bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Phosphoproteomic Dissection of Immunological Signaling Pathways Applied to Mast Cell and T Cell Signaling (42.5)
Authors: Salomon Arthur R. , Nguyen Vinh , Hung Norris , Cao Lulu , Lung Jonathan , Yu Kebing
Published: January 2023

Although many details of immune cell signaling have been described previously, a systematic, quantitative wide-scale analysis of the global tyrosine phosphorylation events that are triggered by activation of the immune cell receptors is lacking. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells and to examine signaling events downstream of FceRI stimulation in mast cells. In our approach, label free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Metabolic labeling of proteins in cell culture (SILAC) enables the calculation of phosphopeptide abundance ratios between Zap-70 null and reconstituted cells across a timecourse of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. Quantitative phosphoproteomic signatures indicative of downstream inhibition, downstream activation, and mutant compensation are revealed and validated with proteomic pathway visual analysis tools using the established T cell signaling pathway structure as a scaffold. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.
Immunorepertoire analysis by multiplex PCR amplification and high throughput sequencing (42.6)
Authors: Han Jian , Wang Chunlin , Sanders Catherine M , Yang Qunying , Wang Elijah , Gharizadeh Baback
Published: January 2023

An immunorepertoire is the sum of functionally diverse B and T cells in one's circulation at any given moment. Three key factors shape the personal immunorepertoire: HLA type, antigen exposure history, and immunoregulation. Two major challenges have made studying the immunorepertoire difficult: the enormous diversity of V(D)Js, and the scarcity of each VDJ in a sample. To overcome these challenges, a powerful multiplex PCR method has been developed to amplify, inclusively and unbiased, all expressed VDJs in a sample. The amplicon mixture was sequenced directly with Roche 454 platform. In one run, for example, more than 73,000 and 119,000 unique V(D)Js were obtained from B and T cells, respectively. High resolution HLA typing was also carried out with the same sequencing run. This novel technology can be used to uncover possible disease mechanisms associated with abnormal immunorepertoires, to evaluate vaccine efficiency, to identify new biomarkers, and to develop new therapeutics. An online dababase and unique analytical tools have been developed for data sharing, deposition, and mining.
Direct and indirect type-1 arginase (Arg1) induction following Mycobacterium bovis (BCG) infection (43.1)
Authors: Qualls Joseph E. , DeFreitas Ashley , Smith Amber M. , Watowich Stephanie S. , Murray Peter J.
Published: January 2023

M. tuberculosis infects lung macrophages (MØs) and evades immune responses by a diverse array of mechanisms. We have recently published that BCG infection triggers a MyD88-dependent Arg1 induction that suppresses NO production from infected MØs. In addition, MØ-specific Arg1 conditional knockout mice were more efficient at clearing M. tuberculosis and BCG. In the present study, we have found that while MyD88 is essential for Arg1 induction following infection, MyD88-/- MØs express robust Arg1 mRNA and protein when stimulated with supernatant from BCG-infected WT MØs. Arg1 induction stimulated with BCG supernatant correlated with enhanced activation of Stat3, but not Stat1, 4, 5, or 6. Two Stat3 activators, IL-6 and IL-10, were present in the supernatants of BCG infected WT MØs. We found the combined treatment of MØs with IL-6 and IL-10 synergistically induces Arg1 in the presence or absence of BCG infection. Consequently, we propose a model by which Arg1 is induced directly by BCG infection via MyD88 signaling, and indirectly through the autocrine/paracrine IL-6/IL-10 activation of Stat3. These data suggest that mycobacteria can condition uninfected neighboring cells to suppress NO production.
The Role of IL-10 in Mice Infected with Neurotropic Murine Coronavirus (43.14)
Authors: Trandem Kathryn A , Perlman Stanley
Published: January 2023

Mice infected with the neurotropic coronavirus mouse hepatitis virus (MHV), attenuated strain J2.2 develop acute and chronic demyelinating disease with histopathological similarities to multiple sclerosis (MS). The process of demyelination is immune-mediated, as RAG1-/- or RAG2-/- (mice deficient in recombination activation enzyme), which lack B and T lymphocytes, do not develop demyelination upon infection with J2.2; however, if RAG1-/- mice are reconstituted with either MHV-immune CD4 or CD8 T cells, demyelination proceeded as in the immunocompetent mouse. While the pathogenic role of virus-specific CD4 and CD8 T cells and bystander CD8 T cells in this model has been demonstrated, less is known about the immune mechanisms that are controlling this anti-viral immune response. The cytokine IL-10 has been shown to be able to suppress an excessive immune response and protect the host against immune-mediated damage in EAE, an autoimmune model of multiple sclerosis. We investigated the role of IL-10 in demyelination using IL-10-/- mice. Additionally, we characterized the time, location, and antigen-specificity of IL-10 producing cells using IL-10gfp mice. Here, we demonstrate that IL-10 has a protective role in an animal model of MS and that the majority of IL-10 producing cells are MHV-specific T-cells located at the site of infection.

Funded in part by grants from the NIH and National Multiple Sclerosis Society.
Inosine-Containing ssRNA Is a Novel Viral-Associated Immune Recognition Element (43.3)
Authors: Imani Farhad , Prater Chrissy , Thakur Sheetal
Published: January 2023

Inflammatory responses that are generated during viral infections are critical for protective anti-viral immune responses. The exact viral-derived structures (also known as pathogen-associated molecular patterns, PAMPs) that activate uninfected cells to induce pro-inflammatory signals are not completely characterized. To date, the viral PAMPs that are identified include dsRNA and 5' triphosphate ssRNA, which are recognized by several cellular pathways such as SR-A, PKR, RIG-I, TLR-3 and MAVS. During viral infections, the cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) catalyzes conversion of adenines to inosines, which is a pathway for blocking viral replication. ADAR-1 activation in the infected cells leads to the presence of inosine-containing viral and cellular RNA (Ino-RNA). Here, we provide in vitro and in vivo data to affirm our hypothesis that the release of Ino-RNA from infected cells and its interaction with surrounding uninfected cells is a potent stimulus for inflammatory innate immune responses. Treatment of primary human bronchial epithelial (PHBE) cells or C57BL/J6 with in vitro synthesized Ino-RNA rapidly and potently induced several markers of innate immune response such as IFN-β, TNF-a, IL-6 and RANTES. Our data uncovers a novel strategy for uninfected host cells to detect viral infections in the surrounding tissue.
IL-10 suppresses influenza-specific Th17 responses and is detrimental during high-dose primary challenge (43.8)
Authors: McKinstry K Kai , Strutt Tara M , Hamada Hiromasa , Dutton Richard W , Swain Susan L
Published: January 2023

Here we study the expression and influence of IL-10 during murine influenza infection. We find that IL-10 has a minimal impact on the outcome of sub-lethal infection but that IL-10-deficient mice display dramatically increased survival compared to wild-type mice when challenged with lethal doses of virus. Administration of IL-10 receptor blocking antibodies to wild-type mice results in a similar survival advantage. Increased survival in the absence of IL-10 does not correlate with increased cellular responses, or with decreased viral titers but does correlate with improved lung function and increased expression of Th17-associated cytokines during the peak of infection.

Our studies identify highly activated, lung-resident virus-specific CD4 T cell effectors that co-produce IFNγ as the major source of IL-10 during primary influenza infection and we show that autocrine IL-10 production by responding CD4 T cells is critical in dampening influenza-specific Th17 responses.

Finally, we show that transfer of virus-specific Th17-polarized CD4 T cell effectors can protect wild-type, B cell-deficient, and T cell-deficient mice against lethal influenza challenge. We further show that Th17-polarized effectors employ novel mechanisms of protection against influenza distinct from previously established modes of protection employed by Th1-polarized CD4 T cells. Our results thus show that production of IL-10 is detrimental during high-dose primary influenza challenge, and furthermore, suggest an unexpected protective role for virus-specific Th17 CD4 T cell effectors.
Regulatory role of IFN-gamma on alveolar macrophage function during influenza infection (43.9)
Authors: Sun Keer , Metzger Dennis W
Published: January 2023

Secondary bacterial infection often follows pulmonary virus infection. We previously reported that pulmonary IFN-γ produced during influenza infection in mice inhibits initial bacterial clearance by alveolar macrophages. As a result, there was a 100-fold increase in bacterial burden in viral-infected WT mice 48 hr after pneumococcal challenge compared to IFN-γ-/- mice. Although IFN-γ production by CD4 or CD8 T cells alone was sufficient to inhibit bacterial clearance, TCR transgenic OT-I and OT-II mice had no detectable IFN-γ following influenza infection. Thus, virus-specific T cells are responsible for significant IFN-γ production. We found that the inhibitory effect of IFN-γ was correlated with down regulation of the class A scavenger receptor MARCO. Indeed, prior influenza infection had no effect on initial bacterial clearance in MARCO-/- mice. Inhibition of MARCO expression following influenza infection could be prevented by IFN-γ neutralization. Preliminary data suggest that IFN-γ inhibits MARCO expression by signaling through STAT1 and there is significant induction of SOCS1 and SOCS3 in alveolar macrophages during influenza infection. Ongoing work is designed to determine whether influenza-induced IFN-γ facilitates activation of a SOCS negative-feedback loop in alveolar macrophages, thereby suppressing innate protection against extracellular bacteria. (NIH grant #AI41715)
Lethal viral infection results from STAT1 but not STAT2 or IRF9 deficiency in mice and is mediated by CD4+ T-cells (44.11)
Authors: Campbell Iain L , Manders Peter , Lim Sue Ling , Terry Rachael L , Getts Meghann T , Getts Daniel R , King Nicholas JC , Hofer Markus J
Published: January 2023

Type I IFN (α/β) signaling is crucial in the antiviral response and is mediated by STAT1, STAT2 and IRF9 while type II IFN (γ) uses STAT1. We studied the role of STAT1, STAT2 and IRF9 in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild type (WT), STAT2 KO and IRF9 KO mice, infection was non-lethal and virus was either cleared (WT) or established persistence (STAT2 and IRF9 KO). In contrast, in STAT1 KO mice, infection was lethal with severe immune pathology with T-cells, macrophages and granulocytes and elevated expression of various cytokines, particularly IFN-γ, present in various organs. However, lethality was unaltered in LCMV-infected STAT1 KO mice that lacked the IFN-γ receptor. While clearance of LCMV in WT mice is mediated by CD8+ T-cells, depletion of these cells in LCMV-infected STAT1 KO mice did not affect lethality. However, depletion of CD4+ T-cells prevented lethality in LCMV-infected STAT1 KO mice with a considerable reduction in tissue immune pathology. In vitro stimulation assays revealed a skewing of the T-cell response to the Th17 phenotype in LCMV-infected STAT1 KO but not WT mice. These findings indicated that STAT1 has a major protective function in LCMV infection, not only being crucial for limiting viral replication and spread, but also preventing the emergence of a lethal CD4+ T-cell response possibly mediated by Th17 cells. Support : NHMRC grant 512407.
Genomic and Proteomic Analysis of Hepatocellular Carcinogenesis in HBV Transgenic Mice (44.14)
Authors: Liu Yugang , Qu Zhonghua , Shi Yongyu , Liang Xiaohong , Sun Wensheng
Published: January 2023

Hepatitis B virus (HBV) transgenic mice invariably developed macroscopically evident tumors within the 20th month of life. Thus HBV transgenic mouse can be used as a natural model of hepatocellular carcinoma (HCC). We confirmed this phenomenon by observation of BALB/c mice harboring HBV complete genome (subtype ayw). To identify molecular changes involved in HCC, we performed both microarray profiling and proteomic analysis between HBV transgenic mice and their syngeneic controls at 6, 12, and 20-month-old. The total liver RNA was hybridized on an oligonucleotide chip containing 35,000 Mus musculus genes. The total liver protein was used for proteomic analysis. Out of all genes and proteins compared and screened, the number of differentially expressed transcripts and proteins was increased along with the growth of mice. We concluded a molecular portrait in different stages of HCC. These molecules were involved in important cellular pathways, including cell growth regulation, apoptosis, immune response, metabolisms, stress, cell adhesion, et al. The dynamics of molecular profiling during hepatocellular carcinogenesis would better elucidate the molecular mechanisms of HCC and help for the discovery of novel therapeutic targets.

Correspondence to Wensheng Sun: wsw@sdu.edu.cn

Supported by the National Nature Science Foundation of China, Grant No. 30440040, 30772031, and 30801036.
Modulation of CD4+ T cells by HCV+ hepatocytes (44.15)
Authors: Hall Caroline H.T. , Hahn Young S.
Published: January 2023

Hepatitis C virus (HCV) infection results in progression to chronic disease in approximately 80% of the infected population. Individuals who clear acute infection have a strong and sustained CD4 Th1 effector response, which is lacking in those who develop persistent infection. Within the liver, an additional immunosuppression is evidenced by a decrease in the percent of IFN-gamma producing CD4+ T cells relative to the peripheral blood. Viral replication occurs primarily within hepatocytes and there is consequently a high concentration of viral products in the liver. Our goal is to determine if HCV-infected hepatocytes have an immunosuppressive effect on CD4+ T cells. We have used a modified HCV cell culture system composed of a human hepatoma cell line, Huh 7.5, containing the full-length HCV genome or the non-structural portion of the genome only. The liver environment is modeled by a co-culture system that allows for direct contact between the hepatocytes and primary CD4+ T cells. We determined that expression of the full HCV genome within hepatocytes has a suppressive effect on CD4+ T cell IFN-gamma production. The CD4+ T cells can further be characterized by a decrease in the Th1 phenotype. These results suggest that local hepatocyte driven immunosuppression may contribute to the inefficient immune responses mounted by HCV infected patients. NIH DK066754 and U19 AI066328 funded this research.
Modulation of memory CD8 T cell function through the inhibitory receptors NKG2A and KLRG1 (44.21)
Authors: Cush Stephanie S
Published: January 2023

The ability of the immune system to establish and maintain functional CD8 T cell memory responses is critical for the control of many persistent viral infections. We used a murine model of g-herpesvirus infection, gHV68, to study the in vivo effects of a low load persistent viral infection on the function of memory CD8 T cells. The aim of this project is to determine how CD8 T cell functions are regulated in the presence of persistent herpesvirus antigens.

Our results show that the inhibitory receptors NKG2A and KLRG1 were consistently up regulated on CD8 T cells during long-term infection with gHV68. At 3 months after infection, more than 90% of the virus specific CD8 T cells expressed KLRG1 while 75% were positive for NKG2A. The expression pattern of NKG2A and KLRG1 during persistent gHV68 infection was fundamentally different than that of memory cells to non-persistent viruses. Our results indicate that the expression of these receptors on CD8 T cells does not impair their ability to produce cytokines or to degranulate. We are currently analyzing the effects of antibody blockade/depletion and of FACS sorting and adoptive transfer of memory populations expressing or not these receptors. Our results suggest that NKG2A and KLRG1 may have a critical role in controlling CD8 T cell responses during persistent herpesvirus infection.
IL-21 is a central component of CD4 T cell help required to purge a persistent viral infection (44.27)
Authors: Brooks David G , Elsaesser Heidi , Sauer Karsten
Published: January 2023

Therapies to cure persistent viral infections have largely been unsuccessful because it is unclear what is required of the immune system to purge virus once it proceeds past acute infection. T cell immunity is crucial to eliminate viral infection. However, CD4 and CD8 T cell functions are rapidly aborted during persistent infection, preventing viral clearance. To date the majority of work examining T cell function during viral persistence has focused on CD8 T cells. Yet, many aspects of immune exhaustion are associated with functions performed by CD4 T helper cells (which are also rapidly exhausted after persistent infection), suggesting that loss of CD4 T cell function is a key mechanism permitting viral persistence. CD4 T cell help is required throughout persistence to sustain CD8 T cell responses and resolve persistent viral infection, suggesting that CD4 T cells maintain some yet undefined, but essential, helper functions. We demonstrate that IL-21 as an essential component of CD4 T cell help during viral persistence. In the absence of IL-21 signaling, despite elevated CD4 T cell responses, help to CD8 T cells is not provided. CD4 T cells utilize IL-21 to prevent CD8 T cell deletion, sustain immunity and resolve persistent infection. Thus, we identify a novel mechanism of CD4 T cell help during viral persistence and propose that CD4 help is not lost during viral persistence, but instead is altered from promoting memory development toward the production of factors that ultimately sustain effector responses to control viral replication.
Cross-protective humoral and cellular immune responses to A/H3N2 drifted influenza viruses following immunization with Fluzone® with a synthetic TLR4 agonist formulation (45.11)
Authors: Shaverdian Narek , Baldwin Susan L. , Reed Steven J. , Raman Vanitha , Goto Yasu , Mompoint Farah , Vedvick Thomas S. , Clegg Christopher H. , Coler Rhea N. , Reed Steven G.
Published: January 2023

Influenza has a worldwide impact with significant human mortality and economic burden. Although the composition of influenza vaccines is reviewed annually to ensure protection against prevalent influenza strains, antigenically drifted strains emerge after the vaccine composition is decided. Vaccine mismatch reduces vaccine efficacy and produces significant epidemiological consequences. Current licensed vaccines poorly stimulate Th1 anti-influenza cellular responses and provide weak protection against drifted influenza strains not included in the vaccine. We investigated the ability of EM005, a synthetic TLR4 agonist formulated in an oil-in-water emulsion, to adjuvant the Fluzone influenza vaccine. EM005 enhanced the potency of the Fluzone vaccine and broadened its serological protection against drifted A/H3N2 influenza variants compared to vaccination with Fluzone alone or Fluzone formulated with emulsion. Equally important, the EM005 adjuvanted vaccine induced a potent Th1 response cross-reactive to all the A/H3N2 strains evaluated. These results suggest that rational adjuvants can enhance both the magnitude and quality of protective humoral and cellular responses of influenza vaccines.

This work was supported in part by Grant #42387 from the Bill & Melinda Gates Foundation
Thalidomide and emergence of Mycobacterium leprae (ML)-specific responses in erythema nodosum leprosum (45.20)
Authors: Pereira Geraldo M B , Esquenazi Danuza de Almeida , Robottom Ferreira Anna Beatriz , Moraes Milton Ozório , Carvalho Daniel Serra , Nery José Augusto da Costa , Alvim Iris Maria Peixoto , Spencer John Stewart , Sampaio Elizabeth Pereira , Pessolani Maria Cristina Vidal , Sarno Euzenir Nunes
Published: January 2023

Acute inflammatory lesions are seen in lepromatous leprosy (LL) during reactions, such as ENL. Necrotic ENL as the initial manifestation of LL was diagnosed at the Souza Araujo Outpatient Unit of FIOCRUZ in Rio de Janeiro (female patient, 26 yrs old). Biopsies and peripheral blood mononuclear cells (PBL) were obtained on days 0, 7 and 30 of treatment with thalidomide (negative pregnancy test). At days 7 and 30 progressive healing of the lesions was observed. The quantification of specific mRNAS for IL-6, TNF-α, ninjurin and IL-10 by qPCR detected an initial high level of IL-6, TNF-α and ninjurin mRNAs, and after thalidomide treatment a decrease of the first 3 mRNAs in the lesion, but IL-10 message increase. ML-specific synthetic peptides induced progressively lower levels of IL-6 in the culture supernatants, but high-level IFN-γ, IL-13 and TNF-α in PBL responses after the healing of the lesions (ML 1419c protein; multiplex assay). CD4+, CD8+, CD45-RO+ and γδ T cells were present in the initial biopsy, but were nearly absent in the last one. Activated (CD69+) CD4, CD8 and γδ T cells were observed in short-term ML-stimulated cultures. Taken together these observations suggest that thalidomide may contribute to the emergence of a ML-specific T cell response with a potential role in the healing process of ENL.
Toll-like receptor 2: An important regulator of the Mycobacterium tuberculosis granuloma (45.23)
Authors: McBride Amanda B , Salgame Padmini
Published: January 2023

Tubercle granulomas are highly organized immune structures that protect the host against Mycobacterium tuberculosis (Mtb) infection. The organization of immune cells around infected macrophages serves to sequester Mtb and enhance macrophage microbicidal responses. In addition, the granuloma localizes inflammation, thereby preventing damage to surrounding lung tissue. Although there is controversy regarding the involvement of Toll-like receptor 2 (TLR2) in host defense against Mtb, our findings indicate that TLR2 may be playing a key role in regulating these two opposing functions of the Mtb granuloma. Comparative studies between wild type and TLR2 gene-deficient mice demonstrated that the absence of TLR2 did not affect the magnitude of the Th1 effector response generated following aerosol infection with Mtb or the induction of recall Th1 memory immunity in response to Mtb challenge. However, the consequence of TLR2 absence was manifested at the level of the granuloma. Analysis of lungs from chronically-infected TLR−/− mice revealed increased bacterial burden and loss of granuloma integrity in comparison with infected WT mice. The infected lungs of TLR2−/− mice also exhibited enhanced inflammation and reduced Foxp3+ regulatory T cells. Together, these findings provide insights into how TLR2 might regulate the tubercle granuloma.
B7.1 and B7.2 are crucial for primary response to Mycobacterium tuberculosis infection but redundant for the induction of memory response (45.24)
Authors: Bhatt Kamlesh , Rafi Wasiulla , Salgame Padmini
Published: January 2023

Costimulation plays a crucial role in priming of naïve T cell to an effector cell type. In a recent study, we have shown that lack of B7.1 and B7.2 (DKO) causes the host to generate a dampened Th1 effector response, disabling it to contain the bacterial burden during chronic phase of infection. Since the development of T cell memory is connected to the effector T cell response, we sought to investigate how a defective primary effector response will affect the induction of the memory. Memory mice were generated by treating infected wildtype (WT) and DKO mice with antibiotics and resting them after the treatment. These memory mice along with age matched naïve controls were then challenged with low dose infection with M. tuberculosis. A significant decrease in bacterial burden was seen in both WT and DKO memory mice at 4 and 6 weeks post infection when compared to naive WT and DKO mice respectively. This decreased bacterial burden was accompanied with a faster Th1 effector response in both WT and DKO memory lungs as indicated by increased number of IFNγ producing cells in the lungs. These results indicate that despite its inability to generate a defective primary response the DKO host is able to mount a memory response against M.tuberculosis infection. Further studies are in progress to study the mechanism of memory induction in the absence of a successful primary response in the DKO mice.

This study was supported by NIH Grant AI 49778, Potts Memorial Foundation Award and UMDNJ Foundation Award.
Parenchymal expression of CD40 exacerbates liver injury in viral hepatitis (45.25)
Authors: Gupta Shalini , Yan Jiabin , Sun yixiao , Chan Tehsheng , Sun Jiaren
Published: January 2023

T cells contribute to immune-mediated liver injuries in viral hepatitis. Although high levels of costimulatory molecules, e.g. CD40 and CD86, are found in activated Kupffer cells and hepatocytes in patients, the precise role of hepatic CD40 expression in antigen presentation, T cell activation and liver injury in viral hepatitis is not very clear. In this study, we generated conditional transgenic mice having the liver-specific expression of CD40, and induced transgene expression by i.v. injection with 2×109 pfu recombinant replication-deficient adenovirus with Cre. The transgenic mice and their non-transgenic littermates were sacrificed, and the serum and tissue samples were collected at 3, 6, 11, 18 and 27 days post infection. In control mice, the serum alanine aminotransferase (ALT) levels were slightly elevated at days 6 and 18, and normal at other time points. Although transgenic mice had relatively normal serum ALT levels at day 3, they displayed a significant elevation of ALT levels to an average of 501 and 366 U/L on days 6 and 11, respectively, followed by decreased levels thereafter. Pathological examinations revealed a relatively mild lymphocyte infiltration in isolated portal areas in control animals at day 6. In contrast, there was significant lymphocyte infiltration in multiple portal areas, as well as in the liver parenchyma, that was accompanied by various degrees of hepatic apoptosis (Councilmen's bodies) and necrosis in transgenic mice at days 6 and 11. These results suggest that CD40 expressed on hepatocytes play a critical role in virus-induced hepatitis.
Impaired Anti-viral Immunity Following Syngeneic Bone Marrow Transplant (45.7)
Authors: Coomes Stephanie M. , Wilke Carol A. , Moore Thomas A. , Moore Bethany B.
Published: January 2023

Patients receiving autologous hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT) as therapy for various malignancies or autoimmune diseases have an increased risk for infectious complications post-transplant, especially in the lung. We have used syngeneic BMT in mice and the murine gammaherpesvirus, MHV-68, to study the efficacy of adaptive immune responses post-BMT. Using this model, we have shown that mice 5 weeks post-transplant have fully reconstituted their hematopoeitic lineages in both the lung and periphery. However, the BMT mice have a reduced ability to clear lytic virus from the lung, despite increased expression of interferon gamma, interleukin-2, and TNF-alpha in the lungs of BMT mice. Recruitment of immune cells to the lungs after MHV-68 infection is similar in BMT mice and untransplanted controls, suggesting functional defects. While bone-marrow derived dendritic cells from transplanted mice are able to stimulate allogeneic T cell proliferation in mixed leukocyte reactions (MLRs), CD4+ T cells from BMT mice show reduced proliferative responses in MLRs. These results suggest defects in CD4+ T cell responses post-BMT may contribute to enhanced susceptibility to viral infections. Additionally, levels of TGF beta are significantly increased in the lungs of BMT mice before infection, and numbers of CD4+Foxp3+ regulatory CD4+ T cells are increased in the BMT lung (but not spleen) both before and at day 7 after viral infection. We speculate that TGF beta-induced expansion of Tregs in the lung limits anti-viral host defense post-bone marrow transplantation.
Reduced c-myc expression levels limit follicular mature B cell cycling in response to TLR signals (46.6)
Authors: Bandaranayake Ashok D , Meyer-Bahlburg Almut , Andrews Sarah F , Rawlings David J
Published: January 2023

The splenic B cell compartment is comprised of two major, functionally distinct, mature B cell subsets, i.e. follicular mature (FM) and marginal zone (MZ) B cells. Whereas MZ B cells exhibit a robust proliferative response following stimulation with the TLR4 ligand, LPS, FM B cells display markedly delayed and reduced levels of proliferation to the identical stimulus. The current study was designed to identify a potential mechanism(s) accounting for this differential responsiveness. In contrast to the delay in cell cycle entry, FM and MZ B cells exhibited nearly identical LPS-driven alterations in the expression level of cell surface activation markers. Further, both the NFκB and mTOR signaling cascades were similarly activated by LPS stimulation in FM vs. MZ B cells, while inducible activation of ERK and AKT were nearly absent in both subsets. MZ B cells, however, exhibited higher basal levels of pAKT and pS6 consistent with a pre-activated status. Importantly, both basal and LPS activation-induced c-myc expression was markedly reduced in FM vs. MZ B cells; and enforced c-myc expression fully restored the defective proliferative response in FM B cells. These data support a model wherein TLR responses in FM B cells are tightly regulated by limiting c-myc levels thereby providing an important checkpoint to control non-specific FM B cell activation in the absence of cognate antigen.
Distinct regulatory roles for Janus Tyrosine Kinase 3 (Jak3) in generation of T-helper 17 (TH17) and inducible regulatory T cells (Tregs) (47.10)
Authors: Khattar Mithun , Chen Wenhao , Stepkowski Stanislaw
Published: January 2023

The development of inducible Foxp3+ regulatory T (iTreg) cells is reciprocally related to that of pro-inflammatory T helper (Th) 17 cells. Both processes depend on transforming growth factor (TGF)-β. Recent studies indicated that interleukin (IL)-2, a Janus kinase 3 (Jak3)-dependent cytokine, is required for the development of iTreg cells, while exhibiting an inhibitory effect on Th17 differentiation. This raises the question about distinct roles of Jak3 in iTreg vs. Th17 differentiation. To address this question, we cultured naïve CD4+ T cells with TGF-β alone for iTreg cell polarization or TGF-β with IL-6 for Th17 cell polarization without or with selective Jak3 inhibitor, NC1153. We found that a dose-dependent inhibition of Jak3 reduced the TGF-β-mediated iTreg differentiation. In contrast, similar NC1153 concentrations under Th17 polarizing condition increased the percentage of Th17 cells. When we examined the proportion of regulatory T (Treg) cells within the CD4+ T-cell population from Jak3-dificient mice, we found that these mice lack Treg cells. Thus, inhibition of Jak3 has differential effects on iTreg/Th17 balance with preferential generation of pro-inflammatory Th17 cells.

This work was supported by grants from NIH (grant number HL 69723)
MicroRNA Profiles of TH Cells (47.11)
Authors: Batts LaKeisha , Coarfa Cristian , Creighton Chad , Gunarante Preethi , Corry David
Published: January 2023

Naïve CD4+ T cells differentiate into two classes of helper effector (TH) cells during exposure to antigen within peripheral lymphoid tissues. TH1 cells are generated in the presence of IL-12 and IFN-γ whereas TH2 cells result from the influence of IL-4 and IL-6. These cells orchestrate adaptive immune responses by regulating: T cell effector differentiation, macrophage activation, and antibody isotype switching. The mechanisms that control CD4+ T cell differentiation are not yet fully defined. These short transcripts regulate gene expression and are capable of defining and altering cell fate. The importance of miRNA function in T-helper cell differentiation and function has yet to be determined. Using Next Generation Sequencing, we have begun to dissect the smallRNAome of TH cells before and after differentiation. We found enhanced induction of miRNAs in TH1 cells (64% vs. 55% in naïve) relative to TH2 cells (45% vs. 55% in naïve) suggesting a role for miRNAs in TH cell differentiation. miRNAs mmu-let-7c and mmu-mir-181a, which have previously been shown to function in TH cell development and activation were increased in expression in TH1 cells. Expression of two additional miRNAs, mmu-mir-199a and mmu-mir-101a, were also enhanced in both TH1 and TH2 cells and based on their putative immune targets STAT6 and IL-4Rα, we propose that they critically regulate TH1 and TH2 cell differentiation. We further discovered novel miRNAs that putatively target GATA3 and several genes related to cell survival and apoptosis. Understanding how differentially expressed miRNAs function in TH cell differentiation will pave the way for novel and highly specific therapies for TH cell-mediated diseases.
Increased number of systemic T regulatory cells but not Th1 or Th2 cells in tobacco smokers (47.14)
Authors: Malmhall Carina Anna , Andersson Anders , Jackute Jurgita , Houltz Birgitta , Lötvall Jan , Lindén Anders , Bossios Apostolos
Published: January 2023

Tobacco smokers with severe chronic obstructive pulmonary disease (COPD) display a local accumulation of T helper cells and it has recently been proposed that smokers have an increased number of local and systemic T regulatory (Treg) cells as well. However, it is not known whether smokers have an altered number systemic T helper (Th) cells matching that of Treg cells. The current transectional study addressed this issue in human subjects.

Blood samples were obtained from 6 chronic smokers with COPD (GOLD stage II-III), 5 chronic smokers with normal lung function and 6 healthy never-smokers. Treatment with cortisone, long acting beta-2-agonists, anticholinergics and NSAIDs, respectively, was not allowed 4 weeks prior to sampling. T cells (CD3+ and CD4+ respectively), Tregs (CD4+FOXP3+), Th1 (CD4+T-bet+) and Th2 (CD4+GATA-3+) cells were determined using immunocytochemistry.

We found no pronounced or statistically significant difference in the number of T cells or Th1 cells, when comparing the three groups of subjects. Systemic Th2 cells were not detectable in either group. However, we confirmed that the mean [SEM] number of Tregs (CD4+Foxp3+) was higher in chronic smokers with and without COPD (3.30 [0.63] x104/ml and 2.72 [0.49] x104/ml, respectively) than in never-smokers (1.36 [0.19] x104/ml) (p = 0.02).

Our results suggest that chronic smoking has no pronounced effect on the number of systemic Th1 or Th2 cells and that chronic smoking rather than COPD per se accounts for the increased number of systemic Tregs.

Funded by the Heart-Lung Foundation of Sweden
The alternative NF-kappa B pathway is essential for OX40-induced CD4 T cell differentiation and effector function (47.19)
Authors: Murray Susan E. , Rowe Alexander M. , Basak Soumen , Hoffmann Alexander , Parker David C.
Published: January 2023

Nuclear factor-kappa B (NF-κB) is an evolutionarily conserved signaling pathway that alerts the immune system to infection and danger. NF-κB can be divided broadly into two pathways-the canonical pathway regulated by IκB and the alternative pathway that depends on NF-κB inducing kinase (NIK) and IKKα. Mediators as divergent as pathogen-associated antigens, reactive oxygen species, UV light, and cytokines can activate the canonical NF-κB pathway. Activation of the alternative pathway seems to be limited to a subset of TNFRs, the best studied of which are LTβR, BAFFR, and CD40. Mice with lesions in the alternative NF-κB pathway have disorganized lymphoid structures and few peripheral B cells, but T cell responses in these mice have been less well-studied. Here, we show that the costimulatory TNFR, OX40 (CD134), activates the alternative NF-κB pathway in primary CD4 T cells. Activation of the alternative NF-κB pathway is necessary for OX40-induced differentiation and acquisition of effector function in vivo. Moreover, inability to activate the alternative NF-κB pathway rendered T cells unable to induce lethal GVHD. These results demonstrate a critical role for the alternative NF-κB pathway in CD4 T cell responses, and suggest that interventions targeting this pathway may have therapeutic potential.

Supported by NIH R21 AI077032 (D.C.P.) and NIH T32 HL007781 (S.E.M.)
Prevalence of NK-like T cells and plasma Th1/Th2 cytokines, but not CMV seropositivity, correspond with health and performance of older adults (47.2)
Authors: Hamel David L , Mueller Robert G , Michel Joshua J , Boudreau Robert , Newman Anne B , Vallejo Abbe N
Published: January 2023

We have reported the accumulation of NK-like T cells with aging. Others reported associations between ill-health and certain cytokines or anti-viral serology. Here, we examined whether health of older adults could be defined by immunologic signatures. Survivors of a long term epidemiological study of aging who had been monitored in the last 20 years (n=140, aged =85 yrs) were recruited, and clinically tested for physical and cognitive performance. Blood samples were examined for T cell phenotypes, and 24 humoral factors including antibodies to CMV, EBV, and VZV. Clinical tests showed 59% of subjects were fully functional, and 41% had physical/cognitive impairments. All subjects had generalized upregulation of cytokines and anti-viral antibodies; but only T cells and cytokines showed significant Spearman correlations (P<0.01). Normalized factor analysis showed distinct immunological profiles between the two groups. The impaired group had TNFα/IL6 cytokine signature, and a cell signature of T cells expressing CD158A/E. In contrast, the functional group had a humoral signature dominated by IFNγ, IL4, IL5, and IL13, and a cell signature of T cells expressing CD56; intact function predicted by IFNγ and IL5 (P<0.05) in logistic models. These data indicate distinct immune physiology of older adults; CD56+ T cells within a Th1-Th2 cytokine environment are likely effectors of favorable health outcomes with aging.
Critical role for the transcription factor T-bet in regulating the development of Th17 responses during parasitic infection (47.20)
Authors: Smeltz Ronald , Guo Siqi , Cobb Dustin
Published: January 2023

CD4+ Th17 cells have emerged as a new T cell subset in the Th1/Th2 paradigm, and efforts have shifted toward understanding the factors that regulate their development in vivo. To analyze the role of the transcription factor T-bet in regulation of Th17 cells, we utilized a murine model of Trypanosoma cruzi infection, an intracellular pathogen that causes Chagas disease in humans. Infection of t-bet-/- mice led to normal, unimpaired development of antigen-specific CD4+ T cells producing IFN-g. However, a robust Th17 response developed concomitant with Th1 responses. Despite significant IFN-g production, the physiological effects of IL-17 prevailed as there was a sharp increase in Gr-1+Ly6G+ neutrophils. Adoptive transfer of T cells from infected t-bet-/- mice into rag-2-/- mice (t-bet+/+) revealed that CD4+ T cells maintained their IL-17-producing phenotype, including those cells capable of producing both IFN-g; and IL-17. Furthermore, and in contrast to the effects of IL-2 on Th17 development, IL-2 had no effect on IL-17 production by primed T cells. Importantly, adoptive transfer of T cells from naive t-bet-/- mice into infected rag-2-/- mice recapitulated the differentiation of T. cruzi-specific Th17 cells observed in infected t-bet-/- mice. Conversely, transfer of wild-type T cells into infected t-bet-/- mice did not reveal an increase in Th17 development. These results demonstrate that T-bet regulates the differentiation of T. cruzi-specific Th17 cells in vivo in a T cell-intrinsic manner. These data provide important insight into the role of T-bet in regulation of parasite-specific Th17 responses.
Role of Interleukin-27 in a vaccination model against Mycobacterium tuberculosis (MTb) (47.25)
Authors: Torrado Egidio , Fountain Jeffrey J. , Martino Cynthia A. , Khader Shabaana A. , Cooper Andrea M.
Published: January 2023

Previous work from our laboratory has shown that vaccination with an I-Ab restricted peptide of an MTb antigen emulsified in Monophosphoryl lipid A/Trehalose dimycolate (MPL/TDM) and Dimethyl dioctadecylammonium bromide (DDA) confers specific protection following an aerosol infection with MTb. This protection is mediated through IL-17+ cells that colonize the lung and recruit protective Th1 cells. Although there is a current appreciation of the pro- and anti-inflammatory properties of IL-27 in different models of inflammation, the role of this cytokine in vaccination models is poorly understood. Since IL-17+ cells are regulated by IL-27, we wanted to clarify the role of IL-27 in our vaccination model. Immunization of mice with MPL/TDM+peptide leads to the development of IFNγ+ cells. On the other hand, DDA is required for the development of IL-17+ cells by inducing expression of TGFβ and IL-6. Although the IFNγ response is similar in WT and IL-27R KO, the latter show a higher frequency of IL-17+ cells both in the draining lymph nodes and lung, even in the absence of infection. Mechanistic analysis of the impact of IL-27 in effector generation after vaccination suggests differences in cell survival and polarization between IL-27R sufficient cells activated in the presence or absence of IL-27R. These data suggest that IL-27 plays a role in the generation of effectors during vaccination.

This work was supported by NHI grant AI069121
PLZF controls the development of innate T cell effector functions. (47.28)
Authors: Sant'Angelo Derek B.
Published: January 2023

Invariant natural killer T cell (iNKT cells) have an innate immunity-like rapidity of response and the capacity to modulate effector functions of other cells. We show that iNKT cells specifically express the BTB-ZF transcriptional regulator PLZF. In the absence of PLZF, iNKT cells developed, but lacked many innate T cell features. PLZF-deficient iNKT cells accumulated in lymph nodes rather than in the liver, did not express NK markers and displayed a non-activated phenotype. PLZF-deficient iNKT cells failed to secrete high amounts of IL-4 and interferon-γ upon activation; however, some cells produced either IL-4 or IFN-γ, but not both. Finally, forced expression of PLZF in conventional CD4 and CD8 T cells results in these cells spontaneously acquiring memory/effector functions. PLZF, therefore, is the determinant of innate T cell effector functions.
Differential modulation of regulatory T cell activities by nonylphenol, octylphenol and diethylstilbestrol (47.3)
Authors: Jung Mi Young , Kim Tae Sung
Published: January 2023

Endocrine disrupters (EDs) are universally present in the environment and may influence health of wildlife and human. These include plastics, detergents, pesticides, industrial chemicals, phytoestrogen and synthetic estrogen for medical purposes. Although biological effects of EDs have largely focused on reproductive tissues, studies on immune system are relatively lacking. Recent cohort studies have showed that individuals and their offspring exposed to EDs had multiple immune disorders, including autoimmune diseases, SLE, and allergic diseases. The fact that EDs modulated interleukin-4 production, Th1/Th2 balance and IgE production suggests their potential effect on allergic immune responses. Regulatory T cells (Tregs) play pivotal role in maintaining immunological unresponsiveness to self-antigens and suppressing excessive immune responses deleterious to the host. Whereas Tregs in normal conditions regulate ongoing immune responses and prevent autoimmunity, imbalanced function or number of Tregs might perturb the immune homeostasis.

In this study, we investigated the effect of various types of EDs on proliferation, differentiation and functional activities of Tregs in vitro. EDs differentially regulated the functions of Tregs. Nonylphenol and octylphenol increased CD4+CD25+Foxp3+ T cell population when combined with TGF-β. Diethylstilbestrol significantly inhibited the TGF-β-induced Treg differentiation and Foxp3 expression. In addition, DES recovered Treg-mediated suppression of CD4+ T cell proliferation, whereas the others had slight or no effects on the differentiation of Tregs. Several EDs like nonylphenol, octylphenol, bisphenol A increased proliferation of activated T cells. These results suggest that endocrine disruptors may modulate inflammation and cancer via Treg cells.
IL-25 promotes TH2 responses through the induction of IL-2 (47.34)
Authors: Zaph Colby , Artis David
Published: January 2023

Dysregulated CD4+ T helper type 2 (TH2) responses are a predominant feature of allergic inflammation, asthma and ulcerative colitis. Unlike TH1 and TH17 cells that employ distinct innate cell-derived cytokines to initiate their differentiation programs (IL-12 for TH1 cells and IL-6 and TGF-β for TH17 cells), TH2 cell development appears to be regulated primarily by autocrine IL-2 and IL-4. However, the factors that regulate expression of IL-2 in developing TH2 cells remain unclear. Here, we demonstrate that the cytokine IL-25 (IL-17E) induces early expression of IL-2 in developing TH2 cells, enhances TH2 cell proliferation and can promote TH2 cell development in the absence of IL-4Rα signaling. Surprisingly, IL-2 was not required for the IL-25-induced increase in CD4+ T cell proliferation, but the ability of IL-25 to promote TH2 responses was lost in the absence of IL-2. In vivo, treatment of resistant mice with an antibody against IL-2 results in impaired TH2 cell-mediated immunity to Trichuris infection. Together, these results identify IL-25 as an upstream differentiation factor for TH2 cells and suggest that manipulation of IL-25 may provide therapeutic benefits for diseases associated with dysregulated TH2 responses.