bluuebunny/crossref_metadata_2025 huggingface dataset:
Using reporter mouse models to track CD4 T cell differentiation and fate based on IL-2 expression (P1199)
Authors: Winstead Colleen , Way Sing Sing , Moon James , Weaver Casey
Published: January 2023

CD4 T cells respond to in vivo activation stimuli, in part, by rapidly producing the homeostatic cytokine IL-2. It is thought that this acute production of IL-2 is necessary for full activation and fate determination of both the conventional CD4 T cells themselves, as well as other components of the adaptive (Tregs/CD8/B cells) and innate immune responses (NK/NKT/DC) to infection. Within a population of responding CD4 T cells, only a percentage of cells will secrete IL-2. Though much is known regarding regulation of IL-2 production by CD4 T cells and the downstream consequences of autocrine and paracrine signaling, little is known about the quantity, quality, or fate of CD4 T cells that produce IL-2. Using L. monocytogenes engineered to express foreign MHCII peptides and peptide-loaded MHCII tetramers, we are able to track and assess the fate of endogenous, antigen-specific CD4 T cells in infected mice. Combining this methodology with novel transgenic IL-2 reporter mouse models, we are able to address questions regarding fate by isolating, depleting, and/or adoptively transferring live, antigen-specific CD4 T cells to secondary hosts based on acute expression of IL-2. Preliminary data suggests IL-2-‘competent’ and -‘incompetent’ cells differ in their ability to traffic to peripheral tissues following an acute, systemic infection, resulting in a potential functional, as well as proliferative and survival advantage for competent cells over those incompetent for IL-2 expression.
Comparison of the T cell surface glycome of activated and naïve T cells (P1210)
Authors: Maverakis Emanual , Ono Yoko , Marusina Alina , Bustos Villalobos Itzel , Rizzo Amilcar , Dimapasoc Lauren , Hua Serenus , Williams Cynthia , Lebrilla Carlito
Published: January 2023

Protein glycosylation plays an important role in a variety of cellular activities including migration, activation, and adhesion. Although the majority of cellular proteins are glycosylated, little is known about the cell surface glycome of the T cell. Here we characterize the glycans expressed on the surface of naïve, activated and malignant T cells. Using a variety of techniques including density centrifugation, PNGase F enzyme glycan release, lectin column affinity chromatography, nanospray HPLC-chip/time-of-flight mass spectrometry, and flow cytometry we were able to characterize the entire surface glycome of naïve T cells and the associated changes following T cell activation with anti-CD3/CD28. The cell surface proteins expressing these glycans were also identified. Using specific-exoglycosidases to remove the glycans of interest, while leaving their associated proteins intact, the effects of these glycans on T cell proliferation, TCR signal transduction, and TCR down-regulation was determined.
CASPASE-12 is not a protective factor for rheumatoid arthritis in African-Americans (P4106)
Authors: Hermel Evan , Marshall Laura , Obaidullah Mohammad , Fuchs Trista , Brinkley Garland , Velasco H. Eduardo , Mikuls Ted , Bridges Jr S. Louis
Published: January 2023

CASPASE-12 (CASP12) modulates inflammation due to its ability to down-regulate inflammatory cytokines, particularly interleukin-1. Most humans possess the inactive pseudogene allele (CASP12p1) but a functional, non-truncated CASP12 allele [single nucleotide polymorphism #rs497116; A->G; ter124R] is found in 20% of persons of African descent. CASP12 is a risk factor for sepsis in persons of African descent. Rheumatoid arthritis (RA) is a serious and complex autoimmune disease that involves significant joint inflammation and destruction, and interleukin-1 contributes to the pathogenesis of RA. To determine if CASP12 may be protective against RA due to its anti-inflammatory functions, we genotyped 742 patients with rheumatoid arthritis and 342 controls from the Consortium for Longitudinal Evaluation of African-Americans with Early Rheumatoid Arthritis and the Veterans Affairs Rheumatoid Arthritis registries. Of RA patients, 17 (2.29%) were homozygous for CASP12, 146 (19.7%) were heterozygous, and 579 (78%) were homozygous for CASP12p1. These numbers did not vary significantly from controls. No significant differences were observed for CASP12 genotype on age of disease onset, seropositivity for rheumatoid factor or anti-CCP, baseline joint erosion, joint space narrowing scores, overall disease scores, C-reactive protein levels or erythrocyte sedimentation rates. Thus, CASP12 does not protect against RA in African-American patients, nor mitigates disease parameters.
Deletion of Snai2 and Snai3 results in impaired physical development compounded by lymphocyte deficiency (P4380)
Authors: Pioli Peter , Dahlem Timothy , Weis Janis , Weis John
Published: January 2023

The Snail family of transcriptional regulators consists of three highly conserved members. These proteins regulate (repress) transcription via the recruitment of histone deacetylases to target gene promoters that possess the appropriate E-box binding sequences, specifically with GC-rich central dinucleotides. Murine Snai1 is required for mouse development while Snai2 deficient animals develop normally but have been reported to have thymic T cell deficiencies. Less is known about the third member of the family, Snai3. To investigate the function of Snai3, we generated a conditional knockin mouse. Utilizing Cre-mediated deletion to facilitate the ablation of Snai3 in T cells or the entire animal, we found little to no effect of the loss of Snai3 suggesting possible compensation with another family member. To test this, we created Snai2/Snai3 double deficient mice. The developmental consequences of lacking both of these proteins was manifested in stunted growth, a paucity of offspring including a dramatic deficiency of female mice, an overgrowth of the eyelids and impaired immune cell development particularly within the lymphoid lineages.
V cells: A novel antigen specific immunoglobulin expressing cell lineage in immunized animals distinct from classical B cell populations (P4386)
Authors: Chronopoulou Efthalia , Uribe-Benninghoff Alejandro , Elia Jeanne , Nguyen Andrea , Rabenstein Jacob , Fisher Janean , Berry Jody
Published: January 2023

Following immunization with protein antigens, an uncharacterized immunoglobulin bearing antigen specific cell population was identified and named as "V cells". This cell population lacks surface expression of B cell lineage specific markers such as B220, CD19, as well as T cell, NK, NKT, basophil and stem cell specific markers as determined by flow cytometric analysis. V cells are characterized as being positive for CD24, CD43, CD45, CD49b, CD48, CD79b and IgG. They are observed in the spleen, bone marrow, and blood of immunized mice but were not detected in other tissues such as the thymus, lymph nodes and peritoneal exudate cells. Antigen specific V cells were generated following immunization of at least 6 different protein antigens and appeared as early as 7 to 14 days following a single immunization. Molecular analysis of isolated mRNA from bulk sorted V cells proved that this novel cell population produces antigen specific Ig. Molecular analysis was also performed on the cDNA of individually and bulk sorted V cells extracted from the bone marrow and spleen. Immunoglobulin VH genes were then sequenced from each cDNA pool with results indicating a higher diversity of VH genes in the bone marrow when compared to the spleen. In addition, presence of shared VH genes between the spleen and bone marrow suggests migration of the V cells between the two compartments. The role of this novel cell population in innate and/or adaptive immunity is currently under investigation.
ZFP521 transcription factor modulates B cell development through interactions with EBF1 (P4392)
Authors: Lei Hong , Lukin Kara , Warming Soren , Hagman James
Published: January 2023

Early B cell factor 1 (EBF1) determines B cell lineage specification and commitment. ZFP521, a transcription factor with 30 zinc fingers, negatively regulates EBF1. We determined that EBF1 accelerates B cell development in the absence of ZFP521. Pre-pro-B and pro-B cells are increased substantially, while late pro-B, pre-B and immature B cells are reduced significantly in Zfp521 KO mice. Interestingly, mature IgM+IgD+ B cells, which accumulate in the bone marrow and spleens of 4-5 week old WT mice, are readily detected in 2-3-week old Zfp521 KO mice. Additionally, RUNX1 plays a role in ZFP521-mediated regulation of EBF1 activity. ZFP521 is detected in the nuclei of B cells and interacts with EBF1 in vivo. In contrast with published reports, ZFP521 and EBF1 co-localize on B cell-specific promoters. This indicates that ZFP521 regulates B cell development through functional interactions with EBF1 on target gene chromatin. Using ChIPseq data we are differentiating between two possible models concerning functions of ZFP521: 1) ZFP521 may modulate EBF1 function through direct protein:protein interactions, and/or 2) ZFP521 may direct transcription of cognate target genes independently of EBF1. Together, our studies provide exciting new information concerning functions of EBF1 and ZFP521 in the B cell regulatory network.
The RAG2 acidic hinge regulates pathway choice, maintains genomic stability, and prevents lymphomagenesis (P4398)
Authors: Coussens Marc , Wendland Rebecca , Lindsay Cory , Deriano Ludovic , Arnal Suzzett , Roth David
Published: January 2023

VDJ recombination-associated DNA double-stranded breaks are normally repaired by the high fidelity, classical NHEJ machinery. Previous studies have implicated the RAG/DNA post-cleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate alternative repair mechanisms such as HR and alternative NHEJ (aNHEJ). We report that RAG2’s acidic hinge is critical for several steps. Mutations reducing the negative charge in this region destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the low-fidelity, translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental data support our hypothesis that flexibility of the acidic hinge is necessary for these functions. Similar human RAG2 sequence variants alter the predicted flexibility and permit repair by aNHEJ, indicating the potential for hazardous physiological consequences. Mice bearing similar neutralizing mutations (RAG2Neut), in the absence of p53, have an increased incidence of lymphomagenesis with hallmarks of VDJ recombination errors. Thus, the acidic hinge, along with stabilizing the PCC and ensuring proper pathway choice, aids in the prevention of lymphomagenesis by maintaining genomic integrity. This novel regulatory role that helps to ensure the safe development of a diverse immune repertoire, is to the best of our knowledge, the first function ascribed to this conserved region of RAG2.
Analysis of illegitimate Notch1 and Bcl11b V(D)J deletion events during mouse development (P4400)
Authors: Champagne Devin , Shockett Penny
Published: January 2023

Previously, others identified hotspots for deletional illegitimate V(D)J rearrangements in normal mouse thymus involving cryptic RSSs in Notch1 and Bcl11b, onco/tumor suppressor genes involved in T cell development. These deletions are associated with radiation-induced and other T cell malignancies. We are conducting expanded frequency and junctional analyses of deletions during mouse development to examine and compare age- and gender-specific detection and occurrence of these events. Deletions in thymus increase in frequency between fetal and neonatal stages, are seen through at least 7 months of age, and are found in spleen. Deletions are more frequent at Bcl11b than Notch1 as previously observed, but both genes show clonality of deletion bearing cells. Junctional processing analysis indicates that nucleotide loss is more frequent and extensive at Bcl11b than Notch1, thus P nucleotide insertions are more abundant in Notch1 junctions. N nucleotide insertions increase between fetal and juvenile stages, with recessed palindromes (Pr nucleotides) corresponding mostly to single-base additions consistent with normal TdT activity. An in depth age and gender junctional processing analysis is in progress as are determinations of postnatal inducibility of deletions using inducible RAG transgenic mice. We present our results in the context of similar studies of V(D)J deletions at the nonimmune HPRT locus and normal V(D)J recombination at antigen receptor loci.
Direct interaction between E47 and p21 controls LT-HSC proliferation and functional longevity (P4471)
Authors: Santos Patricia , Borghesi Lisa
Published: January 2023

Long-term hematopoietic stem cells (LT-HSCs) are important multipotent cells that replenish all immune cells lost due to natural turnover, infection or injury. LT-HSC proliferation is controlled by cell cycle regulators and upstream transcription factors but direct links between the two have not been established. Here, we demonstrate a mechanistic link between the transcription factor E47 and the major cell cycle regulator p21 in controlling LT-HSC proliferation, self-renewal, and functional competence. Gain-of-function and loss-of-function studies show that E47 directly regulates p21 transcription in HSCs. Using E47/p21 compound heterozygotes, a powerful approach for analyzing HSC activity in vivo, we show a 2-fold increase in BrdU+ LT-HSCs compared to WT or single HET controls in both steady state conditions and under transplantation stress. E47HETp21HET HSCs are also more sensitive to mitotoxic challenge with 5-FU resulting in a 45% decrease in HSC numbers compared to treated controls. In serial adoptive transfer assays, a rigorous challenge to HSC self-renewal, E47HETp21HET HSCs were decreased by 40% and 60% in secondary and tertiary recipients, respectively. In addition to hyperproliferation and poor self-renewal, E47HETp21HET adoptive transfer recipients had selective myeloid bias indicating HSC skewing. Together, these data suggest a developmental stage-specific, cell-intrinsic collaboration between E47 and p21 in the regulation of LT- HSC competence in vivo.
Foxp1 and Foxp4 regulate Foxp3+ nTreg generation in the thymus (P4420)
Authors: Wiehagen Karla , Staub Elizabeth , Tucker Haley , Morrisey Edward , Maltzman Jonathan
Published: January 2023

CD4+ Foxp3+ regulatory T cells (Tregs) are fundamental for transplantation tolerance and prevention of autoimmunity. nTreg generation occurs in the thymus and requires both T cell receptor- and cytokine receptor-generated signals. Foxp1 and Foxp4 are closely related transcription factors expressed in both Tregs and conventional T cells. We sought to determine whether Foxp1 and Foxp4 have a role in nTreg development. Using a CD4Cre transgenic conditional knockout approach, we deleted both Foxp1 and Foxp4 in the T lineage. Mice conditionally deficient for both Foxp1 and Foxp4 (cDKO) have normal thymic cellularity. CD4SP and CD8SP thymocytes are present in normal ratios. CD4+Foxp3+ cells are present but express higher levels of receptors for IL-2 and IL-7. Markers of activation CD44, CD69, and GITR are also found at elevated levels on the cell surface. In the competitive setting of mixed bone marrow chimeras, there is a significant reduction in cDKO-derived chimerism in the Foxp3+ nTreg population. The competitive disadvantage occurs despite expression of the high affinity IL-2 receptor alpha chain, CD25. Together, these results suggest that Foxp1 and Foxp4 are necessary for the normal generation of nTreg cells. Ongoing experiments to understand the underlying molecular basis of these findings will be discussed.
Maternal methyl-donor supplementation increases regulatory T-cell-mediated redox remodeling (P4422)
Authors: Yung Raymond , Delaney Colin , Garg Sanjay , Banerjee Ruma
Published: January 2023

Regulatory T cells (Treg) maintain peripheral tolerance by inhibiting the proliferation of effector T (Teff) cells. Previously, we exposed dams to a diet supplemented with methyl donors (MS) or not (control) during mating and pregnancy, and found the MS diet suppresses selected T cell pro-inflammatory chemokines and cytokines in F1 mice. Here, we studied the effects of these prenatal diets on Treg function in F1 mice. Dams were fed either MS or control diets prior to mating, and during pregnancy and lactation. At 4 weeks of age, F1 mice from both groups were either analyzed or placed on the standard NIH-31 chow and allowed to age. We found that prenatal MS diet exposure is associated with lower Treg numbers in F1 mice, and this decrease is sustained into late life while on the standard NIH-31 chow. Both FoxP3 mRNA and protein were decreased in mice on the MS diet, compared to the control diet. The difference in Treg numbers is not due to differences in food consumption or weight gain between dams exposed to either prenatal diet. Lower Treg cell numbers are associated with hyper-methylation of the FoxP3 promoter and enhancer regions. Despite their lower numbers, Tregs from F1-MS mice produce increased IL-10 and suppress Teff proliferation more, compared to Treg from F1-control mice. Together, these results show that a methyl-donor-rich prenatal diet lowers total Treg numbers, though these cells maintain an anti-inflammatory phenotype.
Homeostatic properties and phenotypic maturation of murine CD4+ pre-thymic emigrants in the thymus (P4436)
Authors: Ge Qing
Published: January 2023

After a tightly regulated developmental program in the thymus, “mature” single positive (SP) thymocytes leave the thymus and enter the periphery. These newly arrived recent thymic emigrants (RTEs) are phenotypically and functionally immature, and will complete a dynamic maturation in the peripheral lymphoid organs before being licensed to be resident naïve T cells. To study the early events occurring in the RTE maturation process, we identified the phenotype of CD4+ pre-RTEs, a population of CD4+ SP thymocytes that have acquired the thymus egress capability. Compared to peripheral naïve T cells, CD4+ pre-RTEs displayed superior survival capability in lymphoreplete mice and faster proliferation under lymphopenic condition. The differences in Bcl2/Bim expression and/or heightened IL-7 signaling pathway may account for the pre-RTEs’ better responsiveness to homeostatic signals. Qa2, the expression of which indicates the phenotypic maturation of SPs and RTEs, was found to be upregulated in CD4+ pre-RTEs in thymic perivascular space. Migratory dendritic cells surround this region contribute to Qa2 expression in pre-RTEs. The dendritic cell-driven Qa2 induction of CD4+ pre-RTEs is independent of MHC class II and Aire molecules.
TSLP-mediated extramedullary hematopoiesis promotes allergic inflammation (P4473)
Authors: Siracusa Mark , Tait Wojno Elia , Osborne Lisa , Saenz Steven , Kim Brian , Spergel Jonathan , Comeau Michael , Artis David
Published: January 2023

Extramedullary hematopoiesis (EMH) refers to the differentiation of hematopoietic stem cells (HSCs) into effector cells that occurs in compartments outside of the bone marrow. Previous studies established a link between pattern recognition receptor-expressing HSCs, EMH and innate immune responses to bacterial or viral pathogens. However, the factors that regulate EMH and whether EMH is a conserved mechanism of innate immunity to diverse stimuli remain poorly defined. Here, we demonstrate a previously unrecognized role for thymic stromal lymphopoietin (TSLP) in promoting the population expansion of Lineage-, CD34+, c-Kit+ granulocyte-monocyte progenitor (GMP)-like cells in the periphery. TSLP-elicited progenitors possess the capacity to differentiate into macrophages, mast cells and basophils. Critically, adoptive transfer of TSLP-elicited GMP-like cells was sufficient to promote Th2 cytokine-dependent immunity to an intestinal helminth parasite, demonstrating a role for TSLP-elicited GMP-like cells in promoting inflammation. Further, circulating progenitor cells were also increased in allergic patients with a gain-of-function polymorphism in TSLP, suggesting that the TSLP-EMH pathway may be operational in human disease. These data demonstrate a previously unrecognized role for TSLP in promoting EMH that contributes to the development of Th2-cytokine-mediated inflammation and identify the TSLP-EMH pathway as a possible therapeutic target to treat allergic inflammation.
Human peripheral Vδ1+ γδ T cells can develop into αβ T cells (P4460)
Authors: Schilbach Karin , Ziegler Hendrik , Haarer Jan , Sterk Marco , Rammensee Hans-Georg , Handgretinger Rupert
Published: January 2023

The T cell compartment can be divided in two large classes: αβ and γδ T cells. It is believed that they develop in the thymus from a common progenitor and that the choice between αβ and γδ T cell fate is the first lineage decision made by progenitors after they commit to the T-cell lineage. However, here we show that peripheral Vδ1+ γδ T cells in an inflammatory environment can transdifferentiate into αβ T cells. Upon their extrathymic route of differentiation, that resembles well-characterized molecular program of thymic αβ lineage development, Vδ1+ T cells upregulate CD4+ coreceptor, develop Vδ1+ CD4+CD8+ double positive cells, show heterodimeric CD8αβ, transcribe RAG, preTα and express a particular Vβchain on their surface. Simultaneously inflammation confers controlled initiation of rearrangement in the TCRα locus. Transdifferentiation of Vδ1+ T cells at the clonal and bulk-culture level into functional CD4+ or CD8+ αβ T cells via a Vδ1+TCR/αβ+TCR double positive stage suggests that, upon inflammatory stimuli, Vδ1+ T-cell conversion participates in the induction of adaptive immune responses. Identifying an innate T cell as an αβ T-cell progenitor and showing the developmental steps linking the progenitor to adaptive, thymus-independant αβ T-cell responses as inflammatory conditions is of utmost relevance and will deeply impact evaluation of immune responses in infection, malignancy and autoimmune processes.
Enhancing T lineage production in aged mice: a novel function of Foxn1 in the bone marrow niche (P4475)
Authors: Le Phong , Zook Erin , Zhang Shubin , Gerstein Rachel , Witte Pamela
Published: January 2023

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. While reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with ETP, derived from MPP in the BM. A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1Tg; whether the effect is initiated in the BM with MPP is not known. Here, we report that Foxn1 is expressed in Wt BM and over expressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of HSC. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, CLP and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared to Wt. We identified a novel population of lineageneg/low, EpCAMpos, CD138neg cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, suggesting that the Foxn1 BM niches function in skewing MPP development toward T lineage progenitors.
Nr4a1 regulates thymic resident macrophage development and function (P4468)
Authors: Tacke Robert , Nowyhed Heba , Wu Amy , Hedrick Catherine
Published: January 2023

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, function in tissue homeostasis, and also regulate immune responses. Tissue resident macrophages in a variety of tissues arise from the yolk sac, and are generated independently of hematopoietic stem cells. However, the origin of thymic macrophages remains poorly understood. Nr4a1 is an orphan nuclear receptor that limits pro-inflammatory cytokine production in macrophages, and functions as the master regulator of Ly6C- patrolling monocyte development. Here, we report a significant defect in the development of thymic resident macrophages in Nr4a1-deficient animals. Thymic macrophage levels were reduced over two-fold compared with wild-type controls. However, Nr4a1 does not globally regulate the development of tissue macrophages, as macrophages resident in the spleen, lung, brain, pancreas, and peritoneum from Nr4a1-deficient mice develop normally. Moreover, Nr4a1-deficient thymic macrophages fail to clear apoptotic thymocytes in vivo, and express reduced levels of genes important for apoptotic cell engulfment, including engulfment receptors Mertk, and Axl. Defective thymic macrophage development likely impairs proper T cell selection, thus altering the immune system as a whole. Together, these data show that Nr4a1 regulates the development and functional capacity of tissue resident macrophages in the thymus, and provide a novel mechanism for maintenance of thymic homeostasis.
Muc1 regulates MDSC expansion and suppressive activity in healthy and tumor-bearing mice (P2049)
Authors: Mukherjee Pinku , Sahraei Mahnaz , Curry Jennifer , Das Roy Lopamudra , Gendler Sandra
Published: January 2023

We demonstrate that a membrane-tethered epithelial mucin, Muc1 affects the differentiation of immature myeloid derived cells (IMCs) in the bone marrow (BM). First, in wild-type (WT) mice, BM-derived Lin-Sca1+ hematopoietic progenitor cells (HPCs) express Muc1. Second, in Muc1-/- mice, the percentage of IMCs is higher and these cells express higher levels of c-MYC as compared to IMCs from WT mice. Interestingly, the myeloid derived suppressor cells (MDSCs) in the Muc1-/- tumor-bearing mice are less differentiated and have higher suppressor function as compared to MDSCs from WT mice. Because of the higher suppressive functions of the MDSCs in the Muc1-/- mice, we confirm that subcutaneous tumors develop significantly faster in these mice as compared to the WT mice (Poh 2009). Further, Muc1-/- mice have decreased NK cell function possibly due to increased suppressive function of their MDSCs. Decreased cytotoxic function of NK cells can partially explain the increased susceptibility of Muc1-/- mice to tumor growth. Lastly, we show that MDSCs from Muc1-/- mice have higher levels of phospho-STAT3, which is known to be critical for MDSC expansion. This finding is of great importance since systemic targeting of Muc1 in several carcinomas is being actively explored. It becomes clear that therapeutic targeting of Muc1 must be localized to the epithelial tumors because down-regulating Muc1 expression in hematopoietic progenitor cells may lead to increased MDSCs.
Medroxiprogesterone acetate drives expansion of CD11b+Gr1high myeloid-derived suppressor cells which suppress NK cell effector functions in tumor-bearing mice (P2059)
Authors: Zwirner Norberto , Spallanzani Raul , Dalotto Tomas , Rossi Lucas , Avila Damian , Ziblat Andrea , Domaica Carolina , Fuertes Mercedes , Raffo Iraolagoitia Ximera , Rabinovich Gabriel , Salatino Mariana
Published: January 2023

Hormone replacement therapy with the progesterone analogue medroxyprogesterone acetate (MPA) is widely used in postmenopausal women for the treatment of endometrial conditions, and as a contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Our aim was to explore if MPA threats immunosurveillance to tumors by effecting myeloid-derived suppressor (MDSCs; CD11b+Gr1+) and NK cells in mammary tumor-bearing mice. We used the highly metastatic 4T1 breast tumor which does not express classical progesterone or glucocorticoid receptors. MPA did not affect primary tumor growth in 4T1-tumor bearing mice but promoted lung metastasis burden. This effect was accompanied by expansion of splenic CD11b+Gr1+ cells (mostly CD11b+Gr1high cells). Sorted CD11b+Gr1+ cells from MPA-treated tumor bearing mice showed a more pronounced inhibitory activity of NK cell degranulation in response to YAC-1 cells and IFN-γ production in response to cytokines than those isolated from untreated tumor-bearing mice. Also, MPA significantly increased the percentage of spleen NK cells in tumor-bearing mice with similar lung infiltration of CD11b+Gr1+ and NK cells as compared to untreated tumor-bearing mice. We conclude that in breast cancer-bearing hosts MPA promotes the accumulation of CD11b+Gr1+ which suppress NK-cell mediated anti-tumor activity potentially contributing to tumor progression and metastasis.
Overexpression of βarrestin-2 in prostate cancer cells from African American males may contribute to aggressive tumor growth (P2075)
Authors: Thomas Ariel , Rivers E. , Smith Nikia , Richardson Ricardo
Published: January 2023

Prostate cancer is the second leading cause of death in African American males. Its incidence is ~60% higher in African American males relative to Caucasian American males. More aggressive prostate cancer is attributed to cancer that is androgen independent resulting in lack of response to androgen replacement therapy. βarrestins are scaffold proteins that promote G protein coupled receptor desensitization and internalization. βarrestin-2 was recently shown to reduce Androgen Receptor expression in prostate cancer cells. The goal of this study was to determine the levels of expression of βarrestin-1/2 in prostate cancer cells from both racial groups and assess their roles in tumor aggressiveness. To that end, prostate cancer cell lines from African Americans (MDAPCa2B, E006AA), Caucasian Americans (LNCaP, PC3), and control (RWPE-1) were assayed for βarrestin-1/2 expression and cell proliferation. βarrestin-1 expression was elevated in all prostate cancer cell lines whereas βarrestin-2 overexpression was observed only in MDAPCa2B. Interestingly, MDAPCa2B showed greater cell proliferation relative to LNCaP and PC3. Suppression of βarrestin-2 in MDAPCa2B cells reduced cell proliferation and tumor growth in nude mice. Altogether the data indicate that 1) βarrestins modulate prostate cancer growth, and 2) overexpression of βarrestin-2 in prostate cancer cells from African Americans relative to Caucasian Americans may contribute to the prostate cancer disparity.
Collagen matrix deposition by hepatic stellate cells protects hepatocellular carcinoma from NK-mediated cytotoxicity (P2013)
Authors: Mailloux Adam , Epling-Burnette Pearlie
Published: January 2023

Liver fibrosis (LF), a leading risk factor for hepatocellular carcinoma (HCC), is caused by collagen-producing hepatic stellate cells (HSC) activated during chronic inflammation secondary to hepatitis-C infection, or alcohol liver disease (ALD). NK cells promote disease resolution via RAE1-dependant HSC clearance. A defect in NK function by an ill-defined mechanism contributes to a pro-fibrotic response. Using an engineered bioartifical collagen matrix, the expression of inhibitory leukocyte associated IgG-like receptor-1 (LAIR-1) was increased on dividing NK cells and IL-2-induced NK proliferation was inhibited. Thus, we hypothesized that the accumulation of HSC-derived collagen directly inhibits NK activity and protects both HSC and HCC from innate clearance. We found that deposition of native collagen matrix for 72h protected HSCs from NK lysis in Cr51 cytotoxicity assays. A HCC cell line (HEPG2), which produced no collagen when cultured in the absence of HSCs, was susceptible to activated NK killing and was unaffected by collagenase. To test if HSC-derived collagen protects HEPG2 cells, the populations were differentially labeled with ipophilic dyes and co-cultured for 72h for use in flow-based assays. Collagen matrix protected both targets, while collagenase pretreatment restored NK sensitivity. These results suggest that HSC-derived collagen may contribute to NK dysfunction in LF and HCC, and identifies LAIR-1 as a potential mediator of tumor-induced immune suppression.
Impact of PPV-23 vaccine in HIV patients under long term HAART (P4262)
Authors: Iyer Anita , Khaskhely Noor , Leggat David , Westerink M A Julie
Published: January 2023

HIV infection is characterized by profound changes in the B cell compartment, which includes, impaired antigen specific response and a significant loss of IgM memory B cells, amongst other defects. Despite HAART’s success in reducing the incidence of opportunistic infections, invasive pneumococcal disease in the HIV infected population continues to be high. PPV-23 vaccine is recommended as a prophylactic measure in the HAART treated population; however, its serological benefit is currently unclear. Although, HAART has been highly successful in reconstituting T cell function, the effect of HAART on antigen specific B cell reconstitution remains to be elucidated. We developed a novel method to identify pneumococcal polysaccharide responding B cells in circulation by conjugating vaccine serotype 14 and 23 to fluorescent molecules. Using this method, we investigated the polysaccharide responding B cells in HIV cohorts on long term HAART. Preliminary data suggests severe depletion of polysaccharide responding IgM memory B cells in HAART treated adults comparable to HAART naïve group; a striking contrast to HIV uninfected adults. This is in alignment with the diminished anti pneumococcal antibody response observed in the HAART treated group. HAART is thus unable to fully reconstitute the functional B cell subset required for protection against S.pneumoniae.
Comparative analysis of CD8 T cell response to cytomegalovirus or influenza virus in healthy human adults (P3002)
Authors: Chen Guobing , Solokina Arina , Oelke Mathias , Madara Karen , Wersto Robert , Schneck Jonathan , Weng Nan-ping
Published: January 2023

The competency of antigen specific (Ag+) lymphocytes plays a critical role in the adaptive immune response. However, little is known about the differences in the competency of Ag+ T cells against different viruses. Here, we compared the CD8 T cell responses of 23 healthy adults to CMV (pp65) or Flu (M1) peptides using an in vitro artificial antigen presenting system. We found that CD8 T cell responses to CMV or Flu varied in both proliferation and Granzyme B production and were independent of the frequency of initial CMV+ or Flu+ CD8 T cells. In the comparison of the responses of Naïve (Tn) and central memory (Tcm) CD8 T cells, Tcm exhibited a greater expansion than did Tn for both CMV and Flu antigen. Intriguingly, the variations of the expansion between CMV+ and Flu+ CD8 T cells were observed only in Tcm but not Tn cells, indicating the importance of antigen experience. Furthermore, the expansion ability was correlated with the telomere length in the initial CD8 T cells positively and expanded Ag+ CD8 T cells negatively, suggesting a role for telomere length in cell proliferation. Taken together, this study showed that incompetency of Ag+ CD8 T cells is associated with antigen experience and short telomere length in healthy adults. Currently, we are analyzing the usage of the TCR variable genes (alpha and beta) of CMV+ or Flu+ CD8 T cells by a single cell RT-PCR-Sequencing method to gain additional information about the structure of these TCR against CMV and Flu antigens.
Rate of AIDS progression is associated with gastrointestinal dysfunction in SIV-infected pigtail macaques (P3045)
Authors: Klatt Nichole , Canary Lauren , Vinton Carol , Morcock David , Estes Jacob , Brenchley Jason
Published: January 2023

During HIV/SIV infection, mucosal immune system dysfunction and systemic immune activation are associated with progression to AIDS, however it is unclear to what extent pre-existing gastrointestinal damage relates to disease progression after infection. Pigtail macaques (PTM) are an excellent model in which to assess mucosal dysfunction in relation to HIV/SIV pathogenesis, as the majority of these animals have high levels of gastrointestinal damage, immune activation, and microbial translocation prior to infection, and rapidly progress to AIDS upon SIV infection. Here we characterized the mucosal immune environment prior to and throughout SIV infection in 13 uninfected PTM and 9 SIV-infected PTM, of which 3 were slow progressors. This small subset of slow progressors had limited innate immune activation in mucosal tissues in the periphery, which was associated with a more intact colonic epithelial barrier. Furthermore, we found that pre-infection levels of microbial translocation, as measured by lipopolysaccharide binding protein (LBP), in PTM correlated with the rate of progression to AIDS. These data suggest that pre-existing levels of microbial translocation and gastrointestinal tract dysfunction may influence the rate of HIV disease progression.
Comparison of the early innate immune responses of Vibrio parahaemolyticus and Vibrio cholerae in human intestinal epithelial cells (P1237)
Authors: Winstead Candace , Buck Evan , El Adaimi Jad , Moores Thomas , Slocum Tyler , Moyle Misty
Published: January 2023

Diarrheal disease is one of the leading causes of death in low-income countries. Two pathogens responsible for these diseases are Vibrio parahaemolyticus and Vibrio cholerae. Although from the same genus, they have distinct virulence factors and induce different host responses. This study evaluates the early immune response of human intestinal epithelial cells (HT-29) to Vibrio pathogens by comparing changes in gene expression induced by the bacteria using quantitative PCR (qPCR) arrays. 84 different genes in an anti-bacterial inflammatory response were compared in cells stimulated in triplicate for two hours with V. parahaemolyticus and V. cholerae. Overall, V. cholerae upregulated many more genes (24 total) than V. parahaemolyticus (11 total). Both species induced expression of the pattern recognition receptor TLR1 and the chemokine CCL5 as well as unexpectedly increasing expression of several putative antiviral mediators including DDX58, IFNGR1 and MHC I. Genes upregulated by only V. parahaemolyticus include IL-8, IRAK-1, MAPK-8, and TLR-5, whereas V. cholerae upregulated NFKB1A and ICAM-1. Interestingly, many more genes were downregulated by V.parahaemolyticus (31 total) compared to V. cholerae (15 total), and several genes downregulated by V. parahaemolyticus (CCR4, TLR4, TLR6 and IL13) were upregulated by V. cholerae. This comparative expression approach with related pathogens may allow patterns to emerge that will guide future experiments.
LC3-associated phagocytosis is a critical regulator of innate immunity. (P1261)
Authors: Martinez Jennifer , Oberst Andrew , Devi-Kanneganti Thirumala , Green Douglas
Published: January 2023

The magnitude and quality of the innate immune response is shaped by many factors, both cell-intrinsic and -extrinsic. The process of autophagy is a cell-intrinsic pathway that has been implicated in the suppression of innate pro-inflammatory cytokines, and polymorphisms in autophagy genes (ATG16L) have been linked to inflammatory disease. A similar but distinct pathway, LC3-associated phagocytosis (LAP), is also important in shaping innate immune responses, largely by linking the sensing of PAMPS to the autophagic machinery, a process critical for the clearance of both pathogens and dead host cells. As autoimmunity may arise from a failure to engulf or degrade dying cells, we sought to determine if LAP might function to dampen inflammatory responses by halting otherwise constitutive signals from within the phagosome. Indeed, we show that mice with phagocytes deficient for LAP machinery (such as ATG7) exhibit increased pro-inflammatory cytokines levels in the serum when challenged with exogenous dead cells. Moreover, successful clearance of the pathogen Listeria monocytogenes requires LAP, rather than conventional autophagy. In order to fully understand this distinct and vital mechanism for effective host defense, it will be necessary to identify molecular events that are unique to LAP, which will allow us to further distinguish the physiological and pathological roles for LAP.
LPS-induced autophagy is mediated by nitric oxide in macrophages (P1285)
Authors: Park Hye-Jin , Pyo Suhkneung
Published: January 2023

Activation of macrophages is essential for the host defense system and accumulating evidence suggests that autophagy is an important component of macrophage activity. Lipopolysaccharide activates Toll-like receptor 4 leading to the release of pro-inflammatory cytokines and inflammatory mediators, which are necessary to activate innate immune response. Another downstream response to LPS/TLR4 signaling is the induction of autophagy, but the signal transduction pathway leading to autophagy is unclear. Here we demonstrate that nitric oxide, which is induced by LPS, induces autophagy via a number of mechanisms in macrophages. To verify the role of NO in autophagy, we compared autophagy-related gene expression in LPS-stimulated and SNP-stimulated macrophages by western blotting. Both LPS and SNP stimulate autophagy in macrophages. In addition, inhibition of NO production by NOS inhibitors, L-NMMA and SMT, significantly reduced LPS-induced autophagy. These finding imply that LPS-induced autophagy might be mediated via NO production. The inhibition of MAPK or NF-κB by pharmacological inhibitors also impaired activation of autophagy by LPS in macrophages. Overall, these finding indicate that LPS induces autophagy in macrophages via activating MAPK/NF-κB/iNOS signaling pathway.
Role of STAT1 in Chlamydia-induced type-I interferon production in murine oviduct epithelial cells (P1245)
Authors: Derbigny Wilbert , Hosey Kristen , Hu Sishun
Published: January 2023

We previously reported that C. muridarum-infected murine oviduct epithelial cells (OE cells) secrete IFN-β in a mostly TLR3-dependent manner. However, C. muridarum-infected TLR3-deficient OE cells were still able to secrete detectable levels of IFN-β into the supernatants, suggesting that other signaling pathways contribute to Chlamydia-induced IFN-β synthesis in these cells. We investigated the role of STAT1 as a possible contributor in the Chlamydia-induced type-1 IFN production in wild-type and TLR3-deficient OE cells. Our data show that C. muridarum infection significantly increased STAT1 gene expression and protein activation in wild-type OE cells; however, TLR3-deficient OE cells showed diminished STAT1 protein activation and gene expression. There was significantly less IFN-β detected in the supernatants of C. muridarum-infected OE cells derived from mice deficient in STAT1 when compared to wild-type OE cells; which suggest that STAT1 is required for the optimal synthesis of IFN-β during infection. Quantitative real-time PCR analyses of signaling components of the type-1 IFN signaling pathway demonstrated equal upregulation in the expression of STAT2 and IRF7 in the wild-type and TLR3-deficient OE cells, but no upregulation in these genes in the STAT1-deficient OE cells. Finally, experiments in which INFAR was blocked with antibody revealed that IFNAR-mediated signaling was critical to the Chlamydia-induced syntheses of IFNα-2 and IFNα-4, but not IFN-β.
The essential, non-redundant roles of RIG-I and MDA5 in detecting and controlling West Nile virus infection (P1385)
Authors: Errett John , Suthar Mehul , McMillan Aimee , Gale, Jr. Michael
Published: January 2023

Virus recognition by the innate immune system represents a critical component of host defense against infection. Proper activation of innate immunity and full immune protection to the emerging infectious disease of West Nile virus has previously been shown to be dependent on viral recognition by the host RIG-I-like receptor (RLR) protein family. RIG-I and MDA5 are the two members of the RLR family that have been shown to recognize viral RNA and to activate innate immune programs that suppress infection. In the current study we have expanded our understanding of the role of the RLRs in protection against West Nile virus infection by assessing the individual roles of RIG-I and MDA5 both in vitro and in vivo. The loss of either gene alone results in a deficit of innate immune signaling and virus control in vitro and increased mortality in vivo. Loss of both RIG-I and MDA5 results in a near complete absence of innate immune gene induction in key target cells of infection and a more severe pathogenesis very similar to that of cells and animals lacking the RLR central signaling adaptor MAVS. Additionally, we have shown that cells infected with West Nile virus contain two distinct pathogen associated molecular patterns that are sensed by RIG-I and MDA5 respectively. Our results highlight the necessity of multiple pattern recognition receptors in the same pathway acting in concert to affect a successful immunological outcome.
Differential effects of TGF-β1 on IL-5 and eotaxin-induced chloride currents in human blood eosinophils (P5130)
Authors: Agrawal Devendra , Kim Min-Jung , Gaurav Rohit
Published: January 2023

TGF-β1 is critical in the pathogenesis of chronic asthma and acts on eosinophils and other cells in the airways. Recently, we found TGF-β1 increases CLC3 expression in human blood eosinophils and induces migration. Here, we examined the interaction of TGF-β1 with IL-5 and eotaxin in the activation of chloride currents in human blood eosinophils. Eosinophils were purified (<99% pure, >98% viable) from the blood of healthy volunteers. Chloride current was measured at pH 7.4 by whole cell patch clamping in freshly isolated eosinophils using Axopatch 200B amplifier and analyzed by pClamp 10 software. Both eotaxin-1 and eotaxin-3 at low doses (0.075-10 ng/ml) increased outwardly rectified Cl- current in a time-dependent manner with maximum response at 500msec plus 100 mV. The eotaxin response at low doses (0.075-0.1 ng/ml) was slower in onset than at high dose. SB328437, a CCR3 antagonist (100 nM), inhibited eotaxin-induced Cl- current. Effect of eotaxin-3 was about 7-times larger than eotaxin-1. TGF-β1 (10ng/ml) decreased eotaxin-induced Cl- current. Incubation of eosinophils with IL-5 (10ng/ml) for 3hr decreased basal level of Cl- current. However, the addition of TGF-β1 with IL-5 significantly enhanced Cl- current. These data suggest differential effect of TGF-β1 in the activation and migration of human blood eosinophils in the presence of eotaxin or IL-5 in allergic airway inflammation and chronic asthma.
The role of CCR2 and CCR7 on dendritic cell migration to and from mycobacterial granulomas (P5096)
Authors: Harding Jeffrey , Schreiber Heidi , Clarkson Ben , Sandor Matyas
Published: January 2023

Dendritic Cells have a complex and critical role in mycobacteria-containing granulomas. Understanding the mechanisms that regulate DC responses during mycobacterial infection will aid the generation of novel vaccines and therapies. Using a novel murine model of granuloma transplantation, combined with the use of CD11c-YFP donors or recipients, we have reported on the incoming and outgoing migration of fluorescent inflammatory dendritic cells from acute and chronic BCG-containing granulomas. Here, we extend our studies to dissect the molecular components of DC involvement during murine mycobacterial infection. We examined CCR2, a chemokine that regulates migration into inflamed tissue, and CCR7, which regulates migration of dendritic cells from the granuloma to the lymph nodes. First, we crossbred CCR2 and CCR7 KO mice with CD11c-YFP mice and analyzed the resulting granulomatous response to BCG infection. We measured granuloma size and frequency, recruitment of different cell types, bacterial load of infected tissues, and spatial organization of CD11c-YFP cells in the granuloma. Then, using transplantation of CD11c-YFP CCR2 and CCR7 KO mice, we measured the chemokine-specific regulation on rates of DC migration in and out of tissue, as well as their role in bacterial dissemination.