bluuebunny/crossref_metadata_2025 huggingface dataset:
miR-144 is a negative regulator of the host antiviral response (158.10)
Authors: Rosenberger Carrie , Podyminogin Rebecca , Peschon Jacques , Rodriguez David , Gundapuneni Madhumati , Weiss Mitchell , Aderem Alan
Published: January 2023

Antiviral responses must be regulated to rapidly defend against infection while minimizing inflammatory damage, but the mechanisms for establishing the magnitude of response within an infected cell are not well understood. microRNAs are post-transcriptional regulators that are attractive candidates for fine tuning the magnitude of the immune response. We identify miR-144 as a negative regulator of the host antiviral response in the lung. We observed that miR-144 expression resulted in increased replication of influenza, EMCV, and VSV in primary mouse lung epithelial cells. We employed a systems biology approach to identify the transcriptional network regulated by miR-144 and demonstrate that it controls the TRAF6/IRF7 antiviral response by post-transcriptionally suppressing TRAF6 expression. Infection of IRF7-null mice revealed that IRF7 is necessary for normal expression of the network of genes that is perturbed by miR-144. Ectopic IRF7 expression restored the antiviral capacity of cells over-expressing miR-144 and reducing TRAF6 levels using shRNAs recapitulated the miR-144 phenotype. In vivo ablation of miR-144 resulted in reduced influenza replication within the lung. These data suggest that miR-144 regulates activation of the IRF7 pathway, thereby creating an exquisitely controlled antiviral response.
MicroRNA-155 enhances ocular inflammation after Pseudomonas aeruginosa infection (158.11)
Authors: Huang Xi , Peng Anping , Sun Mingxia , Wu Minhao , Zhang Ping
Published: January 2023

Abstract: As a gene regulator, microRNA (miR)-155 plays an important role in both autoimmune inflammation as well as antiviral innate immunity. However, the role of miR-155 in Pseudomonas aeruginosa (PA) keratitis remains unknown. Our study demonstrated that miR-155 expression levels were significantly up-regulated in PA-infected vs normal human corneas as well as Macrophages derived from PBMC. Furthermore, in vivo studies showed that miR-155 expression was significantly increased in PA-infected corneas of C57BL/6 (B6) mice. Inhibition of miR-155 led to reduced ocular disease after PA infection, by down-regulating pro-inflammatory cytokine (e.g., IL-1β, IFN-γ, IL-6, IL-12) production. In vitro studies also demonstrated that PA challenge induced miR-155 expression in a time- and dose-dependent manner in peritoneal Macrophages and RAW264.7 cells. The induction of miR-155 was dependent on TLR2, TLR4, TLR9, through NF-κB, IKK, JNK and PI3K, but not P38 or ERK pathways. Moreover, overexpression of miR-155 significantly decreased Macrophage-mediated phagocytosis and intracellular bacterial killing of PA, by down-regulating expression of mannose receptor (MR) and complement receptor 3 (CR3). miR-155 overexpression also enhanced the production of pro-inflammatory cytokines including IFN-γ, IL-1β, IL-6, IL-12, IL-17A, but reduced anti-inflammatory cytokine IL-10 expression. Collectively, these data indicated that miR-155 functions as an inflammatory amplifier in PA keratitis.
Haplotype specific activating NK cell receptors provide recognition of virus-infected cells and mediate resistance to infection (158.7)
Authors: Pyzik Michal , Charbonneau Benoit , Gendron-Pontbriand Eve-Marie , Babic Marina , Krmpotic Astrid , Jonjic Stipan , Vidal Silvia
Published: January 2023

The ability of numerous activating Ly49 receptors to recognize mouse cytomegalovirus (MCMV) infected cells of various H2 background was examined. Reporter cells expressing either Ly49P1 from NOD/Ltj mice, Ly49L from BALB mice or Ly49D2 from PWK/Pas mice were activated upon co-culture with MCMV infected cells provided the presence of an appropriate H2 molecule and the m04/gp34 viral peptide. Such recognition was most frequent in the H2k background, yet also occurred in the H2d and H2f contexts. To demonstrate whether Ly49 activating receptors confer MCMV resistance, H2 congenic BALB mice were infected with MCMV. Neither BALB.By (H2b) nor BALB/c (H2d) mice were able to control MCMV spread in target organs contrary to BALB.K (H2k) mice, which demonstrated delayed NK cell-mediated virus control. In BALB mice, the antibodies YE1/48 and 12A8 delineate a Ly49L+ NK cell population (YE1/48INT12A8-). BALB.K resilience to the infection correlated with the expansion of the specific Ly49L+ NK cell fraction in the spleen and secretion of IFNγ 6 days after MCMV infection. Adoptive transfer of Ly49L+ but not Ly49L- NK cells provided partial protection from death in neonate BALB.K mice following MCMV infection. Thus, we have uncovered three activating Ly49 receptors partaking in the recognition of MCMV infection by NK cells through an H2- and m04-dependent mechanism, which we find to be more common than the direct recognition of m157.
Elevated numbers and frequencies of CD56- CD3- NK cells with high perforin occur in healthy donors (160.14)
Authors: Hudig Dorothy , Barao Isabel , Hunter Kenneth , Diamond W. , Redelman Doug
Published: January 2023

Routinely, human natural killer (NK) lymphocytes are identified as CD3eneg CD56bright or dim. CD3neg CD56neg cells with low perforin are found in viremic patients infected with human immunodeficiency virus and hepatitis C virus (reviewed, N.K. Bjorkstorm et al., Trends Immunol. 31:401, 2010). To our surprise, we found by flow cytometry that CD3neg perforin(PRF1) positive NK cells included as many as 50% CD56neg cells within the blood of 97 healthy subjects. Among these subjects (aged 21 to 62 years), the CD3eneg PRF1positive cells included at least 10% CD56neg cells in 24 of 97 (~25%) subjects. CD56neg perforin high CD3neg cells were over 50 cells/mm3 of blood in 14% of the healthy donors. As anticipated, the CD56dim cells had ~7 fold higher perforin MFI than the CD56bright cells. Notably in the healthy donors with high frequencies of CD56neg cells, these cells had bimodal perforin levels comparable to the levels in the CD56bright and CD56dim subsets. The perforin levels indicate that many CD56neg NK cells may have strong cytotoxicity in healthy donors in contrast to the low perforin and cytotoxicity observed in CD56neg NK cells of the viremic patients. We conclude that CD56neg NK cells now warrant attention as potential cytotoxic cells after receiving considerable attention as high chemokine-secreting cells.
Impact of T cell receptor reactivity and HLA phenotype on naive CD8 T cell frequency in humans (160.16)
Authors: Bonneville Marc , Legoux Francois , Saulquin Xavier
Published: January 2023

The impact of major histocompatibility complex (MHC) phenotype on the shaping of the peripheral naive T cell repertoire in humans remains unknown. Here we exploited a pMHC multimer-based sorting approach to analyze the impact of expression of a given human leukocyte Ag (HLA) class I allele on the frequency and quality of peripheral blood and thymic T cells directed against immunodominant self, viral and tumor antigens presented by a human MHC class I allele (HLA-A*02, referred to as A2) in individuals expressing or not this allele. Naive T cell frequencies varied from one antigen specificity to another but were restrained for a given specificity. While A2-restricted T cells showed similar repertoire features and antigenic avidities in A2+ and A2- donors, A2 expression had either a positive, neutral or negative impact on the frequency of A2-restricted naive CD8 T cells, depending on their fine specificity. We also identified in all donors CD4 T cells specific for A2/peptide complexes, whose frequencies were not affected by MHC class I expression, but nevertheless correlated with those of their naive CD8 T cell counterparts. Therefore both selection by self MHC and inherent TCR reactivity regulate the frequency of human naive T cell precursors. Moreover this study also suggests that T cell repertoire shaping by a given self MHC allele is dispensable for generation of immunodominant T cell responses restricted by this particular allele.
Thymocyte specific interleukin-7 restores T cell development but not homeostasis in IL-7KO mice (160.6)
Authors: Kim Grace , Lucas Philip , Gress Ronald , Park J.
Published: January 2023

T cells are critically dependent on interleukin-7 (IL-7) as IL-7-deficient mice fail to generate and maintain a functional T cell compartment. Importantly, IL-7 is not produced by T cells and it is only provided by dendritic cells, stromal cells and other non-T cells in trans. Using an lck-proximal enhancer/promoter driven IL-7 transgene (IL-7Tg), here we tested whether T-lineage specific expression of IL-7 can restore T cell development in IL-7 deficient mice. We found that T cell development was completely restored in IL-7Tg.IL-7KO mice as thymocyte numbers, thymic selection, and CD4/CD8 lineage differentiation were identical to WT mice. Importantly, however, IL-7Tg.IL-7KO mice were peripheral T lymphopenic with less than 20% of T cell numbers compared to WT controls. This was presumably induced by termination of transgenic IL-7 expression upon positive selection as qPCR analysis showed a rapid downregulation of IL-7 mRNA expression in mature thymocytes. Surprisingly, the few surviving peripheral T cell in IL-7Tg.IL-7KO mice did not show upregulation of activated or memory T cell markers,and we did not observe any autoimmunity. Further characterization of the function and phenotype of these T cells are currently under progress. Collectively, these data show that IL-7 expression and signaling in cis is sufficient for normal T cell development, and they show in a new in vivo model of peripheral T lymphopenia that continuous IL-7 is critical T cell homeostasis.
CD40 engagement ablates CTLA-4 expression on Th40 effector cells: a mechanism for breach of tolerance (160.9)
Authors: Wagner David , Carter Jessica , Vaitaitis Gisela , Waid Dan
Published: January 2023

Biomarkers defining pathogenic effector T cells have been slowly forthcoming. Towards this we identified CD4+ cells that express CD40 (Th40), and defined these cells as pathogenic in the NOD type 1 diabetes (T1D) model. Th40 cells are unique in their ability to adoptively transfer T1D. To study the origin of Th40 cells and disease pathogenesis we employed a dual transgenic model expressing OVA323-339 peptide as a neo-self antigen on islet beta cells and medullary thymic epithelial cells (mTEC) and a transgenic TCR recognizing the OVA323-339 peptide. Th40 cells and Tregs that both recognize the same cognate antigen, rather than being deleted through central tolerance drastically expand. DO11.RIPmOVA mice develop an autoimmune-type diabetes very rapidly. In pancreatic lymph nodes Th40 cells are greatly expanded while Tregs remain equivalent to controls and importantly remain functional throughout the disease process, suggesting another breach of tolerance mechanism. DO11.RIPmOVA Th40 cells do not express CTLA-4, a molecule for cell auto-regulation, while DO11 Th40 cells do. CD3/CD28 engagement induces CTLA-4 cell surface expression on DO11 Th40 cells while CD40 co-engagement ablates that induction. These data suggest a unique means for Th40 cells to thwart tolerance.
Lymphatic pump treatment enhances pulmonary immunity and protects against solid tumor formation in the rat lung. (161.14)
Authors: McCauley Lyndsey , Pedrueza Mayela , Zhang Xin , Jones Harlan , Hodge Lisa
Published: January 2023

Lymphatic pump treatments (LPT) are used by osteopathic physicians to treat edema and infection. In animals, LPT increases lymph flow and leukocyte numbers, which may enhance protection against infection and cancer. To determine if LPT would enhance anti-tumor immunity and reduce tumor formation in the lungs, F344 rats were injected intravenously with MADB106. Rats received no treatment (control), 4 min of light touch under anesthesia (sham), or 4 min of LPT under anesthesia, for 7 days. At day 8, tissues were measured for tumors and leukocyte numbers/activities. Serum was measured for vascular endothelial growth factor C (VEGF-C). LPT reduced solid tumors and increased B cell, CD4+ T cell, CD8+ T cell, NK cell and macrophage numbers in the lungs. In addition, Leukocytes isolated from the lungs of LPT rats secreted more IL-2, IL-10 and IFN-γ but did not enhance tumor lysis in vitro. Previously we demonstrated LPT mobilizes gut-derived lymphocytes into lymphatic circulation; therefore, we stained pulmonary lymphocytes for the gut marker α4β7. LPT increased α4+β7+ leukocytes in the lungs, suggesting LPT mobilizes gut-derived leukocytes into the lungs. Finally, there were no differences in the levels of VEGF-C between treatment groups, suggesting that LPT does not promote lymphangiogenesis in this model. Our data suggests LPT inhibits pulmonary tumor formation by enhancing the numbers of leukocytes with anti-tumor activities that migrate into the lungs.
Defining the phenotype, migration, and longevity of true tissue lung CD8 T cells. (161.15)
Authors: Anderson Kristin , Skon Cara , Vezys Vaiva , Masopust David
Published: January 2023

Thus far, CD8 T cell responses to respiratory infections have been restricted to broad tissue analyses, which may not accurately represent the true nature of the tissue, nor the response to infection. In order to thoroughly explore the dynamics of the CD8 T cell response, we used respiratory and systemic infection models to perform a detailed analysis of the anatomic location, phenotype, migration capacity, and longevity of CD8s in the lung. Interestingly, we found that as many as 98% of the T cells that would commonly be perceived to be in the tissue were actually in the capillaries. We show that the phenotype of cells in the vasculature is distinct from cells in the tissue and the airway, and that migration to the tissue is chemokine dependent. Additionally, local infections generate a memory CD8 population with a tissue distribution different than a systemic infection. These findings have implications for establishing and maintaining CD8 T cell dependent immunity within the lung.
Interleukin-33 and its receptor ST2 contribute to inflammation in the lungs of patients with idiopathic pulmonary fibrosis (161.16)
Authors: Pickering Edward , Todd Nevins , Lockatell Virginia , Atamas Sergei , Luzina Irina
Published: January 2023

Interleukin (IL)-33 and its receptor ST2 have been recently implicated as critical regulators of responses to injury and of ensuing inflammation. The roles of IL-33 and ST2 in pulmonary inflammation and scarring remain unclear. We analyzed bronchoalveolar lavage (BAL) from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by inflammation, with known elevated levels of IL-8 and excessive collagen deposition in the lungs. IL-33 was not found in BAL from 5 controls but was found in 7 out of 11 IPF samples, at 10.0 ± 3.2 pg/ml. Soluble ST2 was found in BAL from healthy controls at 93.2 ± 82.1 pg/ml, and in all IPF samples at 352.1 ± 135.2 pg/ml (p < 0.05, Mann-Whitney U test). The elevation of IL-33 in BAL suggested that this cytokine may act directly on pulmonary epithelial cells and through this action contribute to inflammation. Cultured human primary pulmonary epithelial cells were stimulated with rhIL-33, and the levels of pro-inflammatory cytokines IL-6, IL-8, MIP-1α, and TNF-α in cell culture media were analyzed by ELISA. Of the analyzed cytokines, only the levels of IL-8 were increased by 2.0 ± 0.2 fold at 24 h and 1.7 ± 0.2 fold at 48 h. In conclusion, levels of IL-33 and ST2 are elevated in the lungs of IPF patients; IL-33 may contribute to inflammation and fibrosis by activating IL-8 production in pulmonary epithelium.
Prophylactic and therapeutic targeting of the neurokinin-1 receptor limits neuroinflammation in a murine model of streptococcal meningitis (161.7)
Authors: Chauhan Vinita , Marriott Ian
Published: January 2023

Bacterial meningitis is a rapid, severe and highly damaging infection of the central nervous system (CNS) that is accompanied with high degree of inflammation. We have previously determined an important role for the neuropeptide substance P (SP) in the initiation and progression of bacterially-induced CNS inflammation. In the present study, we provide evidence that SP augments inflammatory glial responses to the important Gram-positive CNS pathogen Streptococcus pneumoniae, and show that endogenous SP/NK-1 receptor interactions play a critical role in the development of neuroinflammation and neurological damage in a mouse model of streptococcal meningitis. Moreover, we show that therapeutic intervention with a systemically delivered NK-1 receptor antagonist attenuates increases in inflammatory cytokine levels and decreases in immunosuppressive cytokine production within the CNS associated with an established in vivo S. pneumoniae infection. Furthermore, we have shown that treatment with an NK-1 receptor antagonist reverses infection-associated pathology in the absence of effects on bacterial burden within the CNS. These results provide the first demonstration that the therapeutic targeting of SP/NK-1 receptor interactions can limit progressive neuroinflammation and neurological damage associated with bacterial infections of the CNS. NK-1 receptor antagonists could, therefore, represent a new strategy for the treatment of potentially lethal CNS inflammation.
Identification of a role for TLR3 in the inflammatory response to Chlamydia infection of murine oviduct epithelial cells. (162.11)
Authors: Derbigny Wilbert , Shobe LaTasha , Kamran Jasmine
Published: January 2023

Because epithelial cells are the major cell type productively infected with Chlamydia during genital tract infections, the overall goal of our research is to understand the contribution of the infected-epithelial cells to the host defense. We showed that TLR3 is the critical PRR in oviduct epithelial (OE) cells that is stimulated during Chlamydia infection, resulting in the synthesis of IFN-β. Our ELISA data indicate that expression of other innate immune modulators including IL-6, CXCL-10, CXCL-16, and CCL-5 were induced in the infected wild-type OE cells; however, this induction was not observed in the TLR3-deficient OE cells. Pre-treatment of the TLR3-deficient OE cells with 50 U/ml IFN-β prior to infection restored the ability of Chlamydia infection to induce IL-6, CXCL10, and CCL-5 expression; however CXCL-16 induction was not restored by IFN-β pre-incubation in the TLR3-deficient cells. Our findings were corroborated in pathway-focused PCR arrays which demonstrated a plethora of different inflammatory genes that were defectively regulated during Chlamydia infection of the TLR3-deficient OE cells, and that some of these genes were induced only when IFN-β was added prior to infection. Our OE cell data implicate TLR3 as an essential inducer of INF-β and other inflammatory mediators during Chlamydia infection, and highlights the importance of the role of TLR3 during the inflammatory cytokine response.
Pegylated leptin antagonist ameliorates chronic colitis by inducing regulatory T cells (Tregs) partially mediated by TGF-beta induction (162.4)
Authors: Singh Udai , Singh Narendra , Singh Balwan , Price Robert , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

IIBD is a chronic intestinal inflammation caused by hyper activated effector immune cells that affects millions of people around the world. Several lines of evidence support an important role for TGF-beta in the induction of tolerance and control gut inflammation. Leptin is satiety hormone produced primarily by adipose tissue, which increases during IBD progression. We investigated the effect of pegylated leptin antagonist mediated inhibition of chronic colitis in IL-10-/- mice. In the present study, we report that pegylated leptin antagonist (mutant L39A/D40A/F41A) effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis including a decrease in body weight, systemic serum amyloid A (SAA), and leptin levels. The percentage of Tregs in the spleen, mesenteric lymph node (MLN) and intestinal lamina propria (LP) of colitis mice was increased after leptin antagonist treatment as compared to control mice. Similarly, the percentage of TGF-beta in the MLN and LP of colitis mice increased after leptin antagonist treatment. Leptin antagonist also reduced systemic and mucosal cytokines as well as increased the insulin level as compared to control mice. These findings suggest that leptin antagonist ameliorates chronic colitis by inducing TGF-beta, which mediates the induction of Tregs and reduces both mucosal and systemic cytokines. The intricate interaction among leptin, TGF-beta and Tregs might provide new strategies for therapeutic interventions.
IL-10 production by B cells is not required for B cell-mediated suppression of contact allergy (163.24)
Authors: Surianarayanan Sangeetha , Karp Christopher L. , Muller Werner , Roers Axel
Published: January 2023

B cells play important roles in the activation of T cells. On the other hand, regulatory functions have been ascribed to B cells based on findings in animal models. Enhanced autoimmunity and contact allergy was found in B cell deficient mice. These data supported the concept of distinct subsets of regulatory B cells specialized in the suppression of immune responses. IL-10 was proposed as one of the key suppressive mechanisms of such cells. An IL-10 producing subpopulation of B cells characterized by expression of CD5 and CD1d, B10 cells, was reported to suppress the T cell response to contact allergens and to ameliorate experimental autoimmune encephalitis. Unexpectedly, our analysis of conditional B cell-specific IL-10 deficient mice revealed no deregulation of contact allergy. This was not due to inefficient Cre/loxP-mediated inactivation of the IL-10 gene in the CD5+CD1d+ B cell subset or in conventional B cells as shown by cell sorting and single cell PCR. We conclude that IL-10 production is not required for regulation of contact allergy by B cells.
Novel strategies to suppress NF-κB in experimental autoimmune encephalomyelitis (164.17)
Authors: Janardhanam Srihari , Srinivasan Mythili
Published: January 2023

Biological strategies that selectively harness the therapeutic benefits of glucocorticoids (GC) are highly attractive in the management of inflammatory conditions such as multiple sclerosis (MS). Glucocorticoid induced leucine zipper (GILZ), is a GC induced protein that binds the p65 subunit of nuclear factor kappa B(NF-κB, prevents its nuclear translocation and suppress inflammatory cytokines. GILZ over-expressing mice exhibit reduced T-bet expression and IFN-γ secretion, a canonical Th1 transcription factor and prototype Th1 cytokine respectively. We hypothesized that the intracellular delivery of GILZ protein may suppress delayed type hypersensitivity reaction, a Th1 response that mimic inflammation in MS. IFN-γ and IL-12 secretion was significantly reduced and that of IL-10 was elevated in human peripheral blood mononuclear cells stimulated with protein peptide derivative in the presence of GC or exogenous GILZ. We next investigated the therapeutic potential of GILZ supplement in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Groups of SJL/J mice induced EAE and administered GILZ protein exhibited significantly reduced clinical disease than vehicle treated mice. Upon re-stimulation with CNS antigens lymphocytes from GILZ protein administered mice exhibited significantly lower proliferation and inflammatory cytokine secretion (TNF-α and IL-12). These observations suggest a therapeutic potential for exogenous GILZ in MS.
Identification of new cancer/testis antigens in non-small cell lung cancer (165.16)
Authors: Chiriva Maurizio , Mirandola Leonardo , Yu Yuefei , Kim Minji , Alalawi Raed , Asawa Chompunut , Jumper Cynthia , Cobos Everardo
Published: January 2023

Lung cancer is the leading cause of cancer death in both genders worldwide. The incidence is only second to prostate cancer in men and breast cancer in women. The lethality of the disease highlights the urgent need for innovative therapeutic options. Immunotherapy can afford efficient and specific targeting of tumor cells, reducing the need for invasive conventional treatments. We have previously reported the aberrant expression of cancer/testis antigens (CTA) in multiple myeloma and ovarian cancer. Here, we show that the CTA, SP17, AKAP-4 and PTTG-1 are aberrantly expressed in lung cancer cell lines and primary cells from patients by RT-PCR, immunohistochemistry and flow cytometry. Our results provide for the first time that CTA/SP17-AKAP-4-PTTG-1 are expressed and are highly immunogenic in advanced non-small cell lung cancer. Further work is warranted to develop CTA tailored immunotherapeutic strategies for non-small cell lung cancer.
Development of an attenuated interleukin-2 cytokine fusion protein that can be activated by tumor expressed matrix metalloproteinases (165.19)
Authors: Skrombolas Denise , Puskas John , Sedlacek Abigail , Lord Edith , Frelinger John
Published: January 2023

Cytokines have been used to treat tumors, however, side effects often limit their use when delivered systemically. We set out to develop an approach in which cytokines would be preferentially activated at the site of growing tumors. We have constructed a novel fusion protein (FP) consisting of the cytokine interleukin-2 (IL-2) joined to a specific inhibitor moiety, the interleukin-2 receptor alpha chain separated by a protease cleavage sequence recognized by matrix metalloproteinases (MMPs). The rationale is that the cytokine would be relatively inactive in the intact fusion protein but its biological activity would increase after protease cleavage. We analyzed a baculovirus expressed FP biochemically by immunoblot analyses and functionally using the IL-2 dependent CTLL-2 cell line. IL-2 activity is attenuated over 50 fold in the intact FP compared to the same molar amount of IL-2. MMP cleavage releases IL-2 resulting in an increase in the amount of biologically available IL-2. Additionally, the FP showed efficacy in vivo using a peritoneal tumor model employing Colon 38. This general approach may be applicable to other cytokines as well as other proteases.
PI3K-Akting in crosstalk between Notch1-Jagged2 and CD28-B7 pathways in dendritic cells (165.21)
Authors: Koorella Chandana , Nair Jayakumar , Carlson Louise , Murray Megan , Rozanski Cheryl , Lee Kelvin
Published: January 2023

Survival of myeloma cells is characterized by their critical interaction with bone marrow stromal cells, which produce IL-6. At the cellular level, dendritic cells (DC) in the bone marrow microenvironment and at the molecular level the CD28-B7 and Notch1-Jagged2 pathways were separately implicated by us in myeloma induced IL-6 production. While Notch signaling leading to IL-6 production in DC is well understood, the mechanism of “backsignaling” via B7, a ligand with a short cytoplasmic tail, is largely uncharacterized. Inhibiting Notch signaling using pharmacological inhibitors and blocking antibodies leads to a significant decrease in CD28 mediated IL-6 production by DC suggesting crosstalk between the Notch1-Jagged2 and CD28-B7 pathways. To underline the molecular mechanism of crosstalk, we blocked PI3K and PKC activity using Ly294002 and Bis respectively and observed a significant decrease in IL-6 production upon B7 crosslinking. Additionally, CD28 mediated crosslinking of B7 in DC leads to activation of P-Akt(Thr 308) thus implicating the PI3K-Akt pathway downstream of B7 which is a novel finding. We are examining the potential role of PTEN downstream of Notch signaling in crosstalk in both human and murine DC by testing its effect on P-Akt expression. Targeting IL-6 induced by crosstalk between these two pathways prompts not only clinical evaluation to improve MM patient outcome but also extends to advancing knowledge in T-cell biology and normal plasma cell biology.
Identification of FOXO3A as a regulator of dendritic cell tolerogenicity in human and murine prostate cancer (165.22)
Authors: Watkins Stephanie , Zhu Ziqiang , Ambs Stefan , Hurwitz Arthur
Published: January 2023

The limited success of cancer immunotherapies is often attributed to the loss of antigen-specific T cell function in situ. In this study, we demonstrate a novel mechanism of tolerance induction by tumor-associated dendritic cells (TADCs) in human and mouse prostate cancer. TADCs from human prostate cancer patients and TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice expressed elevated levels of FOXO3A which correlated with expression of suppressive genes that negatively regulate T cell function. Similar results were observed in both a B16 melanoma and a RENCA renal cell carcinoma model. Furthermore, TADC isolated from tumors in Foxo3a-/- mice had reduced tolerogenic function. Additionally, silencing Foxo3a with siRNAs abrogated the ability of TADCs to tolerize and induce suppressive activity by T cells. This was associated with diminished expression of tolerogenic mediators, indoleamine-2,3-dioxygenase, arginase, and TGF-beta and up-regulated expression of co-stimulatory molecules and pro-inflammatory cytokines. In TRAMP mice, provision of tumor-specific CD4+ T cell help abrogated TADC tolerogenicity which was associated with reduced FOXO3A expression. These findings demonstrate that FOXO3A plays a critical role in mediating TADC-induced immune suppression. Moreover, our results identify a novel target for preventing CTL tolerance and enhancing immune responses to cancer by modulating the immunosuppressive activity of TADC found in the tumor microenvironment.
Syk survival signals in Epstein-Barr virus + B cell lymphomas (165.27)
Authors: Hatton Olivia , Lambert Stacie , Krams Sheri , Martinez Olivia
Published: January 2023

EBV latent membrane protein 2a (LMP2a) is a B cell receptor mimic that provides a constitutive survival signal to virally-infected cells through Syk; however, the precise downstream mechanisms executing this survival signal have yet to be elucidated. Previously, we have shown that Syk inhibition induces apoptosis and reduces PI3K/Akt and Erk downstream signals in our EBV+ B cell lymphoma lines. Small molecule inhibitors for the Syk, PI3K/Akt and Erk pathway, or a DMSO vehicle control, were used to interrogate the function of each pathway. Survival of EBV+ B cell lymphoma lines was dependent on PI3K/Akt, but not Erk, as assayed by Annexin-V staining. Akt signaling can promote survival by directly phosphorylating molecules that inhibit apoptosis, including FOXO, Bad, and XIAP. XIAP is degraded after Syk and PI3K/Akt, but not Erk, inhibition, as assayed by Western blot. Treatment of EBV+ B cell lymphoma lines with an inhibitor of the XIAP protease Omi/HtrA2 rescued XIAP degradation and apoptosis induced by Syk inhibition. Our data supports a model in which Syk signaling in EBV+ B cell lymphomas activates Akt, which subsequently stabilizes XIAP and prevents its degradation. Small molecule, peptidic, substrate mimetic inhibition strategies for XIAP, Akt, and Syk are currently in clinical development and present potential new treatment strategies for EBV+ B cell lymphomas that target these EBV-driven survival signals.
Increased expression and cleavage of amyloid precursor-like protein 2 in pancreatic cancer and regulation of fragment stability by major histocompatibility complex class I molecules (165.4)
Authors: Peters Haley , Tuli Amit , Wang Xiaojian , Pan Zenggang , Liu Cuiling , Ouellette Michel , MacDonald Richard , Solheim Joyce
Published: January 2023

Particularly in neurological systems, amyloid precursor-like protein 2 (APLP2) has been linked to cellular growth, adhesion and migration. Our laboratory has demonstrated that several human pancreatic cancer cell lines, as well as pancreatic cancer tissue, express a high level of APLP2. Furthermore, we showed that oncogene-induced transformation of pancreatic cells resulted in increased levels of full-length APLP2 and 12-15 kDa APLP2 C-terminal cleavage fragments. Additionally, in pancreatic cancer cell lines we have noted association of APLP2 C-terminal fragments and major histocompatibility complex (MHC) class I molecules, the proteins responsible for the presentation of tumor antigens. The APLP2 C-terminal fragments associated with MHC class I molecules had prolonged stability compared to unassociated fragments. Incubation with calyculin A, which enhances serine/threonine phosphorylation, increased the rate of APLP2 C-terminus turnover. In agreement with our earlier finding, the APLP2 C-termini associated with MHC class I molecules underwent relatively slower degradation in the presence of calyculin A. Thus, MHC class I molecules have a previously unrecognized function of regulating APLP2 homeostasis. Overall, our discoveries have established that APLP2 expression and cleavage are up-regulated in pancreatic cancer, and have led to a new understanding of the involvement of MHC class I molecules in the regulation of APLP2 C-terminal fragments in pancreatic cancer cells.
Role of CD91 in HSP-mediated innate immunity (165.9)
Authors: Pawaria Sudesh , Binder Robert
Published: January 2023

The immunogenic HSPs gp96, hsp70 and calreticulin are known to chaperone antigenic peptides and elicit strong immunity against tumors and pathogens. The peptide bound form of the HSP bind to and are endocytosed by CD91 on antigen presenting cells leading to the cross-presentation of the chaperoned peptides. We show here that CD91 gets phosphorylated in response to HSPs and results in activation of downstream signals. By selective mutation of the two phosphorylation sites of CD91, we observed that HSPs appear to utilize different sites resulting in different downstream signaling pathways. As a result the cytokine profiles released from stimulated APCs are different for each HSP leading to priming of differential T helper responses including Th17 cells. These results are important in understanding how T cell responses are initiated when HSPs are released into the extracellular environment from dying tumor or infected cells or introduced via vaccination.
Novel model of colitis induced by a non-inflammatory small bowel infection (166.10)
Authors: Dann Sara , Le Christine , Nguyen Kim , Dann Raymond , Hanson Elaine , Eckmann Lars
Published: January 2023

Introduction: Chronic colitis is sustained by aberrant T cells responding to normal microbiota, although how these cells develop is poorly understood. Our aim was to evaluate whether Giardia, a parasite that infects the small intestine but does not elicit inflammation, triggers colitis in a susceptible host and to elucidate mechanisms governing decisions leading to disease. Methods: IL-10-/- mice were infected with Giardia and evaluated up to 12 wks after infection. T cells and DC were isolated and characterized by FACS. Mechanistic studies were performed using neutralizing antibodies and double knockout mice. Results: Within 2 wks of Giardia infection, IL-10-/- mice developed colitis, which persisted after parasite eradication. Infection resulted in Th1 and Th17 expansion and increased expression of neutrophil-recruiting chemokines. Surprisingly, a similar response was measured in infected WT mice, which did not develop colitis. Mechanistic studies revealed that absence of T cells or Myd88 in IL-10-/- mice prevented Giardia-induced colitis. Giardia infection induced expansion of the colonic DC population and ablation of DC led to reduced severity of inflammation. Conclusions: These results suggest that Giardia functions as a microbial trigger of colitis in IL-10-/- mice. This is a powerful new model of chronic intestinal inflammation that can provide fresh insight into mechanisms controlling homeostasis and the development of colitogenic T cells.
Role of Axl receptor tyrosine kinase in the generation of intestinal γδ T cells (166.13)
Authors: Park Min , Lee Eun-Hee , Kang Hyung-Sik
Published: January 2023

Axl receptor tyrosine kinase has been known to play a crucial role in cell survival, proliferation, and migration, but little is yet known about its regulatory role in the generation of γδ T cells. Here we investigated the role of Axl in the generation of intraepithelial γδ T cells. The γδ T cell population was increased in intraepithelial lymphocytes (IELs) of Axl-/- mice compared to them of wild type mice. In addition, upregulaton of IL-7α Rexpression was observed in both IELs and γδ T cells purified from IELs of Axl-/- mice. In electrophoretic mobility shift assay, IELs of Axl-/- mice showed the reduced AP-1 activation due to the dephosphorylation of its upstream signaling molecules, c-Jun and JNK. Taken together, these results suggested that Axl is required for the generation of intestinal γδ T cells by the transcriptional regulation of IL-7Rα via the JNK pathway.
A murine model of the immune responses to trauma and transfusion (169.36)
Authors: Jackman Rachael , Muench Marcus , Heitman John , Utter Garth , Busch Michael , Norris Philip
Published: January 2023

Traumatic injury is immunosuppressive, and we have previously observed major fluctuations in plasma cytokines following trauma in humans with notable differences between those that are transfused or not. It has been difficult, however, to distinguish the effects of blood loss, injury, and other patient characteristics from those of allogeneic transfusion on immune function in human subjects. We have developed a murine model to understand the relative contributions of traumatic blood loss and allogeneic transfusion on immunomodulation. BALB/c mice were bled 25-30% of their estimated total blood volume to model traumatic blood loss, and transfusions of saline-diluted C57B6 blood or saline alone were administered IV into these mice as well as into mice without injury. Serum cytokines and chemokines were evaluated 4 hours after treatment. Several proteins, including IL-6, IL-10, MMP-9, and KC were elevated after traumatic blood loss (p<0.05), with some of these increases reduced or prevented by transfusion. Some proteins were elevated in response to transfusion alone (such as sE-Selectin, p<0.05), and still others responded to both blood loss and/or transfusion (such as IL-5 and MCP-1, p<0.05). Saline administration alone produced intermediate changes compared with allogeneic blood transfusion. These results are consistent with our clinical observations in humans and demonstrate clear roles for both blood loss and transfusion in modulating the early immune response to trauma.
BTLA-HVEM interaction delivers bidirectional signals and modulates the severity of GVHD (169.48)
Authors: Tamada Koji , Sakoda Yukimi , Park JangJune , Zhao Yuming
Published: January 2023

B and T lymphocyte attenuator (BTLA), upon interaction with herpesvirus entry mediator (HVEM), delivers an inhibitory co-signal which regulates immune response in various diseases. In graft-versus-host disease (GVHD), unexpectedly, BTLA mediates positive effects on donor T cell survival, but immunological mechanisms of this function have yet to be fully explored. Our recent studies addressed a role of BTLA in GVHD by employing the newly established agonistic anti-BTLA mAb that stimulates BTLA signal without antagonizing BTLA-HVEM interaction. We revealed that BTLA signal inhibited donor anti-host T cell responses and ameliorated GVHD with a successful engraftment of donor hematopoietic cells. These effects were dependent on BTLA signal into donor T cells but neither donor non-T cells nor recipient cells. On the other hand, expression of BTLA mutant lacking an intracellular signaling domain restored impaired survival of BTLA-deficient T cells, suggesting that BTLA also serves as a ligand and delivers HVEM prosurvival co-signal in donor T cells. Thus, BTLA-HVEM interaction delivers positive and negative bidirectional co-signals to donor T cells via HVEM and BTLA, respectively, and modulates the severity of GVHD. Additional data obtained by recent studies will be also presented.
Survey of HPA and HLA class 1 antibodies in Nigerian antenatal women (169.5)
Authors: Jeremiah Zaccheaus , Atiegoba Anne , Mgbere Osaro
Published: January 2023

Background: The prevalence of antibodies to HPA and HLA class 1 antigens among Nigerian pregnant women has not been reported in our country. Aims: To screen for platelet specific and HLA class 1 antibody among pregnant women. Methods: One hundred and forty four (144) pregnant women attending the obstetric clinic of Military Hospital, Port Harcourt, participated in the study. Their sera were tested for antibodies to HPA and HLA class 1 antigens using GTI PakPlus solid phase ELISA Kit. Results: The total prevalence rate of antibody production was 60.5% (87 out of 144). Among the positive samples, 60 had platelet glycoprotein specific antibodies (41.7%) and 27 had HLA class 1 antibodies (18.8%). In 39.6% of the pregnant women, both platelet specific antibodies and HLA class 1 antibodies appeared. The prevalence of platelet specific glycoprotein antibodies were obtained as follows: GP IIb/IIIa 12 (8.3%), GP Ia/IIa 30 (20.8%), GP Ib/IX 18 (12.5%) and GP IV 9(6.3%). The prevalence of each platelet antibody subgroup was obtained as follows: anti-H PA-1a,-3a,-4a (4.2%), anti-H PA-1b,-3b,-4a (4.2%), anti-H PA- 5a and anti-GP Ib/IX (12.5% each), anti-HPA-5b (8.3%) and anti-GP IV (6.3%).). Conclusion: A high prevalence rate of human platelet and cytotoxic antibodies has been observed in our obstetric population.
Identification of novel immune factors regulating outcome to viral encephalitis. (170.12)
Authors: Taylor Katherine , Borisevich Vika , Kolokoltsova Olga , Smith Jennifer , Smith Jeanon , Estes Mark , Paessler Slobodan
Published: January 2023

Viewed as a public health and zoonotic threat, Venezuelan equine encephalitis virus (VEEV) causes morbidity and mortality in both human and equine populations during naturally occurring epizootic outbreaks. The specific immune factors contributing to lethality or survival following viral invasion of the brain remain largely unknown. Using intranasal infection of the vaccine strain of VEEV (TC83), we established three models of brain infection: (1) Lethal infection in C3H/HeN (C3H) mice (2) Non-lethal infection in C57BL/6 (C57) mice (3) Non-lethal infection followed by chronic viral persistence in α/β T cell-receptor deficient mice (TCR KO) on a C57 background. The three models display similar viral kinetics with each developing a high level viral load in the brain. While absence of a functional T-cell compartment contributes to persistent brain infection, profiling the host response using genomics and multiplex cytokine array identified natural killer (NK) cells and type I interferon (IFN) as potential prognosticators of outcome. The systemic depletion of NK cells enabled C3H mice to tolerate brain infection without developing lethal disease. The trend toward lower INF-β levels in the brains of lethally infected animals compared to surviving strains corroborates a protective role for IFN in the brain. The novel knowledge generated in this model system enabled identification of intervention points that influence a positive outcome to viral invasion of the brain.
Histocompatibility in an invertebrate is controlled by a complex of polymorphic IgSF-like genes. (170.4)
Authors: Nicotra Matthew , Dellaporta Stephen , Buss Leo
Published: January 2023

Animals have evolved sophisticated immune systems to distinguish self from infectious non-self. In addition, several groups of colonial marine invertebrates—animals such as sponges, corals, and sea squirts—have evolved allorecognition systems that allow them to distinguish between their own tissues and those other members of their species. Allorecognition phenomena occur when these animals, which live as encrustations on marine surfaces, grow into contact with each other. Compatible colonies fuse, while incompatible colonies reject and often compete for space. Although allorecognition phenomena have long been known to be under genetic control, the molecular basis of these phenomena remains largely unknown. One model system for the study of invertebrate allorecognition is Hydractinia, a cnidarian related to Hydra and reef corals. Using lines of Hydractinia inbred for >8 generations, we have mapped allorecognition loci to a single genomic interval, called the allorecognition complex (ARC). The ARC contains at least two histocompatibility genes, alr1 and alr2, each of was identified by positional cloning. Both genes encode polymorphic transmembrane proteins similar to immunoglobulin superfamily (IgSF) molecules. Sequence variation between alleles of alr1 or alr2 is concentrated in the extracellular region and is predictive of allorecognition responses in inbred and wild-type colonies. Intriguingly, alr1 is located within a family of at least 10 other IgSF-like genes.
A complex family of diversified immunoglobulin domain-containing proteins (DICPs) includes inhibitory and activating forms and likely plays a role in zebrafish innate immunity (170.9)
Authors: Yoder Jeffrey , Haire Robert , Turner Poem , Mueller M. , O'Driscoll Marci , Litman Ronda , Cannon John , Litman Gary
Published: January 2023

One of the first lines of defense against pathogens and disease is the recognition of foreign or altered antigens by innate immune receptors. A large number of these receptors fall into the large immunoglobulin (Ig) super family of proteins. We have identified a multi-gene family of 29 diversified immunoglobulin domain-containing proteins (DICPs) encoded on zebrafish chromosomes 3, 14 and 16. DICPs typically encode two extracellular Ig domains: an amino terminal variable (V) Ig domain and an additional Ig-like domain. Certain DICPs possess a single Ig domain. DICP variants include membrane bound and secreted forms. Putative inhibitory forms have been identified which possess cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Putative activating forms have been identified which possess a positively charged residue within their transmembrane domain and are predicted to associate with adaptor proteins that possess cytoplasmic activation motifs (e.g. Dap12, Dap10 etc). Database searches reveal that the Ig domains of DICPs are unlike any other Ig domains encoded by the zebrafish genome and are more (albeit very weakly) similar to the V domains of mammalian immunoglobulin genes, than to any other zebrafish or mammalian sequence. DICP annotation, transcript variants, tissue-specific expression and efforts to identify DICP ligands will be discussed.
IL-21/IL-21R interaction on both CD4 cells and B cells is required to promote autoimmune parameters in chronic graft versus host disease. (44.10)
Authors: Nguyen Vinh , Irina Luzina , Chen Qing , Rus Horea , Rus Violeta
Published: January 2023

IL-21 stimulates B cell proliferation, plasma cell (PC) differentiation and germinal center (GC) expansion and it exerts autocrine effects on T follicular B helper cells (TFH) and Th17 cells while downregulating Treg cells. We assessed the effect of IL-21/IL-21R signaling on B cells independent from the effect on CD4 T helper cells using IL-21R-/- or IL-21R+/+ mice as donor or hosts in the P-into-F1 and Bm12-into-B6 models of chronic Graft Versus Host Disease (cGVHD). cGVHD induced by injection of IL-21R-/- CD4 cells from B6 mice into B6D2F1 hosts was characterized by a significant decrease in the expansion of donor CD4, TFH and Th17 cells and an increase in Treg cells compared to IL-21R +/+ donor cells. Furthermore, the number and size of GC, GC B cells and levels of anti-ssDNA antibody (Ab) were significantly decreased. When cGVHD was induced by injecting Bm12 spleen cells into IL-21R+/+ or IL-21R-/- B6 mice, parameters of cGVHD including B cell activation, number and size of GC, percentage of GC B cells and plasmablasts, IgG anti-dsDNA Ab production were significantly decreased compared to IL-21+/+ hosts. Long-term studies showed decreased severity of lupus-like renal disease in the IL-21R-/- hosts. These results suggest that IL-21/IL-21R interaction enhances autoimmune features in lupus by promoting the expansion of TFH and Th17 cells and the downregulation of Treg cells and by promoting the differentiation of autoreactive B cells into autoAb producing cells.