bluuebunny/crossref_metadata_2025 huggingface dataset:
Unique potential of 4-1BB agonist antibody to promote durable regression of HPV+ tumors when combined with an E6/E7 peptide vaccine (TUM2P.1016)
Authors: Bartkowiak Todd , Singh Shailbala , Yang Guojun , Galvan Gloria , Allison James , Sastry Jagannadha , Curran Michael
Published: January 2023

Antibody modulation of T cell co-inhibitory (e.g. CTLA-4) or co-stimulatory (e.g. 4-1BB) receptors promotes clinical responses to a variety of cancers. Therapeutic cancer vaccination, in contrast, has produced limited clinical benefit. The E6 and E7 onco-proteins of human papilloma virus drive the majority of genital and oropharyngeal cancers. We discovered 15-19 amino acid peptides from HPV16 E6/E7 for which T cell immunity correlates with disease-free survival in patients treated for high-grade cervical neoplasia. We report that intra-nasal vaccination with these peptides and the adjuvant alpha-galactosylceramide elicits systemic and mucosal T cell responses leading to reduced HPV+ TC-1 tumor growth and prolonged survival in mice. We hypothesized that the inability of these T cells to fully reject established tumors resulted from suppression in the tumor which could be ameliorated through checkpoint modulation. Combining this vaccine with checkpoint blockade produced only modest benefit; however, co-administration with a 4-1BB agonist antibody promoted durable regression of 100% of established genital HPV+ tumors. The vaccine/α4-1BB combination promoted the highest CD8+ versus regulatory FoxP3+ T cell ratios, elicited much higher infiltration by E7-specific CTL, and evoked higher densities of highly cytotoxic TcEO CD8 (>70-fold) and ThEO CD4 (>17-fold) phenotype T cells. Activation of 4-1BB may have a unique capacity to potentiate therapeutic cancer vaccines.
A monomeric fab fragment capable of inducing CD3 conformational change increases T cell receptor reactivity to poorly immunogenic antigens (TUM2P.1020)
Authors: Hoffmann Michele , Parks Christopher , Hansen Michael , Rajagopalan Govindarajan , Pease Larry , Schrum Adam , Gil Pages Diana
Published: January 2023

TCR interactions with agonist pMHCs induce a conformational change in the tails of the associated CD3 complex (CD3Δc) that contributes to efficient TCR/CD3 triggering and productive T cell responses. In contrast, TCR engagement by antagonistic pMHCs does not induce either CD3Δc or full TCR/CD3 signaling in mature T cells. Here, we have used a monovalent Fab fragment (Mono-Fab) specific for the murine CD3 complex to provide induction of CD3Δc to mature T cells engaging pMHC ligands. This Mono-Fab does not interfere with TCR capacity to bind pMHC and fails to elicit signaling in the absence of TCR/MHC interactions. Using OT-I Tg T cells and a collection of altered peptide ligands derived of the agonist peptide OVA, we show that addition of CD3Δc to OT-I T cells using Mono-Fab enables T cell responses to antagonist ligands that fail to induce CD3Δc on their own. Moreover, using a mouse model of metastatic melanoma, we show that Mono-Fab promotes in vivo T cell dependent anti-melanoma responses that are antigen-specific. This Mono-Fab can also be combined with established therapies targeting signals 2 and 3 involved in T cell activation for a synergistic effect. Our data suggests that in-trans CD3Δc provision decreases the activation threshold of T cells. We propose that targeting the CD3 complex for the induction of CD3Δc could be exploited to achieve efficient TCR/CD3 triggering by poorly immunogenic antigens in the context of vaccination against cancer and infectious diseases.
Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma (TUM2P.1038)
Authors: Hastings Karen , Nguyen Jennifer , Bernert Richard , Ko Christine , Sebastiao Noemi , Hu Chengcheng
Published: January 2023

T cell-mediated immunity has the ability to produce durable anti-melanoma responses resulting in improved survival of patients with advanced melanoma. Antigen presentation in the tumor microenvironment directs anti-melanoma T cell responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells. However, GILT expression in melanoma has not been defined. We evaluated GILT and MHC class II expression in human primary and metastatic melanomas and nevi using immunohistochemistry. Both GILT and MHC class II expression were increased in melanocytes of primary and metastatic melanomas compared with nevi. GILT expression was increased in antigen presenting cells of primary and metastatic melanomas compared to nevi, whereas MHC class II had equivalent high expression in antigen presenting cells of all melanocytic lesions. Keratinocytes in primary melanoma lesions had increased GILT and MHC class II expression compared with nevi. Increased GILT expression in melanocytes, antigen presenting cells and keratinocytes of melanoma demonstrates that changes in the antigen presentation pathway occur in the tumor microenvironment. GILT expression in melanoma is anticipated to result in improved presentation of melanoma antigens and more effective anti-melanoma T cell responses and may be a biomarker of immune recognition of melanoma.
Targeting deposited complement component C3d to potentiate monoclonal antibody cancer therapy (VAC3P.1057)
Authors: Skarzynski Martin , Butera Vicent , Lindorfer Margaret , Vire Berengere , Farooqui Mohammed , Rader Christoph , Taylor Ronald , Wiestner Adrian
Published: January 2023

Treatment-induced loss of targeted cell surface antigens through trogocytosis or internalization reduces efficacy of monoclonal antibody (mAb) therapy of cancer. However, cells that escape therapy mediated by complement-fixing mAbs carry covalently deposited complement activation fragments on their cell surfaces, in particular C3d. We hypothesized that cell-associated C3d constitutes a neoantigen that could be exploited to selectively retarget cells escaping from therapeutic mAbs. We generated an anti-C3d IgG1 human/mouse chimeric mAb specific for human C3d that is not competed by C3 in human serum. For proof of concept, we used cells from chronic lymphocytic leukemia (CLL) patients that had substantially reduced CD20 levels due to treatment with the anti-CD20 mAb ofatumumab (OFA). The chimeric anti-C3d mAb bound cell surface C3d on these CLL cells ex vivo (KD = 6.7nM), and mediated complement-dependent, and antibody-dependent cellular cytotoxicity and phagocytosis in vitro. CLL cells opsonized by C3d in vivo and reacted with the anti-C3d mAb in vitro were further C3d opsonized, resulting in an amplification loop that enhanced anti-C3d mAb binding capacity and killing of target cells. In vivo, the anti-C3d mAb was effective in reducing tumor burden in blood and spleens of NSG mice xenografted with human CLL cells. Our results identify anti-C3d mAbs as a means to specifically and potently augment the therapeutic efficacy of complement-fixing mAbs.
Mammalian target of rapamycin inhibitors are promising T cell lymphoma treatment agents that synergize with ERβ agonists in vitro but not in vivo (VAC3P.1061)
Authors: Liu Yang , Sareddy Gangadhara , Pandeswara Srilakshmi , Dao Vinh , Clark Curtis , Liu Aijie , Vadlamudi Ratna , Curiel Tyler
Published: January 2023

mTOR signals through 2 complexes: mTORC1 and mTORC2. Approved cancer mTOR inhibitor drugs inhibit mTORC1 and are poorly effective against carcinomas and B cell lymphomas. There is thought that dual mTORC1/2 inhibitors could have better clinical efficacy. As mTOR regulates important T cell functions, we tested mTOR inhibitors in mouse EL4 T cell lymphoma. The mTORC1 inhibitor rapamycin potently suppressed EL4 proliferation in vitro, and significantly reduced EL4 tumor growth in vivo. AZD8055, a dual mTORC1/2 inhibitor, also inhibited EL4 cell proliferation in vitro, decreased in vivo tumor burden and improved survival in EL4 challenged mice, but no better than rapamycin. EL4 tumor growth was slower in females versus males, suggesting hormonal effects. As estrogen receptor (ER)β agonists reportedly can treat lymphomas we tested them. We found that ERβ agonists altered EL4 mTOR signals and a novel ERβ/mTOR physical interaction (mTORC2>>mTORC1) in EL4 by GST pull-down. The ERβ agonist S-equol synergized with AZD8055 to inhibit EL4 cell proliferation in vitro but did not alter EL4 tumor growth in vivo nor boost AZD8055-mediated EL4 growth suppression or survival in vivo. EL4 tumors from AZD8055 treated mice had reduced viability and proliferation (Ki67) without affecting CD3+ T cell Ki67. AZD8055 treated ID8 ovarian cancer in vivo but rapamycin did not, suggesting T cell lymphomas could respond to mTOR inhibitors better than carcinomas. ERβ effects also deserve more study.
Infection-permissive immunity provided by NA- and M2e-based vaccines protects against influenza A virus challenge and allows the induction of heterosubtypic immunity during subsequent infections. (VAC5P.1127)
Authors: Schotsaert Michael , Ysenbaert Tine , Smet Anouk , Schepens Bert , Fiers Walter , Saelens Xavier
Published: January 2023

Licensed influenza vaccines protect by inducing neutralizing antibodies against the main viral surface antigens hemagglutinin (HA) and neuraminidase (NA). Influenza infection induces an antiviral T-cell response that correlates with protection against a subsequent infection with a virus of a different subtype (heterosubtypic immunity, HSI). Sterilizing immunity provided by classical vaccines however, may impair the induction of cellular HSI. We previously demonstrated in mice that vaccination with virus like particles containing the ectodomain of Matrix 2 protein (M2e-VLP) induce infection-permissive immunity that not only protects against influenza A viruses of different subtypes, but also allows the induction of cellular HSI. We could recently show that infection-permissive immunity provided by vaccination with recombinantly produced soluble tetrameric N1 NA, alone or combined with M2e-VLP, not only is protective against homologous challenge with pandemic 2009 H1N1 virus but also allows establishment of cellular HSI that protects against challenge with H3N2 virus. NA-immune animals also have an enhanced frequency of circulating functional antiviral memory CD8+ T-cells upon rechallenge with heterologous virus. In contrast, vaccination with recombinant soluble trimeric H1 HA derived from pandemic 2009 H1N1 virus induces strain-specific sterilizing immunity without infection-induced cellular HSI and fails to protect against challenge with H3N2 virus.
PTPN22 deficiency leads to clearance of chronic viral infection (VIR1P.1150)
Authors: Maine Christian , Teijaro John , Sherman Linda
Published: January 2023

Mutations in the phosphatase-encoding gene, PTPN22, are associated with a number of autoimmune conditions, including type I diabetes, rheumatoid arthritis and systemic lupus erythematosus. To study how PTPN22 functions in immune cells we have made extensive use of a PTPN22 deficient (KO) mouse. PTPN22 KO mice exhibit increased T cell numbers, activity and function as well as increased germinal center activity and antibody production with no autoimmune phenotype. As such we are currently investigating whether loss of PTPN22 would be of benefit to host immunity against chronic viral infections. To test this hypothesis we infected PTPN22 KO mice with the persistent lymphocytic choriomeningitis virus (LCMV) cl13 virus. LCMV cl13 infection in WT B6 mice leads to chronic infection lasting over 60 days characterized by dysfunctional ‘exhausted’ virus-specific T cells. PTPN22 KO mice were able to clear the cl13 infection whereas WT mice were not. Clearance is associated with increased viral-specific CD4 T cell cytokine production 8 days post infection, as well as increased numbers of Tfh and plasma cells. At later time points after infection, PTPN22 KO mice have increased numbers of functional CD8 T cells that produce IFNγ and TNFα, whereas CD8 cells in WT mice do not. These results suggest that inhibition of PTPN22 may prove beneficial in helping to clear chronic infection.
Longitudinal study of CD4 T cell responses following Japanese encephalitis virus vaccination (VIR2P.1165)
Authors: DeGottardi Maren , Balaji Hari , James Eddie , Mai Duy , Malhotra Uma , Kwok William
Published: January 2023

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. There are currently a number of vaccines shown to effectively prevent disease. In the United States, an alum adsorbed vaccine, which consists of a formaldehyde-inactivated, attenuated JEV strain is available. Vaccine protection has been shown to be mediated by neutralizing antibody responses. However, the CD4 T cell response associated with vaccine protection has not been fully characterized. In particular, the JEV model also presents an opportunity to study the kinetics of T cell responses elicited by a multi-dose vaccination in human subjects. In this study, we characterized the longitudinal response of JEV-specific CD4 T cells following vaccination using tetramer guided epitope mapping (TGEM), direct ex vivo MHC class II tetramer staining, and intracellular cytokine staining. A total of 67 epitopes restricted by 7 common HLA-DRB1 alleles and one DRB5 allele were identified. Vaccination elicited a robust CD4 T cell response, which was substantially boosted after the second dose of vaccine was administered. JEV-specific CD4 T cells exhibited a memory phenotype and expressed CCR4 and CXCR3. Consistent with this surface phenotype, JEV specific responses were polyfunctional, secreting IFN-g, IL-4 and sometimes IL-17a. These results indicate that JEV vaccination induces a robust, polyfunctional CD4 T cell response that is similar in magnitude to other highly effective vaccines.
Coordinated immune activation by distinct mononuclear phagocytes subtypes against the mismatch of minor antigens
Authors: Atif Shaikh M , Gibbings Sophie L , Gill Ronald G , Murphy Kenneth M , Grazia Todd , Torres Raul M , Henson Peter M , Randolph Gwendalyn J , Jakubzick Claudia
Published: January 2023

Recently, we identified that Batf3-dependent DCs were solely responsible for the rejection of minor antigen-mismatched grafts, and demonstrated in Batf3−/− female mice, lacking Batf3-dependent DCs, the complete acceptance of minor antigen-mismatched male cells, which are normally rejected in WT female mice. Intriguingly, this rejection occurs in the absence of PAMPs, leading to considerable uncertainty of how endogenous APC subtypes become activated and licensed to induce an adaptive immune response against male cells in female mice. Investigating this gap in knowledge is the main goal of this study. Rejection of minor antigen-mismatched grafts involves all branches of the immune system: adaptive immunity (T cells), humoral immunity (B cells) and innate immunity (DCs and antigen-presenting monocytes). Although we showed that depletion of one cell type dampens or omits the inflammatory outcome of minor antigen-mismatched graft rejection, here we show how lymphoid and myeloid cell subtypes act sequentially and in concert for the induction of minor antigen-mismatched graft rejection without the presence of PAMPs. In conclusion, we believe that understanding the mechanisms of action against minor antigen-mismatched graft rejection will also apply more broadly to other diseases such as autoimmunity and cancer, in which an immune response is elicited against self-associated minor antigens.
MHC-I molecules disulfide-linked to antigenic peptides offer a simple approach to stable MHC-I/peptide complexes
Authors: Jiang Jiansheng , Boyd Lisa F , Natarajan Kannan , Margulies David H
Published: January 2023

A variety of engineering approaches have been used to develop MHC/beta2m/peptide complexes with improved stability for structural studies and binding to T cell receptors and other MHC-I ligands. To generate MHC-I molecules covalently linked to antigenic peptides we considered X-ray structures of the H2-Dd molecule bound to the HIV IIIB derived peptide, P18-I10, RGPGRAFVTI. Computer analysis suggested that mutation of an H2-Dd residue to cysteine in the peptide binding groove would provide an MHC heavy chain that could be refolded with P18-I10 derived peptides substituted with cysteine at peptide position P5. Using standard E. coli expression as inclusion bodies and in vitro refolding protocols, we show that a set of cysteine-substituted peptides derived from P18-I10 including the decamer P18-C5-I10 (RGPGCAFVTI) and octamer P18-C5-V8 (RGPGCAFV) could be efficiently refolded with cys-mutated H2-Dd heavy chain and beta2-microglobulin. These molecules were analyzed by antibody binding studies and by X-ray crystallography. The P18-C5-I10/cys-mut-H2-Dd complex has a structure almost identical to that of the parental P18-I10/H2-Dd complex, while the complex with the octamer P18-C5-V8 repositions the peptide such that the C-terminal valine residue occupies the F pocket of H2-Dd. Such a strategy for producing covalently coupled MHC/peptide complexes offers a new approach to generate stable complexes for structural and imaging applications that should be widely applicable.
Chlamydial infection and the efficiency of MHC class I self-antigen presentation in a lymphoblastoid cell line
Authors: Simmons Ryan S , Cram Erik , Palmer Amy , Dolan Brian
Published: January 2023

The direct MHC class I antigen presentation pathway presents cytosolic proteins for immune surveillance. While this pathway is an attractive target for therapeutics, we must first understand the efficiency of antigen presentation. Previous studies have shown the presentation efficiency from pathogen-derived antigens ranges from 0.03–25%. The intracellular lifestyle of Chlamydia makes them an attractive model to study class I presentation. It is likely that Chlamydia have developed mechanisms to evade CD8+ T cells through manipulation of this pathway. Here, we investigate the impact of Chlamydial infection on a recombinant fusion protein expressed in a human lymphoblastoid cell line (JY). The fusion protein contains a destabilization domain, an antigenic peptide, and a reporter. Chlamydial infection led to decreased accumulation of the model protein and increased presentation of model protein-derived antigens. This enhanced self-peptide presentation was only observed when antigen presentation was restricted to the DRiP form of the protein. We also investigated the antigen presentation efficiency of different model recombinant proteins in JY cells. We calculated the antigen presentation efficiency to be 0.35%, much lower than was previously observed in murine cells with a similar protein. The skewed antigen presentation phenotype observed following Chlamydial infection was not statistically different from the efficiency observed in the absence of infection. These data suggest that Chlamydia have evolved a mechanism to skew the host antigen presentation machinery toward presentation of DRiPs, and that the efficiency of direct class I presentation may be mechanistically unique among common immune signaling pathways.
Fetal and adult progenitors give rise to unique populations of CD8+ T cells
Authors: Wang Jocelyn , Wissink Erin , Smith Norah L. , Grimson Andrew , Rudd Brian D
Published: January 2023

Neonates often generate incomplete immunity against intracellular pathogens. As CD8+ T cells are essential in clearing intracellular infections, it is crucial to understand how neonatal CD8+ T cells respond to infection. We showed that neonatal CD8+ T cells fail to form memory. However, the underlying basis for this is unclear. Here, we investigated whether neonatal CD8+ T cells fail to form memory cells because (i) they have undergone more extensive homeostatic proliferation (HP) prior to infection (proliferation model) or (ii) they are created from a distinct hematopoietic stem cell (HSC) population (origin model). We first compared the behavior and gene expression profiles of different aged CD8+ T cells that had undergone similar amounts of HP. Interestingly, neonatal CD8+ T cells still possessed unique gene expression profiles and failed to form memory post-infection. Hence, the proliferation model cannot fully explain the differences. We tested the origin model by comparing neonatal and adult CD8+ T cells at the same stage of thymic development. Again, we observed distinct gene expression profiles, indicating that CD8+ T cells are created differently in neonates. To examine whether different HSCs underlie these differences, we adoptively transferred fetal and adult progenitors into adult thymii and found that fetal progenitor derived CD8+ T cells exhibited a memory phenotype and selectively formed short-lived effectors after infection. The adult progenitor derived cells exhibited a more naïve phenotype and preferentially formed memory precursors after infection. Collectively, our data extends the layered immune hypothesis to CD8+ T cells and suggests that neonatal and adult CD8+ T cells are derived from different HSCs.
Activation of Complement Factor B Contributes to Murine and Human Myocardial Ischemia/Reperfusion Injury
Authors: Zhang Ming , Haddadin Ala , Hou Yunfang , Wong Karen , Liu Junying , Chun Nicholas , Rushbrook Julie , Hines Roberta , Nuno Beatriz Nistal , Pekna Marcela , Patel Foramben , Shevde Ketan , Heeger Peter S
Published: January 2023

The pathophysiology of myocardial injury resulting from cardiac ischemia and reperfusion (I/R) is incompletely understood. Experimental evidence from murine models indicates that innate immune mechanisms including complement activation via the classical and lectin pathways are crucial. Whether factor B (fB), a component of the alternative complement pathway required for amplification of complement cascade activation, participates in the pathophysiology of myocardial I/R injury has not been addressed. We induced regional myocardial I/R injury by transient coronary ligation in WT mice, a manipulation that resulted in marked myocardial necrosis associated with upregulated myocardial expression of fB gene, activation of fB protein, and myocardial deposition of C3 activation products. In contrast, in fB−/− mice, the same procedure resulted in significantly reduced myocardial necrosis and diminished deposition of C3 activation products in the myocardial tissue. To extend the analysis to humans we measured levels of activated fB (Bb) in intracoronary blood samples obtained during cardio-pulmonary bypass surgery before and after aortic cross clamping (AXCL), during which global heart ischemia was induced. Intracoronary Bb increased immediately after AXCL, the levels were directly related to the length of the AXCL and correlated with peripheral blood levels of cardiac troponin I, an established biomarker of myocardial necrosis. Taken together, our results support the conclusion that fB is a crucial pathophysiological amplifier of I/R-induced, complement-dependent myocardial necrosis and identify fB as a potential therapeutic target for prevention of human myocardial I/R injury.
Bisphenol A (BPA) and Bisphenol S (BPS) increase Coxsackievirus B3 myocarditis in female and male BALB/c mice by activating mast cells, increasing immune cell infiltrate and activating the inflammasome
Authors: Bruno Katelyn A , Yang Alex , Mathews Jessica , Greyner Henry , Molina Frank , Bucek Adriana , Fairweather DeLisa
Published: January 2023

Myocarditis is an inflammatory heart disease that is the leading cause of heart failure in young adults. Sex hormones play a vital role in development of myocarditis with testosterone driving disease in males. Whereas, estrogen, via Estrogen Receptor α (ERα) mediates cardioprotection in females. Since myocarditis is influenced by sex hormones, it is highly probable that endocrine disruptors (EDs); which interfere with natural hormones, will play a part in the progression of the disease. The human population is exposed to Bisphenol A (BPA), a known ED that binds the ER, from plastics, such as water bottles and plastic food containers. BPA could increase myocarditis through deleterious actions of the ERβ rather than beneficial effects via ERα. To our knowledge no one has examined the role of EDs like BPA on myocarditis. We found that clinically relevant doses (25 μg/L and 250 μg/L) of BPA increased acute myocarditis compared to control water in females. In females we found BPA significantly decreased ERα, while ERβ was significantly increased. We found that mast cells (cKit) are largely responsible for the increase in inflammation along with CD4 Thelper cells, IL-1β, IFN-γ, TLR4, Caspase-1, Mmp9, and ST2. In male mice, we found that at the high dose of BPA increased myocarditis and increased all immune cell markers and the inflammasome. We also investigated the compound Bisphenol S (BPS) which has replaced BPA in a number of products and found that BPS also increases myocarditis. Interestingly this effect was only seen in Balb/c mice and not BL/6 suggesting that race could be a factor in evaluating ED role in disease. We have found that ED exposure is having a significant effect on myocarditis and could potentially influence a number of human diseases.
Human Gingiva-Derived Mesenchymal Stromal Cells Inhibit Graft-versus-Host Disease through CD39 and IDO
Authors: Wang Julie , Chen Maogen , Chen Weiqian , Olsen Nancy J , Zheng Song Guo
Published: January 2023

Mesenchymal stem cells (MSCs) have the capacity to maintain immune homeostasis and prevent autoimmunity. However, it is unclear whether human-derived gingival mesenchymal stem cells (GMSCs) can suppress human T cell-mediated diseases. GMSCs were isolated from human gingival tissues. GMSCs were cultured with allogeneic T cells which labeled with CFSE, and cell proliferation was evaluated by CFSE dilution in FACS. In other experiments, human PBMCs were stimulated with LPS with a ratio of PBMCs to GMSCs at 1:5. After 3 days, intracellular cytokine including IL-4, IL-17, and IFN-γ in CD4+ cells were determined by FACS. Mouse xeno-GVHD Model was induced in NOD/SCID mice, GMSCs or human fibroblasts (negative control) or human bone marrow mesenchymal stem cells (positive control) were intravenously into mice. Weight loss and symptoms of GVHD were assessed. We observed that GMSCs potently suppress the proliferation of T cells and differentiation of PBMC in vitro, and that co-transfer of GMSC but not fibroblast and human PBMC significantly prolonged xeno-GVHD mouse survival. We also demonstrated that GMSC is even better than BMSC on treating xeno-GVHD. We further revealed that GMSCs inhibit human PBMCs-initiated xenogenic responses via CD39 and IDO signals. GMSCs can suppress human immune responses and immune system-mediated diseases, offering a potential clinical option to be used for modulating GVHD and other autoimmune diseases.
Long-range transcriptional effects and specific epigenetic marks in regulation of transcription from Lck proximal and distal promoters
Authors: Chiang Jeffrey , Hodes Richard J
Published: January 2023

Lck is a critical molecule for T cell development and activation. The T cell-specific expression of Lck is controlled by two distinct promoters separated by 15kb. The proximal promoter contributes the majority of Lck transcripts in immature thymic T cells, while the distal promoter expresses the majority of Lck transcripts in mature T cells. To study the regulation of these two promoters, promoter-mutated mouse models were generated by CRISPR technology, resulting in multiple mouse lines with distinct promoter deletions. The activity of the proximal promoter was reduced by 95% in the DP thymocytes of PROX[mut] mice with ~100bp deletion in the proximal promoter. Expression of distal-promoter-driven Lck mRNA was completely abrogated in immature and mature T cells in Lck distal promoter DIST[mut] mice with partial deletion of the distal promoter and exon 1. Distal-promoter-driven Lck mRNAs were not altered in PROX[mut] mice. However, proximal-promoter-driven Lck mRNAs were dramatically increased in the mature T cells of DIST[mut] mouse, suggesting that there is a long-range transcriptional interaction between the two promoters. To further understand the epigenetic influences in Lck gene expression, we analyzed histone modifications in the regions of Lck promoters. Our preliminary data showed the distal promoter was highly H3K9 acetylated in peripheral T cells but not immature T cells, and that this acetylation was decreased in DIST[mut] mice. Therefore, distal promoter H3K9 acetylation was correlated with promoter activity and may be involved in the developmental stage specificity of Lck promoters. The findings of this study on Lck promoters may provide important information on gene expression and its tissue-specificity.
The histone demethylase LSD1 inhibits PD-1 expression in acute viral infections
Authors: Bally Alexander , Austin James W , Lu Peiyuan , Boss Jeremy
Published: January 2023

During immune responses against chronic viral infection or cancer, expression of the immune-inhibitory receptor Programmed cell death-1 (PD-1) on T cells results in cellular exhaustion, characterized by poor cellular immune functions ultimately leading to a failure to respond appropriately to antigen. Intriguingly, exhausted PD-1hi cells from a chronic infection remain exhausted even after removal from chronic antigen exposure, indicating an active epigenetic mechanism is employed to inhibit PD-1 expression. This study elucidates the role of the histone H3K4me1/me2 demethylase LSD1 in mediating epigenetic changes at the PD-1 locus which lead to gene silencing. Blimp-1, a known inhibitor of PD-1 that associates with epigenetic modifiers, was bound to the locus in CD8 T cells during both acute and chronic modes of infection. However, LSD1 was only recruited during acute infection concurrent with PD-1 silencing. Accordingly, the active histone modifications H3K4me1 and me2, which are removed by LSD1, were absent after acute but not chronic infection. PD-1 expression on T cells was enhanced and prolonged during acute infections in an LSD1 conditional knockout mouse. Expansion of these studies to an in vivo melanoma model also showed that loss of LSD1 resulted in higher expression of PD-1 on tumor-infiltrating lymphocytes, and a corresponding failure to clear tumor cells. Together these data highlight a distinct role for LSD1 as a defining factor mediating the silencing of PD-1 expression following acute immune activation. This study elucidates a key epigenetic mechanism governing the switch in cell fate between acute effector or exhausted T cell, and highlights the overall role that epigenetics play in modulating immune responses.
Zbtb-1 controls NKp46+RORgammat+ innate lymphoid cell (ILC3) development.
Authors: Lu Ying , Zhang Xianyu , Bouladoux Nicolas , Belkaid Yasmine , kovalovsky Damian
Published: January 2023

Innate lymphoid cells (ILCs) play a central role in conferring protective immunity at the mucosal frontier. We identified a new function of the transcription factor Zbtb1 for the development of RORgammat+ ILCs (ILC3s). Zbtb1-deficient mice lacked NKp46+ ILC3 cells in the lamina propria of the small intestine and colon, which was consequence of impaired development of its RORgammat+ CCR6−NKp46− progenitors. This requirement of Zbtb1 was cell intrinsic as it was observed in in vitro developmental co-cultures with OP9-DL1 stroma and in vivo in bone marrow chimeras. The ILC3 developmental defect correlated with inefficient upregulation of T-bet expression and, as consequence, ILC3 cells from Zbtb1-deficient mice failed to acquire IFN-γ and repress IL-22 production upon development into NKp46+ cells. In correlation with a lack of NKp46+ILC3 cells, Zbtb1-deficient mice presented increased susceptibility to C.Rodentium infections. Altogether, these results establish that Zbtb1 is essential for the development of NKp46+ ILC3 cells.
Role of CCR10 and CCL27 in skin resident T cell development and homeostasis
Authors: Davila Micha L , Fu Yaoyao , Yang Jie , Xiong Na
Published: January 2023

The skin hosts numerous immune cell subsets which generate synergistic responses to a variety of stimuli including foreign antigens, pathogens and commensal bacteria. Each stimulus is unique and calls for a specific and balanced response; any disruption of this homeostasis can lead to complications such as inflammatory diseases. T cells in the skin are a key player in the response to infection, maintenance of commensal bacterial, and tissue homeostasis. Thus, elucidating the mechanisms regulating T cell migration, establishment, and maintenance within the skin is a high priority. CCR10, a receptor expressed on the majority of skin resident T cells, has a complex role in the development of skin-resident T cells through interaction with its skin-specific receptor CCL27. Preliminary data from our analysis of T-cell specific phenotypes in CCL27a knockout mice demonstrates a preferential reduction of Treg cells in the skin, highlighting the importance of CCL27a in the establishment of skin resident Treg cells under homeostatic conditions. Furthermore, we found CCR10+ resident CD8+ T cells contribute to the homeostatic maintenance of Treg and CD4+ effector T cells in the skin. Defective establishment of CCR10-knockout CD8+ T cells in the skin was shown to correlate with a reduction of skin-resident Treg cells. Skin-resident CD8+ T cells express high amounts of B7.2, a ligand for CTLA-4 which is highly expressed on Treg cells in the skin. In vitro co-culture of purified skin CD8+ and Treg cells suggests B7-2/receptor interaction is important for CD8+ cell-mediated survival of Treg cells. These findings will greatly aid in further understanding the development and homeostasis of skin resident T cells.
IL-33 mediated expansion of host ST2+ Treg prior to allogeneic hematopoietic cell transplantation decreases macrophage and effector T cell activation reducing acute GVHD
Authors: Reichenbach Dawn K , Matta Benjamin M , Zhang Xiaoli , Koehn Brent , Mathews Lisa , Smith Michelle J , Feser Colby J , Zeiser Robert , Turnquist Heth Roderick , Blazar Bruce R
Published: January 2023

Graft-versus-host disease (GVHD) is the leading complication and cause of mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Recently, we reported that IL-33 released from tissue damaged during allo-HCT conditioning mediates pro-inflammatory responses by Teffector cells resulting in greater acute GVHD lethality and clinical symptoms in mice. However, IL-33 is a pleiotropic cytokine that mediates both pro-inflammatory and anti-inflammatory responses. We hypothesized that the beneficial effects mediated by IL-33 could be harnessed if IL-33 was present prior to inflammatory mediators released during conditioning. Here, we demonstrate that exogenous IL-33 given peri allo-HCT (days −10 to 4) to recipients of MHC-disparate allo-HCT asserts its immunoregulatory properties reduced GVHD lethality and clinical symptoms compared to non-treated controls. IL-33 treatment peri allo-HCT resulted in the expansion of recipient Treg and suppressive myeloid cells that persisted post irradiation. Host Treg depletion in C57Bl/6 FoxP3-DTR mice by diphtheria toxin given concurrently with peri allo-HCT IL-33 treatment accelerated GVHD symptoms and lethality compared to peri allo-HCT IL-33 treatment alone, demonstrating that host Treg were necessary for GVHD protection. Transfer of Treg deficient in ST2, the IL-33 receptor, afforded less GVHD protection than wild-type Treg. ST2+ Treg controlled macrophage activation and prevented accumulation of Teffector cells in GVHD target tissue. Thus, we demonstrate that peri allo-HCT IL-33 expanded and activated host Treg have potential as a therapeutic modality to prevent and treat GVHD.
Serine protease inhibitor 6 protects allogeneic T cells from granzyme B-mediated mitochondrial damage without impacting graft-versus-tumor effect.
Authors: Du Wei , Kumar Sandeep , O’Neill Rachel , Chen Chuan , Mei Lin , McCarthy Philip , Cao Xuefang
Published: January 2023

Allogeneic hematopoietic cell transplantation(allo-HCT) is a potentially curative therapy for hematologic malignancies. Donor T cells are able to eliminate residual tumor cells in the host after allo-HCT, producing the beneficial graft-versus-tumor (GVT) effect, but can also causegraft-versus-host disease (GVHD) when attacking host normal tissues. We previously reported that granzyme B (GzmB) is involved in activation induced celldeath (AICD) of donor T cells and exerts differential impacts on GVHD and GVT effect. Serine protease inhibitor 6 (Spi6) is the sole endogenous inhibitor of GzmB that can protect immune and tissue cells against GzmB-mediated damage. Now this study is aimed to delineate the mechanism by which the GzmB-Spi6 axis regulates allogeneic T cell response. We have found that Spi6 is concentrated in mitochondria during allogeneic T cell activation, while Spi6−/− T cells exhibit abnormal mitochondrial membrane potential, mass, reactive oxygen species (ROS) production and increased GzmB-dependent AICD. Compared with WT T cells, Spi6−/− T cells exhibit decreased expansion in the host and cause significantly reduced GVHD. Notably, however, Spi6−/− T cells demonstrate the same level of GVT activity as WT T cells. These results suggest that Spi6 protects allo-reactive pathogenic T cells from GzmB-mediated mitochondrial damage but is dispensable for tumor-reactive T cells mediating GVT effect, probably due to its differential expression in these T cells. In sum, our data demonstrate that Spi6 playsa novel and critical role in maintaining T metabolism during allogeneic response, and suggest that disabling Spi6 in donor T cells may represent a novel strategy that can alleviate GVHD without sacrificing GVT effect.
Tumor-associated CAIC5 suppresses NKT cell proliferation and cytokine production
Authors: Choi Inhak , Lee Eungchang , Jung Keunok , Kim Heejung
Published: January 2023

In the previous study, we identified cancer-associated immune checkpoint 5 (CAIC5) as a new co-inhibitory molecule belonging to B7-related family. Typically, CAIC5 is expressed in tissue macrophages including Kupffer cells and peritoneal macrophages. We also found out human CAIC5 fusion protein greatly suppressed the proliferation and cytokine production in T cells and NKT cells in a respective stimulation assay. Since CAIC5 expression was identified in tumor microenvironment, we examined the impact of tumor cell-associated CAIC5 on NKT cell function. CAIC5-expressing cancer cells that are pulsed with alpha-galactosylceramide (a-Gal) greatly suppressed NKT cell IFN-g production and proliferation. CAIC5 KO mice that were systemically injected with a-Gal enhanced IFN-g production in NKT cells. Furthermore, WT mice that were injected with CAIC5-expressing tumor cells showed a poor survival compared to those with mock-expressing cells. We also found out human NKT cells were similarly suppressed in IFN-g production in the coculture with CAIC5-expressing cancer cell line. Therefore, tumor CAIC5 can be an important part of mechanisms for cancer immune escape.
In vitro and in vivo characterization of small molecule inhibitors of the anti-inflammatory TAM receptor MerTK
Authors: Owyang Alexander , Holland Sacha , Yi Sothy , Young Chi , Braselmann Sylvia , Frances Roy , Bagos Art , Tai Ernie , Siu Stacey , Park Gary , Lau David , Duan Matt , Yu Jiaxin , Ding Pingyu , Kolluri Rao , Bhamidipati Somasekhar , Darwish Ihab , Singh Raj , Payan Donald G , Masuda Esteban S
Published: January 2023

MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands (Gas6 or Protein S) bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome.

Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK −/− mice exhibit impaired growth and metastasis compared with those implanted in WT mice. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor-induced immunosuppression, or blocking tumor cell survival.

Here we describe novel and potent MerTK-selective and Mer-Axl small molecule inhibitors that block both MerTK in vitro kinase activity and MerTK autophosphorylation and downstream signaling in cells. Moreover, compounds were able to inhibit phagocytosis of ACs by MerTK-expressing human primary macrophages, and they block activation of MerTK in vivo. The MerTK inhibitors were not overtly cytotoxic or antiproliferative, and did not block the activity of TLR or T cell immune effector pathways. Limited off-target activity was observed in an in vitro kinase panel. Activity of these novel MerTK inhibitors in tumor models is currently under investigation.
Macrophage depletion decreases inflammation and tumorigenesis in the AOM/DSS mouse model of colon cancer
Authors: Bader Jackie E , Velazquez Kandy T , Enos Reilly T , Cranford Taryn L , Davis J Mark , Murphy E Angela
Published: January 2023

Macrophages have recently emerged as major players in the connection between inflammation and cancer; they represent up to 50% of the tumor mass and produce a wide array of inflammatory mediators with pro-tumoral functions. We used a macrophage depletion technique to examine the role of macrophages in colon cancer. To induce colon cancer, male C57BL/6 mice (n=20) received a single injection of AOM followed by three cycles of DSS supplemented water at concentrations of 2%, 1%, 1% at weeks 1, 4, and 7, respectively. Prior to the final DSS administration (week 7) and twice weekly thereafter for a duration of 5 weeks, mice (n=10/group) were administered 200μl of clodronate (CL2MDP) or PBS encapsulated liposomes via an intra-peritoneal route to deplete macrophages. Body weight, body composition, and symptom severity score were evaluated throughout the macrophage depletion period. Mice were sacrificed at 12 weeks following the initiation of colon cancer and colon tissue was analyzed for tumor burden, macrophage markers, and inflammatory mediators. There were no differences in body weight, body composition, or symptom severity score between the groups. Macrophage depletion decreased tumor number by ~25% and specifically medium tumor number by 50% (p<0.05). This was consistent with a decrease in expression of EMR1 (overall macrophage marker) as well as expression of markers associated with M2 macrophages (IL-13, IL-10, TGFβ & CCL17) (p<0.05) but not M1 markers (NOS2, IL-12a & IL-23). In addition, expression of the inflammatory mediators IL-6 and TNF-α were decreased following macrophage depletion. We demonstrate that macrophages are an important mediator of tumor growth and inflammation in colon cancer.
Ly6G+ neutrophils and polymorphonuclear-myeloid derived suppressor cells promote the survival of tumor cells
Authors: Herlihy Sarah E , Ramachandran Indu , Condamine Thomas , Lin Cindy , Garfall Alfred , Vogl Dan , Gabrilovich Dmitry , Nefedova Yulia
Published: January 2023

Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells which suppress the immune system. Collectively a MDSC population is comprised of monocyte-like MDSC and polymorphonuclear MDSC (PMN-MDSC). The number of MDSC is increased with the presence of tumors. Our lab has previously shown that PMN-MDSC represent the majority of MDSC in tumor-bearing mice. We found that depletion of myeloid cells in vivo results in increased survival in a mouse model of multiple myeloma (MM). Direct co-culture of neutrophils (CD11b+Gr1+ cells from tumor-free mice) or PMN-MDSC (CD11b+Gr1+ cells from tumor-bearing mice) and MM cells increases the survival of the MM cells from chemotherapeutic-induced death. The protective effect is not due to cell-cell contact as neutrophil and PMN-MDSC supernatants also protect MM cells from cell death. This suggests that the factor protecting tumor cells is soluble. Results in the mouse were confirmed using human MM cells. Similar results have been replicated on breast cancer cells with neutrophils and PMN-MDSC. Several cytokines and growth factors, such as IL-6 and fibroblast growth factor, are implicated in chemoresistance. The role of these neutrophil and PMN-MDSC soluble factors and the identification of novel contributors to chemoresistance are under investigation.
Oxidized lipid bodies block antigen cross-presentation in cancer by preventing trafficking of peptide-MHC class I complexes
Authors: Veglia Filippo , Tuyrin Vladimir A , Mohammadyani Dariush , Murphy Maureen , Gabrilovich Dmitry I
Published: January 2023

Background

Antigen cross presentation is a unique feature of dendritic cells (DCs) and plays an important role in the development of specific CD8+ T cell responses. It is established that the defect in cross-presentation by dendritic cells (DCs) in cancer results in ineffective antitumor immune responses and favors tumor escape. However, the mechanisms behind this phenomenon are unclear.

Results

In this study we found that tumor-derived factors impaired cross-presentation by differentiated conventional DCs without affecting expression of MHC class I, class II, or costimulatory molecules. The ability of DCs to stimulate antigen-specific T cells after loading with short peptide (directly bound to MHC class I) was fully preserved. We found that tumor-derived factors blocked trafficking of peptide-MHC class I (pMHC) complexes to the cell surface. These complexes were found to be entrapped into the lysosomes. This effect was caused by the accumulation of lipid bodies (LB) containing oxidized (ox) triglycerides (TAG) in DCs. Importantly LB containing non-oxidized TAG did not affect cross-presentation. Further experiments have identified the specific molecular mechanism responsible for oxTAG containing LB mediated block of cross-presentation in DCs.

Conclusions

Our results suggest a novel mechanism of regulation of cross-presentation in cancer
Glycolysis promotes MDSCs and cancer stemness through the interaction between Sirt1 and C/EBPb
Authors: LI WEI , Kryczek Ilona , Maj Tomasz , Wu Ke , Peng Dongjun , Zhao Ende , Xia Houjun , Lei Zhang , Tao Kaixiong , Wang Guobin , Zou Weiping
Published: January 2023

Glycolysis is considered a hallmark of cancer. However, its involvement in tumor immune regulation and breast cancer stem cell biology is poorly understood. Here we investigated how glycolysis affected the development of myeloid derived suppressor cells (MDSCs) and in turn controlled breast cancer stemness in breast cancer. G-CSF promotes MDSC development in 4T1 breast cancer model. We found that knock down of LDH-A in 4T1 cells resulted in decreased G-CSF production and reduced MDSC numbers. Accompanied with these findings was the inhibited tumor glycolysis, reduced breast cancer stemness potential, and increased mouse survival. Molecular mechanistic studies demonstrated that glycolysis regulated G-CSF expression, MDSCs and cancer stemness by controlling the interaction between Sirt1 and C/EBPb via NAD+-dependent manner. Moreover LDH-A expression is positively associated with breast cancer patient outcome. Collectively, our study has revealed that glycolytic metabolism is associated MDSCs and linked to breast cancer stemness.