bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of BCL6 and PD-1 in CD4 memory T cell development (P1165)
Authors: Hollister Kristin , Wu Hao , Dent Alexander
Published: January 2023

Follicular T helper (TFH) cells have been shown to play an indispensable role within the germinal center (GC). To study the effects of TFH cell loss, our lab has developed a mouse line wherein BCL6, the master transcription factor behind TFH cell differentiation, is conditionally deleted in specific cell subsets (BCL6fl/fl). When mated to Cre-CD4 mice, loss of BCL6 specifically in CD4 T cells resulted in a complete absence of GCs. Data from control mice confirmed the correlation between BCL6 and PD-1 expression, with the highest levels of both found in TFH cells. Further analysis showed the few CD4+ CXCR5+ ICOS+ PD-1hi cells found in BCL6fl/fl Cre-CD4 mice have higher rates of apoptosis than those in control mice. Because previous studies have shown that Bcl6 regulates T cell memory, we investigated whether there were any effects on CD4 T cell memory development and found BCL6fl/fl Cre-CD4 mice have an altered memory cell phenotype, with higher levels of central memory cells and lower effector memory cells than control mice. Furthermore, memory cells in these mice show significantly lower rates of apoptosis as well as surface expression of PD-1. Taken together, this data suggests a complex regulatory network involving BCL6 and PD-1 expression, which effects not only TFH cell differentiation, but the development of CD4 T cells into effector versus central memory cells as well.
Bioenergetics of stomatin-like protein-2-deficient T cells reveal a regulatory role for this protein in immunometabolism (P1173)
Authors: Mitsopoulos Panagiotis , St-Pierre Julie , Dunn Stanley , Madrenas Joaquín
Published: January 2023

Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein identified in proteome analysis of glycolipid-enriched membrane microdomains of mammalian cells, and modulates T cell activation. To study SLP-2 function we generated T cell-specific SLP-2 knockout (SLP-2 T-K/O) mice since conventional SLP-2 knockout mice were embryonic lethal at the pre-implantation stage. These mice showed reduced CD4+ T cell responses and decreased respiratory complex I and II+III activities. Our mechanistic studies suggest SLP-2 recruits prohibitins to cardiolipin (CL)-enriched domains in the mitochondrial inner membrane to form specialized CL-enriched membrane microdomains, required for optimal cellular respiration. We hypothesized SLP-2 functions in this manner to regulate the bioenergetics of T cells. Seahorse XF analysis revealed that although SLP-2 T-K/O cells showed unaltered total cellular and mitochondrial oxygen consumption rates (OCRs), they had significantly greater non-mitochondrial OCR, mitochondrial OCR uncoupled from ATP synthesis, and greater reliance on glycolysis (71% of mice). Surprisingly, SLP-2 T-K/O cells exhibited greater spare respiratory capacity, a phenomenon not due to differential sensitivities of cells to uncoupling agents. This new immunometabolic phenotype suggests SLP-2 may regulate assembly of functional respiratory supercomplexes. Bioenergetic and biochemical analyses of mitochondria are underway to establish the regulatory role of SLP-2 in T cell metabolism.
Th17 cells differentiated without TGF-β undergo FasL-dependent AICD (P1183)
Authors: Zhang Jimin , Ren Guangwen , Shi Yufang
Published: January 2023

Interleukin (IL)-17-producing T helper (Th17) cells can be differentiated under the combination of cytokines: IL-6, a proinflammatory cytokine, and transforming growth factor (TGF) beta, an antiinflammatory cytokine. We previously reported that TGF-beta is acting indirectly for Th17 cell differentiation by blocking expression of the transcription factors signal transducer and activator of transcription (STAT) 4 and GATA-3, thus preventing Th1 and Th2 cell differentiation. From T cells derived from Stat-6(-/-)T-bet(-/-) mice, which are unable to generate Th1 and Th2 cells, IL-6 alone can induce robust differentiation of Th17 cells. Here we report that Th17 cells derived from Stat-6(-/-)T-bet(-/-) mice, which were differentiated without TGF-β, underwent FasL-dependent activation-induced cell death (AICD). FasL was up-regulated upon TCR reactivation of those Th17 cells and the addition of FasL blocking antibodies abolished those Fas induced Th17 cell death. These features were comparable to wild type Th17 cells differentiated under the traditional combination condition of IL-6 and TGF-β. In all, these data demonstrate that Th17 cells differentiated without TGF-β undergo FasL-dependent AICD in a similar way to those wild type Th17 cells differentiated with TGF-β, indicating that Th17 cells derived from these two different conditions share common characteristics in AICD.
Role of Aryl hydrocarbon receptor and the effect of selective AhR modulators on T cell differentiation (P1201)
Authors: Mohinta Sonia , August Avery , Perdew Gary
Published: January 2023

The physiological role of AhR, particularly in immune response remains a key question after identification of AhR as an essential transcription factor for Th17 differentiation. Selective AhR modulators (SAhRMs) are a recently identified class of compounds that are capable of binding to AhR and repress cytokine- driven gene expression. These compounds are interesting therapeutically as they can be used to inhibit Dioxin Response Element (DRE)-mediated while inducing anti-inflammatory responses. Under non-skewing conditions, AhR agonist beta-naphthoflavone (BNF) enhances IL-17A production whereas SGA-360 an inhibitor suppresses it. Under Th17 skewing condition, AhR agonist BNF enhanced Th17 differentiation whereas AhR antagonist GNF and partial antagonist SGA-360 suppressed it. Interestingly, under Treg skewing condition the antagonists enhanced the Foxp3 expression, suggesting that such compounds have therapeutic potential in autoimmune disease by modulating the Th17/Treg balance. We have also examined AhR antagonists after disease development in a murine model of Th17 mediated disease, hypersensitivity pneumonitis mediated by Staphylopolyspora rectivirgula (SR). We find that these antagonists suppress AhR activity and induce IL-4, suggestive of a shift in T helper lineage. This suggests that SAhRMs may be useful in modulating AhR activation to regulate Th17/Treg balance, and that AhR selective ligands have potential for use as therapeutics in Th17 mediated diseases.
Complex role for Interleukin-2 inducible T-cell kinase in T helper differentiation and effector function in vivo (P1204)
Authors: Kannan Arun , Butcher Barbara , Kim Do-Geun , Lee Yong , Bynoe Margaret , Denkers Eric , August Avery
Published: January 2023

T helper responses are critical for a productive immune response but an inappropriate response results in inflammatory and autoimmune disorders. The tyrosine kinase ITK has been shown to regulate the development of Th2 responses. Here, we show that ITK regulates Th1/Th17 responses as well. We find that Itk-/- mice are more susceptible to infection by Th1 inducing intracellular pathogen T. gondii, with generation of fewer antigen specific Th1 cells in vivo and fewer effector cells ex vivo upon antigen re-challenge. Transfer of WT CD4+ T cells confers protection against infection in Itk-/- mice. Furthermore, WT but not Itk-/- CD4+ T cells can promote the survival of Rag-/- mice infected by T. gondii. Additionally, the absence of ITK is protective during Th1/Th17 mediated Experimental Autoimmune Encephalomyelitis (EAE) with fewer pathogenic Th1 and Th17 effector cells in the periphery. Furthermore, transfer of Itk-/- CD4+ T cells into Tcra-/- mice results in lower severity of EAE compared to mice that receive WT cells suggesting that Itk-/- CD4+ T cells are intrinsically less pathogenic. Finally, using mice that express an analog sensitive mutant of ITK (ITKas) we show that temporally inhibiting the kinase activity of ITK with a genetically selective small-molecule inhibitor impairs development of Th1, Th2, and Th17 lineages. This work has implications for understanding ITK as a therapeutic target for Th2 mediated inflammatory and Th1/Th17 mediated autoimmune disorders.
Early effector T helper cells are precursors to both terminal effectors and memory T cells (P1207)
Authors: Opata Michael , Carpio Victor , Obiero Joshua , Dillon Brian , Stephens Robin
Published: January 2023

Effector memory CD4+ T cells (Tem) are important for protection from many chronic infections, but their development is poorly understood. While IFN-γ+ effector T cells (Teff) can survive into the memory phase in infection, it is not clear if mature Teff generate Tem, or if memory T cells diverge from Teff early after activation, as suggested for central memory T cells (Tcm) by data in model antigen systems. However, we have shown that both Tem and Teff are continuously generated from Tcm in chronic malaria infection. In the current study, we identified phenotypic subsets defining the gamut of activation of Teff using malaria-specific B5 TCR transgenic and IFN-γ reporter mice. We demonstrate that all Effector subsets (Early, Middle and Late) divide, and make IFN-γ in Plasmodium chabaudi infection, with IFN-γ+ TeffMid dominating expansion. We have shown by adoptive transfer that only TeffEarly have the potential to differentiate into the Middle and Late subsets, while TeffMid express low levels of anti-apoptotic Bcl-2, and high levels of Fas and PD-1, suggesting terminal differentiation. TeffLate express high levels of the Th1 factors T-bet and RUNX3, but are highly apoptotic. Of these subsets, only the less-differentiated TeffEarly subset survives to become memory cells (both Tcm and Tem) in naïve recipients. Our data suggest that TeffEarly are the elusive CD4+ precursor to memory and will accelerate the study of T cell mediated vaccine protection from the earliest stages.
Spatial and temporal regulation of interleukin 4 and interleukin 13 expression identified by dual reporter mice. (P1129)
Authors: Kyle Ryan , Robinson Marcus , Plunkett Catherine , Mearns Helen , Ochiai Sotaro , Tang Shiau Choot , Forbes-Blom Elizabeth , Chen Xi , Paul William , Le Gros Graham
Published: January 2023

Type II immune responses are associated with protection against helminth infections, as well as the pathology of allergic diseases that are initiated against innocuous antigens. Two integral cytokines associated with type II immunity are interleukin (IL-)4 and IL-13. The 4C13R transgenic dual reporter mouse has been created to allow identification of IL-4 and IL-13 producing cells by the production of two intracellular fluorescent molecules, AmCyan and DS-Red respectively. The expression of the distinct reporter proteins are under the normal transcriptional control of the Il4 or Il13 genes. This technology allows for the analysis of in situ IL-4 and/or IL-13 production by the relevant differentiated immune cell types without any effect on the endogenous cytokine genes or their effector activities in the mouse. Using this reporter system we have identified that in vitro generated CD4+ Th2 cells have divergent expression of IL-4 and IL-13, suggesting cytokine specific Th2 subsets. Additionally, IL-13 expression is delayed compared with IL-4 in this system. Reporter expression in vivo has also highlighted that IL-4/IL-13 double producers are only a subpopulation of CD4+ T cells, and cytokine expression profiles differ significantly between lymph node and effector tissues during type II responses. Studying the expression and regulation of these cytokines will allow us to understand their contribution in both disease and protection.
Gfi1 couples maturation of DP thymocytes and generation of nTreg cells (P4418)
Authors: Shi Lewis Zhichang , Kalupahana Nishan , Turnis Meghan , Neale Geoffrey , Vignali Dario , Chi Hongbo
Published: January 2023

Double-positive (DP) thymocytes respond to intrathymic TCR signaling by undergoing positive selection and lineage differentiation. Concomitant with these well-characterized events is the acquisition of mature T-cell gene-expression programs, notably the upregulation of effector molecules that are essential for their proper survival and trafficking, a process that is much less understood. nTreg cells also develop from immature thymocytes, but how their development is linked to the thymocyte maturation remains unclear. Here we report that Gfi1 has a central role in coupling DP cell maturation and thymic generation of nTreg cells. Using a conditional Gfi1 deletion system, we observed that specific ablation of Gfi1 in DP cells led to premature expression of mature effector genes and transcription factors, thereby facilitating developmental maturation. Associated with this “expedited” maturation of DP cells was expansion of nTreg cells. Mechanistically, we identified that elevated IL-2 signaling in the absence of Gfi1 drove the expansion of nTreg cells via a non-cell-autonomous mechanism. Finally, we provide evidence that deficiency of Gfi1 dampened anti-tumor immunity. Our results establish a Gfi1-dependent mechanism that actively maintains DP cell fate for proper developmental maturation and generation of nTreg cells.
Role of Dβ germline sequence on constraining TCR CDRβ3 diversity (P4426)
Authors: Levinson Michael , Silva Sanchez Aaron , Zhuang Yingxin , Schroeder Harry
Published: January 2023

Diversity of the T cell receptor is mainly developed through V(D)J rearrangement and N addition during TCR development. The product of V(D)J rearrangement is the CDRβ3, a region of high variability that recognizes antigen and includes all of the D gene. The Dβ sequence is highly conserved across various species, from trout to mouse to humans. This suggests that there are some natural constraints on the TCR; these constraints are thought to limit deleterious T cells from reaching the periphery. We hypothesize that altering the D region will have an effect on the development of thymocytes. To do this we have created Dβ altered mice. These mutants are a Dβ2 KO (Dβ1); a replacement of the Dβ locus with a charged Dβ (DβDKRQ); and a replacement of the Dβ locus with a hydrophobic DH (DβYTL). When compared to WT mice, the mutant Dβ mice have an altered T cell repertoire in both CDRβ3 amino acid composition and length and these differences can be attributed to the changes in the germline sequence. Changing the Dβ also changes the total T cell number in both developing and mature T cells, with a charged Dβ being selected against. Ongoing experiments will elucidate the role of the Dβ sequence on the development of T cells and the role it plays in infection and inflammation.
CD55 expression is induced during T cell maturation and protects recent thymic emigrants from complement-mediated killing (P4433)
Authors: Hsu Fan-Chi , Chen Meibo , McWilliams Douglas , Seaburg Lauren , Tangen Sarah , Shapiro Virginia
Published: January 2023

Newly generated T cells must undergo phenotypic and functional maturation. Previously, we showed that the absence of NKAP severely curtailed T cell maturation, and NKAP-deficient recent thymic emigrants (RTEs) fail to enter the long-lived naïve peripheral T cell pool. The paucity of peripheral T cells in CD4-cre NKAP conditional knockout (cKO) mouse is not due to apoptosis as neither loss of Bax nor Bcl-2 or Bcl-xL transgenes rescued the maturation defect. Here, we show that CD55, a complement regulatory protein functioning to disassemble C3 convertases, plays a role in T cell maturation. The expression of CD55, but not Crry or CD59, increases concurrently with T cell maturation by two orders of magnitude. NKAP-deficient peripheral T cells display a diminished expression of CD55 and an increase in complement C3 deposition, indicating CD55 is required for peripheral T cell fitness of RTEs. Reconstitution of CD4-cre NKAP cKO bone marrow into C3-deficient host partially rescues the maturation defect as measured by upregulation of Qa2 and CD55, and an increased proportion of mature naïve T cells. Thus, successful T cell maturation results in increased expression of CD55 that protects RTEs from complement-mediated killing.
The thymoproteasome subunit β5T generates pMHC specialized for positive selection of CD8 T cells (P4445)
Authors: Xing Yan , Hogquist Kristin
Published: January 2023

Recent data show that a thymoproteasome subunit β5T is uniquely expressed in cortical thymic epithelial cells (cTECs) and plays an essential role in the development of CD8 T cells. The thymoproteasome may generate peptides that are specialized for positive selection or it may simply serve to generate peptides that are distinct from other APCs that cause negative selection, thereby promoting an overall larger number of surviving clones to mature and function in the immune system. To distinguish these models, we engineered mice to express distinct peptide repertoires in cTECs versus other APCs, but not using β5T. We did this by generating β5t5i knock-in mice in which the immunoproteasome subunit-β5i replaced β5T in cTECs. When such animals were crossed to β5i-/- mice, β5i was exclusively expressed in cTECs, whereas β5 was expressed in other cells. However, this did not support normal positive selection, suggesting that β5T generates peptides that are intrinsically “better” for positive selection, and not merely because these peptides are distinct from peptides presented by other APC. Finally, using a Nur77GFP reporter, we demonstrate that the T cells generated in the absence of β5T have higher reactivity to self; generating predominantly CD44hi “memory phenotype” peripheral CD8+ T cells. Altogether our results suggest that the thymoproteasome supports positive selection by generating peptides that are optimized for the selection of weakly self-reactive, naïve T cell clones.
Genome-wide profiling of estrogen receptor alpha-bound chromatin in differentiating myeloid progenitors reveals regulation of inflammatory pathways by estradiol (P4470)
Authors: Kovats Susan , Turner Sean , Wren Jonathan , Dozmorov Mikhail
Published: January 2023

In inflammation, elevated GM-CSF directs the development of dendritic cells (DCs) from myeloid precursors that infiltrate tissues. These inflammatory DCs play a central role in infection and autoimmunity. We previously showed that estradiol and estrogen receptor alpha (ERα) act in murine Flt3+ myeloid progenitors to promote GM-CSF-mediated DC differentiation. ERs are ligand-dependent transcription factors that form complexes with chromatin-modifying coregulators near gene promoters. Thus, we hypothesized that ERα regulates chromatin structure at loci important for DC differentiation. We used anti-ERα chromatin immunoprecipitation and next generation sequencing to identify genome-wide sites of ERα binding in myeloid progenitors stimulated with GM-CSF -/+ estradiol. Bioinformatics analyses identified unique ERα binding sites in the presence of estradiol near the transcription start site of >900 genes. Binding of ERα was enriched at CpG islands and consensus binding sites for SP1 and GABPA transcription factors. ERα bound to genes in pathways related to ER, GM-CSF and ERK/MAPK signaling, protein ubiquitination and cell cycle progression. ERα also bound to multiple genes in the NF-κB and Toll-like receptor pathways. These data suggest that ERα regulates genes involved in the NF-κB and GM-CSF signaling pathways during GM-CSF-mediated differentiation, and primes myeloid progenitors or mature DCs for responses to inflammatory stimuli.
Role of inhibins in dendritic cells differentiation and maturation (P4482)
Authors: Olguín Alor Roxana , Ambriz Peña Xochitl , Aleman-Muench Germán Rodrigo , Bonifaz Laura , Macías-Silva Marina , García-Zepeda Eduardo , Soldevila Gloria
Published: January 2023

Dendritic cells (DC) play a central role establishing the balance between tolerance and immunity. Inhibins and Activins, members of the TGFβ superfamily, have been implicated in the immune system as keys regulators of cellular functions, such as proliferation, apoptosis and differentiation. Several reports have previously shown the role of Activins in DC maturation and function, however the role of their closely related cytokines, the Inhibins, in those processes remains unclear. In this study, we compare the differentiation and maturation of bone marrow derived DC (BMDC) of wild type (WT) and Inhibin α deficient (KO) female mice. Our data showed that the percentage of immature BMDC was similar between WT and KO mice but, interestingly, analysis of mature BMDC obtained after lipopolysaccharide (LPS) stimulation (mBMDC) showed a significant reduction in the surface expression of MCHII, CD80 and CD86 in KO BMDC compared to WT mBMDC. In agreement, KO mBMDC retained the morphology characteristic of iDC, consisting in cellular spreading and presence of podosome like structures. Moreover, the chemotaxis of KO mBMDC towards CCL19 was significantly diminished in comparison to WT mBMDC. Collectively, these data show an impairment in the maturation of KO BMDC mediated by LPS, suggesting a role of Inhibins in the regulation of DC mediated functions. This data contribute to a better understanding of the mechanisms involved in DC maturation and the balance between tolerance and immunity.
Leucine rich repeat containing 8a is essential for lymphocyte development and function (P4372)
Authors: Kumar Lalit , Chou Janet , Yee Christina S.K. , Borzutzky Arturo , Vollmann Elisabeth , von Andrian Ulrich , Park Shin-Young , Hollander Georg , Manis John , Poliani P. Luigi , Geha Raif
Published: January 2023

LRRC8a encodes a ubiquitously expressed leucine rich repeat containing protein. Absence of B cells and agammaglobulinemia has been reported in a patient with a balanced translocation mutation affecting an LRRC8a allele. We have generated LRRC8a deficient mice in order to define the role of LRRC8a in B- and T-cells development and function. LRRC8a deficient mice exhibited a modest block in B cell development, decreased peripheral B cell numbers but intact intrinsic B cell function. In contrast, they exhibited a cell autonomous severe block in early thymic development with decreased proliferation and increased apoptosis of thymocytes, and severely impaired peripheral T cells function. The thymus had a ~ten-fold reduction in cellularity. TdT-mediated dUTP nick end labeling (TUNEL) staining demonstrated significantly increased numbers of apoptotic cells. LRRC8a deficient thymi had diminished content of phosphorylated Akt. LRRC8a constitutively associated with Grb2 and its ligation caused PI3-kinase dependent activation of Akt. These findings reveal a critical role for LRRC8a in the early development and peripheral expansion of B- and T-cells and in T-cell function.
Trp53 inactivation in the tumor microenvironment promotes tumor progression by enhancing MDSC differentiation (P2073)
Authors: Cui Yan , Guo Gang , Marrero Luis , Rodriguez Paulo , Ochoa Augusto
Published: January 2023

Inactivation of the tumor suppressor p53 is one of the leading causes of cancer as p53 inactivation via somatic mutations occurs in 50% of human cancers and sometimes in other cells of the tumor microenvironment (TME). Recent studies by our laboratory and others showed that innate and adaptive immunities are skewed towards pro-inflammation in hosts lacking p53 function. As chronic inflammation plays a vital role in tumor initiation, progression, and metastases, we hypothesized that p53 inactivation in the TME favors tumorigenesis by promoting inflammation. In this study, we compared the growth of subcutaneously inoculated B16F1 melanoma in p53null and WT mice. Tumor growth in p53null mice was greatly accelerated, correlating with marked increases in CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs), FoxP3+ regulatory T cells, and a loss of effector function, compared with those in WT mice. This augmented immunotolerance in tumor bearing p53null mice was associated with an enhanced MDSC proliferation, an increased frequency of myeloid progenitors in the peripheral lymphoid tissues, and a marked elevation of serum inflammatory cytokines/chemokines, compared with those of WT mice. Together, these results support our hypothesis and reveal a previously unappreciated function for p53 in maintaining an immunological microenvironment that suppresses tumor initiation and progression.
Thyroid status modulates the in vivo antitumor response to T lymphoma cells (P2079)
Authors: Sterle Helena A , Barreiro Arcos Maria L , Valli Eduardo , Paulazo Maria A , Colombo Lucas , Rabinovich Gabriel A , Cremaschi Graciela A
Published: January 2023

We have previously observed the regulation of immunity by thyroid status but little is known on how the levels of thyroid hormones (THs) influence antitumor responses. Our aim was to evaluate the antitumor immune response in hyper- and hypothyroid C57BL/6 mice (Hyper and Hypo respectively) which develop solid tumors and metastases in response to inoculation with EL-4 T lymphoma. Hyper showed increased rate of tumor growth compared to Hypo and control mice (C), while Hypo showed more metastases than other groups. Hyper Solid tumors had increased expression of cyclin D1, D3, E1, while Hypo had higher levels of cell cycle inhibitors p27, p16 and p53. At day 10th after EL-4 cell inoculation, when differences in tumor growth were significant, Hyper displayed higher number of cells in lymph nodes and spleens, a lower percentage (%) of myeloid-derived suppressor cells and an increased % of NK cells in the spleen accompanied by an augmented NK cell cytotoxic activity. On the other hand, Hypo displayed a reduction of CD8+ T cells and a decrease in tumor-specific cytotoxic activity. However, there were no differences among the groups in the % of T, B or Treg cells in secondary lymphoid organs. Our results suggest that THs modulate tumor growth through mechanisms involving regulation of proteins related to the progression and/or arrest of the cell cycle, and through the control of the anti-tumor immune response that could limit tumor dissemination.
FcγRIIB limits protection against F. tularensis LVS challenge induced by inactivated F. tularensis immunogen (P4294)
Authors: Franz Brian , Li Ying , Bitsaktsis Constantine , Iglesias Bibiana , Pham Giang , Rosa Sarah , Gosselin Edmund
Published: January 2023

We have previously shown that intranasal immunization with inactivated Francisella tularensis LVS targeted to FcR induces partial protection against a mucosal challenge with F. tularensis SchuS4 and that this protection is Ab and IFN-γ dependent. FcγRIIB, the only inhibitory FcγR, has been reported to regulate IgG production, which plays a major role in protection against F. tularensis LVS and SchuS4 challenge. Thus, we sought to determine the impact of FcγRIIB on immune protection against F. tularensis infection. We utilized inactivated F. tularensis as an immunogen. Naïve or inactivated F. tularensis-immunized FcγRIIB knockout versus wildtype C57BL/6 mice were challenged with varying doses of F. tularensis LVS. While we observed no difference in survival between naïve knockout versus wildtype mice, knockout mice immunized with inactivated F. tularensis showed significantly increased survival as compared to similarly immunized wildtype mice. Also, both F. tularensis-specific IgG and IgA production were elevated in serum and bronchoalveolar lavage fluid from inactivated F. tularensis-immunized knockout versus wildtype mice, as was IFN-γ production. In summary, our studies show that FcγRIIB limits the level of protection against F. tularensis challenge generated by inactivated F. tularensis immunization, by limiting the production of F. tularensis-specific IgG, IgA, and IFN-γ, which have been shown to be important for protection against F. tularensis infection.
Protective immunity against invasive Streptococcus pneumoniae infections by tissue-specific memory Th17 cells (P4266)
Authors: Wang Yan , Jiang Bin , Weiser Jeffrey , Shen Hao
Published: January 2023

Pneumonia caused by Streptococcus pneumoniae (Sp) remains a leading cause of serious illness and death worldwide. Current vaccine is highly effective in preventing colonization by inducing serotype-specific antibodies. However, there is an increasing prevalence of infection by serotype strains not included in the vaccine; this highlights the need for a universal vaccine that protects against all serotypes. The first step in developing a universal vaccine is to elucidate the immune mechanism that can provide broad protection against different serotype Sp strains. We found that intranasal immunization of mice with Sp resulted in a strong CD4+T cell response in the lung that consisted of mostly Th17 cells but also IFNγ producing Th1 cells. These immunized mice were protected against lethal challenge with a different serotype strain of Sp and cleared bacteria in lung by day 2 post-challenge. Adoptive transfer of T cells from immunized mice also provided protection against a heterologous challenge, with a dominant Th17 recall response in the lung from donor memory T cells. Furthermore, immunization of mice with Listeria monocytogenes (LM) provided protection against a recombinant Sp strain expressing a CD4 epitope from LM (LLO190), as shown by decreased CFU in the lung, and elevated LLO190-specific Th17 response. Our results suggest that memory Th17 cells may play a key role in providing broad protective immunity against invasive Sp infection in a serotype independent manner.
Susceptibility of NOD/scid IL-2Rγnull mice inoculated with alcoholic patient white blood cells to Pseudomonas aeruginosa infection (P3029)
Authors: Nakamura Kiwamu , Asai Akira , Ohama Hideko , Tsuchimoto Yusuke , Tsuda Yasuhiro , Higuchi Kazuhide , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

We have previously demonstrated that CCL2+ neutrophils (PMN-II) and IL-12−IL-10+ monocytes (M2 monocytes) are predominated in WBC of alcoholic patients. Since these cells are inhibitory on the generation of major antibacterial effector cells, in this study, we investigated the susceptibility of alcoholic patients to P. aeruginosa infections in a humanized murine model. To create an experimental model of alcoholic patients, 5 x 106 cells of WBC of alcoholic patients were adoptively transferred to X-irradiated-NOD/scid IL-2Rγnull mice. As a control, these mice were inoculated with healthy donor WBC. Then, they were infected s.c. with P. aeruginosa (10 CFU/chimera) and bacteria in liver and kidneys of these chimeras were quantified. In the results, bacteria grew (> 103 CFU/g organ, 24 hrs after infection) in liver and kidneys of the chimeras created with alcoholic patient WBC, while bacteria disappeared within 24 hrs after P. aeruginosa infection from these organs of the control chimeras. CD14+ cells isolated from WBC of alcoholic patients were shown to be IL-10+IL-12−CCL1+ cells, while IL-10−IL-12+CCL1- cells were isolated from healthy donor WBC stimulated with heat-killed P. aeruginosa. These results indicate that chimeras created with alcoholic patient WBC are carriers of M2b monocytes and susceptible to P. aeruginosa infection, while chimeras created with healthy donor WBC are carriers of M1MΦ and resistant against these infections.
HLA-G 14bp polymorphism on HIV-1 perinatal transmission (P3041)
Authors: Fernandes Ana , Sanches Roberta , Matos Francielly , Massaro Juliana , Donadi Eduardo
Published: January 2023

Background/aims: HLA-G is expressed at maternal-fetal interface and is strongly involved on maternal-fetal tolerance. The 3' untranslated region of HLA-G gene presents an insertion (INS) and/or deletion (DEL) of a 14bp fragment, which is associated with stability and expression levels of HLA-G mRNA. The aim of this study was to analyze the hypothesis that the 14bp polymorphism can influence the HIV vertical transmission. Methods: Blood samples were obtained from 49 mother-child pairs (26 pairs with and 23 without vertical transmission). All children were born from HIV-positive mothers who did not receive antiretroviral therapy during pregnancy. The 14 pp polymorphism was detected by PCR-amplified DNA using specific primers. Statistical analyses were made by Fisher's exact test. This study was approved by the local ethics committee. Results: We did not detect significant differences in allele and genotype frequencies between HIV-infected and HIV-uninfected child; however, the presence of the D/D homozygous genotype was more frequent among mothers with HIV-infected child (p=0.05). In addition, the 14pb similarity among mother and child pairs was more frequent on vertical transmission (69%) than on its absence. Conclusion: The presence of the genotype (DEL/DEL), associated with high production of HLA-G, and the similarities of the 14bp DEL/INS genotypes between mother and child may favor HIV mother to child transmission.
SIV induced modulation of the enteric serotonergic response contributes to chronic immune activation (P3047)
Authors: Grishina Irina , Hirao Lauren , Sankaran-Walters Sumathi , Capitanio John , Dandekar Satya
Published: January 2023

Immune activation is a hallmark of SIV infection, which induces immune dysregulation through the course of infection. Serotonin (5HT) is a neurotransmitter, >90% of which is made in enteric enterochromaffin (EC) cells, where it modulates gut motility and immune function. Though immune cells are functionally responsive to 5HT, its role in the gut has not been studied in context of SIV infection. We hypothesize that SIV infection alters 5HT regulation and signaling, contributing to chronic immune activation. During chronic SIV infection elevated levels of mucosal 5HT, decreased expression of 5HT reuptake transporter (SERT) and elevated numbers of EC cells were indicative of elevated extracellular 5HT availability. SERT expression correlated positively with CD4+ T-cell numbers, and inversely with viral replication and CD8+ T cell Interferon-gamma (IFNγ) production. Increase of 5HTRs 1A and 2A expression demonstrated potential involvement of 5HT signaling in cell activation and proliferation. Treatment of LPLs from SIV+ animals with 5HT resulted in increased percentages of CD14+ macrophages and CD11c+ mDCs producing IFNγ. Changes in expression of 5HT regulatory molecules, EC cell numbers, 5HTR profile and immune cell response to 5HT stimulation during chronic SIV infection implicate the intestinal serotonergic response as a potential mechanism contributing to chronic immune activation and highlight the virally disrupted interface between enteric neuronal and immune homeostasis.
Intrinsic CXCR5 expression is required to maintain long-lived antigen specific CD4 T cells during chronic Mycobacterium tuberculosis infection (P3013)
Authors: Moguche Albanus , Shafiani Shahin , Dihn Crystal , Larson Ryan , Clemons Corey , Urdahl Kevin
Published: January 2023

Chronic antigenic stimulation is known to cause T cell functional exhaustion and death. During Mtb infections, antigen specific CD4 T cells are subject to chronic antigenic stimulation but a sizeable population of these cells survives and maintains the ability to produce IFN-γ. How these cells are maintained is not yet understood. Mtb-specific CD4 T cells can be classified based on the surface expression of the inhibitory receptors PD1 and KLRG1. The PD1+ cells proliferate robustly but produce low amounts of IFN-γ while KLRG1+ cells proliferate less but produce large amounts of IFN-γ. Significantly, the PD1+ cells are capable of differentiating into KLRG1+ cells and not vice versa indicating that a self-renewing progenitor population may be contained within the PD1+ cells. On further characterization we have found that PD1+ cells are heterogeneous containing three different subsets based on expression of the chemokine receptor CXCR5 i.e. PD1+CXCR5hi cells that resemble Tfh cells, PD1+CXCR5int and PD1+CXCR5lo cells. We found that B cells were not required to maintain the Tfh like cells or overall Mtb-specific CD4 T cells. However, using mixed bone marrow chimeras we found that intrinsic CXCR5 expression on CD4 T cells is required to maintain Mtb-specific CD4 T cells. Taken together, these studies indicate that during Mtb infections, a subset of CD4 T cells capable of self-renewal is induced and intrinsic CXCR5 is important for its maintenance.
Neutrophil extracellular traps exhibit no bactericidal activity against Pseudomonas aeruginosa (P1255)
Authors: Gadjeva Mihaela
Published: January 2023

In CF lungs, PMN are the dominant cell responding to bacterial infection in the lungs. However, PMNs are incapable of preventing chronic colonization and contribute to host damage by excessive release of granule contents and formation of neutrophil extracellular traps (NETs) predominantly composed of DNA. It is currently unclear whether NETs serve to trap bacterial pathogens and, hence, facilitate bacterial colonization, or whether they have weak bactericidal activities. We investigated the kinetics, composition, and functional consequences of NET release in response to Pseudomonas aeruginosa, the most frequent bacterial colonizer of Cystic Fibrosis patients. We found that both non-mucoid and mucoid strains of P. aeruginosa were not sensitive to NET-mediated killing. This was not due to the inability of P. aeruginosa to induce NET formation by PMNs as NETs, released in response to P. aeruginosa, exhibited the typical NET composition. Overstimulation of NET release by PMA treatment prior to infection also failed to reduce the ability of P. aeruginosa to survive. Importantly, the presence of NETs hindered the bactericidal activity of complement. These findings suggest that targeting NETosis could provide an effective therapy for CF. To this end, we identified a host factor, termed, macrophage migration inhibitory factor, which facilitated PMN’s ability to NETose. Therefore, inhibiting MIF in this setting may be a significant innovation in therapy of CF lung disease.
Adverse role of influenza A virus-induced Nox1 in neutralizing antibody responses and survival against lethal viral challenge in mice. (P1407)
Authors: Hofstetter Amelia , De La Cruz Juan , Patel Jenish , McCoy James , Cao Weiping , Kim Jin , Diebold Becky , Belser Jessica , Tumpey Terrence , Katz Jacqueline , Sambhara Suryaprakash , Lambeth J. , Gangappa Shivaprakash
Published: January 2023

Highly pathogenic influenza viruses can elicit a severe cytokine storm, leading to acute lung injury (ALI), or in its more severe form, acute respiratory distress syndrome (ARDS). Reactive oxygen species (ROS) produced by NADPH Oxidase (Nox) enzymes of both the lung infiltrating cells and the lung epithelial tissue can contribute to lung injury directly or via inflammatory signaling pathways. Here, we present evidence that Nox1 expression is enhanced in vitro (A549 lung epithelial cells, 33-fold; THP-1 monocytic cells, 633-fold; HULEC lung endothelial cells, 27-fold) and in vivo (mouse lung tissue, 17-fold) in response to infection with influenza A virus. Notably, in A549 cells, Nox1 expression levels were enhanced by laboratory strains A/X31 (H3N2) and A/WSN/33 (H1N1), as well as A(H1N1)pdm09 clinical isolates (A/California/08/2009, A/Mexico/4108/2009, and A/Texas/15/2009) of influenza A virus. Nox1 induction was virus dose- and replication-dependent. However, Nox1-deficient mice had a higher survival rate (p=0.008) compared to wild-type controls in response to a lethal dose of A/PR8/1934 influenza. Improved survival of Nox1-deficient mice corresponded with increased (p=0.0006) flu-specific neutralizing antibody responses. These results suggest that therapeutic blockade of Nox1 deserves further attention as a possible adjunct therapy for influenza A-associated ALI/ARDS.
Expression of TREM-1 is triggered by flaviviruses: possible role in virus-associated innate immune responses (P1381)
Authors: Roe Kelsey , Nelson Jacob , Orillo Beverly , Verma Saguna
Published: January 2023

Activation of Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), an immunoreceptor expressed mainly on immune cells results in a cascade of inflammatory effects including cytokine production. In addition, TREM-1 also modulates signaling cascades of other PRRs such as TLRs and NLRs. Engagement of the TREM-1 receptor via adaptor protein DAP12 leads to the activation of downstream kinases, mobilization of intracellular calcium and production of pro-inflammatory cytokines. Innate immune responses are essential for the clearance, dissemination and neurovirulence of West Nile virus (WNV), a neurotropic flavivirus, which has emerged in the U.S. as a significant cause of viral encephalitis in humans. However, the role of TREM-1 signaling pathway in immunity to flaviviruses is unclear. In this study we investigated the changes in the expression kinetics and activation of TREM-1 following WNV infection. Expression of TREM-1 was markedly increased in WNV-infected mouse immune cells and embryonic fibroblasts (MEFs) at 48 hrs after infection and in the mouse brain at day 8 after infection. Further, activation of TREM-1 using agonist antibody resulted in an increase in the levels of IFN-β and key cytokines in WNV-infected MEFs. Our results so far indicate a possible role of TREM-1 in the induction of inflammatory innate immune responses to WNV and further studies are ongoing to characterize unexplored mechanisms of TREM-1 associated with the pathogenesis of flaviviruses.
IKKϵ is targeted by pathogenic West Nile virus to evade host immune control (P1387)
Authors: Huang Albert , Perwitasari Olivia , Ramos Hilario , Suthar Mehul , Brassil Margaret , Blahnik Gabriele , Gale, Jr. Michael
Published: January 2023

Innate antiviral immunity and interferon (IFN) defenses are essential for the control of West Nile virus (WNV) infection. A pathogenic strain of the virus (WNV-TX) can suppress IFN actions by blocking JAK-STAT signaling, thus evading host IFN defenses. It was recently shown that IKKϵ directly regulates STAT1 activity by phosphorylating STAT1 on S708 to enhance IFIT2 expression and suppress WNV infection. However, the spectrum of interferon-stimulated gene (ISG) expression that is regulated by IKKϵ/STAT1 S708 signaling undergoing WNV infection and IFN signaling is not defined nor is it known if pathogenic WNV can evade these actions as a basis of disease. Here, we compared the transcriptomes of cells infected with pathogenic (WNV-TX) and non-pathogenic (WNV-MAD) strains of WNV to identify the genes specifically regulated by pathogenic WNV. Further, we compared the transcriptomes of IFN treated WT and IKKϵ KO cells to identify IKKϵ-dependent ISGs. These analyses allow us to identify genes that are shut down by pathogenic WNV in an IKKϵ-dependent manner to evade host immune response. Importantly, we also found IKKϵ-mediated induction of ISGs to be blocked by the anchored capsid portion of WNV-TX but not WNV-MAD. The anchored capsid of WNV-TX physically interacts with IKKϵ to mediate kinase suppression and a block in STAT1 S708 phosphorylation. Thus, IKKϵ is preferentially targeted by the anchored capsid of the pathogenic WNV to evade antiviral innate immunity and IFN actions.
CpGB DNA activates dermal macrophages and highly recruits inflammatory monocytes into the skin (P5120)
Authors: Mathes Allison , Christmann Romy , Affandi Alsya , Stifano Giuseppina , Lafyatis Robert
Published: January 2023

Toll-like receptor 9 (TLR9) recognizes DNA with unmethylated CpG motifs and evidence suggests that it is highly involved in several autoimmune skin diseases. This study investigated the effects of chronic TLR9 activation in the skin by identifying upregulated cytokines and chemokines and their roles in cell recruitment after subcutaneous treatment of TLR9 ligand, CpGB. Also, it established the importance of circulating and skin resident immune cells in potentiating this inflammatory response. CpGB DNA skin treatment dramatically induced an influx of CD11b+ F4/80+ macrophages, markedly increasing over 4 weeks of treatment, and an induction of IFNγ and TNFα expression. Chemokines, CCL2, CCL4, CCL5, CXCL9, and CXCL10, were highly induced in CpGB DNA-treated skin, although abrogation of these signaling pathways individually did not alter macrophage recruitment. TLR9 activation in the skin led to an increase in circulating CD11b+ CD115+ Ly6Chi inflammatory monocyte population following 1 week CpGB DNA treatment. Lastly, skin resident CD11b+ cells initially take up subcutaneous CpGB DNA and propagate the subsequent immune response. Overall, this study demonstrates that chronic TLR9 activation induces selective monocyte recruitment into the skin, likely derived from the population of inflammatory monocytes strikingly increased in the blood. Furthermore, it shows that macrophages are the key cells recognizing immune stimuli and initiating an inflammatory response in the skin.
Cholesterol crystal production by endothelial cells and its impact on early atherosclerosis development. (P5128)
Authors: Baumer Yvonne , William Boisvert
Published: January 2023

Atherosclerosis is an inflammatory disease that begins with a dysfunctional endothelial phenotype caused by endogenous factors such as blood lipid and cytokine/chemokine levels or exogenous factors like cigarette smoke and certain microorganisms. While performing experiments involving endothelial cells (EC) and lipoproteins we observed cholesterol crystal (CC) formation by the EC and decided to study if this phenomenon would contribute to early stages of atherogenesis. When EC were incubated with high but physiologic levels of LDL we observed CC formation within multivesicular bodies of EC and their subsequent transport and excretion to the basolateral side of EC. We then cultured human aortic endothelial cells on CC coatings and detected several pro-atherogenic changes in endothelial function such as increased permeability due to incomplete inter-endothelial junction formation, increased transmigration of mononuclear cells and decreased regeneration potential, indicating that the formation of subendothelial CC may be important for endothelial dysfunction in early atherogenesis. Interestingly, in ldlr-/- mice fed a high fat diet for only 1 week we could detect subendothelial CC deposition in the earliest stages of atherosclerosis. Our findings indicate the important role of EC in possibly creating the first “subendothelial space” through cholesterol metabolism and subsequent CC formation that may be critical for the very beginning of atherosclerotic plaque formation.
Selenoproteins regulate oxidative stress and inflammatory phenotype of endothelial cells (P5132)
Authors: Sordillo Lorraine , Corl Chris , Gandy Jeffery
Published: January 2023

Oxidative stress contributes to the pathology of inflammatory-based diseases by increasing the expression of vascular-derived adhesion molecules, cytokines and eicosanoids. Certain selenoproteins, such as glutathione peroxidase (GPX) and thioredoxin reductase, possess important antioxidant defense functions in endothelial cells that can counteract the damaging pro-inflammatory effects of oxidative stress. Using an oxidative stress model based on selenium (Se) deficiency in bovine aortic endothelial cells (BAEC), we sought to determine whether specific selenoproteins may contribute to the differential regulation of adhesion molecules, cytokines and eicosanoids during pro-oxidant challenge. Se supplementation reduced adhesion molecule expression, cyclooxygenase 2, and pro-inflammatory cytokine expression by BAEC when compared to Se-deficient BAEC. Subsequent results using GPX4 siRNA showed that this selenoprotein is at least partially responsible for controlling adhesion molecule expression and cytokine production during oxidative stress. These data provide evidence for the involvement of certain selenoproteins in the regulation of adhesion molecule expression, eicosanoid biosynthesis, and cytokine production in BAEC during pro-oxidant challenge. Further characterization of the role of selenoproteins in endothelial cell metabolism may provide new insights as to how Se may provide specific health benefits in combating inflammatory-based diseases.