SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.
Authors: COTE Marjorie , Fos Camille , Canonigo-Balancio Ann Janette , Ley Klaus , Becart Stephane , Altman Amnon
Published: January 2023

SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca2+ signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and LFA-1 affinity maturation in CD4+ T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1. This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4+ T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6−/−) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling.
C-Myc regulation by Notch Signaling Modulates T Cell Differentiation
Authors: Song Jim Jianxun , Haque Rizwanul , Song Xinmeng , Lei Fengyang
Published: January 2023

Notch signaling is essential for T lineage commitment and further required during early phases of thymocyte differentiation. Active Notch signaling during early stages of T cell development inhibits other lineage potentials, such as B cell and myeloid cell (e.g., dendritic cell) potentials. However, the intracellular signaling pathways by which Notch signaling regulates T cell differentiation remain unknown. Here we show that the transcriptional factor c-Myc is controlled by Notch signaling which regulates T cell differentiation. In a well-established in vitro differentiation of T lymphocytes from stem cells, Notch signaling directly controls c-Myc expression. Overexpression of active c-Myc promotes while dominant-negative (dn) c-Myc inhibits early cell differentiation. Moreover, c-Myc expression mediated by Notch signaling modulates survivin, an inhibitor of apoptosis (IAP) protein, which is crucial for T cell development. Overexpression of active c-Myc increases while dn c-Myc reduces survivin expression, which corresponds to T cell differentiation within the in vitro differentiation system. These results identify c-Myc, together with survivin, as regulators of the differentiation of T cells from Notch signaling.
Temporal expression of Bim limits the development and repertoire of TCR+ double negative thymocytes and CD8αα intestinal intraepithelial lymphocytes.
Authors: Li Kun-Po , Fähnrich Anke , Roy Eron , Cuda Carla , Grimes H Leighton , Perlman Harris , Kalies Kathrin , Hildeman David A
Published: January 2023

DN T cells (CD4−CD8α−TCRαβ+) and CD8αα T cells compose a major lymphocyte population in the intestine and promote gut homeostasis. Their thymic development, i.e. agonist selection, has been shown to be controlled by the pro-apoptotic factor Bim. In addition to its role in the thymus, Bim also controls peripheral T cell survival. However, the role of Bim in thymic and peripheral homeostasis of DN T and CD8αα T cells remain unclear. Here, we found that T cell-specific expression of Bim during early, but not late, thymic development limits the levels of DN T and CD8αα T cells in the spleen and iIEL compartments. The loss of Bim altered the TCR repertoire of DN T cells, but not conventional CD4 or CD8 T cells. As IL-15 controls CD8αα cell homeostasis, and antagonizes Bim in other T cells, we examined whether the additional loss of Bim would restore CD8αα T cells in IL-15−/− mice. Strikingly, the additional loss of Bim restored splenic, but not intestinal, DN T and CD8αα T cells in IL-15−/− mice. Instead, IL-15 signaling via Stat5 synergized with TCR signaling in peripheral DN T cells to increase expression of CD8α. Furthermore, adoptive transfer of splenic DN T cells gave rise to CD8αα cells in immune intact recipients. Combined, these data show that Bim functions temporally to limit the development and TCR repertoire of CD8αα precursors in the thymus. Moreover, Bim limits IL-15-driven homeostasis of CD8αα cells in the secondary lymphoid organs; but does not restrict their IL-15-driven maturation that is critical for intestinal homeostasis.
Analysis of Ikaros tumor suppressor function in BCR-ABL1+ pre-B ALL reveals conserved target genes and biological pathways
Authors: Schjerven Hilde , Ayongaba Etapong Fonabei , McLaughlin Jami , Cheng Donghui , Eggesbø Linn Margrethe , Lindeman Ida , Park Eugene , Witte Owen N , Smale Stephen T , Frietze Seth , Muschen Markus
Published: January 2023

Inactivation of the transcriptional factor Ikaros (IKZF1) correlates with poor prognosis in progenitor B-cell acute lymphoblastic leukemia (pre-B ALL), and is a hallmark of the BCR-ABL1+ subgroup of pre-B ALL. Ikaros is a critical regulator of hematopoietic development and required for B-cell development, however the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. We analyzed recently developed mouse models of BCR-ABL1+ pre-B ALL containing targeted deletions of Ikaros DNA-binding zinc finger domains together with a new model of inducible expression of WT Ikaros in IKZF1-mutant human BCR-ABL1+ pre-B ALL. We found that both the mouse and human Ikaros-mutated leukemic cells displayed a less mature cell surface phenotype and failed to downregulate the developmentally restricted cell surface receptors c-kit and CD34, respectively. In addition, Ctnnd1, a gene that is also expressed in earlier hematopoietic progenitor cells and normally downregulated as cells differentiate down the B-cell lineage, was found to be a conserved Ikaros target gene, with increased expression in Ikaros-mutated leukemic cells. RNA sequencing defined the Ikaros target genes in both mouse and human Ikaros-mutated pre-B ALL cells and revealed additional conserved target genes and biological functions. Loss of Ikaros tumor suppression was associated with deregulated adhesion pathways and stem-cell signatures. Furthermore, our results presented herein suggest that Ikaros mediates tumor suppressor function, at least in part, by enforcing proper developmental-stage specific expression of multiple genes involved in a network of cadherin-dependent, Rho-regulated Wnt/b-catenin pathways.
Kainate receptor deficient mast cells exhibit increased activation in vivo and in vitro.
Authors: Elkovich Andrea , Sturgill Jamie L , Conrad Daniel H
Published: January 2023

Allergies and asthma are common ailments that are on the rise around the world, especially in young children. Mast cells play a direct role in the signs and symptoms characteristic in allergic patients. Kainate is an ionotropic member of the glutamate receptor family and is mainly studied in the brain. Recent investigations, however, have shown the presence of the Kainate Receptor (KAR) in peripheral tissues, including cells of the immune system. Our lab has previously studied the effects of KAR deficient mice and have found that these mice have reduced Th2 responses in a house dust mite extract model of allergic airway inflammation. We sought to determine if KAR deficient mast cells played a role in the phenotype observed. Paradoxically, these mast cells exhibited increased, rather than decreased mediator release in vitro by antigen–dependent degranulation. In vivo, KAR KO mice release significantly more histamine during a model of passive systemic anaphylaxis. Finally, KAR KO mice exhibit an exacerbated temperature drop in response to administered histamine. These results stand in contrast to the inhibition of the asthma model, but indicate that KAR agonist, rather than antagonist treatment of mast cells would be a treatment modality to decrease mast cell activation. Such studies are currently underway.