bluuebunny/crossref_metadata_2025 huggingface dataset:
Exosomes released from Doxorubicin-treated breast tumor promote lung metastasis through miR126 mediated induction of MDSCs (TUM10P.1060)
Authors: Deng Zhongbin , Teng Yun , Rong Yuan , Zhuang Xiaoying , Mu Jingyao , Zhang Lifeng , Samykutty Abhilash , Zhang Huang-Ge
Published: January 2023

Chemotherapy induces myeloid-derived suppressor cells (MDSCs) which promote tumor metastasis. However, the molecular basis for chemotherapy induced MDSCs remains poorly understood. Here, we demonstrate that Doxorubicin-derived MDSC promote cancer progression via their effect on angiogenesis and the formation of a premetastatic niche. miRNA-containing exosomes from doxorubicin-treated MDSC exert more potent proangiogenic effects. We also demonstrate high level of MiR-126 was detected in Doxorubicin-derived exosomes, and inhibition of miR126 encapsulated in exosomes impaired proangiogenic effects of Doxorubicin-derived MDSC. Furthermore, nanoparticle-mediated targeting delivery of anti-miR-126 to MDSCs improves Doxorubicin-based therapeutic responses, leading to inhibition of lung metastasis. Collectively, our finding provides a compelling rationale to develop therapeutic strategy for overcoming chemotherapy resistance.
Gender and alcohol differentially influence DC signaling and ability to initiate an anti-tumor immune response. (TUM10P.1026)
Authors: Watkins Stephanie , Thompson Matthew
Published: January 2023

Gender disparity and effect of alcohol has been observed in cancer in relation to incidence, disease aggressiveness and prognosis. However, the role of the immune system and the activation of an anti-tumor immune response based on these parameters are unclear. Ethanol is known to reduce the DC’s ability to present antigen in the setting of an infection, but the signaling mechanisms effected to regulate DC functions which maybe critical in cancer are unclear. Some of the most successful cancer therapies to date have focused on hormone receptor blockade in tumor cells. Hormones, including androgen and estradiol, can dampen immune responses, but immune suppression or activation has not been studied during hormone deprivation therapy or upon previous exposure to alcohol. Tumor-associated dendritic cells (TADC) are key regulators of cytotoxic T-cell (CTL) function and may serve as a critical target for immunotherapy. Therefore, in the current study, we sought to determine the effect of alcohol on hormone receptor signaling, androgen receptor (AR) and estrogen receptor (ER), in TADC. The changes in regulation of these signaling pathways have a critical impact on the transcriptional and translational regulation of factors known to induce immune suppression and may be a key factor to consider when designing immune therapies for cancer.
Simulation of B cell affinity maturation explains enhanced antibody cross-reactivity induced by the polyvalent malaria vaccine AMA1 (VAC11P.1063)
Authors: Chaudhury Sidhartha , Wallqvist Anders , Reifman Jaques
Published: January 2023

Polyvalent vaccines use a mixture of antigens representing distinct pathogen strains in order to induce a broadly reactive immune response. However, it is unclear how polyvalency alters the fine specificity of the antibody response and what those consequences might be for protection. Here, we present a coarse-grain model of B cell affinity maturation during monovalent and polyvalent vaccinations that predicts the fine specificity and cross-reactivity of the antibody response. We stochastically simulate affinity maturation using a population dynamics approach where the host B cell repertoire is represented explicitly, and individual B cell sub-populations undergo rounds of stimulation, mutation, and differentiation. Antigens contain multiple epitopes and are present in subpopulations of distinct pathogen strains. This epitope and strain-specific model enables us to simulate the polyclonal response in granular detail and identify immune mechanisms driving serum specificity and cross-reactivity. We applied this approach to study the antibody response to a polyvalent vaccine based on the polymorphic malaria antigen AMA1. Our simulations show that polyvalent AMA1 vaccination induces an enhanced cross-reactive antibody response, primarily through a shift in affinity maturation that favors B cells specific to shared and cross-reactive epitopes, and demonstrates how a polyvalent vaccine with a small number of strains and only moderate allelic coverage may be broadly neutralizing.
Flagellin’s hypervariable D2/D3 domain is required for robust anti-flagellin primary antibody responses (VAC11P.1065)
Authors: Smith Kelly , Lopez-Yglesias Americo , Lu Chunchi , Zhao Xiaodan , Strong Roland
Published: January 2023

Bacterial flagellin is a well-known agonist of the innate immune system. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity towards influenza virus, which has been largely attributed to flagellin activation of TLR5. Our lab recently demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces antibody responses in mice through a third pathway that is independent of TLR5, Casp1, and MyD88. Herein, we define the structural features of FliC that contribute to this third pathway. By destroying the Naip5/6 (D0 domain) or TLR5 (D1 domain) recognition sites, we demonstrate that neither affected the TLR5-, inflammasome- and MyD88-independent antibody responses towards FliC. In contrast, deletion of the D2/D3 domain eliminated primary anti-flagellin antibody responses. Optimal primary and secondary anti-flagellin antibody responses required TLR5- and inflammasome-mediated recognition, and FliC’s D2/D3 domain. We demonstrate FliC’s D2/D3 domain is required for induction of primary antibody responses and robust secondary antibody responses. Our results indicate that FliC’s D2/D3 domain is essential for flagellin’s robust immunogenicity and suggest that the D2/D3 domain interacts with the host immune system to augment antibody production. Thus, the D2/D3 domain of FliC may also be critical for optimal immunogenicity and antibody responses in flagellin based vaccines.
Simulation explains the effect of preexisting memory on variable immune responses to 2009 pH1N1 influenza vaccination (VAC11P.1067)
Authors: Anderson Christopher , Topham David
Published: January 2023

Although it is generally assumed older individuals respond poorly to vaccination, the pandemic 2009 H1N1 vaccine demonstrated that this is not always true. Interestingly, there is considerable antigenic likeness between the pandemic 2009 H1N1 virus and the H1N1 viruses that circulated between 1918-1957. During the 2009 H1N1 pandemic the majority of individuals vaccinated with 2009 H1N1 mounted an immune response to the conserved stalk region of the influenza hemagglutinin. Older individuals alive during 1918-1957, however, mounted a response primarily to the variable head region of hemagglutinin. Memory B cells seem to play the dominant role in both of these responses. A proposed explanation for this difference is that older individuals were exposed to 1918-like viruses and contained preexisting memory B cells capable of cross-reacting with the 2009 H1N1 head epitopes. To test this hypothesis, we have developed a course-grain theoretical model using the theory of Shape Space to simulate the human humoral response. By simulating the variant influenza strains each age group would be exposed to over their lifetime, we can directly test the effect of exposure to 1918-like viruses on the response to pandemic 2009 H1N1. Understanding how differences in exposure history influence the B cell memory repertoire is crucial to predicting how individuals will respond to the future influenza vaccines.
PD-1 expression on peripheral blood lymphocytes enables direct enrichment of mutation-specific lymphocytes (VAC12P.1117)
Authors: Gros Alena , Tran Eric , Pasetto Anna , Parkhurst Maria R , Prickett Todd , Gartner Jared , Yu Zhiya , Wunderlich John , Robbins Paul , Yang James , Rosenberg Steve
Published: January 2023

Identification and isolation of mutation-specific T-cells is a major obstacle to the development of effective cancer immunotherapies. The tumor is a natural sink for neo-antigen specific cells, but these are far less frequent in peripheral blood, a more accessible and abundant source of T cells. Previous studies have revealed that PD-1 expression in the tumor microenvironment can identify the patient-specific repertoire of tumor and mutation-specific cells infiltrating human tumors. Here, we evaluated the potential utility of PD-1 expression on circulating lymphocytes to identify and enrich for mutation-specific cells. Selection of CD8+PD-1+ lymphocytes circulating in peripheral blood, but not the bulk or CD8+PD-1- cells, led to the direct enrichment of mutation-specific cells in three melanoma samples tested, with at least 1, 3 and 5 unique patient-specific neo-antigens recognized, respectively. The PD-1+ derived cells also displayed autologous tumor recognition. Furthermore, CD8+PD-1+ and PD-1hi cells isolated and expanded from the blood from a patient with colorectal cancer led to the identification of mutation-specific cells targeting two tumor neo-antigens. These findings provide a novel strategy to harness naturally-occuring mutation-specific T cells present in peripheral blood for the development of personalized T -cell based therapies to treat cancer.
B7-H1/PD-1 signals in carcinoma and lymphoma immunopathogenesis and immunotherapy (VAC12P.1118)
Authors: Clark Curtis , Liu Yang , Murthy Kruthi , Sareddy Gangadhara , Vadlamudi Ratna , Hurez Vincent , Curiel Tyler
Published: January 2023

αB7-H1 (αPD-L1) reportedly treats cancer by blocking PD-1 signals, but few formal data are reported. We showed B16 melanoma is CD80-PD-1-B7-H1+. αB7-H1 slowed subcutaneous (SQ) B16 in wild type (WT), B7-H1 KO and CD80 KO, with little difference between groups. αB7-H1 did not slow B16/B7-H1lo tumors in B7-H1 KO mice, formally showing tumor B7-H1 mediates αB7-H1 effects. αPD-1 slowed B16 in WT or B7-H1 KO, but not B16/B7-H1lo in B7-H1 KO, formally showing B7-H1/PD-1 effects in αB7-H1. Tumor B7-H1 variably predicts αB7-H1 efficacy, possibly due to variable tumor B7-H1 expression. To model variable B7-H1, we gave B16 or B16/B7-H1lo on both flanks, or each on opposite flanks of the same WT mice. αB7-H1 slowed B16, but not B16/B7-H1lo, unless B16 was on the other flank, suggesting spread of anti-tumor immunity, helping explain variable αB7-H1 effects by tumor B7-H1 status. We found EL4 T cell lymphoma is CD80loB7-H1loPD-1hi. αB7-H1 and αPD-1 did not slow SQ EL4. Unexpectedly, EL4/B7-H1hi exhibited significant growth delay after SQ or intravenous challenge in WT. EL4/B7-H1hi grew well in vitro, but in clusters as opposed to EL4. αPD-1 or αB7-H1 in vitro blocked clusters but such pre-treated cells still grew slowly in vivo suggesting negative inter-cell B7-H1/PD-1 signals. Our data formally show B7-H1/PD-1 signals in αB7-H1 treatment effects, help explain variable αB7-H1 effects by tumor B7-H1 status, and suggest novel and specific effects in T cell lymphomas requiring more study.
One-shot sustained release nanoparticle vaccines (VAC13P.1133)
Authors: Hook Sarah , Bobbala Sharan , Mitra Ashim
Published: January 2023

Sustained release of vaccines over an extended period of time may reduce or negate the need for multiple vaccinations (boosters) and also effectively enhance the immunogenicity of subunit vaccines. We have investigated the physcial, chemical and immunological properties of polymeric thermogelling formulations loaded with nanoparticulate protein and peptide based vaccines. The formulations were developed to be free-flowing liquids at room temperature and to form stable gels at body temperature. The vaccine components were loaded into nanoparticles before incorporation into the polymers in order to achieve synchronous release of antigen and adjuvant. The ability of the vaccines to stimulate long lived cellular and humoral responses after a single dose of antigen was investigated in mice. A single dose of formulations could induce detectable responses 50 days post immunisation and could protect mice in a tumor challenge model.
Use of a cytotoxic peptide as an immunotherapeutic agent in the treatment of metastatic breast cancer (VAC13P.1134)
Authors: Khaled Annette , Bassiouni Rania , Limaye Arati , Oyer Jeremiah , Igarashi Robert , Flores Orielyz , Perez J. , Copik Alicja
Published: January 2023

Breast cancer is a challenge for immunotherapy because of its genetic heterogeneity and decreased immunogenicity, which impedes the use of immune checkpoint blockade. A promising approach is to generate dying cancer cells that operate like a vaccine to induce a tumor-specific immune response and eradicate residual cancer cells. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature that stimulates immunity. To develop a novel ICD agent for cancer immunotherapy, our group discovered a therapeutic peptide, CT20p, and a nanotechnology-based platform to concentrate CT20p in breast tumors. Treatment of mice with nanomolar amounts of CT20p caused tumor regression, and dying breast cancer cells expressed markers characteristic of ICD: calreticulin (Crt) and heat shock protein (HSP)-70. The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT) that folds actin and tubulin. CCT was overexpressed in breast cancer cells susceptible to CT20p. In contrast, normal epithelial cells, macrophages or NK cells, which have low levels of CCT, were unaffected by CT20p. Breast cancer cells treated with CT20p displayed alterations in IRE1 and PERK, mediators of the unfolded protein response (UPR) that are upstream drivers of ICD signals. Hence, CT20p-treated breast cancer cells were more effectively killed by NK cells and ingested by phagocytes, demonstrating that CT20p is a robust stimulator of innate immunity.
HPV specific immunotherapy for cervical intraepithelial neoplasia using VGX-3100 induces regression of cervical lesions and potent T cell responses: results from a randomized, double-blind, placebo-controlled phase II study (VAC13P.1135)
Authors: Morrow Matthew , Trimble Cornelia , Shen Xuefei , Dallas Michael , Weiner David , Boyer Jean , Yan Jian , Kraynyak Kimberly , Sylvester Albert , Giffear Mary , Marcozzi-Pierce Kathleen , Shah Divya , Broderick Kate , Khan Amir , Lee Jessica , Humeau Laurent , Sardesai Niranjan , Bagarazzi Mark
Published: January 2023

Here we report the results of a Phase II study assessing the safety and efficacy of VGX-3100 in 167 women with biopsy-proven CIN 2/3 with concurrent HPV16/18 infection. The randomized, placebo-controlled, double-blind study evaluated cervical tissue changes after three 6 mg intramuscular doses of VGX-3100 followed by electroporation (EP) with Inovio’s CELLECTRA®2000 device at weeks 0, 4, and 12. Cervical tissue was examined before treatment and 9 months later. The study met its primary efficacy endpoint; the percentage of patients who had regression of CIN 2/3 to CIN 1 or no disease at 6 months post third dose was significantly higher in the VGX-3100 group compared to the placebo group (p=0.017). In addition, the trial demonstrated the ability of VGX-3100 clear HPV infection concurrent with regression of CIN lesions. The study also explored humoral and cellular immune responses to VGX-3100 in blood samples taken periodically during the trial. ELISA as well as IFN-g ELISpot results revealed significantly higher responses in the VGX-3100 treated group than in the placebo group, suggesting that VGX-3100 was able to robustly engage the patients’ immune system. Altogether, the successful phase 2 results represent a significant milestone in the development of active immunotherapies not only to robustly engage the immune system but also to treat cancer and infectious diseases and have the potential to provide physicians an important alternative to surgery to treat CIN 2/3 disease.
mRNA loaded CMRF-56+ dendritic cells: a novel anti-cancer vaccination strategy (VAC13P.1122)
Authors: Papadimitrious Michael , Anguille Sebastien , Bryant Christian , Kupresanin Fiona , Verma Nirupama , Lo Kevin , Clark Georgina , Newman Elizabeth , Bradstock Kenneth , Berneman Zwi , Fromm Phillip , Hart Derek
Published: January 2023

Current dendritic cell therapeutic vaccinations using tumour associated antigen (TAA) loaded monocyte derived dendritic cells (MoDC) has demonstrated anti-cancer immune responses. However, functional and transcriptional differences between blood dendritic cells (BDC) and MoDC have emerged. Using the GMP human CMRF-56 IgG4 chimeric monoclonal antibody, we are able to obtain clinical-grade BDC appropriate for loading TAA mRNA. We evaluated hCMRF-56 as an improved clinical preparation for BDC immune therapy by 1) their ability to initiate anti-tumour antigen immune response following loading of TAA mRNA 2) comparing CMRF-56+ BDC and MoDC capacity to migrate to draining lymph nodes in a preclinical xenograft model. Characterisation of the proposed clinical formulation identified up-regulation of CD40 and CD80 post GM-CSF activation and GM-CSF activation with mRNA nucleofection, respectively. Nucleofected CMRF-56+ BDC migrated towards CCL21 in vitro. CMRF-56+ BDC and MoDC were administered to SCID mice to monitor their in vivo migration to inguinal lymph nodes. Flu Matrix Protein 1 IFNγ responses were observed by CD4+ and CD8+ lymphocytes. Stimulation of CMRF-56+ BDC loaded with Wilms Tumor 1 antigen mRNA was able to induce CD4+ and CD8+ T cell responses. Our data suggests BDC can be effectively nucleofected with TAA mRNA. This demonstrates the efficacy of mRNA nucleofected BDC to warrant further clinical investigation
Pro-inflammatory cytokine production by activated monocytes and macrophages in response to cowpox virus infection in cynomologus macaques (VIR9P.1144)
Authors: Janosko Krisztina , Cohen Melanie , Josleyn Nicole , Zhang Tengfei , Cong Yu , Jahrling Peter , Johnson Reed
Published: January 2023

Monocytes and macrophages play pivotal roles in host defense against microbial and viral pathogens through production of inflammatory and cytotoxic mediators, phagocytic activity, and activation of T cells. In previous studies, we reported significant expansion of CD14+ cells following intravenous cowpox virus Brighton Red (CPXV) infection of cynomologus macaques accompanied by increased levels of pro-inflammatory cytokines in plasma. Here, monocytes were assayed for production of pro-inflammatory cytokines and chemotactic factors during CPXV disease progression at days 3, 4, 6, and 7 post-infection in a serial sacrifice study of cynomologus macaques intravenously inoculated with 5 x 106 PFU CPXV. We measured monocyte absolute cell numbers in blood, frequencies of monocytes and macrophages in blood, and compared normal and infected monocytes through production of pro-inflammatory cytokines IL-6, IL-8, MCP-1, and IFNγ. Our results demonstrated an early increase in frequency and absolute numbers of total monocytes in blood with a rapid decline in moribund animals. Cytokine and chemokine analysis demonstrated an increase in IL-8 and MCP-1 expression over the course of disease but little IFNγ or IL-6. Pox antigen positive monocytes also expressed IL-8, but were not observed to express MCP-1. This suggests pox infection may alter cytokine production profiles of monocytes and may play a role in disease progression.
Role of IFN-α signaling in T cell pool dysregulation during chronic exposure to type-I IFN under lymphopenic conditions: impact on HIV pathogenesis (VIR9P.1145)
Authors: Le Saout Cecile , Hasley Rebecca , Imamichi Hiromi , Tcheung Lueng , Hu Zonghui , Smith Mindy , Rupert Adam , Sneller Michael , Lane H. , Catalfamo Marta
Published: January 2023

HIV infection and its associated chronic immune activation alter T cell homeostasis leading to CD4 T cell depletion and CD8 T cell expansion. The mechanisms behind these outcomes are not totally defined and only partially explained by the direct cytopathic effect of the virus. We established a murine model of immunpoathogenesis in which adoptive T cell transfer into lymphopenic hosts chronically treated with IFN-α leads to CD4 T cell depletion and CD8 T cell expansion, recapitulating the alteration of the T cell pools observed in HIV infected patients. In these conditions, CD4 T cells undergoing IL-7/lymphopenia induced-proliferation upregulate their level of the Signal Transducer and Activator of Transcription 1 (STAT1) resulting in enhanced CD4 T cell responsiveness to IFN-α. In this setting, CD4 T cell depletion resulted from an indirect effect of IFN-α driven inflammatory environment, since animals with IFNAR deficient T cells underwent the same degree of CD4 T cell depletion than wild type animals. In contrast, direct IFN-α signaling through IFNAR on CD8 T cells was necessary for their expansion induced by chronic exposure to Type-I IFN under lymphopenic conditions. These findings suggest that direct and indirect effects of IFN-α signaling may play a role in the pathogenesis of HIV infection. Further analysis of this pathway may contribute to the development of new strategies to reverse the dysregulation of the T cell pools seen in patients with HIV infection.
Using gel centrifugation to determine the monomer-dimer equilibrium of the HIV-1 5’-leader (VIR9P.1162)
Authors: Bolden Nicholas , Van Verna , Swanson Canessa , Monti Sarah , Summers Michael
Published: January 2023

An estimated 35 million people live with Human Immunodeficiency Virus (HIV-1), with approximately 1.7 million associated deaths per year. HIV-1 is a retrovirus that infects the T cells of the host’s immune system. After infection, the virus reverse-transcribes its RNA genome into DNA and integrates the viral DNA into the host DNA, which is eventually transcribed into RNA by the host cell. The 5’ untranslated region (UTR) of the RNA, is the most conserved region of the genome. It exists in equilibrium between a monomeric form, important for translation, and a dimeric form, important for genome packaging. While gel electrophoresis is usually used to determine the equilibrium of monomer and dimer, we have developed a new technique that uses gel centrifugation. We are developing this technique as it may allow us to look at the monomer-dimer equilibria of different RNAs, such as HIV-2, which have weaker “kissing” dimer interactions. When gel electrophoresis is used on these dimers, the magnesium that stabilizes the dimer is drawn towards the opposite pole as the RNA, providing inaccurate data by dissociating the dimers and causing more monomer to be visualized. Magnesium containing gels are used to combat this, but it is difficult to determine how much magnesium is needed to obtain accurate results. Future work includes testing this method on retroviruses with a “kissing dimer” interaction and eventually determining a way to quantify the amount of monomer and dimer present.
Mechanisms of regulatory T cell activation and expansion utilizing virus-specific CD8+ T cells (VIR10P.1172)
Authors: Myers Lara , Joedicke Jara , Carmody Aaron , Messer Ron , Dittmer Ulf , Kim Hasenkrug
Published: January 2023

The CD8+ T cell response to acute Friend virus (FV) infection is critical for recovery from lethal disease. However, in the late acute phase of infection this response becomes suppressed by regulatory T cells (Tregs), thereby leading to chronic infection. A major question regarding these events is how does FV infection induce the Treg response. We discovered a novel subset of natural Tregs that expand disproportionately in response to FV infection. These Tregs bear Vβ5+ T cell receptors, which are unique because of their known specificity for a superantigen (Sag) encoded by an endogenous retrovirus. Our results demonstrate that these Sag-reactive Tregs are independent of IL-2 but dependent on TNFα. Studies revealed that the expansion of these Tregs is actually driven the CD8+ T cell response. Activated, FV-specific CD8+ T cells upregulate membrane bound TNFα, which induces Vβ5+ Treg expansion by binding to TNFR2 receptors on the Tregs. Thus the expansion of the Vβ5+ Tregs is dependent on activated CD8+ T cells, the eventual targets of Treg-mediated suppression. These results demonstrate a new pathway of Treg expansion and two-way intercellular communication between Tregs and activated CD8+ T cells. Control by Tregs appears to be a natural mechanism to prevent immunopathological damage from CD8+ T cell responses that are too vigorous or too sustained. Modulation of this Treg subset has implications not only for the treatment of viral infections but also autoimmune diseases.
MHC alleles influence CD8 T cell exhaustion and host survival during chronic viral infection (VIR10P.1164)
Authors: Christiaansen Allison , Varga Steven
Published: January 2023

Specific HLA alleles have been associated with enhanced control of chronic viral infections such as hepatitis C and HIV. However, the exact mechanism by which HLA alleles result in enhanced viral clearance is currently unknown. Infection of inbred C57BL/6 (B6) with the clone 13 (Cl-13) strain of lymphocytic choriomeningitis virus (LCMV) is widely used as a model of chronic viral infection. To determine the impact of MHC alleles on the progressive loss of CD8 T cell effector functions, MHC congenic mice were used to delineate the role of MHC alleles from those of non-MHC genes. B10.D2 x B6 F1 (D2B6F1) mice were bred that express H-2d/b on a B6 background and compared to H-2b B6 mice. Following Cl-13 infection, D2B6F1 mice maintain higher levels of CD8 T cell effector function and exhibit more rapid viral clearance than B6 mice. Strikingly, D2B6F1 mice exhibited 75% mortality following Cl-13 infection. CD8 T cell depletion ameliorated the Cl-13 induced disease in D2B6F1 mice indicating a critical role for CD8 T cells in mediating the immunopathology. The frequency of CD49d+ CD11ahi antigen-experienced CD4 T cells was also increased in Cl-13-infected D2B6F1 mice relative to B6 controls. Our results indicate that increasing MHC heterogeneity can have a critical impact on the breadth and effectiveness of the antiviral T cell response resulting in more rapid viral clearance at the cost of enhanced immunopathology following a chronic viral infection.
Partial reversal of exhausted T cell phenotype and memory re-differentiation in chronic HCV-infected patients receiving direct-acting antivirals (VIR10P.1165)
Authors: Burchill Matthew , Golden-Mason Lucy , Wind-Rotolo Megan , Rosen Hugo
Published: January 2023

Chronic infection with Hepatitis C Virus (HCV) results in T cell exhaustion and subsequent impairment of T cell immunity. Recently, novel therapies of direct acting antiviral (DAA) regimens were developed which induce rapid and sustained clearance of HCV. These DAAs have provided the unique opportunity to determine whether successful treatment-induced eradication of viral antigen leads to reversal of T cell exhaustion and reconstitution T cell effector function. Herein we found that treatment with DAAs resulted in the reconstitution of the immune compartment in the peripheral blood. Furthermore, DAA-mediated viral clearance induced the re-differentiation of the memory T cell compartment to a more effector like state. Finally, DAA treatment caused the down modulation of markers associated with T cell chronic exhaustion, most intriguingly, the co-inhibitory T cell immunoglobulin and ITIM domain (TIGIT). The down modulation of TIGIT was unique to DAA treatment, as this effect was not observed when patients were treated with standard pegylated IFN and ribavirin therapy. Collectively, our data demonstrates that rapid HCV viral clearance following treatment with DAAs resulted in the partial restoration of global and viral specific T cell immunity. These findings suggest that T cell chronic exhaustion can persist long term in the absence of viral antigen and that complete reversal of this phenotype requires interventions that are independent of the persistence of viral antigen.
B-lymphocytes expressing an immunoglobulin specificity recognizing the natural pancreatic beta cell autoantigen, peripherin, are rendered proliferatively anergic but still accelerate type 1 diabetes development in NOD mice. (BA3P.107)
Authors: Leeth Caroline , Chapman Harry , Arpa Berta , carrillo jorge , Carrascal Jorge , Rosell Estela , Verdaguer Joan , Serreze David
Published: January 2023

Type 1 diabetes (T1D) results from the destruction of insulin-producing pancreatic ß-cells by autoreactive CD4+ and CD8+ T-cells culminating in glucose dysregulation in both humans and the NOD mouse model. It is clear in the NOD mouse model and also likely humans that B-lymphocytes also play a key pathogenic role in T1D. A sizable proportion of B-lymphocytes isolated from pancreatic islets in NOD mice recognize the autoantigen, peripherin, a type II intermediate filament expressed widely in the peripheral and central nervous system. To further understand the role peripherin-reactive B lymphocytes play in the pathogenesis of T1D, we created NOD mice transgenically expressing non-secretory heavy and light chain transgenes encoding an immunoglobulin (Ig) recognizing peripherin derived from islet-associated B lymphocytes. These mice overwhelming produce B-lymphocytes expressing the transgenic peripherin-binding Ig molecule and develop T1D at a significantly accelerated rate compared with the standard NOD strain. Islet infiltration by lymphocytes also occurs earlier in these mice with the vast majority of islet-associated B-lymphocytes expressing the transgenic Ig. While these peripherin-reactive B lymphocytes are diabetogenic, they are also proliferatlively anergic and appear to require frequent replenishment from hematopoietic stem cells. These findings lend credence to the targeting of peripherin autoreactive B-lymphocytes as a possible T1D intervention.
Disruption of tolerance by ER stress in type 1 diabetes (BA4P.131)
Authors: Marre Meghan , Profozich Jennifer , Coneybeer Jorge , Geng Xuehui , Bertera Suzanne , Trucco Massimo , James Eddie , Piganelli Jon
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease in which autoreactive T cells target and destroy islet β cells. The events that break peripheral tolerance in patients genetically predisposed to autoimmunity are poorly understood. Many physiological and environmental triggers associated with T1D cause endoplasmic reticulum (ER) stress, which may increase abnormal protein post-translational modification (PTM). We hypothesized that β cell ER stress generates neo-antigens that activate autoreactive T cells in T1D. Chemical (Thapsigargin) induction of ER stress in murine islets or insulinomas increased their recognition by diabetogenic BDC2.5 T cells (28-1500 fold) through activation of the PTM enzyme tissue transglutaminase 2 (Tgase; 18 fold). Indeed, reduced Tgase expression decreased stress-induced immunogenicity (58%). Also, physiological conditions in vivo increased ER stress (300 fold) and Tgase activity (44 fold) in primary islets and transplanted insulinomas, increasing BDC2.5 IFNγ responses (22 fold). Thus, ER stress leads to PTM-dependent murine β cell immunogenicity. We are currently translating these findings to a human T1D model. Preliminary data confirm that ER stress in human β cells significantly increased IFNγ secretion by T cells isolated from T1D patients that recognize Tgase-modified β cell antigens (24-360 fold). Thus, Tgase-dependent PTM may also cause immunogenicity in human β cells, demonstrating the relevance of our murine studies to human disease.
Susceptibility to IBD is associated with with specific KIR and HLA ligand genotypes (BA4P.142)
Authors: Trachtenberg Elizabeth , Udell Julia , Osoegawa Kazu , Ladner Martha , Noonan David , McGovern Dermot , Rotter Jerome , Taylor Kent , erlich henry
Published: January 2023

Adaptive and innate host immune responses to intestinal microbes have been implicated in the pathogenesis of Ulcerative Colitis and Crohn’s Disease, the subtypes of Inflammatory Bowel Disease (IBD). Polymorphisms of the Killer Ig-like Receptors (KIR) genes, which encode receptors on natural killer cells, influence immune mediated diseases. Here we study the genetic contribution of KIR and their HLA ligands in UC and CD. HLA genotyping was performed by next generation sequencing and KIR genotyping with our MALDI-TOF assay. The KIR HLA ligands, Bw4/6 and C1/C2 were assigned using HLA-B and HLA-C residue positions 77-83. The case-control analysis consisted of 1232 Caucasian IBD patients (461 with UC, 771 with CD) and 203 Caucasian controls. The homozygous genotypic combination of KIR2DL3, KIR3DL1 (homozygous KIR A/A haplotype) and Bw6 (absence of Bw4) with a heterozygous C1/C2 genotype was protective (cf, C1/C1 and C2/C2) against ulcerative colitis (OR = 0.29, p < 0.001). No significant association was found with KIR loci and their HLA-ligands with CD. In a C1/C2 heterozygote, signaling would be expected via both the KIR2DL3 receptor and the KIR2DL1 receptor. These data reveal a central role for a specific combination of KIRs and HLA ligands (i.e., homozygous KIR A haplotype, absence of Bw4 and C1/C2 heterozygosity) in protecting against UC disease and underscore the importance of analyzing KIR genotypes in the context of their HLA ligands in disease association studies.
Hypoxia promotes C5a-associated inflammation in rheumatoid arthritis via upreglating the microRNA miR-342-3p to inhibit IL-4R, STAT5b, ubiquitin specific peptidase 13 and EP300 (BA4P.123)
Authors: Liu Shuxun
Published: January 2023

Hypoxia-inducible miRNAs (hi-miR) are identified in hypoxia-associated physiological and pathological processes. Hypoxia plays a pivotal role in the pathogenesis of several inflammatory diseases, particularly rheumatoid arthritis (RA). However, the roles and underlying mechanisms of hi-miR in autoimmune diseases remain poorly understood. Considering that synovial dendritic cells (DCs) and C5a-associated inflammation are critical for RA pathogenesis, here we investigated what hi-miR regulated C5a-mediated inflammatory responsiveness of DCs. Hypoxia upregulated the expression of C5aR1 on DCs from human monocytes, consequently promoting C5a-mediated inflammatory responsiveness of DCs. Furthermore, hypoxia-upregulated miR-342-3p targeted STAT5b, EP300 and ubiquitin specific peptidase 13 (USP13), thus maintaining the high expression of C5aR1 on DCs via arresting STAT5 and STAT6-mediated downregulation of C5aR1 during monocytic differentiation into DCs. Moreover, blockade of miR-342-3p abnormally increased in RA models significantly represses the progression of RA models. So, our study identifies hypoxia-inducible miR-342-3p as an important mediator for DC and C5a-associated inflammatory pathogenesis in authoimmune diseases.
MCPIP1/Regnase is a negative feedback inhibitor of IL-17-mediated signaling and inflammation (CCR3P.200)
Authors: Gaffen Sarah , Garg Abhishek , Amatya Nilesh , Chen Kong , Cruz Juan , Grover Prerna , Whibley Natasha , Conti Heather , Hernandez Mir Gerard , Childs Erin , Smithgall Thomas , Biswas Partha , Kolls Jay , McGeachy Mandy , Kolattukudy Pappachan
Published: January 2023

IL-17 came to prominence with the discovery of Th17 cells. IL-17 induces inflammatory pathology in autoimmunity and certain infections, and thus constraint of this pathway is an essential part of its regulation. However, to date our understanding of IL-17 signaling checkpoints remains quite limited. Here, we identify MCPIP1 (Regnase-1, Zc3h12a) as a novel negative feedback inhibitor of IL-17 signaling. IL-17 treatment of fibroblasts stimulated increased MCPIP1 expression, whereas MCPIP1 silencing enhanced IL-17-mediated signal transduction. Conversely, reconstitution of MCPIP1-/- cells restricted IL-17 signaling, which was dependent on the MCPIP1 endonuclease domain, not its deubiquitinase domain. In vivo, MCPIP1 deficiency enhanced IL-17-dependent autoimmunity and host resistance to infection. Namely, susceptibility to candidiasis was reduced in MCPIP1 haploinsufficient mice, whereas IL-17-dependent pathology in EAE and pulmonary inflammation was enhanced. In addition to its well-documented capacity to degrade cytokine mRNA transcripts such as Il6, we found that MCPIP1 regulates some, though not all, IL-17-dependent promoters. In addition, we identified a novel function for MCPIP1 in mediating decay of mRNA transcripts encoding inflammatory receptors, including IL-17R subunits, in a manner independent of their 3’ UTR elements. This is the first demonstration that MCPIP1 restricts IL-17R signal transduction, and that this enzyme regulates inflammatory receptor expression.
Regulation of granulocyte colony stimulating factor by Fli-1, a novel transcriptional activator of inflammatory mediators (CCR3P.213)
Authors: Lennard Richard Mara , Brandon Danielle , Sato Shuzo , Zhang Xian
Published: January 2023

The Fli-1 transcription factor plays a significant role in the pathogenesis of systemic lupus erythematosus (SLE). Fli-1 expression is significantly increased in patients with lupus nephritis and lupus-prone mice with reduced Fli-1 expression (Fli-1+/-) live longer and have significantly less nephritis compared to wild type controls. Recently, Fli-1 has emerged as a critical regulator of inflammatory mediators, including MCP-1, CCL5, and IL-6. The cytokine, granulocyte colony stimulating factor (G-CSF) regulates neutrophil precursor maturation and survival, and activates mature neutrophils. In clinical settings, G-CSF increases the inflammatory response and worsens tissue damage. Previously, a significant decrease in neutrophil infiltration into the kidneys of Fli-1+/- lupus-prone mice was observed. In this study, a significant decrease in G-CSF protein expression was detected in stimulated endothelial cells transfected with Fli-1 specific siRNA. The murine G-CSF promoter contains numerous putative Fli-1 binding sites and five regions within the proximal promoter were found to be significantly enriched for Fli-1 binding. Transient transfection assays indicate that Fli-1 drives transcription from the G-CSF promoter and mutation of the Fli-1 DNA binding domain resulted in a 94% loss of transcriptional activation. In conclusion, we have discovered that Fli-1 plays a major role in the regulation of inflammatory cytokines and chemokines and inflammatory disease pathogenesis.
EZH2 in B lymphocyte commitment (HEM1P.229)
Authors: Jacobsen Jennifer , Kee Barbara
Published: January 2023

EZH2 is the histone methyltransferase subunit of the Polycomb Repressive Complex 2 that catalyzes the repressive histone modification, H3K27me3. We hypothesize that EZH2 is required to repress alternative lineages during B cell development. Loss of EZH2 in hematopoietic cells leads to blocks in B and T cell development. Here we use an Il7racre mouse model to delete Ezh2 at the common lymphoid progenitor stage to study the role of EZH2 in B lymphocyte commitment. We confirm that Il7racreEzh2fl/fl mice have a cell-intrinsic block at the pro-B cell stage of development. Although there are normal numbers of pro-B cells in Il7racreEzh2fl/fl mice, EZH2-deficient pro-B cells are outcompeted by WT cells in competitive chimeras. By qPCR we find that there is modest upregulation of Gata3 and Nfil3, which regulate innate lymphocyte development. Future studies will investigate the developmental plasticity of EZH2-deficient pro-B cells.
A CTCF-dependent regulatory element in the mouse TCRα/δ locus shapes the TCRα/δ repertoire (HEM2P.233)
Authors: Chen Liang , Carico Zachary , Shih Han-Yu , Krangel Michael
Published: January 2023

The diversity of antigen receptors on T and B lymphocytes is generated by the assembly of variable (V), diversity (D), and joining (J) gene segments. At the TCRα/δ locus, Vδ-Dδ-Jδ rearrangement occurs in CD4−CD8− double-negative (DN) thymocytes, whereas Vα and Jα are joined in CD4+CD8+ double-positive (DP) thymocytes. We previously showed that CTCF is an important regulator of chromatin conformation and Vα-Jα recombination in DP thymocytes. Here we show that INT1-2, a pair of CTCF-binding elements (CBEs), plays an important role in shaping the TCRδ repertoire in DN thymocytes. INT1-2-/- mice have a restricted TCRδ repertoire with Vδ4 being strongly predominant. Chromatin conformation capture assays showed that INT1-2-deletion disrupts a stable loop between INT2 and TEA CBEs and promotes the formation of new interactions between TEA and CBEs that are upstream of INT1-2. This alteration of chromatin loops disrupts a locus conformation designed to grant diverse Vδ segments an opportunity to pair with Dδ and Jδ segments. Additional analysis of INT1-2-/- DP thymocytes revealed a restricted TCRα repertoire, which we believe occurs as a consequence of the restricted TCRδ repertoire.
RORgT modulates key features in adult thymopoiesis (HEM2P.235)
Authors: Guo Yanxia , MacIsaac Kenzie , Hawwari Abbas , Miller Richard , Ferguson Heidi , Vilas Deshmukh Sujal , Mudget John , Engstrom Laura , Piers Kyle , Baltus Gretchen , Barr Kenneth , Mehmet Huseyin , Correll Craig , Cua Daniel , Jones Dallas
Published: January 2023

Recent studies have elucidated the molecular mechanism of RORgT transcriptional network controlling Th17 cell lineage fate and effector function. RORgT was initially identified as a transcription factor required for thymopoiesis by maintaining survival of CD4+CD8+ (DP) thymocytes. There are increasing efforts to target RORgT for the treatment of autoimmune disorders, but it remains unclear how inhibition of RORgT in adult animals may impact thymic T cell development. Here, we show that RORgT is required to maintain adult thymopoiesis using both ERT2-CRE-mediated conditional genetic ablation and pharmacological inhibition with a selective small-molecule antagonist. RNA-SEQ analysis demonstrated that RORgT regulates thymocyte migration, positive selection, proliferation, inflammation and DNA synthesis involved in thymopoiesis beyond DP cell survival. RORgT also directly engages multiple target DNA in close proximity to key transcription factors such as T cell factor-1, Ikaros family zink finger protein 1 and others required for thymocyte development. Our work illustrates a new perspective in thymic development by showing that RORgT modulates adult thymopoiesis via cooperation with other major transcription factors.
HDAC3 is required for thymocyte positive selection (HEM2P.236)
Authors: Philips Rachael , Chen Meibo , McWilliams Doug , Belmonte Paul , Constans Megan , Hiebert Scott , Shapiro Virginia
Published: January 2023

In order to generate functional peripheral T cells, proper gene regulation during T cell development is critical. Development involves both the activation and repression of genes as cells progress through each developmental checkpoint. Histone acetylation/deacetylation is one mechanism regulating gene expression. Acetylation by histone acetyl transferases (HATs) allows for gene expression and deacetylation by histone deacetylase (HDAC) enzymes promote gene silencing, and their role in regulating T cell development is not clear. In this study, our lab focused on HDAC3. We examined the role HDAC3 plays during T cell development by utilizing a CD2-Cre-mediated loss of HDAC3 (CD2icre HDAC3-cKO), which deletes early in thymocyte development. When HDAC3 is absent, there is a developmental block during positive selection, leading to the production of very few CD4 and CD8 SP thymocytes leading to a severe defect in peripheral T cell numbers. During positive selection, HDAC3-deficient DP thymocytes fail to down-regulate RORγt as they progress to the SP stage. Consistent with the failure to down-regulate RORγt, thymocytes post-positive selection express high levels of Bcl-xl and fail to express Bcl-2, which is required for their survival. Moreover, HDAC3-deficient thymocytes show increased apoptosis. These results suggest that HDAC3 is required for the down-regulation of RORγt during positive selection.
Histone deacetylase 3 is necessary for T cell maturation (HEM2P.238)
Authors: Hsu Fan-Chi , Belmonte Paul , Constans Megan , Chen Meibo , McWilliams Doug , Hiebert Scott , Shapiro Virginia
Published: January 2023

Recent thymic emigrants (RTEs) are newly generated T cells that need to undergo post-thymic maturation in the periphery. The mechanism of T cell maturation remains unclear. Histone deacetylase 3 (HDAC3) controls gene regulation, chromatin accessibility and genomic stability. However, the function of HDAC3 in T cell has yet to be elucidated. As we previously demonstrated that HDAC3 is a binding partner to NKAP, we hypothesize that HDAC3, like NKAP, may regulate T cell maturation. We generated CD4-cre HDAC3 cKO mice, which have similar numbers of DN, DP, CD4 SP and CD8 SP thymocytes as compared to WT mice; however, the peripheral numbers of CD4+ and CD8+ T cells are dramatically decreased in CD4-cre HDAC3 cKO mice. When we examined Rag1-GFP CD4-cre HDAC3 cKO mice, the majority of peripheral naïve T cells are Rag1-GFP+ RTEs indicating a block in T cell maturation. There is no defect in the positive selection, negative selection, migration, or homeostasis in CD4-cre HDAC3 cKO mice. However, expression of the complement inhibitor CD55, which is induced during maturation, is severely decreased. In the periphery, similar to NKAP-deficient T cells, HDAC3-deficient T cells are targeted for elimination by complement. In addition, HDAC3-deficient T cells have decreased sialic acid modifications on the cell surface, mainly due to α2-3, and α2-8 linkages, that recruit natural IgM to initiate the classical complement pathway. Thus, HDAC3 is required for T cell maturation.
A role for CD31 (PECAM-1) during T cell differentiation in the human thymus (HEM2P.239)
Authors: Douaisi Marc , Resop Rachel , Jamieson Beth , Uittenbogaart Christel
Published: January 2023

Although CD31 expression on human thymocytes has been reported, a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. Here we address the evolution of CD31 expression from the CD34+ hematopoietic precursor to the CD45RA+ mature CD4+ and CD8+ single positive (SP) T cells. Using 9-color flow cytometry, we show that CD31 is highly expressed on CD34+ progenitors and stays high until the early double positive stage (CD3- CD4+ CD8α+β-) when CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3high CD4+CD8+ double positive thymocytes. However, following positive selection, CD31 expression differs dramatically between CD4 and CD8 lineages: homogenously high on CD8 SP but low to negative on CD4 SP. Remarkably, there is a substantial subset of semi-mature (CD45RA-) CD4 SP thymocytes that lack CD31 expression. Moreover, FoxP3+ and ICOS+ cells are over-represented in this CD31- subpopulation. Despite the CD31- CD45RA- subpopulation, the majority of egress-capable mature CD45RA+ CD4 SP thymocytes expresses CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: TCRβ selection, as well as positive and negative selection. Considering the ability of CD31 to modulate the TCR’s activation threshold via the recruitment of tyrosine phosphatases, our results suggest a likely significant role for CD31 during T cell development.