bluuebunny/crossref_metadata_2025 huggingface dataset:
Secretory IgD has an evolutionarily conserved role in respiratory mucosal defense (160.18)
Authors: Bromage Erin , Ramirez-Gomez Francisco , Greene Whitney , Rego Katherine , Hansen John , Boardman Timothy , Dupras Danielle
Published: January 2023

Recently, we discovered secreted IgD in rainbow trout, which is produced by a novel exon-splicing mechanism. Molecular and cellular analysis revealed that secIgD plasma cells were distributed within the primary and secondary immune tissues. The concentration of serum IgD (2-80µg/ml) was ~400x lower than serum IgM (800-9000µg/ml). Two dominant secIgD heavy chain isoforms were identified in serum of trout which were associated with antibody light chains to form 240 or 370 kDa IgD-monomers. Antigen-specific secIgD was produced in response to vaccination with either TI or TD antigens. Interestingly, the distribution of antigen-specific IgD plasma cells within mucosal tissues revealed that they were preferentially located in the gills of rainbow trout. Analysis of gill mucosal secretions revealed that secIgD was the dominant antibody isotype, which contrasts the profile observed in serum. However, our data suggests that the number of IgD plasma cells in the gills of rainbow trout could not account for the elevated IgD concentration observed in the gill mucus. We hypothesize that serum IgD is preferentially transported across the gill epithelium to neutralize pathogens at this important site. The receptor responsible for trans-epithelial transport of IgD has proved elusive. However, the analogous distribution of IgD plasma cells and secreted IgD in the respiratory mucosa of trout and mammals would suggest that IgD provides an evolutionarily conserved function within this tissue.
Potential role of TSLP and IL-16 for migration and proliferation in Mycosis Fungoides T cells (161.15)
Authors: Tuzova Marina , Richmond Jillian , Wolpowitz Deon , Curiel-Levandrowsk Clara , Kupper Thomas , Cruikshank William
Published: January 2023

Mycosis Fungoides is a cutaneous T cell lymphoma disease characterized by CCR4+ T cells in skin plaques. While CCL22/CCR4 has been implicated, the mechanism for recruitment and expansion is not well understood. We have now identified elevated levels of IL-16 and thymic stromal lymphopoietin (TSLP) in the sera and skin of MF patients and investigated the potential role of these cytokines. Sera levels showed a stage-dependent increase of TSLP (3 fold versus control), and consistently, stage independent, elevated IL-16 levels. The source appears to be the skin, as both were positive compared with staining of normal skin. MF T cells expressed higher levels of TSLP receptor and expression correlated with CCR4 expression. In vitro migration studies indicated a 210% and 230% increase in migration to IL-16 and TSLP respectively. Pre-treatment of IL-16 prior to TSLP induced migration increased the migratory response to 280%, suggesting an additive effect. Addressing T cell proliferation, we determined that TSLP and IL-16 separately had minimal effects on proliferation however in combination proliferation increased 3 fold versus stimulated control T cells. Taken together our findings indicate the potential role of IL-16 and TSLP to recruit and expand MF T cells at sites of plaque formation in MF. Co-expression of TSLPR and CCR4 suggests a possible mechanism that at least in part would account for the prevalence of CCR4+ T cells in the skin of MF.
IL-7Rα deficiency in p53null mice exacerbates thymocyte telomere erosion and lymphomagenesis (161.6)
Authors: Cui Yan , Kibe Ryoko , Zhang Shuzhong , Guo Darcy , Marrero Luis , Tsien Fern , Rodriguez Paulo , Zieske Arthur , Huang Jiaqiang , Li Wenqing , Durum Scott , Iwakuma Tomoo
Published: January 2023

IL-7 is essential for T cell survival and development. IL-7 receptor (IL-7Rα) efficiency manifests SCID due to substantial thymocyte apoptosis. We hypothesized that IL-7Rαnull-induced apoptosis is partially mediated by an elevated p53 activity. To investigate the genetic association of IL-7Rα signaling with the p53 pathway, we generated IL-7Rαnullp53null (DKO) mice. DKO mice exhibited a marked reduction of apoptosis in developing T cells and an augmented thymic lymphomagenesis with telomere erosions and exacerbated chromosomal anomalies, including chromosome duplications, breaks, and translocations. In particular, Robertsonian translocations, in which telocentric chromosomes fuse at the centromeric region, and a complete loss of telomeres at the fusion site occurred frequently in DKO thymic lymphomas. Cellular and molecular investigations revealed that IL-7/IL-7Rα signaling withdrawal diminished the protein synthesis of POT1 (protection of telomere 1), a subunit of telomere protective complex shelterin, leading to telomere erosion and the activation of the p53 pathway. Blockade of IL-7/IL-7Rα signaling in IL-7-dependent p53null cells reduced POT1 expression and caused telomere and chromosome abnormalities similar to those observed in DKO lymphomas. This study underscores a novel function of IL-7/IL-7Rα during T cell development in regulating telomere integrity via POT1 expression and provides new insights into cytokine-mediated survival signals and T cell lymphomagenesis.
Sinonasal epithelial cell derived IL-33 induces IL-13 in ST2+ innate lymphoid cells in chronic rhinosinusitis. (161.8)
Authors: Shaw Joanne , Fakhri Samer , Citardi Martin , Corry David , Kheradmand Farrah , Liu Yong-Jun , Luong Amber
Published: January 2023

Chronic rhinosinusitis (CRS) is an inflammatory process of the nasal and paranasal sinus mucosa that affects more than 40 million people annually. CRS without nasal polyps (CRSsNP) or with nasal polyps (CRSwNP) are associated with Th1 and Th2 cytokine polarization respectively. Although the pathophysiology of CRS remains unclear, recent studies have implicated a role for infectious agents including fungus, bacteria and bacterial superantigens in this chronic disease. Here we investigated the role of the epithelial cell derived cytokine IL-33 in the pathophysiology of CRS. Using quantitative real-time PCR we found elevated expression of the IL-33 receptor ST2 in inflamed sinonasal mucosa from CRSwNP patients compared to CRSsNP patients and healthy controls. Further, we found significant induction of IL-33 from epithelial cells derived from inflamed mucosa of CRSwNP patients as compared those derived from CRSsNP patients in response to stimulation with an extract of the filamentous fungus Aspergillus fumigatus. We discovered that IL-33 promotes the secretion of IL-13 from CD45+ cells isolated from inflamed mucosa of CRSwNP patients and subsequently discovered a unique population of ST2+ innate lymphoid cells responsible for the production of IL-13 in response to IL-33 stimulation. Collectively these data provide evidence for a role for sinonasal epithelial cell derived IL-33 and an IL-33 responsive innate lymphoid cell population in the pathophysiology of CRS.
Efficiency of colon cancer RNA-transfected DC vaccine is enhanced by previous treatment of tumor cells with low concentration of 5-fluorouracil (162.11)
Authors: Kaneno Ramon , Camargo Marcela , Gorgulho Carolina , Frederico Juliana , Ikoma Maura
Published: January 2023

We have recently observed that human DCs treated with low doses of antitumor chemotherapeutic agents became them more efficient to stimulate T lymphocytes, whereas treatment of tumor cells with low concentrations of drugs made them more immunogenic than wild type cells. In this study we aimed to evaluate whether transfection of DC with drug-treated tumor cells RNA, enhances the effectiveness of DC-based vaccine, and if modulatory effects of drugs can be observed in vivo. To achieve these goals, C57/Bl-6 mice were s.c inoculated with MC-38 cells and 10 days later they were treated with DC transfected with RNA from MC-38 cells pre-treated with the minimum effective concentration of 5-fluorouracil (5-FU). Vaccination with these DC decreased the tumor growth, being the tumor size 40% lower than the Control. DC phenotyping showed that transfection increased the percentage of CD86+ (55% higher than control), CD40+ (57% higher), and MHC class II+ cells (58% higher). Spleen cells of tumor-bearing mice were co-cultured with MC-38 target cells and the supernatants evaluated for IFN-γ. Vaccination with transfected-DC was able to increase the in vitro production of IFN, compared with control. Our results suggest that treatment of tumor cells with minimum effective concentration of 5-FU induces transcriptional changes that can be transfered to DC by RNA transfection, enhancing their ability to stimulate the antitumor response.
Spatiotemporal analysis of illegitimate V(D)J deletions in Notch1 and Bcl11b genes (162.14)
Authors: Champagne Devin , Shockett Penny
Published: January 2023

Aberrant V(D)J recombination events in oncogenes and tumor suppressor genes are implicated in development of T-cell leukemias and lymphomas. Previously, others identified hotspots for deletional DNA rearrangements adjacent to cryptic recombination signal sequences in Notch1 and Bcl11b genes in thymus of adult mice with radiation-induced thymic lymphomas. Interestingly, these rearrangements were also found at detectable frequencies in thymus of unirradiated WT animals suggesting that factors in addition to deletions contribute to development of lymphomas, but the frequency of deletions at different developmental stages or in peripheral lymphoid organs was not analyzed. To determine the timing, frequency, and tissue distribution of Notch1 and Bcl11b deletions during development, we are examining tissues harvested at different developmental stages from C57BL/6 mice. We detect these deletions in thymus at all ages examined from fetus to 6 months. Deletions are also detected in spleen, consistent with the presence of T cells bearing deletions in the circulating lymphocyte pool. We are analyzing sequences of deletion junctions for clonal relationships in individual animals and performing broken DNA end analysis for ongoing deletions. We are also using an inducible tetracycline-regulated RAG transgenic mouse system to determine if these rearrangements can occur at all ages with induced RAG expression or are limited to specific developmental windows.
Modulation of immunostimulatory and inhibitory signals augments T cell function by distinct mechanisms in the ovarian cancer microenvironment (162.15)
Authors: Duraiswamy Jaikumar , Freeman Gordon , Coukos George
Published: January 2023

Infiltrating T cells (TILs) with anti-tumor potential exist in patients with ovarian cancer. However, tumor microenvironment mediates induction of immunosuppressive molecules, such as PD-1 and CTLA4, on ovarian TILs. We assessed the interaction between PD1:PD-L1, PD-1:PD-L2, PD-L1:B7.1 and CTLA4:B7.1 pathways and their relative contribution in modulating TIL function. First, we evaluated the functional impact of PD-1 and CTLA4 on antigen-specific TILs in vitro from ovarian cancer patients. For in vivo studies, we used two mouse models: i) ID-8, a syngeneic model of ovarian cancer, and ii) a humanized model (HLA-A2+ NSG mice reconstituted with human CD34+ cells). PD-1 and CTLA4 blockade in ID-8 model resulted in partial tumor rejection. However, combining one or more of the following immune modulators, i) whole tumor vaccines expressing either GM-CSF or Flt3-ligand, ii) CpG-ODN, a TLR9 agonist, iii) anti-4-1BB agonistic antibody or iv) murine IL-15, added value to inhibitory blockade. In case of humanized mice, immune checkpoint blockade alone caused regression of human ovarian tumors. Our study shows that combining immunomodulators reject ovarian tumors by i) elevating T cell infiltration into tumors, ii) decreasing intratumoral regulatory T cell population and iii) increasing inflammatory cytokine production. We thank Drs. James P. Allison and Michael Curran (GM-CSF and Flt3L plasmids), Dr. Rafi Ahmed and Dr. John D. Altman (tetramers).
Cytomegalovirus-based vaccine induces protective immunity against melanoma (162.19)
Authors: Qiu Zhijuan , Maher Leigh , Adler Barbara , Khanna Kamal
Published: January 2023

Cytomegalovirus (CMV), which belongs to herpesviridae family, infects 40-60% of the population in developed country and more than 90% in developing country. It can establish life-long, asymptomatic infection in immunocompetent individual. CMV infection elicits strong adaptive immune responses, and superinfection is observed despite of the presence of CMV-specific immunity. Those unique characteristics make CMV a potentially promising vaccine vector. Here we used recombinant CMV that expresses ovalbumin (CMV-OVA) and examined its efficacy in protecting mice from OVA-expressing B16 melanoma (B16-OVA). We showed that in both therapeutic and prophylactic settings CMV-OVA vaccination significantly reduced tumor burden in mice in an antigen-dependent manner. We further designed CMV-based vectors that express the B16 endogenous antigen gp100 (CMV-gp100) and a mutant (CMV-gp100KGP), and evaluated their ability to protect mice against highly metastatic lung B16-F10 melanoma. Our results demonstrated that both CMV-gp100 and CMV-gp100KGP provided strong protection against B16-F10 melanoma. However, CMV-gp100KGP, which elicited an antigen specific CD8 T cell response against the self-protein gp100, provided better protection when compared to the CMV-gp100 that failed to generate gp100-specific effector CD8+ T cells. In conclusion, our studies demonstrated the efficacy of CMV-based vaccines to prevent and treat tumors.
CD40L expression by CD8+ T cells is essential for successful rejection of SV40 T antigen expressing tumor cells in the absence of CD4+ T cells (162.38)
Authors: Stark Regina , Frentsch Marco , Listopad Joanna , Schulz Axel , Meier Sarah , Blankenstein Thomas , Thiel Andreas
Published: January 2023

Cytotoxicity is considered to be the main effector function of CD8+ T cells in the immune system. However, we demonstrate here that CD40L, one of the central effector molecules of CD4+ helper T cells, is expressed by a substantial part of human and murine CD8+ T cells after activation. Human central as well as effector memory CD8+ T cells comprise up to 50% CD40L+ cells after polyclonal activation. These CD40L+ CD8+ T cells display a non-cytotoxic phenotype and resemble functionally various distinct CD4+ T helper cell subtypes with respect to expression of IL2 and IFNγ or IL4 as well as with respect to their potential to induce activation of B cells and maturation of DCs in vitro. To investigate the functional relevance of CD40L expression by CD8+ T cells we analyzed a murine anti-tumor response. During the whole course of the response 50% of the tumor-specific CD8+ T cells expressed CD40L. While application of CD40L competent wt CD8+ T cells to Rag1ko mice challenged with the tumor cells prevented the establishment of solid tumors, injection of CD40Lko CD8+ T cells resulted in a non-controlled tumor progression similar to non-treated tumors. Our results disclose an essential functional relevance of CD40L expressed by CD8+ T cells. Particularly in situations where CD4+ T-cell help is diminished, MHC-II antigen presentation is reduced or limited danger signals are present, CD40L+ CD8+ T cells may exert essential helper responsibilities for a competent cellular immune response.
Increased Th17 cells in the tumor microenvironment is mediated by tumor-secreted PGE2 (162.42)
Authors: Liu Jianguo , Ning Huan , Qian Xuesong
Published: January 2023

Tumor microenvironment can convert the host immune responses to favor tumor growth. However, the underlying mechanisms of how tumor cells educate host immunity are still largely unknown. We found that Th17 cells were increased in the peripheral blood, spleen and tumor tissues of mammary gland tumor-bearing mice. Th17 cell survival factor, IL-23 was overexpressed in tumor tissues isolated from both mouse and human breast cancer patients. Soluble molecules secreted from tumor cells but not normal breast epithelial cells induced IL-23 protein secretion in DCs via induction of the p19 mRNA expression. Our data further indicates that tumor-secreted PGE2 via its receptor EP2 and EP4 enhanced IL-23 p19 gene transcription through binding to the cAMP-response element in the p19 promoter. Blocking PGE2 synthesis by NS398, a Cox2 inhibitor, abrogated the enhancement of p19 expression both in vitro and in vivo. Furthermore, blocking PKA by H89 completely abrogated the inductive effects of tumor conditioned medium and PGE2 on p19 transcription. In line with this finding, cAMP active analog, Forskolin mimics the PGE2 effect. Taken together, our results indicate that tumor-secreted PGE2 differentially induce IL-23 but not IL-12 production in the tumor microenvironment which could promote Th17 cell expansion. This inductive effect is mediated through cAMP/PKA signaling transduction pathway by increasing p19 transcription.
TLR5 mediated regulation of tumor growth in an inducible model of cancer (162.48)
Authors: Rutkowski Melanie , Escovar-Fadul Ximena , Sarmin Fahmida , Stephen Tom , Allegrezza Michael , Gil-Guerva Oswaldo , Tesone Amelia , Conejo-Garcia Jose
Published: January 2023

It has been reported that up to 25% of the human population carries a dominant point mutation in TLR5 resulting in the loss of TLR5 signaling. TLR5 expression is paramount in modulating the balance of regulatory FoxP3+ and proinflamatory IL-17+ T cell lineages, two cellular subsets with potentially divergent roles in cancer. However, in the context of cancer, the significance of this mutation remains unknown. Sequencing of cDNA isolated from human ovarian tumor specimens revealed that 6% of tumor specimens harbor a functional mutation in TLR5, suggesting that a loss in TLR5 signaling results in “resistance” to cancer. To further investigate the role of TLR5 signaling in cancer progression, we utilized a novel p53 and KRas dependent model of inducible cancer. TLR5-/- mice had a remarkable delay in tumor onset and a dramatic reduction in tumor burden. Corresponding to the delayed tumor onset, we observed that T cells sorted from the tumor draining lymph nodes of TLR5-/- mice were able to vigorously recall against tumor antigens compared to a profound paralysis of effector function in T cells sorted from the draining lymph nodes of WT mice. These data indicate for the first time that pattern recognition receptors may influence the elimination and/or equilibrium phases of tumor progression at extraintestional locations, pinpointing potentially novel pathways to target for immunotherapy.
Serum amyloid A3 mediates inflammation-related tumor growth by fortifying the immunosuppressive mechanism of myeloid-derived suppressor cells (162.7)
Authors: Lee Jung-Mi , Chae Min-Ji , Park Young-Jun , Kim Yun-Sun , Kim Eun-Kyung , Kang Chang-Yuil
Published: January 2023

Myeloid-derived suppressor cells (MDSCs), which suppress diverse innate and adaptive immunity and thereby provide a tumor evasion mechanism, are emerging as a key population linking inflammation to cancer. Although many inflammatory factors inducing MDSCs have been known, the crucial components and the underline mechanisms remain elusive. In this study, we demonstrated for the first time a novel mechanism of serum amyloid A (SAA) 3, a well-known inflammatory marker, connecting inflammation and MDSCs. We found that tumor progression or hSAA treatment specifically acted on Mo MDSCs in subsets of MDSCs to induce SAA3 expression. These resulted in enhancing the survival and suppressive function while inhibiting the GM-CSF-induced differentiation of Mo MDSCs to activated macrophages through TLR2 signaling. Therefore, it suggests a novel mechanism that inflammation exacerbates cancer progression with the mediation of Mo MDSCs through SAA3. This study also provides new insight into increasing immunosuppressive activity of Mo MDSCs with tumor growth, demonstrating that SAA3 is specifically increased in Mo MDSCs as tumor grows.
B7-CD28 costimulation blockade via CD28Ig differentially modulates helper T cell differentiation without affecting Tregs (163.1)
Authors: Cho Hyoung-Soo , Kim Yong-Hee , Park Chan-Sik , Iwakura Yoichiro , Kim Sang-Joon , Park Chung-Gyu
Published: January 2023

B7-CD28 costimulation plays a prominent role in T cell full activation, and its blockade renders T cell anergic. CTLA-4Ig has been known to bind B7 ligands and effectively suppress T cell responses. However, CTLA-4Ig has been also reported to have a negative effect on Tregs. In the present study, we used CD28Ig to inhibit B7-CD28 engagement while sparing Tregs. CD28Ig dose-dependently suppressed alloreactive T cell proliferation, and it did not exert any harmful effect on the cellularity, frequency, and suppressive function of Tregs in mice. Next, we applied CD28Ig in mouse islet allograft model, and graft survival was significantly prolonged upon treatment in comparison to untreated control without negatively affecting Tregs. Interestingly, CD28Ig effectively controlled Th1 response in treated recipients while concomitantly upregulated both Th17 and Th2 response. Also, we further confirmed that Th17 is responsible for an eventual allograft rejection. CD28Ig differentially affected helper T cell differentiation by modulating lineage-specific transcription factor expression. Moreover, both expression of p-STAT3 and 6 were increased in response to CD28Ig, but Th1-associated p-STAT1 and 4 expression was repressed. Altogether, we here demonstrate that B7-CD28 blockade via CD28Ig differentially modulates helper T cell differentiation without dampening Treg integrity, and this implicates a new therapeutic strategy for regulating T cell response in autoimmunity or transplantation.
CD25 signaling via STAT5 is necessary for development of cytolytic CD4 T cells (163.13)
Authors: Brown Deborah , Workman Aspen , Jacobs Ashley , Lee Sarah , Condon Shirley
Published: January 2023

Cytolytic CD4 cells have been identified in vivo during viral infections; however, the factors necessary for driving the cytolytic phenotype have not been characterized. Previously, we found that IL-2 is necessary and sufficient to drive the in vitro differentiation of naïve CD4 cells into cytolytic effectors. To further dissect the role of IL-2 in CD4 CTL generation, TCR transgenic mice deficient in IL-2 production or IL-2Rα (CD25) were used. In vitro, CD25 deficient cells do not express granzyme B (GrB) or acquire cytolytic activity when cultured in the presence of IL-2. In contrast, CD25+/- cells exhibit half maximal expression of GrB and reduced cytotoxicity when compared to WT cells. In vitro, IL-2 activates STAT5 phosphorylation, peaking at 18-24 h, correlating with GrB expression and cytotoxicity by 72-96 h. Inhibition of the JAK3/STAT5 pathway resulted in a dose dependent reduction of GrB expression. Surprisingly, other common γc cytokines such as IL-7 and IL-15 induce STAT5 phosphorylation but do not induce cytotoxicity, most likely due to the timing of STAT5 activation. Studies determining whether STAT5 activation synergizes with other signaling pathways to induce optimal CTL activity are underway. Further, the role of CD25 signaling in the induction of cytolytic activity in vivo during influenza infection will be discussed. These data have implications in the design of vaccines and in adoptive immunotherapy against certain tumors.
T cell proliferative and apoptotic responses in conditional Daxx knockout mice (163.16)
Authors: Li Jinghe , Zhang Jianke
Published: January 2023

Apoptosis is essential for maintaining homeostasis in the immune system. Fas is a single transmembrane domain protein capable of inducing apoptotic death in lymphocytes. Mutations in the Fas receptor or it’s ligand, FasL, lead to autoimmune-lymphocproliferation diseases characterized by lymphadenopathy, splenomegaly and autoantibody production. The Fas-associated death domain (FADD) protein is an adaptor protein which binds to Fas, recruits and activates the initiator caspase 8. In addition to FADD, proteins including Daxx have also been indicated to bind to Fas. It has been proposed that Daxx mediates a parallel apoptotic pathway that is independent of FADD and Caspase 8, but is dependent on ASK1-medaited signaling. Deletion of FADD blocks Fas-induced apoptosis in T and B cells, which, however, does not lead to lymphoproliferation diseases as present in Fas mutant animals. Recent studies indicate that absence of FADD unleashes a potent necrosis-like death pathway. The in vivo function of Daxx in lymphocytes has not been properly analyzed as deletion of Daxx in germ cells lead to embryonic lethality. In this study, we used a conditional mutant mouse in which Daxx is deleted specifically in T cells. Our data revealed a novel function of Daxx in the regulation of proliferation and death in primary T cells.
Localized p:MHCII presentation maintains CD4+ T cell stability and avoids exhaustion (163.19)
Authors: Nelson Ryan , McLachlan James , Jenkins Marc
Published: January 2023

Recent work has demonstrated that memory CD4+ T cells decay following clearance of some acute bacterial infections. However, this issue has not been addressed for persistent bacterial infections where CD4+ T cells are the protective cell type. During persistent Salmonella infection in mice, CD4+ T cells control pathogen burden by activating infected macrophages and limiting bacteria to the gut draining mesenteric lymph nodes. Using a peptide:MHCII (p:MHCII)-tetramer based enrichment strategy, we tracked a polyclonal Salmonella p:MHCII-specific CD4+ T cell response to oral infection. Salmonella p:MHCII-specific T cells expanded and were maintained at a numerically stable level throughout persistent infection but decayed if the infection was cleared. This population consisted of highly functional Th1 cells capable of producing IFN-γ and TNF-α upon re-stimulation. Using adoptive transfer of memory cells into mice persistently infected with Salmonella lacking the relevant peptide, we showed that p:MHCII dependent proliferation by a small number of cells accounted for stable memory. However, transfer of memory cells into transgenic mice systemically expressing the relevant p:MHCII complexes induced high PD-1 expression and functional exhaustion. These results indicate that localized p:MHCII presentation in the chronically infected mesenteric lymph nodes is the key to maintaining this naturally stable CD4+ T cell response to Salmonella and simultaneously avoiding exhaustion.
TGF-β-dependent suppression of IL-22 production in TH17 cells is mediated by the transcription factor c-Maf (163.2)
Authors: Rutz Sascha , Noubade Rajkumar , Eidenschenk Celine , Singh Harinder , Ouyang Wenjun
Published: January 2023

T helper (TH) cells modulate immune responses to better cope with various types of invading pathogens through the production of different effector cytokines. TH17 cells produce Interleukin(IL)-17, and IL-22, and play an indispensable role in host defense against infections of extracellular pathogens. IL-22, a member of the IL-10 cytokine family, enhances proinflammatory innate defense mechanisms from epithelial cells, and provides crucial tissue protection from damages caused by inflammation and infection. Transforming growth factor (TGF)-β differentially regulates IL-22 and IL-17 expression. IL-6 alone promotes T cells to only produce IL-22, whereas the combination of IL-6 with high concentrations of TGF-β results in production of IL-17 but not IL-22. Here we identify the transcription factor c-Maf, which is induced by TGF-β, as a downstream repressor of Il22. c-Maf binds to the Il22 promoter, and is both necessary and sufficient for the TGF-β-dependent suppression of IL-22 production in TH17 cells. While a number of factors, such as RORγt, AhR and BATF, have been identified as positive regulators of IL-22 expression, c-Maf is the first transcription factor shown to be involved in its negative regulation. This is even more surprising since c-Maf at the same time is critical in promoting the expression IL-10 and IL-21 in T cells.
The induction of FoxP3 in naive T cells is dependent upon the transcription factors TFE3 and TFEB. (163.4)
Authors: Bettinger Patricia , Roman Christopher
Published: January 2023

TFE3 and TFEB are members of the basic helix-loop-helix leucine zipper MiTF/TFE (Microphthalmia-associated transcription factor) family of transcription factors. They are ubiquitously expressed in human and murine tissue and form homo- and/or heterodimers to bind the E-box core sequence CAYGTG. Using a transgenic mouse line expressing a trans dominant negative (TDN) inhibitor of TFE3 and TFEB in T cells, we previously showed that CD40L is a key target of both TFE3 and TFEB and that inactivating these molecules causes hyper IgM syndrome. Unexpectedly, we now show that naïve T cells from these mice have a significant defect in the in vitro differentiation into the inducible T regulatory (iTreg) phenotype, demonstrated by reduced induction of the hallmark Treg transcription factor, FoxP3. Although Smad7, a negative regulator of TGFβ signaling, is a target gene of TFE3 in non-lymphoid cells, preliminary results indicate its expression is unaffected in these cells, as is the phosphorylation of Smad3, indicating TGFβ signaling pathway is intact. This suggests TFE3/B are important in Treg differentiation, possibly as direct regulators of the FoxP3 gene. This combined evidence demonstrates a multiplicity of roles for TFE3 and TFEB in T cell function.
Malaria-induced IL-10 contributes to the systemic burden of non-typhoidal Salmonella (164.10)
Authors: Mooney Jason , Butler Brian , Lokken Kristen , Xavier Mariana , Schaltenberg NIcola , Chau Jennifer , George Michael , Dandekar Satya , Luckhart Shirley , Tsolis Renee
Published: January 2023

Non-typhoidal Salmonella serotypes (NTS) in pediatric patients with severe malaria can develop a life threatening bacteremia, a major source of child mortality in Sub-Saharan Africa. Here, we use a mouse model, mimicking severe anemia seen in humans, to address mechanisms by which an underlying malaria infection contributes to the increased risk of NTS bacteremia. Plasmodium yoelli infected red blood cells were administered i.p. in CBA or B6 mice. At peak parasitemia, Salmonella Typhimurium was administered by g.g. in a streptomycin-induced colitis model. Characterization of inflammation to NTS in the cecum was assessed by histopathology, microarray and qRT-PCR. Bacterial loads in systemic tissues were followed up to 4 days post inoculation. Our results indicate that the malaria parasite infection causes a global suppression of proinflammatory responses in the intestine, blunts intestinal neutrophil influx and increases the bacterial burden at systemic sites. Blunting of intestinal inflammatory responses was independent of antibody-induced anemia, but required induction of the immunoregulatory cytokine IL-10. Blocking IL-10 activity reduced systemic NTS in coinfected mice and administration of exogenous IL-10 was sufficient to increase the systemic burden of NTS in the absence of parasite infection. However, an increase in circulating NTS required both anemia and IL-10. Thus, we have identified two factors that synergize to increase bacteremia using a murine model.
Influence of viral specific memory cells on Leishmania major infection (164.12)
Authors: Crosby Erika , Wherry E. , Scott Phillip
Published: January 2023

One of the hallmarks of adaptive immunity is the development of a pathogen specific immune response that is maintained long term. Despite this specificity, an important question is whether these persistent memory cells can influence the immune response to an unrelated infection. Here we utilized lymphocytic choriomeningitis virus (LCMV) and Leishmania major (L. major) as our model infections to investigate the influence of viral specific memory cells on L. major infection. Using a transgenic P14 CD8 T cell transfer system, we assessed the ability of LCMV activated cells to migrate to an L. major lesion. While naïve P14 T cells were unable to migrate to an L. major lesion, P14 T cells from a mouse that had been infected with LCMV 30 days prior migrated readily to the lesion. Having seen that LCMV memory cells were able to enter the lesion, we asked whether viral CD8 memory T cells could influence the course of L. major infection. Mice were infected with LCMV and 30 days later infected with L. major. From 2 weeks post infection on, there was a significant increase in the pathology of the coinfected mice compared to mice infected with L. major alone. Corresponding with the increased lesion size was an increase in the number of CD8 T cells making granzyme, but not IFN-γ, within the lesions of coinfected mice. Ongoing studies are focused on determining the mechanism by which these non-specific memory cells increase the pathology associated with L major infection.
Modulation of immune responses to Clostridium difficile by peroxisome proliferator-activated receptor γ and miRNA-146b (164.14)
Authors: Viladomiu Monica , Hontecillas Raquel , Pedragosa Mireia , Michalak Pawel , Michalak Katarzyna , Guerrant Richard , Roche James , Warren Cirle , Bassaganya-Riera Josep
Published: January 2023

Clostridium difficile is typically a harmless anaerobic bacterium but has recently re-emerged as a pathogen that can cause nosocomial diarrhea, colitis and death. To investigate the role of peroxisome proliferator-activated receptor (PPAR) γ in modulating immune responses to C. difficile we have performed several studies using a mouse model of antibiotic-induced C. difficile-associated disease (CDAD). The loss of PPARγ in T cells increased disease activity, body weight loss, and colonic inflammatory lesions after infection. It also resulted in upregulated IL-17 and MCP-1, and downregulated IL-10 expression, suggesting that a Th17 phenotype predominates during CDAD in mice lacking T cell PPARγ. Treatment of C. difficile-infected mice with PPAR γ agonists ameliorates disease severity and colonic lesions. RNA-seq results in colonic specimens indicated that three miRNAs were significantly overexpressed in infected mice: mmu-miR-146b, mmu-miR-1940, and mmu-miR-1298 (FDR P < 0.05). Real-time PCR results validated that infection results in overexpression of miR-146b, a molecule involved in regulating immunity and inflammation. Furthermore, NCOA4, a co-activator of PPARγ and target of miR-146b, was down-regulated in colons of infected mice. We provide novel evidence supporting a role for PPARγ in regulating C. difficile-related inflammation and immunopathology and explore the potential role of miRNA in modulating host responses to C. difficile.
Probiotics-containing yogurt ingestion modulates systemic cellular and humoral immunity in children (164.20)
Authors: Huang Shu-Ching , Yang Yao-Jong
Published: January 2023

Background: This study aimed to investigate whether short-term probiotics-containing yogurt ingestion benefits to systemic cellular and humoral immunity in healthy schoolchildren. Methods: Healthy schoolchildren aged 4-12 years without major systemic or allergic history were studied. Subjects had diarrheal diseases or taken antibiotics within one month were also excluded. The Ethical Committee of our institute has approved our study design (ER-98-050). All of them have provided the serum samples before and after 4-week ingestion of probiotics-containing yogurt. The serum samples were tested for the TNF-α, IL-17, IL-10, IL-6, Immunoglobulin (Ig) A, G, and E levels. Results: A total of 60 children completing at least 80% consumption of total designed amount of yogurt were enrolled. Their mean age was 9.3±1.6 years with a female to male ratio 0.54. For systemic cytokines production, yogurt ingestion significantly decreased serum IL-6 level (12.8±4.2 vs. 24.2±6.3 pg/ml, P = 0.002) and marginal significantly increased TNF-α (66.4±14.6 vs. 44.5±10.9 pg/ml, P = 0.11). For immunoglobulin levels, children consumed yogurt can significantly increase serum IgA level (178.8±8.2 vs. 167.1±7.9 mg/dl, P = 0.001). However, yogurt consumption did not affect the levels of IL-17, IL-10, IgG, and IgE. Conclusions: Short-term probiotics-containing yogurt ingestion benefits on an increment of serum IgA and decrease of IL-6 levels in healthy schoolchildren.
Differential regulation of pathogenesis and immune reactions in C57BL/6 and CBA/N mice infected with Clonorchis sinensis (164.22)
Authors: Jin Yan , Wi Hae Joo , Choi Min-Ho , Hong Sung-Tae , Bae Young Mee
Published: January 2023

The current knowledge of immunological response to clonorchiasis, a major Asian endemic helminthic disease, is insufficient. To develop a mouse model of clonorchiasis, CBA/N and C57BL/6 mice were infected with Clonorchis sinensis metacercariae and analyzed host responses at 2, 4, and 8 weeks post infection. CBA/N mice infected with C. sinensis developed more severe gross and histopathological changes than C57BL/6 mice. In addition, more pronounced inflammatory cell infiltration and hepatocyte necrosis were noticed in CBA/N mice, especially at 2 weeks post-infection. Serum biomarkers of liver injury (ALT and AST) were increased significantly in CBA/N mice after C. sinensis infection as compared with those of C57BL/6 mice. We also analyzed the proliferation of splenocytes stimulated with crude C. sinensis antigen and cytokine production in the culture supernatant of activated splenocytes. We found that splenocytes from CBA/N mice were highly proliferative and the IL-17 level was increased. Additionally, an increased IL-4 level was sustained until 8 weeks post-infection in CBA/N mice. The serum levels of C. sinensis-specific IgG1 and IgG2a were increased in CBA/N mice, whereas IgE was increased in C57BL/6 mice. Furthermore, the level of IL-17 was increased in intrahepatic lymphocytes in CBA/N infected groups. Present study suggest that CBA/N could be a appropriate mouse model for clonorchiasis and might be useful for studying early immune responses in liver fluke infection.
Identifying host genes that determine disease outcome to Toxoplasma infection through expression QTL analysis (164.23)
Authors: Jensen Kirk , Hassan Musa , Hu Kenneth , Boedec Erwan , Saeij Jereon
Published: January 2023

Disease outcome is significantly influenced by one’s genetic background. Here we explore the genetic basis for differences in host susceptibility to infection with Toxoplasma, an orally acquired parasite that chronically infects 2 billion people worldwide. The survival of 30 recombinant inbred (RI) mouse strains, derived from crosses between susceptible C57BL/6 and resistant A/J mice, following oral challenge with Toxoplasma has previously been reported and genetic loci on mouse chromosomes 1, 9, 11 and 15 control susceptibility. However, it is not clear what genes within these loci predispose B6 mice to infection. To identify genes within these loci that determine disease outcome, we sequenced the RNA of bone marrow derived macrophages from each RI mouse strain after Toxoplasma infection or TNFα + IFNγ stimulation. The macrophage cell type was chosen because it elicits many of the host’s immuno-regulatory and toxoplasmacidal mechanisms following oral infection and/or cytokine stimulation. The gene expression of each gene was considered a quantitative trait (expression QTL) that was subsequently mapped to mouse genetic loci. By analyzing both trans- and cis-regulation of gene expression, key regulators of the macrophage response to cytokine stimulation was uncovered, including the differential expression of a single candidate gene that correlates well with host susceptibility loci on mouse chromosomes 3, 9 and 15. Results from ongoing experiments will be discussed.
Treatment with Lm-LLO immunotherapies lead to decreased Tregs and MDSC in the tumor microenvironment (165.12)
Authors: Singh Reshma , Malinina Inga , Ramos Kimberly , Wallecha Anu
Published: January 2023

Bioengineered Listeria monocytogenes (Lm), which secrete antigens fused to a fragment of listeriolysin O (LLO) have been shown to be effective cancer immunotherapeutics in several mouse models of cancer, and are currently being evaluated in Phase 2 clinical trials for HPV-associated dysplasia and malignancies. Treatment with Lm-LLO immunotherapies results in a marked increase in tumor infiltrating lymphocytes, in particular CD8+T cells. The antigen-specific CD8+T cells have been observed to kill tumor cells in vitro, showing the functionality of the Lm-stimulated T cells. Lm-LLO immunotherapies also have an impact on suppressor cells in the tumor microenvironment. In various tumor models, treatment results in a decrease in the ratio of regulatory T cells (Tregs) to effector T cells in the tumors. Here, we report a corresponding decrease in the ratio, and number, of monocyte-derived suppressor cells (MDSCs) after Lm-LLO immunotherapy. These change are only seen in the tumor microenvironment. Further, both Tregs and MDSCs isolated from the tumor have a decreased ability to suppress the division of T cells after Lm-LLO immunotherapy. However, there is no change in functional ability of splenic Tregs or MDSCs. Thus, the efficacy of Lm-based immunotherapies on tumors is likely due to a combined increase in antigen-specific effector cells and a tumor specific decrease in the numbers and function of suppressor cells in the tumor microenvironment.
Intracellular signaling “fingerprint” in CD4 T cells in response to vaccination (165.14)
Authors: Reichenbach Dawn , Finn Olja
Published: January 2023

Early biomarkers of vaccine efficacy need to be established. We hypothesize that intracellular signaling within CD4 T cells as early as 24 and 48 hours post vaccination could yield a “fingerprint” that is predictive of the ultimate effectiveness of the elicited immune response. In our model, there are reduced responses to MUC1 peptide (MUC1p) vaccination in T cells in human MUC1 transgenic mice (MUC1.Tg) mice compared to wildtype (WT) mice. We are examining phosphorylation downstream of TCR, cell survival, proliferation, and apoptosis signaling cascades post vaccination with either DC loaded with MUC1p (DC:MUC1p) or soluble MUC1p in conjunction with poly:ICLC (sMUC1p:ICLC). Adoptively transferred TCR transgenic CD4 T cells specific for MUC1p displayed no statistically significant differences in signaling during priming in WT vs MUC1.Tg mice at 1, 6, 12 and 24 hrs post vaccination with DC:MUC1p. However, there were trends towards decreased phosphorylation in STAT1, STAT4, and increased pLCK in WT mice. We are currently examining signaling differences in the endogenous memory response in MUC1.Tg and WT mice 1 and 24 hrs post secondary boost of either DC:MUC1p or sMUC1p:ICLC vaccines to determine if differences exist upon secondary exposure and if signaling can be altered. Vaccinated MUC1.Tg and WT mice have been challenged with B16.MUC1 to correlate early T cell signaling with control of tumor burden and intracellular signaling occurring in the tumor microenvironment.
Novel cancer immunotherapy using rational combinations of B7-H1 blockade, Treg depletion and estrogen receptor beta signaling (165.17)
Authors: Curiel Tyler , Kious Mark , Sun Lishi , Vadlamudi Ratna , Hurez Vincent , Murthy Kruthi
Published: January 2023

We recently showed that B7-H1 blockade in mouse models for B16 melanoma and ID8 ovarian carcinoma worked better in females versus males through reduced regulatory T cell (Treg) function. We now show that B7-H1 blunts estrogen-mediated mTOR activation in differentiating Tregs as the mechanism. When we differentiated naïve CD4+ T cells into Tregs in vitro, estrogen reduced Treg numbers and function only if B7-H1 was blocked. The effect was equivalent in males and females. Treg depletion for cancer immunotherapy is promising but hampered by rapid Treg regeneration. Because estrogen signals reduced Treg generation when B7-H1 was blocked, we hypothesized that B7-H1 block during therapeutic Treg depletion would improve clinical efficacy of either individual approach, and that estrogen signals would further boost combination effects. We then showed that estrogen effects were through estrogen receptor beta, suggesting that estrogen receptor beta agonists (in human clinical trials with promising results) could boost Treg depletion with B7-H1 blockade as cancer immunotherapy. In a proof-of-concept experiment, in B16 melanoma challenge, estrogen improved tumor resistance in B7-H1 KO males but not WT males through reduced Treg function. B7-H1 blockade plus estrogen also improved anti-CTLA-4 effects, suggesting rational combinations of immune blockade, Treg depletion and estrogen signals as novel cancer immunotherapies.
Comparative Efficacy of miniKexin DNA versus protein vaccination against Pneumocystis infection. (166.13)
Authors: Elsegeiny Waleed , Zheng Mingquang , Yu Amy , Kolls Jay
Published: January 2023

Pneumocystis Pneumonia (PCP) is a leading cause of death in individuals with AIDS or other immunocompromised conditions. We have previously shown that mice vaccinated with DNA encoding full-length PC antigen Kex1 have approximately a three-log reduction in PC organism burden after challenge. Full length Kex1 is poorly expressed so to improve on this we developed codon optimized vectors expressing a truncated conserved form of Kex1, mini-kexin. This also allowed investigation of a prime boost strategy since mini-kexin could be efficiently packaged into recombinant adenovirus. Mice were vaccinated with DNA encoding mini-kexin followed by boosting with adenovirus, CD4+ T cells were then depleted by GK1.5, and finally the mice were challenged with live Pneumocystis. The results showed significantly reduced PC burden in vaccinated mice. We then proceeded to generate a protein-based, E coli produced vaccine, however, this time vaccine elicited similar anti-Kex1 titers but lacked protection. We hypothesize that post-translational modification such as glycosylation could play a role in the efficacy of mini-kexin vaccination.
Memory T cells convert tissue resident myeloid cells into APCs during clearance of a persistent viral infection (166.28)
Authors: Herz Jasmin , McGavern Dorian
Published: January 2023

Persistent viral infections are often very difficult to treat in humans. Adoptive immunotherapy is an approach that involves administration of anti-viral T cells and has shown some promise in the clinic. Our laboratory models adoptive immunotherapy by transferring anti-viral memory T cells into mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV). Here, we demonstrate that memory T cells can completely purge the brain of persistently infected mice without causing immunopathology or blood brain barrier breakdown. Memory T cells accomplish this by inducing a tailored release of chemoattractants that recruit adaptive immune cells, but few pathogenic innate immune cells (e.g. neutrophils and inflammatory monocytes) into the nervous system. Memory T cells also enlist the support of nearly all brain resident myeloid cells (referred to as microglia) by converting them into CD11c-expressing antigen-presenting cells (APCs). Following conversion, CD11c+ microglia release CCL5 and promote interactions with therapeutic memory T cells in the brain parenchyma. Our two photon imaging studies revealed that anti-viral CD8 T cells are more likely than CD4 T cells to decelerate and form stable interactions with brain-resident APCs. We propose that non-cytopathic viral clearance from the brain by therapeutic memory T cells results from tailored chemoattractant production and conversion of resident myeloid cells into CD11c+ APCs.