bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and regulation of dermal γδ T cells (P3219)
Authors: Cai Yihua , Fleming Christopher , Yan Jun
Published: January 2023

Recently, others and we have identified a unique subset of γδ T cells in dermis that is capable of producing IL-17. Dermal γδ T cells have been demonstrated to play a critical role in the psoriasis pathogenesis. However, it is still unclear how these cells are developed and regulated. In this study, we found that dermal γδ T cells were mainly developed from the thymus. Surprisingly, there is also an extrathymic pathway for these cells development as our data showed that bone marrow cells alone could also reconstitute the dermal γδ T cells in the mice absence of thymus (Nude mice). Although most of dermal γδ T cells are skin bona-fide residents, there were a part of γδ T cells migrated from the periphery. Such trafficking process required chemokine receptor 6. In the periphery, we found that both IL-23 and IL-1β promoted dermal γδ T cells proliferation. However, IL-23-induced dermal γδ T cell proliferation was mainly dependent on IL-1β. Additionally, IL-1β synergized with IL-23 to produce large amounts of IL-17 by dermal γδ T cells. Moreover, mice with IL-1RI deficiency had significantly decreased epidermal thickness and neutrophil infiltration induced by IL-23 or Imiquimod. Taken together, we demonstrate the development, peripheral trafficking and regulation of dermal γδ T cells, which provides new insights into understanding the biology of this newly discovered cell population and implies new mechanisms involved in the pathogenesis of skin inflammation such as psoriasis.
Interleukin-21 drives intestinal inflammation by bridging the adaptive and innate immune compartments (P3181)
Authors: Poholek Catherine , Harrington Laurie
Published: January 2023

Th17 CD4 T cells have been cast as pathogenic in the context of many autoimmune diseases, including Inflammatory Bowel Disease (IBD); however, the mechanism for this pathogenicity has yet to be elucidated. Based on the observation that IL-21 expression is increased in biopsies from patients with ulcerative colitis compared to healthy controls, we posit that IL-21 produced by Th17 cells mediates inflammation in the intestine. Using models of both spontaneous and CD4 T cell-dependent colitis, we show that a large number of IL-21-producing CD4 T cells are present in the intestines of mice with colitis and importantly, that IL-21 production is required for full disease progression. We find that although IL-21-/- CD4 T cells are unable to initiate disease, they produce elevated levels of IL-17A and IL-17F and are not selectively converted into Foxp3+ regulatory T cells, indicating that IL-21 is both crucial to the induction of colitis and surprisingly, is not acting via effects on the activation and differentiation of T cells. In direct support of this idea, IL-21R-/- CD4 T cells are capable of inducing disease unlike their IL-21-/- counterparts. In fact, our data demonstrate that IL-21 has an impact on the innate lymphoid cell compartment, which has previously been implicated in IBD pathogenesis. Taken together, our data show an important and previously unrecognized role for IL-21 that links CD4 T cells to innate lymphoid cells in the induction of chronic intestinal inflammation.
The magnitude of acute hepatitis is more limited by regulation of cytokine signalling than apoptosis of liver-infiltrating effector CD8 T cells (P4174)
Authors: Vo Michelle , Holz Lauren , Benseler Volker , McGuffog Claire , Hilton Douglas , McCaughan Geoffrey , Bowen David , Bertolino Patrick
Published: January 2023

Acute hepatitis is often mediated by CD8 T cells that kill target cells or secrete hepatotoxic cytokines. However, the parameters regulating CD8 T cell-mediated acute hepatitis are not well understood. We used the well-characterized Met-Kb Tg mouse model of acute hepatitis, in which hepatitis is mediated by lymph node activated TCR Tg CD8 recognizing their cognate antigen in the liver. To investigate whether cytokine regulation or apoptosis of CD8 T cells were critical in limiting acute hepatocellular injury, adoptive transfer experiments were performed using TCR Tg T cells deficient for either suppressor of cytokine signaling (SOCS-1) or the pro-apoptotic molecule Bim. Although there was a substantial accumulation of Bim-/- Tg cells, the outcome of hepatitis remained unchanged, suggesting that effector T cell survival was not a critical parameter in limiting liver damage. In contrast, SOCS-1-/- Tg T cells induced a heightened severity of hepatitis than their wt counterparts. SOCS-1-/- Tg cells isolated from the liver displayed upregulated IL-2Rα, required for the high affinity IL-2 receptor, and was associated with higher levels of IFN-γ expression, CTL activity, and proliferation rates, consistent with enhanced effector function. These data support a critical role for cytokines in CTL function, and demonstrate that the propensity of CD8 T cells to mediate acute hepatitis is determined by the quality rather than the number of CTLs infiltrating the liver.
Neuropathy in a mouse model of CD8+ T cell-mediated CNS demyelination (P4176)
Authors: Rainone Anthony , Fournier Sylvie
Published: January 2023

Several lines of evidence suggest that CD8+ T cells could contribute to the pathogenesis of many autoimmune diseases of the nervous system. Our group studies a mouse model that spontaneously develops neurological symptoms associated with demyelinated lesions in the CNS (herein referred to as L31 mice). We have shown that the demyelinating disease in L31 mice is T cell-dependent with CD8+ T cells acting as effector T cells in the pathological process while CD4+ T cells play a regulatory role. Because L31 mice exhibit motor dysfunction indicated by clasping of the limbs and difficulty walking, we wanted to determine whether the PNS was also affected. A flow cytometry approach was used to determine T cell kinetic accumulation in the CNS and PNS. Data demonstrate that CD8+ T cells accumulate in the CNS of L31 mice from early on in life while they only appear in the PNS once mice display neurological symptoms. Furthermore, immunofluoresecent studies of the CNS and PNS of these mice recapitulate the flow cytometry data and demonstrate the large extent of demyelination in the PNS of symptomatic L31 mice, which is associated with CD8+ T cell accumulation. We hypothesize that epitope spreading during the course of this CD8+ mediated demyelinating disease is the cause of PNS pathology. It will be of further interest to determine the T cell receptor specificity of the CNS and PNS infiltrating lymphocytes.
Interferon-beta therapy increases concentration of soluble interferon alpha/beta receptor in multiple sclerosis patient serum. (P4156)
Authors: Fatakdawala Taher , Skawinski Michael , Ferreira Jonathan , Stauffer Tara , Lavoie Thomas
Published: January 2023

Interferon-beta is a frontline treatment for Multiple Sclerosis and yet the molecule’s mechanism of action in this disease is not fully understood. We have examined the serum/plasma concentrations of soluble Interferon alpha/beta receptor 2 (sIFNAR2) using a newly developed IFNAR2 ELISA in 35 normal human donors, 23 MS patients not on IFN therapy, and 29 MS patients on IFN therapy. Other biomarkers, IFN-beta, and Type I IFN activity were also assessed in most samples. In most patients receiving IFN-beta therapy, IFN beta mass as well as IFN bioactivity can be detected in the serum. Consistent with previous literature, IFN bioactivity is lower than that which would be predicted based on IFN beta mass. For patients on IFN-beta therapy, we observe that sIFNAR2 concentration is significantly (p=0.0001) higher than that in MS patients on IFN-free therapy and relative to normal donors (p=0.0007). Normal donor sIFNAR2 concentration does not significantly differ from that of MS patients on IFN-free therapy. These results expand upon previous literature which suggests that the mRNA encoding a soluble form of the IFN receptor is increased by long term IFN-beta therapy. Data from additional samples, potential mechanisms, and correlations to other biomarkers will be discussed.
Use of gingiva-derived MSC ameliorates CIA via suppressing Th1 and Th17 and enhancing Treg cell differentiation (P4158)
Authors: Zheng Song Guo , Chen Maogen , Wang Julie , Le Anh , He Xiaoshun
Published: January 2023

Accumulating evidence has revealed that manipulation of bone-marrow mesenchymal stem cells (BMSCs) may have the potential to treat RA. While BMSC-based therapy faces many challenges, we reveal that substitution of gingival-derived mesenchymal stem cells (GMSCs) results in significantly improved therapeutic effects on established collagen-induced arthritis (CIA). CIA was induced with the immunization of type II collagen (CII) and CFA in DBA/1J mice. GMSCs were injected i.v. into mice on day 14 after immunization. In some experiments, injection of PC61 (anti-CD25 antibody) i.p. was used to delete Tregs in arthritic mice. Infusion of GMSCs to CIA mice significantly decreased the severity of arthritis and pathology scores, and down-regulated inflammatory cytokine (IFN-γ, IL-17A) production. Infusion of GMSCs resulted in an increase in CD4+CD39+Foxp3+ cells in arthritic mice. These increases were noted early in spleen and LN and later in synovial fluid. The increased frequency of Foxp3+ Treg cells consisted of cells that were mainly Helios negative. Infusion of GMSCs partially interfered with the progress of CIA when Treg cells were depleted. Pre-treatment of GMSCs with CD39 or CD73 inhibitor significantly reversed the protective effect of GMSCs on CIA. The role of GMSCs in controlling CIA pathology mostly depends upon CD39/CD73 signals and partially upon the induction of CD4+CD39+Foxp3+ Treg cells. GMSCs provide a promising approach for the treatment of autoimmune diseases.
Quantitative and qualitative differences in CD4+ T cell responses for subjects with symptomatic and asymptomatic West Nile virus infection (P6125)
Authors: James Eddie , Gates Theresa , May Duy , LaFond Rebecca , Malhotra Uma , Kwok William
Published: January 2023

The West Nile virus (WNV), a mosquito-borne flavivirus, induces a wide range of clinical manifestations in humans. While the majority of infections are essentially asymptomatic, a small percentage of inflections lead to neuroinvasive disease with symptoms ranging from mild disorientation to paralysis and death. Increased risk of symptomatic infection in immunosuppressed subjects could suggest that lack of effective immune responses against WNV may lead to worse outcomes. In this study, we utilized HLA class II tetramers to characterize the epitope specificity, frequency, and phenotype of WNV-specific T cells in subjects who had previously documented WNV infection. CD4+ T cell epitopes were present within all WNV fever proteins allowing broad characterization of WNV-specific responses. Ex vivo tetramer analysis revealed that WNV-specific CD4+ T cells were present at significantly higher frequencies in subjects with symptomatic infection than in those with no symptoms. CD4+ T cells had a memory phenotype in all WNV infected subjects, exhibited varying levels of activation, and produced multiple cytokines, including interferon-γ and IL-17. T cells from symptomatic subjects had a distinct phenotype, producing higher levels of Th2 cytokines after re-activation in vitro. Therefore, quantitative and qualitative differences in CD4+ T cell responses toward WNV are associated with neuroinvasive disease.
Natural IgM is produced by B1a cell-derived bone marrow plasma cells that do not share a survival niche with IgG AFC. (P1467)
Authors: Reynolds Alexander , Kuraoka Masayuki , Kelsoe Garnett
Published: January 2023

Natural antibody makes up the majority of serum IgM and provides protection against both infection and autoimmunity. The B1 lineage is required for natural IgM, but the location and identity of the cells that secrete natural IgM are uncertain. B1-8i VHDJH knock-in mice lack virtually all B1a cells, but have normal B2 cell development and support robust splenic B2 populations. The loss of B1a cells is associated with a 10-fold reduction in serum IgM, whereas serum IgG levels are normal. We examined B1-8i mice and found a 10-fold reduction in the number of IgM AFC in the BM, while BM IgG AFC populations were normal. As B1 cells themselves do not secrete substantial amounts of antibody, our observation indicates that the BM is the major source of natural serum IgM, and that BM IgM plasmacytes require the presence of B1a cells. By FACS and histology, we determined that IgM-secreting cells in BM are CD5- CD138high and appear as differentiated plasma cells, not small B1a cells. Examination of BM revealed that while cytoplasmic IgG+ cells preferentially co-localize with eosinophils, cytoplasmic IgM+ cells do not. This localization correlated with an enhanced survival of IgG AFC, but not IgM AFC, from sorted CD138high BM cells cultured with IL-6. We propose that B1a cell-derived BM plasma cells produce natural serum IgM, and that these cells occupy a survival niche distinct from IgG AFC.
Primary repertoire expansion by junctional diversity in Bos taurus (P1445)
Authors: Liljavirta Jenni , Ekman Anna , Niku Mikael , Iivanainen Antti
Published: January 2023

B cell development varies significantly between species. In many domestic species fetal and neonatal gut-associated lymphoid tissue is essential for the generation of the primary antibody repertoire due to limited range of Ig genes. In fetal ruminants, the repertoire is expanded by somatic hypermutation (SHM) driven by activation-induced cytidine deaminase in the absence of external antigens. In addition to SHM also other mechanisms are likely to contribute to the repertoire expansion. Junctional diversity (JD) created during the recombination process might play a substantial role in bovine fetal IgH. To further characterize the significance of JD, we studied the tissue-specific expression of terminal deoxynucleotidyl transferase (TdT) using RT-qPCR and immunostaining. We also constructed and sequenced several IgH cDNA libraries of different tissues and analysed the junction formation between V(D)J sequences. Our work demonstrates that in fetal bovine B lymphocytes TdT is primarily expressed in the bone marrow. B lymphocytes in other lymphoid tissues did not show elevated TdT expression levels. V(D)J junctions were heterogeneous in sequence, suggesting the addition of N-nucleotides by TdT. In addition, spectratyping revealed significant differences in the distribution of CDR3 lengths between tissues. Our results suggest that along with SHM, TdT-mediated JD contributes significantly to the bovine primary antibody repertoire in fetal life.
Nr4a1 (Nur77) regulates B cell survival and activation. (P1453)
Authors: Park Kiwon , Nowyhed Heba , Hedrick Catherine
Published: January 2023

NR4A1 (Nur77) is a member of the NR4A family of orphan nuclear receptors that function as transcription factors. Nur77 is expressed in immune cells, including macrophages, T cells and B cells. However, the role of Nur77 in B cells is not understood. In bone marrow, Nur77 is expressed in early lymphoid precursors and is nearly absent (or maintained at low level) in Pre-pro B cells and in all stages of development through small pre B cells. However, newly formed immature B cells in bone marrow start to express low levels of Nur77 and Nur77 expression increases during transitional stages of B cell development and is maximal in mature B cells in periphery. To investigate the role of Nur77 in B cells, we compared B cell functionalities from normal C57BL/6 and congenic Nur77-deficient mice. We found that Nur77-deficient mice have significantly elevated levels of all four classes of antibodies, which implicates autoimmunity. Nur77-/- mice have 10% more BAFFR+ T1 (transitional stage 1) B cells than WT. Nur77-deficient naïve mice show a 3-fold increase of GL7+ germinal center B cells and a 60% increase of CD38hi memory B cells, compared to those from WT. Those data indicated Nur77-/- mice undergo autoimmunogenic B cells response. In addition, we found that Nur77-/- B cells that are stimulated by anti-IgM and anti-CD40 survived better than WT B cells. Our data suggest that Nur77 plays a primary role in B cell survival and activation.
CD40 exon 8 deficiency causes a novel form of immunodeficiency with hyper-IgM (P3327)
Authors: Li Yi , Li Shiwu , Wang Hai , Sun Yu , Segal Emma , Xu Yuan , Zhuang Haoyang , Han Shuhong , Sobel Eric , Satoh Minoru , Yang Li-Jun , Reeves Westley
Published: January 2023

Introduction: The germinal center (GC) reaction generates switched memory B cells and plasma cells. Mutation of CD40L or other genes involved in the GC reaction causes hyper-IgM syndrome (HIM). Hypothesis: We evaluated a 56-year-old woman who had an immunological phenotype resembling HIM but only mild immunodeficiency. The age, gender, and clinical presentation were not readily explained by known HIM mutations and we hypothesized that the patient might have a novel mutation. Method: The patient’s immune function was evaluated and CD40 DNA/RNA sequences were determined. The patient’s CD40 cDNA lacking exon 8 (Δexon8) was cloned and transfected into 293 cells. Signaling downstream of CD40 was assessed. Results: By flow cytometry, the patient’s peripheral blood lacked switched memory B cells. The patient mounted a robust IgG response to tetanus toxoid immunization, but antibody levels dropped precipitously suggesting that there was a problem generating long-lived plasma cells. Although CD40 expression was mildly decreased, cDNA sequences showed a deletion of the entire exon 8 of CD40 in 67% of her cDNA clones. This was rare in controls. Expression of CD40-Δexon8 in 293 cells resulted in surface expression of CD40, but signaling downstream of CD40 upon sCD40L stimulation was impaired. Conclusion: This patient has a novel immunodeficiency syndrome due to deletion of exon 8 of CD40, which encodes a cytoplasmic domain critical for signal transduction.
Deep sequencing of the IgH locus in CVID reveals global alterations in central and peripheral B cell development (P3329)
Authors: Simchoni Noa , Roskin Krishna , Boyd Scott , Cunningam-Rundles Charlotte
Published: January 2023

Common Variable Immunodeficiency (CVID) is the most common clinically important primary immune deficiency, affecting roughly 1:25,000 persons and characterized by hypogammaglobulinemia and loss of specific antibody (Ig) production. The causes are poorly understood, but defective B cell activation has been demonstrated. Examining the Ig repertoires of patients could help elucidate early and late B cell maturation. Rearranged Ig heavy chains were sequenced on the Roche 454 for 78 well-characterized patients, with or without non-infectious complications, and 27 healthy controls. Reads were parsed into component genes and structural segments using the iHMMune-align algorithm. Comparison of the repertoires was done in R, or with a combination of SQL and Prism. CVID subjects consistently showed altered central B cell development, with fewer in-frame sequences (85 v 86%, p<0.01), shorter in-frame CDR3 segments (48.1 v 49.6bp, p<0.0001), and VDJ recombination biased away from nontemplated and towards germline-encoded nucleotides. Peripheral somatic hypermutation was also defective in CVID, with fewer mutations both overall (0.6 v 1.5% of V bases, p<0.0001) and in mutated sequences (3.1 v 5.8%, p<0.0001), and fewer in-frame mutated sequences (83.4 v 89.2%, p<0.0001). CVID subjects with <0.55% circulating isotype switched memory B cells showed more severe defects. Defective B cell activation has been shown in CVID; here we demonstrate intrinsic differences in early B cell development.
Sub-phenotypes of Systemic Lupus Erythematosus are associated with genetic sequence polymorphisms in the IRF5-TNPO3 region of chromosome 7 (P3317)
Authors: Zoller Erin , Kottyan Leah , Hawtrey Samantha , Kaufman Kenneth , Harley John
Published: January 2023

Systemic Lupus Erythematosus (lupus) is a systemic autoimmune disorder with a strong genetic etiology. A recent fine-mapping study of the IRF5-TNPO3 lupus association revealed two genetic effects that independently increase lupus risk. We tested the hypothesis that variants in the IRF5-TNPO3 region are also associated with the clinical presentation of lupus. We performed a case-only sub-phenotypic association study using 101 genetic variants in the IRF5-TNPO3 region that were genotyped in 8,395 lupus patients across four ancestral populations. In Europeans Americans with lupus, the age-of-onset (p-value=2.44x10-16, OR=0.68) and the presence of anti-Ro (p-value=2.09x10-7, OR=0.67) and anti-dsDNA (p-value=4.15x10-7, OR=0.75) autoantibodies are associated with genetic variants in the IRF5-TNPO3 locus. In a step-wise logistic regression analysis of age-of-onset, variants are associated in both the IRF5 promoter and the independent genetic effect spanning IRF5 and TNPO3. In contrast, the presence of anti-Ro and anti-dsDNA autoantibodies is only associated with genetic variants in the IRF5 promoter. No other sub-phenotypic analysis in any ancestry demonstrated association after permutation tests were applied to correct for multiple hypothesis tests. In conclusion, genetic variants in the two genetic effects of the IRF5-TNPO3 locus independently affect the age-of-onset and production of anti-dsDNA and anti-Ro autoantibodies in European American lupus patients.
4-1BBL costimulation enables CD4 T cells expressing a CD28/zeta chimeric antigen receptor to eliminate aggressive leukemia. (P4338)
Authors: Condomines Maud , Zhao Zeguo , Wang Xiuyan , Kloss Christopher , Davila Marco , Sadelain Michel
Published: January 2023

Adoptive T-cell therapy using chimeric antigen receptor (CAR) engineered T cells is a promising cancer treatment. First generation CARs include an scFv targeting a surface antigen and the CD3ζ chain to direct tumor cell killing. CAR transduced T cells however need appropriate costimulation to proliferate and persist. Here we assessed in a B-cell leukemia xenograft model, the anti-tumor activities of human T cells transduced with an anti-CD19 CAR harboring the zeta chain alone (19z1+), fused to the CD28 or 4-1BB cytoplasmic domain (1928z+), (19BBz+), along with 4-1BBL (19z1-4-1BBL+), or with CD28 and 4-1BBL (1928z-4-1BBL+). We observed total tumor regression in NALM-6 bearing NSG mice treated with 1928z-4-1BBL+ T cells and no or partial regression in the other T-cell groups, as measured by bioluminescence imaging. 1928z-4-1BBL+ T cells showed the highest bone marrow accumulation and persisted the most. Experiments utilizing purified T cell subsets revealed that naïve and central memory 1928z-4-1BBL+ CD4+ T cells are sufficient to trigger leukemia eradication and are necessary to support CD8+ T-cell in vivo expansion. 1928z-4-1BBL+ CD4+ T cells displayed reduced apoptosis and increased cytotoxicity compared to 1928z+ CD4+ T cells. Thus, constitutive 4-1BBL expression and CAR-mediated CD28 signaling afforded the most efficient costimulatory combination to generate long-lived T cells capable of eradicating aggressive leukemia.
CAR mediated redirection of Teff and Treg cells for the treatment of cancer and autoinflammation (P4340)
Authors: Eshhar Zelig , Waks Tova , Globerson-Levine Anat , Blat Dan
Published: January 2023

T cells expressing chimeric, antibody based receptor (CART cells or T bodies) recognize their target and execute their destined function in none MHC restricted manner. Effector T bodies lyse their target and secrete effector cytokines whereas regulatory T cells produce suppresive cytokines. In an attempt to optimize the performance of Teff and Treg T bodies, we used murine models with spontanous or induced pathology; mammary cancers in the HER2NG mice, over expressing Her2/neu, or the CEABAC transgenic mice expressing low levels of CEA in its colon. In the first system we found that i.v. injections of Her2/neu Teff T bodies delayed the apperance of mammary tumors. When injected to tumor bearing mice, Tbodies accumulated in the mammary lesion. Yet, to eradicate the tumors, repeated injections were needed. A single intratumoral injection of Teff T bodies rejected large tumors, nevertheless, most tumors relapsed. In the second system, naïve Tregs were modified to express a CEA-specific CAR and transferred into colitic CEA transgenic mice. Colitis was induced by azoxymethane-DSS that develop colitis and later on colorectal tumors. The other colitis model was induced by i.v.injection of CEA-specific Teff T bodies. The adoptive transfer of the Treg T-bodies markedly reduced the severity of colitis as monitored by endoscopy. Moreover, in the AOM-DSS model CEA-Treg T-bodies significantly decreased the subsequent tumor burden.
Novel immunotherapy for AML using DNT cells (P4348)
Authors: Zhang Li , Li Xujian , Schuh Nicholas , Joe Betty , Allen Christopher , Chen Claire , Der Sandy , Minden Mark , Dick John , Schuh Andre
Published: January 2023

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with very poor survival due to high relapse rates after chemotherapy. New therapeutic approaches that can effectively prevent relapse are needed. Human CD56-CD3+CD4-CD8- double negative T cells (DNTs) are a small subset of peripheral T cells. We have developed a protocol enabling ex vivo expansion of DNTs from both healthy donors and AML patients and showed recently that these DNTs have potent anti-leukemia activity in vitro. Here we further characterized the anti-leukemia properties of DNTs in vivo using a murine xenograft model. Following infusion into NSG mice, human DNTs proliferated and persisted for at least 14 days, and were detectable in spleen and bone marrow. DNTs did not kill normal bone marrow cells in vitro or in vivo. Unlike infusion of CD4+ or CD8+ T cells, DNT infusion did not cause graft-vs-host disease. Pre-treatment of AML blasts with allogeneic DNTs in vitro led to a 4-fold reduction in subsequent engraftment of CD45+CD33+ AML blasts in NSG mice. Further, infusion of DNTs expanded from an AML patient resulted in a 75% reduction in autologous CD33+ and CD34+ leukemia cell engraftment in NSG mice. These studies demonstrate that expansion of DNTs to therapeutic quantities is possible and that DNTs may be used as a novel immunotherapy to reduce AML burden by targeting primary leukemic blasts and potentially leukemic stem cells.
Human blood Tfh cells (P4534)
Authors: Crotty Shane , Locci Michela , Havenar-Daughton Colin , Kroenke Mark , Haddad Elias
Published: January 2023

Follicular helper (Tfh) CD4 T cells are the specialized T cells for B cell help, required for development of germinal centers and most B cell memory. T cell help is frequently a limiting factor for these processes. Therefore understanding Tfh cells is important for developing rational vaccine strategies. Tfh cells are also potentially extremely useful biomarkers in human vaccine clinical trials as predictors of long-term humoral immunity. We have therefore pursued an understanding of the phenotype, transcription factors, and functions of Tfh cells in humans. CXCR5+ CD4 T cells are present in human blood, and constitute ~20% of CD45RO+ cells. There are differences of opinion as to whether these “blood Tfh” are memory cells or not. We have performed a series of experiments subsetting the blood CXCR5+ cells into 4 groups based on differential expression of Tfh associated surface markers. Gene expression profiling indicates that one of the four groups is the most related to active Tfh in human lymphoid tissue. All four populations have significant gene expression differences from CXCR5lo memory CD4 T cells in blood. Furthermore, a series of functional studies indicates that these four subsets of CXCR5+ cells differ in their capacity to help B cells. We are assessing whether long term memory cells are present in one or more of these blood Tfh subpopulations. These studies examining the functions and ontogeny of blood Tfh cells are important for the field of vaccine design.
A novel platform to generate peptoid-based mimetic vaccines (P4504)
Authors: Case Allison , Desmond Angela , Yi Kate Jiyeun , Chakravarty Pavitra , Smallshaw Joan , Collins Angela , Dye Kelly , Mapes Kelly , Ruback Steve , Yang Jue , Tran Helen , Connolly Michael , Zuckermann Ronald , Chirayil Sara , Luebke Kevin , Astle John , Reddy M. , Kodadek Thomas , Vitetta Ellen
Published: January 2023

The aim of this research is to generate vaccine candidates for any virus for which a neutralizing monoclonal antibody exists without prior knowledge of the protective epitope. We have developed a platform to generate such vaccine candidates and are working on a device to automate their identification. The platform consists of libraries of B cell epitopes prepared by displaying peptoid sequences on beads, screening with neutralizing monoclonal antibodies to select peptoids bound by the antigen binding site of the monoclonal antibody, and retaining the antibody-bound peptoids with protein G dynabeads and a magnet. Peptoids are similar to peptides but with the R group attached to the nitrogen instead of the carbon. They are haptens that can be attached to protein carriers to elicit anti-peptoid antibody responses. We have conducted platform optimization and proof-of-principle testing using FLAG peptide and anti-FLAG monoclonal antibody. We then applied the optimized platform to screen peptoid libraries with monoclonal anti-FLAG and identified two potential mimetics. Mice immunized with the peptoids have made FLAG peptide-reactive antibodies, thereby demonstrating proof of concept. Implementation of this platform with neutralizing monoclonal antibodies we have in hand against HIV, West Nile virus and hepatitis C virus can identify potential mimetic vaccine candidates without prior knowledge of important epitopes, and could represent an entirely new way to generate safe vaccines.
Mechanisms of soluble program death 1 fusion DNA vaccine with antigen-specific enhanced adaptive immunity (P4497)
Authors: Zhou Jingying , Cheung Allen , Tan Zhiwu , Chen Zhiwei
Published: January 2023

Previously a well-defined dendritic cell (DC)-targeting strategy of anti-DEC205 based vaccine shows highly enhanced CD4+ T cell immunity. We have demonstrated that a DNA vaccine with soluble (s)PD1 fused with HIV-1 p24 antigen can greatly enhance p24-specific humoral and cellular immune responses in mice by targeting DCs. Consequently, we compared the efficacies of the two vaccines in our system and found that sPD1-based DNA vaccination induced higher functional CD8+ T cell responses. Although the binding and uptake of encoded fusion proteins are similar between sPD1-p24 and anti-DEC205-p24 in DCs in vitro, transfer of DCs pulsed with these proteins into mice resulted in the sPD1-p24 group eliciting higher IFN-γ releasing CD8+ T cells. These data suggests that different mechanistic pathways exist between the two DC-targeting strategies. We examined the route of antigen processing/presentation by confocal microscopy and found that sPD1-p24 protein co-localized to both Rab14 and Lamp1 endosomal compartments used for MHC class I and II presentation, respectively, while anti-DEC205-p24 was only found in the latter. In addition, sPD1 DNA vaccination resulted in draining lymph node DCs with elevated CD40 and MHC class II expression with higher production of IL-12 compared to anti-DEC205. The mechanism of sPD1-based vaccination in enhancing CD8+ T cells immunity is likely dependent on DC-targeting and activation, production of Th1 cytokines and antigen cross-presentation.
The impact of IL-4 on the CD8 T cell response to infection (P4483)
Authors: Hamilton Sara , Lee June-Yong , Lee You Jeong , Hogquist Kris , Jameson Stephen
Published: January 2023

IL-4 influences the development of memory-phenotype, IFNγ-producing, CD8 T cells and plays a critical role in the generation of antigen-specific memory cells during liver-stage malaria infection. Elevated IL-4 levels, which can be efficiently created by treatment with IL-4 cytokine-antibody complexes (IL-4C), enhances the number of CD8 memory T cells displaying high expression of CXCR3 and Eomes, but low levels of T-bet. This is in contrast to treatment with IL-2 cytokine-antibody complexes (IL-2C) that generate T-bet high, Eomes and CXCR3 low, and granzyme B expressing cells. IL-2C and IL-4C also caused differential responses to an acute pathogen. Whereas IL-2C treatment of naïve C57BL/6 mice produced a significant improvement in clearance of Listeria monocytogenes(LM) infection, IL-4C failed to have an effect, despite a large expansion of the CD8 memory pool. Interestingly, BALB/c mice, which are known as a “Type-2” or naturally IL-4 high mouse strain, demonstrated better clearance of LM after IL-4C treatment. This suggests that depending on the mouse strain, there may be intrinsically different CD8 T cell signals generated in response to γc cytokines, with global functional consequences for the host. Future studies will explore both the functional significance of IL-4 at different stages of the CD8 T cell response to LM and malaria and ask whether IL-4C treatment is able to augment immunity.
The Ron receptor tyrosine kinase, macrophage heterogeneity and obesity associated disease. (P5070)
Authors: Hankey Pamela , Yu Shan
Published: January 2023

Macrophages play a central role in the progression of obesity associated disease. Macrophages are generally classified into two distinct programs, termed M1 and M2 macrophages, polar ends of a spectrum of potential macrophage activation states. The ability to tip the balance of macrophage activation away from inflammatory M1 cells and toward a reparative M2 phenotype could be beneficial in the treatment of chronic inflammation associated with obesity. The Ron receptor is expressed on tissue resident macrophages and plays a critical role in suppressing inflammation while promoting hallmarks of M2 macrophage activation. Here we show that Ron plays a protective role in the progression of atherosclerosis and hepatic steatosis in obese animals, associated with decreased inflammatory cytokine expression and decreased lipid deposition in these tissues. In lean animals, Ron is highly expressed on resident macrophages in adipose tissue. Upon weight gain, the expression of Ron is decreased and there is shift in the relative percentages of M1/M2 cells in Ron deficient mice, favoring an inflammatory environment. The percentage of Ron+ cells was also reduced in the liver and atherosclerotic plaques, while pro-inflammatory cytokine expression in isolated macrophages was enhanced in the absence of Ron. We predict that Ron is a potential marker for reparative macrophages and that the gene expression profile of macrophages from obese animals will correlate with Ron expression in these cells.
Aging subverts immune function by dictating alternative STAT responses to cytokine signaling (P5078)
Authors: Mirza Noweeda , Zorro Manrique Soraya , Cohen Skylar , Dominguez Ana , Cohen Peter , Gendler Sandra
Published: January 2023

Chronic inflammation in the aged closely resembles tumor-induced immune suppression, manifested as a progressive T1→T2 shift and an increased presence of MDSCs. We hypothesize that reversing these age-related phenomena should reduce the elderly’s heightened susceptibility to malignancy. We have now optimized real-time analyses of phosphorylated STATs in murine T cells and MDSCs in order to correlate STAT activation with immune suppression. Both young and old T cells responded to IL-4 stimulation with STAT6 activation and to IFN-γ stimulation with STAT1 activation. Uniquely, however, young T cells also phosphorylated STAT1, STAT3 and STAT5 in response to IL-4, and responded to IL-6 with STAT3 activation, all consistent with a greater susceptibility to T1/T17 differentiation. Regarding MDSCs, both old and young displayed constitutive STAT3 activation which could be enhanced by G-CSF or IL-6 treatment. Both young and old MDSCs activated STAT1 briskly in response to IFN-γ, STAT5 in response to GM-CSF and STAT6 in response to IL-4. However, younger MDSCs, like younger T cells, displayed more diverse responsiveness to IL-4, activating not only STAT6 but also STAT1 and STAT3. While chronic in vivo exposure to T2 cytokines may explain the decreased responsiveness of older MDSCs and T cells to IL-4, paradoxically IL-4 still robustly activated STAT6 in these cells. Such selective responsiveness may be required to preserve the immune suppressive potential of older MDSCs.
Pro-resolving lipid mediators attenuate lung inflammation (P5080)
Authors: Phipps Richard , Thatcher Thomas , Hsiao Hsi-Min , Serhan Charles , Sime Patricia
Published: January 2023

Introduction: Cigarette smoking is the major world-wide cause of COPD and other inflammatory diseases. COPD persists after smoking cessation, suggesting that this may be a failure to resolve inflammation. Resolution of inflammation is an active process regulated by small lipid mediators derived mainly from omega-3 polyunsaturated fatty acids, including the resolvins, and others. We hypothesized that resolvin D1 (RvD1) would have anti-inflammatory and pro-resolving effects in cigarette smoke-induced lung inflammation. Methods: Primary human lung cells (e.g. fibroblasts, etc) were treated with cigarette smoke extract and RvD1 in vitro, and production of pro-inflammatory mediators was determined. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effect on lung inflammation was assessed. Results: RvD1 suppressed production of pro-inflammatory mediators by human cells. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines. RvD1 promoted differentiation of alternatively activated (M2) macrophages. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure. Conclusions: RvD1 has potent pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins may be a novel therapeutic approach to resolve lung injury and disease.
Novel IL-9-Secreting innate helper cells promote oral antigen-induced anaphylaxis (P6249)
Authors: Lee Jee-Boong , Chen Chun-Yu , Liu Bo , Wang Pin-Yi , Burwinkel Karen , Finkelman Fred D , Hogan Simon P , Wang Yui-Hsi
Published: January 2023

IgE-mediated food allergy is manifested by an overactive T-helper 2 immune response to dietary antigens in the gastrointestinal (GI) tract. Interleukin (IL)-9 promote intestinal mastocytosis which allow the development of oral antigen-induced anaphylaxis. However, little is known about the primary cellular sources of IL-9 or the mechanisms that promote intestinal mastocytosis. Here, we report the identification of multifunctional IL-9-producing innate helper cells (IHC9) that secrete prodigious amounts of TH2 cytokines and mast cell protease-1 (MCPt-1) in response to IL-33 and antigen/IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induces significant accumulations of both intestinal IHC9 and CD4+TH2 cells, which correlate positively with symptoms and susceptibility to oral antigen-induced anaphylaxis. IHC9 development requires T cells and IL-4R, not IL-9R, signaling. The failure of irradiated mice reconstituted with STAT6-deficient bone marrow to generate IHC9 was accompanied by reduced serum MCPt-1, intestinal TH2 inflammation, mastocytosis, and goblet cell hyperplasia and loss of allergic diarrhea. Furthermore, inhibition of food allergy symptoms in mice ablated of IHC9 by anti-FcϵRIα antibody treatment could be reversed by adoptive transfer of IHC9. Thus, IHC9 represent a new type of innate helper cell lineage and function in promoting intestinal TH2 inflammation and mastocytosis that drive IgE-mediated anaphylaxis.
Allergic airway inflammation to cockroach allergen is dependent on thymic stromal lymphopoietin (P6251)
Authors: Sheih Alyssa , Ziegler Steven
Published: January 2023

Environmental allergens are a major culprit in the development and exacerbation of asthma. Particularly, exposure and sensitization to cockroach allergen has been highly associated with asthma severity and morbidity. Currently, there is very little information regarding how exposure to cockroach allergen leads to allergic sensitization and airway inflammation. TSLP (thymic stromal lymphopoietin) is an epithelial cytokine that has been linked to the development of allergic inflammatory diseases. Levels of TSLP are elevated in the lungs of asthmatics and correlates with the severity of the disease. Using a mouse model of allergic airway inflammation to cockroach allergen, we’ve identified an important role for TSLP in the response to cockroach allergen. In this model, intranasal sensitization and challenge to cockroach extract results in a robust TH2 airway inflammatory response along with mucus production. This inflammatory response is significantly reduced in TSLP receptor-deficient mice and depends on the presence of the TSLP receptor on specific immune cell populations. Results from these studies will help expand our knowledge of how exposure to cockroach allergen triggers the development of an allergic TH2 response.
Modulation of dendritic cells by Japanese encephalitis virus: A mechanism of immune evasion (P6344)
Authors: Gupta Nimesh , Hegde Pushpa , Bayry Jagadeesh , Vrati Sudhanshu , Lacroix-Desmazes Sebastien , Kaveri Srini
Published: January 2023

The mechanisms of Japanese encephalitis virus pathogenesis need to be explored to delineate appropriate therapeutic approaches. It is believed that, JEV manipulate the innate and adaptive compartments of the host’s immune system to evade immune response and cross blood-brain barrier to reach the CNS, for pathogenesis and clinical manifestations. The study was designed to explore the functional modulations of dendritic cells and their consequences on CD4+ T lymphocyte polarization after exposure to JEV. Human monocyte-derived DCs were either infected with 1 MOI of virus or mock infected. JEV induced the maturation of DCs with a significant increase in the expression of maturation markers 48 hours post infection, along with inducing the expression of PD-L1 and PD-L2 ligands. Importantly, the JEV-EDIII domain alone was able to reproduce the increased expression of PD-L1 by DCs. JEV-infected DCs induced the generation of Tregs in allogenic mixed lymphocyte reactions. The induction of Tregs by JEV-infected DCs was significantly reduced upon blocking PD-L1 using an antagonist. In addition, JEV-infected DCs significantly altered the proliferation and polarization of T helper cells to ‘Th1/Th2/Th17’ subsets. The results, for the first time, suggest that JEV may evade the host’s immune system by inducing regulatory T cells via PD-L1/PD1 pathway and altering the polarization of T helper cells. The EDIII domain of JEV envelope is implicated in this mechanism of immune evasion.
Memory-like CD8+ T cells that persist throughout MCMV infection have the capacity to self-renew and recapitulate memory inflation (P6312)
Authors: Quinn Michael , Turula Holly , Snyder Christopher
Published: January 2023

Cytomegalovirus (CMV) is a herpesvirses that generates a typically asymptomatic primary infection followed by a life-long latent infection. Episodic viral reactivation events during latency are thought to repeatedly stimulate the immune system and drive the life-long accumulation of functional antigen-specific CD8 T cells, a process termed “memory inflation.” How inflation is homeostatically maintained over time remains unclear. Although the majority of the inflationary pool appears to be terminally differentiated short-lived “effectors” (TEFF) that undergo minimal division during CMV latency, a subpopulation of inflating T cells retains a memory-like phenotype (TEM). We have proposed a model where TEM cells formed early during CMV infection maintain life-long immunity. Utilizing the mouse model, murine (M)CMV, we adoptively transferred TEM cells from chronically-infected mice into naïve mice and rechallenged these cells with MCMV. Interestingly, TEM cells recapitulated inflation by expanding and differentiating asymmetrically to produce TEFF and TEM progeny. This process appears influenceable by factors known to modulate terminal differentiation (i.e. antigen load and inflammation). Importantly, the TEM progeny retained their “memory capacity” and could reproduce memory inflation even after repeated rechallenges.
Invariant natural killer T cells recognize and restrict the transformation of B cells by Epstein-Barr virus (P4363)
Authors: Chung Brian , Tsai Kevin , Gibson William , van den Elzen Peter , Priatel John , Tan Rusung
Published: January 2023

Individuals with X-linked lymphoproliferative disease (XLP) lack invariant natural killer T (iNKT) cells and are uniquely susceptible to Epstein-Barr virus (EBV). To determine whether iNKT cells recognize or regulate EBV replication, human B cells were infected with GFP+ EBV 95.8 in the presence or absence of iNKT cells. We observed that iNKT cells significantly reduced early EBV titres and GFP+ B cell expansion but that within 72 hours of infection, B cells transformed into lymphoblastoid cell lines (LCL) by EBV had downregulated the iNKT antigen receptor CD1d, completely abrogating iNKT recognition of the lipid agonist α-galactosylceramide (αGalCer). To determine whether ectopic CD1d expression could restore iNKT recognition, LCL were treated with a synthetic retinoic acid receptor-α agonist AM580 known to modulate CD1d expression by regulating the nuclear protein lymphoid enhancer-binding factor-1 (LEF-1). AM580 treatment significantly reduced LEF-1 at the CD1d promoter, induced surface CD1d expression and enabled iNKT recognition of LCL loaded with αGalCer. Unexpectedly, AM580-treated LCL also activated iNKT function even in the absence of αGalCer suggesting that EBV infection and transformation triggers endogenous iNKT antigen expression. These observations indicate that iNKT cells may be important for the early recognition and control of EBV-infected B cells prior to CD1d downregulation and the loss of CD1d may allow EBV to circumvent iNKT-mediated immunosurveillance.
A CpG triggered Syk-Calcium-CaMKII signal directs the secretion of TNFα by innate immune cells (P6297)
Authors: Behrens Edward , Liu Xiaohong , Freedman Bruce , Rao Sheila
Published: January 2023

The kinase Syk is reported to regulate cytokine secretion in multiple and conflicted manners. We therefore examined Syk in regulating CpG DNA stimulated TNFα secretion. Secreted cytokines were measured by ELISA and surface cytokines by flow cytometry. Gene deletion was achieved with both Cre-Lox and shRNA methods. Deletion of Syk decreases CpG induced TNFα secretion while TNFα mRNA and intracellular protein levels remain normal. In contrast, IL-6 secretion is unaffected. CpG induced plasma membrane levels of TNFα are decreased in Syk deficiency, suggesting that Syk is important for TNFα intracellular trafficking. A downstream effector of Syk is calcium (Ca) flux mediated by PLCγ activation. Supporting a role for Ca in Syk mediated TNFα release, we show: 1) CpG stimulation triggers calcium flux only in Syk-sufficient cells, 2) silencing PLCγ2 phenocopies the TNFα defect of Syk-deficient cells, 3) forced Ca mobilization using ionomycin rescues the TNFα secretion defect in Syk-deficient cells. CpG-triggered phosphorylation of the calcium dependent kinase CaMKII is defective in Syk-deficient cells. CpG-induced TNFα secretion is defective in CaMKII-deficient cells, and is not rescued by ionomycin, suggesting that CaMKII is downstream in the pathway initiated by Syk signaling. These data suggest that CpG provides active signals for TNFα transcription and translation as well as intracellular trafficking, via a Syk-Ca-CaMKII axis, allowing for the fine-tuning of TNFα secretion.