bluuebunny/crossref_metadata_2025 huggingface dataset:
Imbalanced adoptive immunity in the diabetic nephropatic BTBR ob/ob mouse (IRC4P.474)
Authors: Rosendahl Alexander , Pedersen Maiken , Fink Lisbeth
Published: January 2023

Human diabetic nephropathy (DN) has extensive kidney fibrosis and inflammation. The BTBRob/ob mouse model has kidney fibrosis, but the inflammatory status is not known. Herein, we investigated the systemic and local lymphocytic immunity in the BTBRob/ob mouse. The BTBRob/ob mouse developed hyperglycaemia and albuminuria. The diabetic kidney had similar numbers of CD4+, enhanced CD8+ T cells and reduced B cells as the ob+ control. The BTBR ob/ob had moderately enhanced spleen size without significant modulation of the T or B cells numbers. Interestingly, the BTBRob/ob CD4 cells had an activated phenotype while the CD8 T cells seemed naïve. An expanded regulatory T cell population was present in the spleen. BTBR ob/ob splenocytes had significantly enhanced intrinsic proliferation, while signal1/2 proliferation was significantly reduced. No significant modulation of the intrinsic capacity to produce cytokines was present although a marked modulation of the cytokine signature was present upon activation. These data demonstrate that the T2D and DN in the BTBR ob/ob mice is associated with changes in both the local and systemic lymphocyte populations and their functions strengthening that inflammation may contribute to the disease progression.
The role of estrogen in atrazine-mediated inhibition of CD4+ T cell function and induction of CD4+CD25+Foxp3+ regulatory T cells (IRC4P.479)
Authors: Emmons Tiffany , Wetzel Scott
Published: January 2023

The herbicide Atrazine (ATR) is the most common drinking water contaminant in the US. Biochemically, ATR is a phosphodiesterase (PDE) inhibitor and also elevates estrogen via induction of aromatase. Based upon these biochemical activities and several previous studies, ATR is considered potentially immunotoxic. We have previously shown that when activated in the presence of ATR CD4+ T cell proliferation and expression of CD25 and CD69 are significantly reduced. This is accompanied by a 2-5 fold increase in the frequency of Foxp3+ CD25+ Treg. While this phenotype is partially mimicked by treatment with the PDE inhibitor pentoxifylline, the ATR phenotype is more pronounced in cells derived from male mice. This suggests the possibility that estrogen is playing an important role in the observed CD4+ T cell phenotype. To examine the potential role of estrogen in the ATR-associated phenotype, we have investigated the role of the G-protein coupled estrogen receptor, GPER1/GPR-30, in the ATR-mediated inhibition of CD4+ T cell activation and induction of Treg. Treatment with estrogen and the GPER1 agonist G1 mimic the effects of low ATR concentrations. Studies to further define the role of estrogen and GPER1 in the ATR inhibition of T cell activation and iTreg conversion are currently underway.
The human CD8β isoforms differentially affect signal transduction pathways and monoubiquitination of the M4 isoform regulates signaling through the mTORC1/2/pathway (IRM4P.497)
Authors: Kavathas Paula , Coman Maria
Published: January 2023

The CD8αβ coreceptor contributes to T cell signaling by bringing the tyrosine kinase p56lck associated with CD8α to theTCR-CD3 complex. Four isoforms (M1 to M4) of the CD8β chain exist in the great apes and humans that differ in the amino acid sequence of their cytoplasmic tails and their expression pattern. We previously found that the M1 isoform, equivalent to murine CD8β, is the predominant isoform in naïve CD8+ T cells, while the M4 isoform is predominant in effector memory T cells. We expressed each isoform in either T cell tumor lines or peripheral blood T cells, stimulated the cells by crosslinking with anti-CD8 or anti-CD3 antibodies and determined effects on signal transduction pathways using phosphoflow. Differences were observed with anti-CD8 crosslinking. For instance, the M1 isoform showed greater amounts of phospho-Erk (2-3 fold) and the M4 isoform showed greater amounts of phospho-Akt (2-3 fold). In addition, because the M4 isoform uniquely binds the adaptor protein AP-2 and is mono-ubiquitinated on lysines, we analyzed mutant forms of the M4 isoform in signal transduction as well. Mono-ubiquitination appears to be a regulator of the mTORC1/2/pathway important in metabolism as the lysine mutant demonstrated a large increase in phosphorylation of the ribosomal protein S6, in the mTORC1 pathway. This supports the hypothesis that the different CD8β isoforms evolved to make unique contributions to CD8 coreceptor function.
TGF-β1 enhances T-cell PD-1 expression through a Smad3-dependant increase in transcription. (IRM4P.499)
Authors: Park Benjamin , Chattergoon Michael , Pan Fan , Pardoll Drew , Cox Andrea
Published: January 2023

Programmed cell death-1 (PD-1) is an inhibitory receptor highly expressed on T cells specific for viral and tumor antigens. TGF-β1 affects the tumor microenvironment and high serum levels are associated with both malignancy and pathogenesis in some chronic viral infections. However, the effect of TGF-β1 on PD-1 expression is unknown. PD-1 expression requires nuclear factor of activated T cells (NFATc1) activation through T cell receptor (TCR) signaling. We found that TGF-β1 signaling increases NFATc1-induced pdcd-1 mRNA and PD-1 expression on human peripheral CD4+ and CD8+ T cells. Smad2 and Smad3 are phosphorylated following TGF-β Receptor I binding and ultimately mediate gene transcription. We found that inhibition of TGFβRI kinase activity or Smad3 phosphorylation blocked TGF-β1-dependent PD-1 enhancement on T cells. Similarly, when compared to TCR stimulation alone, TGF-β1 increased PD-1 expression on murine wild-type (WT) but not on Smad3 knock-out (KO) CD4+ T cells. In contrast, Smad2 KO CD4+ T cells maintain increased PD-1 expression in the presence of TGF-β1, demonstrating a specific role for Smad3. We show that Smad3 increases pdcd-1 transcription by directly binding to a proximal region of the human PD-1 promoter and stabilizing NFATc1 binding. In addition to TGF-β1’s previously known effects on T cell function, our findings suggest that TGF-β1 mediates suppression via PD-1 upregulation. Smad3 may represent an additional target in therapeutic modulation of PD-1.
Vitamin D regulates CD8 T cell expansion and immune memory to infections (IRM4P.502)
Authors: Yuzefpolskiy Yevgeniy , Baumann Florian , Kalia Vandana , Sarkar Surojit
Published: January 2023

Epidemiological studies implicate a potential role of vitamin D in modulating immune responses to infections and vaccines. In this study we analyzed the contribution of vitamin D signals to the development and maintenance of antigen-specific memory CD8 T cell responses during a viral infection. Using the vitamin D receptor (VDR)-deficient murine model, we found that lack of vitamin D signals resulted in a dysregulated CD8 T cell expansion program, which was largely attributable to cell-extrinsic effects. Investigation of events proximal to TCR activation demonstrated a delayed yet sustained priming of antigen-specific CD8 T cells in the absence of vitamin D signals. In spite of a larger ensuing effector CD8 T cell pool generated in the absence of vitamin D signals, the final memory pool was smaller. Interestingly, within both effector and memory CD8 T cell pools, we observed a bias towards CD8 T cells specific for immunodominant determinants, resulting in a restricted repertoire of antigen-specific CD8 T cells in the absence of vitamin D signals. Collectively, these studies delineate a critical role of vitamin D in regulating the quantity and quality of adaptive T cell immunity. Importantly, our studies have direct impact on the efficacy of vaccines, owing to disparate nutritional states of vitamin D that are currently seen cross-sectionally around the world.
Investigating the mechanism of TLR signaling crosstalk in mouse macrophages (IRM5P.702)
Authors: Lin Bin , Fraser Iain
Published: January 2023

It is well established that the TRIF- and MyD88-dependent signaling branches in the TLR4 pathway cooperate to induce a strong innate immune response. It is also known that certain toll-like receptor (TLR) ligand combinations can induce synergistic production of some cytokines. However, the molecular mechanisms underlying this signaling pathway cooperation remain elusive. We conducted a microarray analysis of mouse bone marrow-derived macrophages stimulated with all pairwise combinations of the TLR ligands LPS, Pam3CSK4, R848 and poly I:C to characterize synergy in transcript induction. We find that the crosstalk between the different TLR pathways is most prevalent in combinations which induce both the TRIF and the MyD88 pathways, which is essentially a mimic of the TLR4 pathway. Genes synergistically induced under dual TLR stimulation are mainly cytokines and immune effectors, including IL-6, IL-10, IL-1α, IL-1β, haptoglobin, lipocalin 2, and SOCS3. Based on these data, over 200 synergy factor candidates were picked and targeted using RNAi screening, with IL-6 and IL-12p40 secretion as read outs. A total of 32 hits were identified in the primary screening, including well-known regulators of secondary response gene transcription such as Nfkbiz, Irak2, Tnfaip3 and Tnip1. Secondary screening is ongoing to verify what role the 32 identified genes play in the MyD88/TRIF-dependent synergistic induction of immune regulators.
Protein kinase C delta (PKCδ)−extracellular-signal regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition−mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)−induced endotoxemia (IRM5P.704)
Authors: Noh Kyung Tae
Published: January 2023

Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)−induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular-signal regulated kinase 1/2 (ERK1/2) activation under LPS−stressed conditions via protein kinase C delta (PKCδ) activation. Furthermore, PKCδ−induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS−treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition restored in ERK1/2−dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition−induced ERK1/2 activation via PKCδ relieved LPS−mediated endotoxemia. This finding suggests that IL-10 hyper-expression resulted from GSK-3 inhibition−induced ERK activation could be a new therapeutic pathway for endotoxemia.
Critical role for extracellular-regulated kinase signaling in endotoxin priming of human monocyte NLRP3 inflammasome (IRM5P.705)
Authors: Ghonime Mohammed , Shamaa Obada , Eldomany Ramadan , Srabani Das , Gavrilin Mikhail , Wewers Mark
Published: January 2023

Inflammasomes play key roles in the innate immune response to pathogens and tissue injury. Inflammasome assembly involves two distinct steps; priming and activation, which together trigger caspase-1 to process and release IL-1β, IL-18 and in some settings to induce pyroptosis. Current understanding suggests that priming is the de novo synthesis of proIL-1β and the up-regulation of NLRP3. However, since proIL-18 is constitutively present in monocytes, we studied the mechanisms and timing of endotoxin induced priming events that precede new protein synthesis. Within 15 min of endotoxin priming, exogenous ATP induces caspase-1-dependent mature IL-18 release. This priming is not impaired by inhibition of gene transcription or protein translation. Knockdown of NLRP3 supports a role for constitutive NLRP3 in this rapid IL-18 processing. To dissect the early signaling events we investigated upstream signaling pathways. Interestingly, ERK phosphorylation was found to be essential for priming as proven by chemical inhibition and knockdown approaches. Scavenging of reactive oxygen species blocked both priming and ERK phosphorylation. These findings suggest that priming involves ERK-mediated posttranslational modifications which license the NLRP3 inflammasome to respond to a second, activation signal. Thus, understanding what these modifications are may provide new opportunities to modulate inflammasome activation in disease.
Transcription repressor Hes1 is a selective regulator of TLR-induced CXCL1 expression and neutrophil responses (IRM5P.708)
Authors: Shang Yingli , Au Karmen , Hu Xiaoyu
Published: January 2023

Chemokine-mediated neutrophil recruitment to sites of inflammation is a critical initial step in host defense and inflammatory responses. However, neutrophil recruitment must be tightly regulated to avoid inappropriate immune activation which may lead to autoimmune and inflammatory diseases. Here we show that transcription repressor hairy and enhancer of split 1 (Hes1) suppresses TLR-induced expression of CXCL1, a chemokine crucial for recruiting neutrophils. Interestingly, Hes1 deficiency does not affect expression of other pro-inflammatory cytokines such as Tnf and Il1b. Hes1 negatively regulates Cxcl1 expression and neutrophil recruitment in vivo in LPS-induced peritonitis. In addition, deficiency of Hes1 exacerbated severity of K/BxN serum-induced arthritis in which neutrophils play a key pathogenic role. Mechanistically, regulation of Cxcl1 expression by Hes1 is at the transcriptional level but not via modification of TLR-induced canonical signaling events. Instead, Hes1 represses Cxcl1 productive transcription by attenuating occupancy of a positive-transcription elongation complex P-TEFb at the Cxcl1 gene locus and thus preventing subsequent recruitment of serine 2-phosphorylated RNA polymerase II. In summary, our results identify Hes1 as a novel homeostatic regulator of CXCL1 expression and neutrophil responses and suggest that targeting Hes1-mediated repressive pathway may represent a new therapeutic approach to selectively curbing neutrophil-mediated inflammation.
Selective ROCK2 inhibitor down-regulates pro-inflammatory T cell responses via shifting Th17/Treg balance (IRM6P.711)
Authors: Zanin-Zhorov Alexandra , Mo Rigen , Scher Jose , Nyuydzefe Melanie , Weiss Jonathan , Schueller Olivier , Weiss Sara , Poyurovsky Masha , Dustin Michael , Abramson Steven , Waksal Samuel
Published: January 2023

Targeting pro-inflammatory cytokines is a promising approach to the treatment of certain autoimmune diseases. However, the signaling mechanisms involved in the initiation and effector phases of auto-aggressive pathways are still an enigma. Rho-associated kinase 2 (ROCK2) regulates the secretion of inflammatory cytokines interleukin (IL)-21 and IL-17 and the development of autoimmunity in mice. Our data from a phase 1 clinical trial demonstrates that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects significantly down-regulates the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g in response to TCR stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, leads to down-regulation of STAT3 phosphorylation, interferon regulatory factor 4 (IRF4) and steroid receptor-type nuclear receptor RORgt protein levels in T cells derived from healthy subjects or rheumatoid arthritis (RA) patients. Simultaneously, ROCK2 inhibition induces phosphorylation of STAT5 and SMAD2/3, and subsequently increases the percentage of Foxp3+ T cells. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between pro-inflammatory and regulatory T cell subsets in man. Targeted inhibition of ROCK2 may therefore have a role in the treatment of autoimmune disease with disturbed immune homeostasis.
Identification of molecular mechanisms that regulate T helper 1 cell plasticity (IRM6P.715)
Authors: Oestreich Kenneth , Read Kaitlin , McDonald Paul
Published: January 2023

In order to mount an effective adaptive immune response, the body relies on a number of specialized CD4+ T cells to perform a variety of functions. These specialized cell types arise from a common naïve CD4+ T cell progenitor and include Th1, Th2, Th17, Treg, Th9, Th22, and Tfh populations. Recent work, including our studies, has demonstrated that a considerable amount of flexibility between T helper cell subsets exists to allow the cells to appropriately respond to changing environmental conditions. Initially, we demonstrated that in response to low IL-2 signaling, Th1 cells upregulate Bcl-6 expression, shifting the gene expression program away from a Th1 profile in favor of a Tfh-like program. In this setting, the interaction between T-bet and Bcl-6 acts as a regulatory switch that senses changing environmental conditions to fine-tune the immune response. We have now extended these findings by using RNA-seq to examine the gene expression signatures of both Th1 and Tfh-like cells. This approach has led to the identification of novel transcriptional regulators that may play roles in promoting Th1-to-Tfh cell flexibility. Furthermore, we have demonstrated that in addition to IL-2, IL-15 signaling is a regulator of Th1 cell flexibility. These findings are critical first steps in elucidating the mechanistic players in Th1-to-Tfh plasticity and the environmental signals they respond to, so that we may be able to therapeutically manipulate T helper immune cell potential.
A novel population of interleukin 21-producing pre-follicular T helper cells develop spontaneously in young naïve mice (LYM3P.743)
Authors: Adkins Elisabeth , Wang Xulong , Sproule Thomas , Christianson Gregory , Park Giljun , Carter Gregory , Morse Herbert , Roopenian Derry
Published: January 2023

Interleukin 21 is a cytokine vital for driving the proliferation of antigen-stimulated B cells within the germinal center and their subsequent differentiation into antibody-secreting plasmablasts and plasma cells. IL21 is predominately produced by a subset of activated CD4+ T cells known as T follicular helper cells. These cells develop at the T cell-B cell border and express ICOS, PD1, CD44, CXCR5 and BCL6. While this cell type has been identified, the processes that ultimately result in TFH differentiation are poorly understood. Here we use a novel IL21-venus fluorescent protein reporter mouse to investigate the signals and pathways involved in the earliest stages of development of TFH cells and production of IL21. We identify and characterize a naturally occurring population of ICOShi CD44hi CD4+ T cells that produce IL21 and are present within 4 weeks of age in naïve healthy C57BL/6J mice. These T cells show characteristics of TFH cells, including the upregulation of Bcl6 and Sostdc1, but lack surface staining for CXCR5 and PD1 and, surprisingly, develop in the absence of B cells. Therefore, these spontaneously arising cells actively producing IL21 appear to identify the earliest precursor to the mature TFH state.
The TNF family member TL1A promotes Th9 differentiation and Th9-mediated immunopathology (LYM3P.733)
Authors: Richard Arianne , Tan Cuiyan , Hawley Eric , Gomez-Rodriguez Julio , Goswami Ritobrata , Yang Xiangping , Cruz Anthony , Penumetcha Pallavi , Hayes Erika , Pelletier Martin , Gabay Odile , Walsh Matthew , Ferdinand John , Keane-Myers Andrea , Choi Yongwon , O'Shea John , Al-Shamkhani Aymen , Kaplan Mark , Gery Igal , Siegel Richard , Meylan Francoise
Published: January 2023

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells through its receptor DR3 (Tnfrsf25). Currently known effects of TL1A on T cell differentiation or systemic immune responses cannot account for the resistance of Tnfrsf25-/- mice to diverse animal models of autoimmune and allergic inflammation. IL-9 is a cytokine that promotes inflammatory and allergic immune responses and is expressed by a distinct subset of T cells early after T cell activation or restimulation. We have found that TL1A potently promotes generation of T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. Unlike the TNF family member OX40 ligand, TL1A increases Th9 differentiation independently of STAT6 and the TNF-signaling adaptor TRAF6. Instead, TL1A affects Th9 differentiation through enhancing IL-2-dependent STAT5 activation and allowing increased binding to the IL-9 promoter. In two models of allergic asthma, we demonstrate that Tnfrsf25-/- mice have reduced disease severity accompanied by fewer IL-9 producing T cells in the lung. In addition to promoting Th9 differentiation, we find that TL1A enhances pathogenicity in Th9 transfer models of both uveitis and allergic asthma, where endogenous TL1A signaling is required for maximal pathology and IL-9 production at the site of inflammation. Taken together, these data identify TL1A as a novel cytokine that promotes Th9 differentiation and pathogenicity and as a possible therapeutic target in diseases dependent on IL-9.
The forkhead transcription factor, Foxo1, may coordinate naïve CD8+ T cell homeostasis with energy availability in mice. (LYM4P.758)
Authors: Gubbels Bupp Melanie , Litvin Seth , Edwards R. , Mihalcoe Brittany , Byrne Frances , Hoehn Kyle , Anoff Joshua
Published: January 2023

The naïve CD8+ T cell population must be large enough so that sufficient diversity exists to respond to potential pathogens, yet not so large as to unnecessarily drain an organism’s resources. Thus, we propose that a molecular network links the size of the naïve CD8+ T cell pool with energy availability. We report that short-term calorie restriction in C57BL/6 mice resulted in reduced numbers of naïve CD8+ T cells which efficiently engaged survival signals and minimized energy usage. For example, calorie restricted naïve CD8+ T expressed higher levels of CD8 and CD127 than ad libitum cells, both of which are responsible for receiving homeostatic survival signals in vivo. Naïve CD8+ T cells isolated from calorie restricted mice also experienced less death-by-neglect and engaged in less glycolysis than cells isolated from mice fed ad libitum. Finally, neither the size of the naïve CD8+ T cell pool nor the sensitivity of those cells to death-by-neglect were affected by calorie restriction in the absence of the forkhead transcription factor, Foxo1, indicating that Foxo1 may be involved in synchronizing the size of the naïve CD8+ T cell pool with energy availability.
NKT cells require IL-7, not IL-15, for survival and homeostasis (LYM4P.761)
Authors: Park Joo-Young , Keller Hilary , Sato Noriko , Park Jung-Hyun
Published: January 2023

NKT cells are thymus-generated T lineage cells with shared phenotypic and functional characteristics of innate NK cells. Both T and NK cells require γc cytokines for their survival and homeostasis with T cells being dependent on IL-7 while NK cells being dependent on IL-15. As NKT cells carry features of both T and NK cells, it has been a long standing question which γc cytokine would drive the survival and homeostasis of NKT cells. In contrast to the current view, here we report that NKT cells survival is not dependent on IL-15 but requires IL-7. Specifically, we found that IL-15-deficient mice have intact numbers of peripheral NKT cells and that overexpression of IL-15 failed to expand NKT cell numbers even as it significantly increased CD8 T cell numbers. On the other hand, using a new in vivo model of IL-7-deficiency selectively in peripheral lymphoid tissues (K7 mice), we demonstrated that IL-7 is a non-redundant survival factor for NKT cells. Furthermore, transgenic IL-7 expression dramatically increased peripheral NKT cell numbers, suggesting that IL-7 availability determines size of the NKT cell pool. Mechanistically, we identified STAT5 as the transcriptional effector molecule downstream of IL-7 signaling to promote survival and maintain NKT cell homeostasis. Collectively, these data provide a new perspective on γc cytokine requirements for NKT cell survival, proposing a new role for IL-7 in NKT cell biology.
ITK signals tune CD8+ T cell homeostatic proliferation and anti-tumor immunity (LYM4P.747)
Authors: Huang Weishan , Luo Junjie , Huang Lu , Huang Fei , August Avery
Published: January 2023

IL-2 inducible T cell kinase (ITK) is a Tec family non-receptor tyrosine kinase. Loss of ITK perturbs CD8+ T cell homeostasis and leads to spontaneous acquisition of innate memory/effector phenotype. However, Itk-/- naïve CD8+ T cells develop on an OTI Rag-/- background, and we use these cells to show here that ITK tunes lymphopenia-induced proliferation (LIP) and antigen sensitivity during CD8+ T cell homeostatic expansion. Experimental analysis and computational simulations revealed that in a lymphopenic environment, naïve Itk-/- CD8+ T cells exhibit massive and immediate homeostatic expansion, accompanied by significant death and followed by population collapse. The enhanced immediate expansion of Itk-/- cells is CD8+ T cell-intrinsic and is independent of recipient MHCI, but dependent on donor CD8+ T cell-T cell interaction. The lack of ITK resulted in enhanced antigen sensitivity and effector program in homeostatically expanded (HP) CD8+ T cells. These Itk-/- HP CD8+ T cells exhibit robust anti-tumor immunity in an antigen specific manner, independent of CD4+ T cell help. These data suggest that ITK intrinsically regulates TcR tuning of homeostatic cytokines during homeostasis of CD8+ T cells and that targeting ITK may have clinical benefit in cancer therapy by generating superior anti-tumor responses.
Translational control of memory CD8 T cell differentiation (LYM4P.752)
Authors: Araki Koichi , Konieczny Bogumila , Morita Masahiro , Sonenberg Nahum , Ahmed Rafi
Published: January 2023

CD8 T cells play an essential role in controlling viral as well as intracellular bacterial and parasitic infections. Memory CD8 T cells provide protective immunity upon re-encounter with the same pathogens. A better understanding of memory CD8 T cell development is critical for the rational design of vaccines. Transcriptome analyses in antigen-specific CD8 T cells have made a significant contribution to characterize global re-programming of gene expression during memory CD8 T cell differentiation. However, there has been minimal emphasis on understanding translational control of gene expression in antigen specific CD8 T cells. To examine mRNA translation during memory CD8 T cell differentiation, we performed longitudinal analyses of polysome profiles in antigen-specific CD8 T cells after acute LCMV infection. We found that mRNA translation was dynamically regulated during CD8 T cell responses; translation activity was significantly enhanced during clonal expansion phase, and it immediately returned to basal level at the peak of CD8 T cell responses. Genome-wide analyses of mRNA translation showed that a significant number of mRNAs were translationally regulated during CD8 T cell responses. Further experiments revealed that translational control of gene expression plays an important role in memory CD8 T cell formation. Thus, our studies provide a framework for understanding translational regulation that occurs during memory T cell differentiation.
Neutrophil elastase regulates both type 1 and type 2 immune responses to enteric infection (MPF1P.800)
Authors: Sun Rex , Smith Allen , Desai Neemesh , Cheung Lumei , Grinchuk Viktoriya , McLean Leon , Wang Anjiang , Urban Joseph , Zhao Aiping , Shea-Donohue Terez
Published: January 2023

Local neutrophil influx occurs in response to enteric bacterial as well as nematode infection. Neutrophilic release of the serine protease, elastase (NE), is implicated in host defense as well as tissue injury. The aim of this study was to determine the contribution of elastase in host defense against enteric pathogens that induce a Th1/Th17 or a Th2 immune response. Elastase deficient (ELA1-/-) or C57BL/6 (WT) mice (n=5-7) were inoculated with Nippostrongylus brasiliensis (Nb) and studied 5 and 9 days later or infected with Citrobacter rodentium (Cr) and studied 14 days later. Gene expression was determined by real-time PCR in samples of small intestine or colon. Nb infection in WT mice induced a 21-fold STAT6-dependent increase in elastase expression. ELA1-/- mice had accelerated Nb expulsion coupled with significantly (p<0.01) augmented elevation in IL-4, IL-13, and M2 marker (e.g arginase-1) expression when compared to WT mice. Both strains showed similar increases in smooth muscle hypercontractility and epithelial permeability. In contrast, ELA1-/- mice had delayed clearance of Cr infection with impaired upregulation of IFNγ and IL-17A expression when compared to WT mice. These data indicate that during nematode infection, the upregulation of elastase negatively regulates the Th2 response and could be a novel target for modulating type 2 immunity. During bacterial infection, elastase is essential for the upregulation of type 1 cytokines and pathogen clearance in the gut.
Extraintestinal helminth infection reduces the development of colitis-associated colorectal cancer (MPF1P.763)
Authors: Terrazas Luis I. , Callejas Blanca , León-Cabrera Sonia , Rodriguez-Sosa Miriam , Perez-Plasencia Carlos , Chirino Yolanda , Gitierrez-Cirlos Emma , Avila-Moreno Federico , Ledesma-Soto Yadira
Published: January 2023

Whereas a causal association between viral and bacterial infections with tumor development has been well established in animal and epidemiological studies, the impact of parasitic infections in cancer development is almost unknown. Evidences indicate that cancer can be triggered by chronic or deregulated inflammatory processes, such as colitis-associated colorectal cancer (CAC). Using a helminth infection model, we have analyzed the role of helminth-induced anti-inflammatory response in determining the outcome of CAC. Following Taenia crassiceps infection and development of CAC, mice displayed inhibited colonic inflammatory responses and reduced circulating and colonic CD11b+Ly6C+CCR2+ monocytes. Instead they increased the expression of IL-4 and alternatively activated macrophages (AAMs) markers at the colonic tissue. Strikingly, T. crassiceps-infected mice developed few tumors and reduced pathology. In contrast, uninfected mice mounted strong inflammatory responses, did not express AAMs markers, developed severe pathology and displayed multiple tumors. Thus, immunomodulatory activities induced by helminth infections may have a role in the resistance and progression of CAC.
HIV-1 alters pathology and bacterial dissemination of Mycobacterium tuberculosis in co-infected humanized mice (MPF1P.765)
Authors: Nusbaum Rebecca , Calderon Veronica , Huante Matthew , Sutjita Putri , Vijayakumar Sudhamanthi , Goez-Rivillas Yenni , Hunter Robert Hunter , Actor Jeffrey , Cirillo Jeffrey , Valbuena Gustavo , Endsley Janice
Published: January 2023

Over a third of the world’s population is infected with Mycobacterium tuberculosis (M.tb). HIV infection significantly increases risk for M.tb infection and reactivation and TB is the most common cause of death among people with HIV. We have developed a humanized mouse model of HIV-1/M.tb co-infection as an investigative tool to study co-infection biology. Humanized mice were infected intravenously with a macrophage-tropic strain of HIV-1 (JR-CSF) or mock (BSA), and 3 weeks later were infected intranasally with M.tb (H37Rv). Animals became productively infected with HIV-1, as confirmed by serum ELISA for HIV-1 p24 capsid protein and detection of viral RNA by using qPCR. We further observed that HIV infected cells were localized to sites of inflammation and bacterial replication in the lung of HIV-1/M.tb co-infected animals as determined by immunohistochemistry staining for p24. The number of CD4+T cells in peripheral blood did not differ among M.tb- and M.tb/HIV-1-infected groups, indicating that the immune impairments due to HIV-1 were independent of peripheral T cell loss at this stage of co-infection. Consistent with clinical observations in co-infected human patients, greater mycobacterial dissemination was observed in M.tb/HIV-1 co-infected mice compared animals infected with M.tb only. These results suggest promise for use of the humanized mouse as a model to understand co-infection pathobiology and as a translational tool to develop vaccines and therapies.
Chronic infection with trypanosoma cruzi alters the phenotypic profile of circulating memory B cells in humans (MPF2P.812)
Authors: Postan Miriam , Quebrada Palacio Luz , Hernandez-Vasquez Yolanda , Fernandez Esteban
Published: January 2023

Trypanosoma cruzi was shown to induce the loss of parasite-specific IgG-producing B cells during the acute infection of mice, which was proposed to hamper B cell responses and favor parasite replication and chronicity (Zuñiga et al. 2002). Herein, we measured the levels of circulating memory and MZ-like B cells in 12 individuals chronically infected with T. cruzi and 9 healthy controls based on the expression of CD19, IgM IgD, IgG, and CD27. T. cruzi-infected individuals had significantly lower percentages of IgM+IgD-CD27+ (IgM-only), IgM-IgD-CD27+ (isotype-switched) and IgG+IgD-CD27+ memory B cells compared to controls (p<0.05). Also, the percentage of IgM+IgD+CD27+ (MZ-like) B cells was reduced in infected individuals while the proportion of IgG+IgD-CD27- memory B cells was increased (p<0.05). The levels of memory B cells were further analyzed according to ECG findings in infected individuals (asymptomatic [ASY] and chronic Chagas heart disease [CHD]). IgG+IgD-CD27+, IgM+IgD-CD27+ and IgM-IgD-CD27+ B cells were significantly reduced in the ASY group compared to controls (p<0.05); CHD individuals had decreased levels of IgM-IgD-CD27+ and IgM+IgD-CD27+ B cells (p<0.05). Altogether, our data indicate that chronic exposure to T. cruzi modulates the phenotypic profile of the peripheral blood CD19+ B cell compartment in humans, and that some of the defects develop early in the infection, prior the onset of heart symptoms. Supported by Argentinian MINCYT/FONCYT PICT 2010-2148
DH reading frame usage influences HIV-1 epitope recognition (MPF2P.804)
Authors: Wang Yuge , Sanchez-Silva Aaron , Liu Cunnre , Haynes Barton , Schroeder Harry
Published: January 2023

Background: Some HIV-1-specific neutralizing antibodies, such as 2F5 and 4E10, contain a long H chain complementarity determining region 3 (CDR-H3) includes patches of hydrophobic and charged amino acids, but B cells bearing Igs with charged or hydrophobic CDR-H3s are normally culled from mature B cell subsets. Thus, DH influence on neutralizing antibody generation is very worth to explore. Methods: Mice-BALB/c mice cohort limited to the use of single DH gene segments were generated. ΔD-DFL is a human-like repertoire control. ΔD-DµFS promotes the use of hydrophobic amino acid repertoire. The ΔD-iD is charged amino acid enriched repertoire. Immunization-each strain of 10 mice was immunized with HIV-1 JR-FL gp140 protein. Epitope Identification, serum Assay were conducted. Results: As a general rule, the heterogeneity of the anti-JR-FL gp140 response varied by DH genotype with ΔD-DFL>Wild Type (WT)>>ΔD-DμFS, ΔD-iD. Conversely, the intensity of the response was greatest in the ΔD-iD. However, linear response was not identified with the response to natural HIV-1 epitope. Anti-JR-FL gp140 antibodies present in the sera of the DH altered did not block HIV-1 envelope CD4, 2F5, 2G12 binding site. Conclusion: The pattern of epitope recognition and antigen binding in the response to T dependent antigen, in part, on DH gene segment sequence. Restrictions imposed by natural selection of DH sequence may underlie some of the difficulty that patients generate neutralizing antibody.
Factor H binds to Trypanosoma cruzi trypomastigotes via its C-terminus and contributes to parasite resistance against the alternative pathway of complement (MPF2P.809)
Authors: Ferreira Viviana , Molina Claudia , Tapia Valentina , Nieto Vanesa , Galdames Pablo , Cortes Claudio , Ramirez-Toloza Galia
Published: January 2023

Trypanosoma cruzi (T. cruzi), the causal agent of devastating Chagas disease, possesses multifactorial resistance to the alternative pathway (AP) of complement. AP regulatory protein factor H (fH) uses its C-terminal 19-20 domains to recognize polyanions (i.e. sialic acid) and C3b on host cells, facilitating decay of C3 convertases and inactivation of C3b via the N-terminus of fH. We have shown that recombinant C-terminal fH domains 19-20 (rH19-20) compete with fH for binding to C3b and host cell polyanions, inhibiting protection from the AP. T. cruzi tripomastigotes resist the AP by capturing sialic acid from host cells. The specific role that fH plays in protecting T. cruzi from the AP and the fH domains involved remain unknown. Here we assessed the susceptibility of T. cruzi (MF, Y and Brenner strains) to AP-mediated killing, and the ability of fH to bind directly to the parasite by FACS. For determining the fH domains needed for the interaction with T. cruzi, trypomastigotes were incubated with normal human sera in the presence or absence of equimolar amounts of overlapping recombinant fH proteins (2 to 3 domains each) spanning domains 7 to 20. The ability of each protein to increase AP-mediated killing of T. cruzi was assessed. Only rH19-20 effectively competed with serum fH, inducing a significant decrease in parasite survival (p<0.0001), indicating that the C-terminus of fH is essential for fH-mediated recognition and protection of T. cruzi trypomastigotes from the AP.
IFN-γ is required for spontaneous colitis-associated colon cancer in mice with a lineage-restricted deletion of the Smad4 gene in T cells. (MUC2P.821)
Authors: Kim Byung-Gyu , Choi Sung Hee , Letterio John
Published: January 2023

Immune cells and the cytokines they produce are important mediators of the transition from colitis to colon cancer, but the mechanisms mediating this disease progression are poorly understood. We previously reported that selective deletion in T cells of the TGF-β signaling intermediate in mice, Smad4 (Smad4TKO mice) leads to spontaneous gastrointestinal inflammation which progresses to mucosal epithelia cancers. IFN-γ expression is significantly increased in the T cells and colon of Smad4TKO mice, thus we established a ‘double knockout’ (Smad4/IFN-γDKO) mouse model in which a germ line IFN-γ gene deletion (IFN-γKO) is combined with the Smad4TKO. Smad4/IFN-γDKO mice had a significantly reduced incidence and progression of colitis-associated colon cancer, and a decrease in the number of mucosal CD4+ T cells, compared to those of Smad4TKO. We observed impaired in vitro induction of Foxp3+CD4+ T cells (iTregs) by TGF-β in T cells derived from both Smad4TKO and Smad4/IFN-γDKO. This phenotype is associated with a decrease in the mucosal natural Foxp3+CD4+ T cells (nTregs) in vivo. The colon epithelia of Smad4/IFN-γDKO mice exhibited a non-oncogenic signature with a decrease in the expression of iNOS and p-STAT1 and a restoration of the tumor suppressor gene, 15-PGDH, compared to Smad4TKO mice. Thus, our data indicate the critical role of IFN-γ in promoting carcinogenesis associated with spontaneous inflammation in mice with a T cell-restricted deletion of the Smad4 gene.
image cytometry method for measuring concentration and viability of primary cells used in cellular therapy (TECH1P.846)
Authors: Chan Leo , Lee James , Kuksin Dmitry , Qiu Jean
Published: January 2023

Cellular therapy has become a major clinical research field that creates tailor-made medical treatments for many human diseases. Primary cells obtained from patients and mouse models often contain nonspecific particles such as red blood cells (RBC), platelets, and cellular debris, which can make the cell sample analysis difficult. To remove nonspecific particles that can interfere with analysis, a ficoll gradient separation or RBC lysis is routinely performed. Measurements of concentration and viability of the cell sample are necessary for clinical researchers to qualify the collected patient samples for research and downstream processing. In this work, we validated a fluorescence-based image cytometry method using acridine orange (AO) and propidium iodide (PI) to rapidly measure concentration and viability without the need for tedious purification protocols. Using the Cellometer Vision instrument we performed the viability and concentration analysis and validated our results using the traditional hemacytometer method for samples including but not limited to peripheral blood mononuclear cells, mononuclear cells, Leuko Pac, bone marrow, cord blood, whole blood, bronchoaveolar lavage, and primary murine samples. This image-based cytometer method can increase clinical research efficiency by eliminating the need for purification steps and for manual counting. Furthermore, it can eliminate the user-to-user variation, thus improving the accuracy of the cell analysis.
New multiplex assay panels for simultaneous quantification of 13 Th cytokines in human and mouse samples (TECH1P.852)
Authors: Sun Binggang , Yao Jingjing , Hu Jiaming , Ji Shaoquan
Published: January 2023

T helper or “Th” cells, play important roles in regulating the responses of the immune system. They secrete cytokines to stimulate various effector cells, such as cytotoxic T cells, B cells and macrophages. Accurate measurement of Th cytokine expression is critical to identify the corresponding Th cell responses and to develop in depth understanding of the immune responses. We have developed two multiplexed assay panels, i.e., the human and mouse Th panels, using fluorescence-encoded beads that are suitable for use on various flow cytometers. Both panels allow simultaneous quantification of 13 human and mouse cytokines including interleukins (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22), IFN-g and TNF-α, which are collectively secreted by Th1, Th2, Th9, Th17, Th22 or T follicular cells. Each antibody pair was carefully selected for assay specificity, sensitivity, accuracy and reproducibility. The assays provide higher detection sensitivity and broader dynamic ranges than traditional ELISA methods. The panels have been validated by detecting expected changes in biological samples. Further advantages include small sample volume, flexible assay configurations, and time- and cost-effectiveness. The Th cytokine panels can be used for serum, plasma, cell culture supernatant and other sample types, offering useful tools for biomedical research and drug discovery.
Simultaneous detection of IgG antibodies associated with viral hemorrhagic fever by a multiplexed luminex-based immunoassay (TECH1P.855)
Authors: Zhang Shuo , Wu Wei , Qu Jing , Zhang Quanfu , Li Chuan , Li Jiandong , Jin Cong , Liang Mifang , Li Dexin
Published: January 2023

Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs. Corresponding author: Prof. Dr. Dexin Li; Email: lidx@chinacdc.cn
Close proximity between ICAM-1 and agonist pMHC-I ligands facilitates T cell integrin activation advancing local cytoskeleton remodeling (TECH1P.856)
Authors: Anikeeva Nadia , Sykulev Yuri
Published: January 2023

To mimic clustering of ICAM-1 and pMHC-I molecules, we assembled pMHC-I and ICAM-1 proteins on fluorescent nanoparticles (NP) and compared the binding of ICAM-1/NP, pMHC/NP and ICAM-1/pMHC-I/NP conjugates to live T cells. The binding of the ICAM-1/NP was poor, but it has been significantly enhanced in the presence of Mg2+ that induce high-affinity conformation of LFA-1. The addition of agonist pMHC-I/NP or anti-CD3 antibody did not influence the binding of the ICAM-1/NP to the T cells. However, when both agonist pMHC-I and ICAM-1 were assembled on NP, a profound increase in the binding of the ICAM-1/pMHC-I/NP was observed. The amplification effect was non-additive suggesting that ICAM-1 and pMHC-I displayed on the NP cooperate in their binding to TCR and LFA-1 on T cells. In control experiment, NP bearing ICAM-1 and null pMHC did not reveal the cooperative binding to the T cells. We conclude that the close proximity of agonist pMHC-I and ICAM-1 facilitates conversion of LFA-1 to a high affinity form upon productive TCR engagement. We propose that this cooperative affect advances initial local cytoskeleton remodeling enhancing the sensitivity and kinetics of TCR signaling.
Using quantitative imaging to link immune profile and tumor grade in breast cancer. (TECH1P.861)
Authors: Blenman Kim , Andersen Emily , Lee Peter
Published: January 2023

Tumor grade determines how quickly a tumor proliferates and spreads. It is one of the factors used to determine the best treatment approach for cancer patients. The Nottingham Histological Score is used to determine tumor grade in breast cancer. Three factors comprise this score: amount of gland formation, nuclear features, and mitotic activity. These factors tell how close the cancer cells and the tumor tissue structure resemble normal cells and tissue. Well-differentiated (grade 1) tumors closely resemble normal cells and tissue and grow and spread slowly. Moderately (grade 2) - and poorly (grade 3) - differentiated tumors have abnormal cell appearance, lack normal tissue structures, and grow and spread faster. In this pilot project, we were interested in determining if we could link tumor microenvironment immune profiles to tumor grade. We used our quantitative imaging process including multiplexed immunohistochemistry, multispectral imaging, and quantitative analysis software to generate immune profiles of CD3+ T cells, CD20+ B cells, CD1a+ dendritic cells, and cytokeratin+ cancer cells in primary tumor and tumor draining lymph nodes of breast cancer patients. Tumor grades were collected from pathology reports. Our preliminary data suggests that we can link immune profiles and tumor grade. Our next steps are to determine if combination of immune profiles and tumor grade can improve selection of treatment approach for breast cancer patients versus tumor grade alone.