bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of Kruppel-like factor 2 (KLF2) in thymic emigration and trafficking of non-conventional T cell lineages (82.16)
Authors: Odumade Oludare A , Weinreich Michael A. , Takada Kensuke , McCaughtry Tom , Carlson Corey M. , Lingrel Jerry , Elewaut Dirk , Jameson Stephen C. , Hogquist Kristin A.
Published: January 2023

Immune function is dependent on the proper development of T cells in the thymus and emigration of these cells to the periphery. We recently showed the transcription factor KLF2 is required for emigration via regulation of S1P1 (sphingosine 1-phosphate receptor 1) and CD62L in conventional αβ T cells. However, the role of KLF2 and S1P1 in trafficking of non-conventional T cell subsets (γδ T cells, Treg, NKT, and Gut intraepithelial lymphocytes -IEL) has yet to be described. We show here that KLF2 is differentially expressed within the T cells subsets studied using both mRNA expression via quantitative PCR and/or a novel KLF2-GFP reporter mouse model. While KLF2 and S1P1 can be detected in sorted non-conventional thymic T cells and Gut IEL αβ T cells, we report no detectable KLF2 in Gut IEL γδ T cells. Using several KLF2 deficient mouse models, trafficking defects were observed in Treg, NKT, and Gut-IEL (αβ and γδ) populations. Finally, the data suggest that lack of KLF2 alters both the distribution and the phenotype of γδ T cells in secondary lymphoid organs. Overall, our results suggest that KLF2 is a common means by which different T cell lineages regulate trafficking and/or entry.

Funding provided by the National Institutes of Health (AI038903 to SCJ and AI039560 to KAH).
The energy sensitive kinase mTOR regulates T-bet mediated heritable type 1 effector cell fate of CD8 T cells (83.1)
Authors: Rao Rajesh  R , Shrikant Protul A
Published: January 2023

The inflammatory cytokine; IL-12, provides an essential third signal for CD8 effector cell fate determination. The molecular mechanisms by which IL-12 regulates STAT4 and T-bet activity for instructing effector cell fate remains unclear. The energy sensitive kinase; mTOR (mammalian target of rapamycin), a target of both the PI3K-Akt and/or AMPK signaling pathways has recently been shown to affect trafficking of effector CD8 T cells in vivo, however its role in cytokine determined effector cell fate remains untested. Using latex microspheres; H-2Kb/SIINFEKL and mB7.1, as surrogate antigen presenting constructs, we have established that the presence of IL-12 enhances and sustains antigen/B7.1 induced mTOR activity in naïve OT-1 T cells. The augmented mTOR activity requires PI3K as well as STAT4 and inhibition of mTOR; rapamycin or temsirolimus, leads to the loss of IL-12 induced sustained T-bet expression and subsequent loss of type I (IFNg) CD8 effector cell fate. Interestingly, inhibition of mTOR in IL-12 conditioned OT-1 cells produced only marginal decreases in cytolytic activity and increases in CD69, L-selectin and Eomesodermin expression. Hence, we have demonstrated a novel role for mTOR in integrating instructions to regulate effector cell fate in naïve CD8 T cells and characterized its mechanisms of action.

Supported by NIH and RPCI Alliance Foundation
The expansion of parasite-specific CD8+ T cells during infection is regulated by the transcription factor T-bet (83.7)
Authors: Smeltz Ronald , Cobb Dustin , Guo Siqi , Lara Ana , Manque Patricio , Buck Gregory
Published: January 2023

T-bet is a transcription factor that promotes Type I immunity to pathogens through effects involving T cells and dendritic cells. However, many of the functions of T-bet in CD8+ T cells can be compensated for by Eomes. Therefore we investigated the role of T-bet in immunity to T. cruzi, an intracellular pathogen that causes Chagas disease and which requires IFN-γ-secreting CD8+ T cells for resistance. T-bet-deficient mice (t-bet-/-) were highly susceptible to T. cruzi infection. However, susceptibility was not secondary to a lack of T cell activation, as T cells displayed typical markers of activation, including CD44, CD62L, and CD25. In contrast, we observed a profound reduction in the number of antigen-specific CD8+ T cells in t-bet-/- mice as determined by MHC tetramer analysis. Failure to generate sufficient numbers of antigen-specific T cells was not due to impaired APC functions, as we observed no differences in co-stimulatory molecule expression or in antigen-driven proliferation of wild-type CD8+ T cells in infected t-bet-/- mice. However, transfer of naïve T cells from t-bet-/- donors into infected rag-2-/- mice (t-bet+/+) demonstrated a similar quantitative defect, as CD8+ T cells failed to expand in vivo. These data demonstrate that T-bet contributes to resistance to T. cruzi infection partially via regulating the expansion of T. cruzi-specific CD8+ T cells in a T cell-intrinsic manner. They also serve to further illustrate the multi-faceted functions of T-bet in controlling quantitative aspects of T cell immunity, in addition to qualitative components such as Th1/Th17 immunity.
Deltex1: a novel regulator of T cell activation and T cell anergy (84.12)
Authors: Liu Wen-Hsien , Hsiao Huey-Wen , Lai Ming-Zong
Published: January 2023

Deltex1 (DTX1) is a target of Notch signaling, but its physiological function in T cells is unclear. Here we show that Dtx1 is a novel transcription target of NFAT involved in T cell anergy. DTX1 was upregulated during both in vitro and in vivo T cell anergy induction. Transgenic expression of Dtx1 attenuated T cell activation but does not alter T cell development. DTX1 transgenic mice were resistant to EAE induction. In the present study, we generated DTX1-deficient mice to study the function of DTX1 in T cell anergy and T cell development. Dtx1 knockout did not interfere with thymocyte development. Deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation and autoimmunity. DTX1 therefore represents an interactive component downstream of calcium/NFAT that regulates T cell anergy.
Protein phosphatase 2A (PP2A) is involved in the age-related down-regulation of E47 mRNA stability (84.14)
Authors: Blomberg Bonnie B. , Landin Ana Marie , Diaz Alain , Riley Richard L. , Frasca Daniela
Published: January 2023

We have previously shown that the transcription factor E47, which regulates activation-induced cytidine deaminase (AID) and Immunoglubulin class switch in splenic B cells, is down-regulated in aged murine B cells due to increased E47 mRNA decay. At least part of the decreased stability of E47 mRNA seen in aged B cells is mediated by tristetraprolin (TTP), a physiological regulator of mRNA stability. We found that TTP mRNA and protein expression are higher in old than in young B cells. Both TTP protein expression and function in B cells are dependent upon p38 MAPK and there is less phospho-p38 in old activated B cells. Also there is less phospho-TTP in old than in young splenic activated B cells which leads to more binding of TTP to the 3'-UTR of E47 mRNA, therefore decreasing its stability. PP2A is a serine/threonine protein phosphatase that plays an important role in the regulation of a number of major signaling pathways. We found that not only the amount but also the activity of PP2A is increased in old B cells. By specific inhibition of PP2A we have shown less phospho-p38 MAPK and phospho-TTP in old B cells. PP2A dephosphorylation of either p38 MAPK and/or TTP may account for more binding of the hypophosphorylated TTP to the 3'-UTR of the E47 mRNA, inducing its degradation. This mechanism may be at least in part responsible for the age-related decrease in antibody class switch.

This work is supported by NIH AG-23717 (BBB) and by NIH AI-064591 (RLR).
Th2 specific immunity and function of peripheral T cells requires the p56Lck SH3 domain (84.15)
Authors: Straus David B. , Finkelman Fred D , McCoy Margaret E.
Published: January 2023

T-cell activation and effector function is essential for immunity. T-cell antigen receptor (TCR) signals can regulate the outcome of differentiation, but the mechanisms are unclear. We have found that the Src family tyrosine kinase, p56Lck, specifically links TCR signals to activation of the Mitogen Protein Kinase (MAPK) pathway through the action of its SH3 domain and controls T cell activation and Th2 immunity. We have utilized an Lck SH3 mutant knock-in mouse line, LckW97A, to investigate the potential role of this regulatory domain in regulation of T-lymphocyte activation and effector function. Our results demonstrate that the p56Lck SH3 domain is required for normal activation of T-lymphocytes. LckW97A T-cells have reduced IL-2 production, CD69 induction and proliferation following TCR stimulation. Biochemical studies show that activation of the MAPK pathway is selectively altered as P-ERK1/2 induction is significantly reduced but phospho-PLCγ1 induction is unaffected. In vivo experiments demonstrate significantly impaired Th2 immunity with reduced serum levels of IgG1, IgE and IL-4 following immunization with DNP-KLH or infection with the helminth Nippostrongylus brasiliensis. These data indicate that p56Lck SH3 domain regulates activation of T-lymphocytes by controlling MAPK pathway induction and demonstrate a critical role for p56Lck in the regulation of Th2-type immunity.
A conserved salt bridge in the G-loop of multiple protein kinases is essential for catalysis, in vivo Lyn function and prevention of autoimmune disease (84.17)
Authors: Sauer Karsten , Che Jianwei , Lee Christian C. , Yang Yating , Herman Ann , Jia Yong , Velentza Anastasia , Watson James , Sternberg Luise , Kim Sunjun , Ziaee Niusha , Miller Andrew , Jackson Carie , Young Mike , Batalov Serge , Liu Yi , Warmuth Markus , Wiltshire Tim , Cooke Michael P. , Barouch-Bentov Rina
Published: January 2023

The glycine-rich G-loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases from plants to humans. By limiting G-loop flexibility, it controls ATP binding, catalysis and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn-/- mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G-loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.
The tyrosine kinase c-Abl regulates CD4+ T-cell differentiation by catalyzing T-bet phosphorylation (84.5)
Authors: Lee Sang-Myeong , Chen An , Gao Beixue , Fang Deyu , Shannon Stephen , Zhang Jinping , Ye Shui Q , Calhoun Karen
Published: January 2023

The tyrosine kinase c-Abl is required for full activation of T cells, while its role in CD4+ T-cell differentiation has not been characterized. We report that c-Abl-deficiency skews CD4+ T cells to Th2 differentiation, and c-Abl-/- mice are more susceptible to allergic lung inflammation. c-Abl interacts with and phosphorylates T-bet, a Th1-lineage transcription factor, indicating that c-Abl is a tyrosine kinase for T-bet in T cells. c-Abl-mediated phosphorylation enhances the transcriptional activation of T-bet. Interestingly, the tyrosine residues within the T-box domain of T-bet are mapped as the predominant sites of c-Abl. Mutation of these tyrosine residues inhibits the promoter-binding activity of T-bet. Therefore, c-Abl regulates CD4+ T cell differentiation by targeting T-bet phosphorylation.
Expression of Sca-1 (Ly-6A/E) defines functionally different developmental stages of plasmacytoid dendritic cells (PDC) (84.9)
Authors: Kurig Stefanie , Fischer Jens , Dzionek Andrzej
Published: January 2023

Stem cell antigen-1 (Sca-1), a phosphatidylinositol-linked membrane protein is one of the most prominent stem cell markers. Functionally, Sca-1 is involved in cross-talk between lymphocyte precursors and stromal cells and plays an important role in the thymic development of T-cells and TcR-dependent activation.

Here we show that expression of Sca-1 on PDC defines functionally different developmental stages of PDC in mice. The proportion of the Sca-1+ PDC showed an organ specific variation ranging from 10-20% in BM to >80% in LN. Sca-1- PDC appeared earlier in the development and give rise to Sca-1+ PDC without the need for activation. In addition to the naturally occurring up-regulation, Sca-1 expression could be further induced by TLR7 and TLR9 mediated activation. Interestingly, when exposed to CpG-oligonucleotides (TLR9), Sca-1- PDC produced significantly higher amounts of IFN-alpha. This was further confirmed by two observations: (1) after stimulation with TLR9-ligands purified PDC from BM (mainly Sca-1-) produced up to 100-fold higher amounts of IFN-alpha when compared to PDC from LN (predominantly Sca-1+), (2) Sca-1- PDC expressed significantly higher amount of genes involved in TLR mediated IFN-alpha induction, including MyD88, IRF3, IRF5, IFNAR1 and IFNAR2.

Taking together, our results identify Sca-1 as a developmental marker of PDC, which defines two functionally heterogeneous subsets of PDC.
Notch and IL-7 regulate cell fate decisions in TCRβ+ DN and ISP thymocytes (85.10)
Authors: Xiong Juan , Yankee Thomas M.
Published: January 2023

During the CD4-CD8- double negative (DN) stage of T cell development, the cells express TCRβ for the first time. TCRβ+ cells activate signaling pathways that promote survival, proliferation, and differentiation, but the receptors required for these signals are unknown. While Notch and IL-7 are critical for early T cell development, their function in TCRβ-expressing DN and immature single positive (ISP) CD8+ thymocytes are unclear. Here, we cultured purified DN3E (CD44-CD25hi), DN3L (CD44-CD25lo), DN4 (CD44-CD25-) and ISP CD8+ thymocytes with OP9 cells that express either the Notch ligand Delta-like 1 (DL1) or control protein. To some samples, IL-7 was also added. DN and ISP thymocytes were able to differentiate into CD4+CD8+ double positive (DP) thymocytes independently of Notch and IL-7. However, Notch promoted the proliferation and survival of the thymocytes, whereas IL-7 promoted survival and slowed differentiation. These data demonstrate that Notch and IL-7 continue to function beyond the time at which TCRβ is expressed and regulate survival, proliferation, and differentiation of TCRβ-expressing thymocytes.

This research is supported by the America Cancer Society grant RSG-08-182-01-LIB.
A full complement of CD3 ζ ITAMs is necessary for NKT cell development and function (85.11)
Authors: Becker Amy M. , Blevins Jon S. , Wight-Carter Mary , Medeiros Jennifer J. , Murray Sean , Richardson James A. , Norgard Michael V. , van Oers Nicolai S.C.
Published: January 2023

Invariant natural killer T (iNKT) cells express a canonical αβ TCR that recognizes glycolipids embedded in non-classical MHC molecules. These cells develop following TCR interactions with CD1d/glycolipid complexes expressed on thymocytes. The αβ TCR is non-covalently associated with six signaling proteins, CD3 ζζ and CD3 γε/δε, that signal through ITAMs. It is unknown whether a full complement of ITAMs is required for iNKT cell development and function. We previously generated CD3 ζ transgenic mice with mutations in the first (YF1,2) or all three CD3 ζ ITAMs (YF1-6) to reduce ITAM signaling. iNKT cell development was analyzed in these mice. Compared to C57BL/6 controls (0.43%; 1.04%), the percentage and absolute numbers of iNKT cells were significantly reduced in the thymus and spleen of YF1,2 (0.008%; 0.023%; 11- and 19-fold) and YF1-6 (0.044%; 0.11%, 50- and 100-fold) mice. Comparative phenotyping suggested that the iNKT cells had an early developmental block in the YF1,2 and YF1-6 mice as evidenced by an increased percentage of iNKT cells expressing CD24 compared to C57BL/6 mice. These data indicate that a full complement of CD3 ζ ITAMs is required for proper iNKT cell development. We are currently assessing the functionality of iNKT cells in the various mice in response to glycolipids or following infections with the pathogenic bacteria Borrelia burgdorferi .
The function of zbtb7b in regulating CD4 vs. CD8 T linage commitment is evolutionarily reserved (85.17)
Authors: He Xiao , Jensen Peter
Published: January 2023

Immature thymocytes develop into mature T lymphocytes, including 2 major functional subpopulations: CD4+ helper and CD8+ cytotoxic T cells, prior to emigration from thymus. Previously we discovered a zinc finger transcription factor, ThPOK (T helper inducing POK factor, also known as zbtb7b, a member of the zbtb7 transcription factor subfamily), functioning as a master regulator in regulating CD4 vs CD8 T cell linage commitment process. However, the molecular mechanisms underlying this process still remain to be elucidated. Intriguingly, when mouse ThPOK was ectopically expressed as a transgene in T cell lineage, all Tg mice developed fatal T lymphomas. Here we reported that when the human zbtb7b sharing an 87% identity with the mouse counterpart at DNA level and a 97% homology at protein level, was expressed in mouse T cell lineage under the CD2 promoter, some Tg mice not only could correct the ThPOK deficient phenotype (MHC class II restricted T cells are CD8+), but also developed T lymphomas. Furthermore, when another zbtb7 transcription factor subfamily member, the mouse zbtb7a sharing an 83% homology with the mouse zbtb7b at protein level, was expressed in T cells, some Tg mice again corrected the ThPOK deficient phenotype and developed T lymphomas as well. Taken together, these results indicate the function of zbtb7b in regulating CD4 vs CD8 T linage commitment is evolutionarily reserved (supported by AI-33614).
AGAPE, a new T cell specific Grb2 binding protein that plays a critical role in T cell development (85.7)
Authors: lesourne renaud , Love Paul
Published: January 2023

We report here the identification of a T-cell lineage protein designated AGAPE (ä-gä-pa; Another Grb2-Associated Protein Enigma). AGAPE contains a proline rich sequence that associates with the adaptor protein Grb2. It lacks any consensus catalytic domains but contains a nuclear localization sequence required for its translocation to the nucleus. AGAPE deficient mice were generated by homologous recombination in ES cells. AGAPE-/- mice have normal numbers of DN and DP thymocytes but markedly reduced numbers of CD4 SP and CD8 SP thymocytes and pheripheral T cells. Positive selection is dramatically reduced in both class II- and class I-restricted TCR transgenic AGAPE-/- mice. The proportion of CD4+CD8lo thymocytes was reduced in AGAPE-/- MHCII-/- mice suggesting that AGAPE plays and important role at the DP to SP transition stage. Interestingly, TCR induced activation responses are unaffected in AGAPE-/- thymocytes indicating that the developmental block is not due to a defect in signaling downstream of the TCR? Although DP thymocytes from AGAPE-/- mice show enhanced susceptibility to antibody-induced cell death, the introduction of the Bcl-2 transgene did not rescue T cell development. Expression of the lineage specific transcription factors ThPok and Gata-3 (but not Tox or Runx1/3) was reduced in CD69+ thymocytes purified from AGAPE-/- mice. However, enforced expression of ThPok or Gata-3 did not rescue CD4 SP development in AGAPE-/- mice. Thus, AGAPE is a new T cell specific nuclear protein that has a critical function during late thymocyte selection prior to CD4/CD8 lineage specification.
An in situ hybridization screen reveals new transcriptional regulators of third pharyngeal pouch and thymic epithelial cell differentiation. (86.12)
Authors: Condie Brian G , Wei Qiaozhi
Published: January 2023

Thymic epithelial cells (TECs) are the major component of the non-lymphoid framework of the thymus and are essential for normal T cell development. In mice, TECs develop from a domain of the embryonic third pharyngeal pouch endoderm marked by Foxn1 expression. Although Foxn1 is necessary for proliferation and differentiation of TECs, it is not required for specification of TECs from the pouch endoderm. Also, some aspects of TEC differentiation are Foxn1-independent. This suggests that there are additional transcription factors expressed within the third pouch endoderm that are responsible for early specification and differentiation of TECs. To identify additional transcriptional regulators of third pouch and TEC differentiation, we performed an in situ hybridization screen on a set of candidate transcription factors identified through a systematic analysis of gene expression databases. We found that most of the genes selected for detailed characterization were expressed in the third pharyngeal pouch endoderm and many of them were specifically expressed in the thymus domain of the pouch. These transcription factors are highly likely to be involved in the earliest steps of TEC development. Our work also shows that data mining of gene expression databases is an efficient way to identify genes involved in the development of a specific tissue or organ.
Estrogen receptor signaling promotes langerin+ dendritic cell differentiation by increasing expression of the transcription factor IRF4 (87.1)
Authors: Carreras-Margalef Esther , Turner Sean , Frank Mark Barton , Knowlton Nicholas , Osban Jeanette , Centola Michael B , Alberola-Ila Jose , Kovats Susan
Published: January 2023

During inflammation, elevated GM-CSF promotes the development of new dendritic cells (DC). We previously showed that estrogen receptor alpha (ERα) signaling in response to estradiol is required for the GM-CSF mediated differentiation of CD207/langerin+ CD11c+ tissue DC. DC development from ERα+ flt3+ committed myeloid progenitors (MP) was increased significantly after a 12 hr exposure to estradiol. We now have identified the transcription factor interferon regulatory factor 4 (IRF4) as an estradiol-induced gene in a microarray experiment comparing MP incubated -/+ estradiol. The IRF4 gene is induced by GM-CSF and critical for myeloid DC development in vivo. Quantitative PCR analysis showed that IRF4 mRNA in MP was increased two-fold within 6 hr of estradiol exposure. Differentiating CD11c- precursors also had two-fold higher levels of IRF4 protein after a 48 hr exposure to estradiol; this increase in IRF4 expression was not observed in differentiating ERα-/- CD11c- cells. Retrovirus mediated delivery of the IRF4 cDNA to ERα-/- bone marrow cells differentiating in the absence of estradiol restored the development of the estradiol-dependent DC population. Thus, ERα signaling in response to estradiol promotes the GM-CSF mediated differentiation of a langerin+ DC population by increasing expression of IRF4 protein in myeloid progenitors.

Supported by OCAST HR06-157 and NIH RR020143.
cFLIP is required for macrophage survival and neutrophil homeostasis (87.3)
Authors: Gordy Claire Lee , Pua Heather , He You-Wen
Published: January 2023

The anti-apoptotic protein cellular FLICE-like inhibitory protein (cFLIP) has been shown to play an important role in survival of T and B lymphocytes; however, the function of cFLIP in other immune cells is unknown. To examine the role of cFLIP in myeloid cell survival and function, we generated mice expressing a loxP-flanked cFLIP allele in the presence of Lysozyme M-Cre (cFLIPf/f Lysm-Cre). cFLIPf/f Lysm-Cre mice lack splenic marginal zone macrophages and thioglycollate-elicited peritoneal macrophages, while inflammatory monocyte and neutrophil numbers are increased. Using this model, we find that loss of cFLIP expression in macrophages and neutrophils results in a severely inflammatory phenotype, with cFLIPf/f Lysm-Cre mice displaying splenomegaly, decreased body weight, profound neutrophilia, increased production of IL-1β and GCSF, and extramedullary hematopoiesis. These defects are secondary to the loss of macrophage populations, as mixed bone marrow chimeric mice do not develop inflammation. Together, these data demonstrate a requirement for cFLIP expression for macrophage survival along with a requirement for mature macrophage populations to maintain neutrophil homeostasis in vivo.

This work is supported by an NSF Graduate Research Fellowship and NIH grants.
Crucial roles of Sp1 in the transcriptional regulation of LIGHT gene by Axl in T cell lymphoma (88.13)
Authors: Lee Eun-Hee , Park Areum , Kim Eun-Mi , Yi Ja-woon , Lee Hwa-youn , Choi Harim , Lemke Greg , Kang Hyung-Sik
Published: January 2023

To elucidate the role of Axl on the regulation of LIGHT expression, we established the stably over-expressing Axl T lymphoma cells in mouse EL4 (EL4-Axl) and human Jurkat T lymphoma cells (Jurkat-Axl). The expression of LIGHT and its receptor, herpes virus entry mediator (HVEM) was remarkably increased both in EL4-Axl and Jurkat-Axl compared with the individual controls. Furthermore, the expression of LIGHT, HVEM and IFN-γ was reduced in the various tissues of Axl-/- mice. The proliferation of EL4-Axl cells was inhibited by treatment of Axl-Ig in a concentration and LIGHT expression-dependent manner. In LIGHT promoter assay using a transient transfection of Jurkat-Axl cells with 5¡ deletion mutants, luciferase reporter activity showed the highest transcriptional activity in the gene segment -190/+1. Electrophoretic mobility shift assay (EMSA) and Western blot analysis revealed that Sp1 was able to directly bind to its binding site on LIGHT promoter upon triggering AKT/PI3K signaling pathway.

These results suggest that Sp1 plays a crucial role in the regulation of LIGHT expression by Axl signaling in T lymphoma.

This work was supported by the SRC/ERC program of MOST/KOSEF (RESEARCH CENTER FOR WOMEN'S DISEASES)
Increased tumorigenicity and metastasis of cervical cancer cells by HPV 16 E6-mediated blockade of lymphotoxin signaling pathway (88.14)
Authors: Kim Dong-Hern , Lee Eun-Hee , Kim Eun-Mi , Park Chang-Eon , Lee Hwa-youn , Yi Ja-woon , Yun Chul-Ho , Park Yong-Wook , Kee Seung-Jung , Kang Hyung-Sik
Published: January 2023

To elucidate the regulatory role of LT on human papillomavirus (HPV) E6-mediated tumorigenecity in cervical cancer, HPV 16 E6-overexpressing HeLa (HE6F) and suppressing Caski cervical cancer cells (CE6R) were established. LT gene expression was markedly suppressed in HE6F cells compared to HeLa cells, which led to a significant augmentation of HE6F cell growth via the reduction of apoptosis-associated gene expression. HPV 16 E6-induced HE6F metastasis was further amplified by silencing of LTβR using small interference RNAs. The blockade of LT signaling by LTβR-Ig treatment in HE6F cells significantly suppressed cytotoxicity mediated by NK cells. Recovery of LT by administration of LT DNA in mice challenged with TC-1 cells expressing HPV16 E6 and E7 resulted in the suppression of tumor mass, and the prolonged survival rate. Furthermore, IgA production in both fecal extracts and vaginal washes of mice administrated with LT DNA was significantly higher than that of control mice.

Taken together, these results demonstrated that progress rate of cervical cancer could be promoted by HPV E6-mediated blockade of LT signaling pathway. Regulation of LT might be an effective way to achieve stable suppression of HPV E6 oncogenesis in cervical cancer.

This work was supported by the SRC/ERC program of MOST/KOSEF (RESEARCH CENTER FOR WOMEN'S DISEASES) and by a grant from Biogreen 21 project, Rural Development Administration.
Immune-mediated depletion of pre-leukemic cells delays disease onset in a murine spontaneous leukemia model (88.17)
Authors: Reid Gregor , Seif Alix , Grupp Stephan
Published: January 2023

In pediatric acute leukemia, the occurrence of initiating chromosomal translocations prior to the development of leukemia provides a window of opportunity for prophylactic intervention. Successful targeting of pre-leukemic cells during the prolonged latency period may reduce or prevent the incidence of disease. We have previously reported that treatment with immunstimulatory DNA containing CpG motifs (CpG ODN) generates protective immune responses against leukemia outgrowth in a transplantable tumor model. Using the E-mu-ret transgenic mouse model of pediatric leukemia, which possesses several key features of human disease, we demonstrate a significant delay in disease onset following immune intervention in the pre-leukemic period. Specifically, the prolonged administration of CpG ODN significantly reduced the size of the pre-leukemic cell population in peripheral blood of treated mice (p = 0.028). This reduction correlated with a significant delay in disease onset in CpG ODN-treated compared to control-treated mice (p<0.0001). While no mice in the control group survived beyond 250 days of age, several CpG ODN-treated mice remained disease free at one year of age and showed no sign of an expanded pre-B cell population at sacrifice. These results indicate that immune-based prevention of pediatric leukemia may be a feasible approach to reduce the impact of this disease.
Role of TRAF6 as a Regulator of Endothelial Cell Activation and Angiogenesis (88.20)
Authors: Cejas Pedro Joel , Walsh Matthew C , Pearce Erika L , Harms Gretchen , Choi Yongwon
Published: January 2023

Tumor angiogenesis and metastasis are dependent on tumor-secreted growth factors and pro-inflammatory signals that target receptors on endothelial cells. The signaling cascade that emanates from many of these receptors is mediated by the adaptor molecule and signal transducer TRAF6. To date, however, the intrinsic effect of TRAF6 in endothelial cell biology has not been examined in detail. Most importantly, its effect on angiogenesis, tumor growth and metastasis has not been investigated in vivo. We hypothesize that, by acting as a molecular convergence point in the signaling downstream of different pro-inflammatory and pro-angiogenic stimuli, TRAF6 is a chief regulator of endothelial cell activation and angiogenesis to drive tumor growth and metastasis.

To test this hypothesis, we have generated transgenic mice that lack TRAF6 specifically in endothelial cells (TRAF6-ΔE) to use as a novel in vivo model of angiogenesis and metastasis. Using in vitro angiogenic tests, we have found that TRAF6-deficient endothelial cells have impaired angiogenic properties, and using two different tumor models we have demonstrated that endothelial cell-expressed TRAF6 has an essential role in tumor growth.
The tyrosine kinase Syk regulates survival of chronic lymphocytic leukemia B cells through PKCδ phosphorylation and proteasome-dependent regulation of Mcl-1 expression (88.22)
Authors: Baudot Alice D , Jeandel Pierre Yves , Mouska Xavier , Maurer Ulrich , Tartare-Deckert Sophie , Raynaud Sophie D , Cassuto Jill Patrice , Ticchioni Michel , Deckert Marcel
Published: January 2023

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature monoclonal CD5+ B cells. The disease results mainly from a failure of cells to undergo apoptosis, a process largely influenced by the existence of constitutively activated components of BCR signaling and the deregulated expression of anti-apoptotic molecules. Recent evidence pointing to a critical role of spleen tyrosine kinase (Syk) in ligand-independent BCR signaling prompted us to examine its role in CLL cells survival. Using primary leukemic cells, we show that pharmacologic inhibition of constitutive Syk activity and depletion of its expression by siRNAs led to a dramatic decrease of cell viability in all tested CLL samples (n=44/44), regardless of clinical and biological status, and induced typical apoptotic cell death, with mitochondrial failure followed by caspase 3-dependent cell death. These events were accompanied by a decreased phosphorylation of PKCδ, but not PKCα/β, and by a down-regulation of Mcl-1, but not XIAP. Moreover, the degradation of Mcl-1 following Syk inhibition relied on a caspase-independent, but proteasome-dependent process, and was rescued by the expression of a proteasome-resistant Mcl-1 mutant. Together, our results show that Syk is a key regulator of B-CLL cell viability, and represents an attractive therapeutic target.
The Function of Cancer-Induced Myeloid-Derived Cells (88.25)
Authors: Shin Tahiro , Tomihara Kei , Guo Miao , Zhang Bin , Curiel Tyler
Published: January 2023

Cancer is one of the most common life-threatening diseases, particularly in developed countries, and causes more than one third of all human deaths every year. The effects of standard treatments are still far from satisfactory. Thus, immunotherapies have been under investigation. The greatest merit of immunotherapy is its specificity, compared to non-specific standard treatments. However, one of the most important issues impeding cancer immunotherapy is the recruitment of the immune suppressor cells by cancer cells in the cancer microenvironment. These immune suppressors, represented by regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs), efficiently attenuate the CTL activities against cancer. By using the mouse ovarian cancer cell line, we have found that a large population of the CD11b+ and Gr-1+ cells instead of Tregs accumulated in ascites and the spleen. CD11b and Gr-1 are the typical markers for MDSCs, for instance, induced by the subcutaneous injection of mouse thymoma cell line. They were well characterized with its strong suppressive property and consist of heterogeneous population. However, the CD11b+ and Gr-1+ cells in ascites induced by the mouse ovarian cancer cell line were not suppressive and highly homogeneous population of granulocyte-like cells. We investigated the detail function of this population comparing with it of MDSCs.
Rapid change of PO2 promoted dendritic cells to induce a strong Th1 cell immune response with decreased Foxp3+Tregs (89.11)
Authors: Liu Chunmei , Wang Qun , Liu Fengming , Zhang Lining
Published: January 2023

Ischemia-reperfusion injury (IRI) can be found in transplantation and other ischemic syndromes in multiple organ systems including kindey, brain, heart and liver, this pathogenesis of IRI is complex and incompletely understood. It has been suggested that reoxygenation after hypoxia is a crucial mechanism mediating organ injury in IRI. Well, dendritic cells (DCs), the critical immune cells linking innate and adaptive immunity, are often exposed to various oxygen tensions under physiological and pathological conditions, such as ischemia-reperfusion. However, the effects of various oxygen tensions on DCs remain unclear. In this study, we showed that hypoxia-differentiated DCs expressed lower levels of MHC-|| molecule, co-stimulatory molecules (CD80, CD86) and pro-inflammatory cytokines (IL-1 β, IL-6, TNF-α and IL-12), but higher levels of immunoregulatory cytokine transforming growth factor-beta (TGF-b) than did normoxia-differentiated DCs. However, reoxygenation of the hypoxia-differentiated DCs dramatically upregulateed the aforementioned molecules leading to a mature phenotype. Significantly, these reoxygenated DCs induced syngeneic or allogeneic naïve CD4+ T cells to differentiate into Th1 effector cells. The generation of CD4+CD25+Foxp3+ regulatory T cells (Treg cells) induced by reoxygenated DCs was however down-regulated. These data indicate that hypoxic microenvironment suppresses the maturation and function of murine DCs. Re-exposure of the hypoxia-differentiated DCs to sutured oxygen however results in complete recovery of their mature phenotype and function and drives them to initiate Th1 cell differentiation, suggesting DCs play a critical role in inflammation in ischemia-reperfusion injury (IRI).
The ratio of Th1 /Treg or Th17/Treg is more important in the onset of acute coronary syndrome (89.2)
Authors: zhang lining , Zhu Faliang , Ma Tian , Gao Qi
Published: January 2023

Considerable evidence supports that CD4+ T lymphocyte, a highly heterogenous subset including Th1, Th2, Th17 and Treg, is the major lineage of T lymphocytes that involve in atherosclerosis, a chronic inflammatory disease of artery wall. Some reports show that Th1, Th17 and Treg may play a role in the plaque destabilization of atherosclerosis. However, the status and significance of Th1, Th17 and Treg in same patient remains largely unknown. In present study, we detected percentage of Treg, Th1 and Th17, related cytokines ( IFN-γ , IL17 and TGF-β ) and analyzed the ratio of these three subsets in same patient with acute coronary syndrome (ACS). Although increased Th1 or Th17 and decreased Tregs revealed statistical difference in group of patients with acute cortical syndrome compared with group of healthy individuals, percentage of Treg or Th17 or Th1 also did not show significant change in some patients. Our results further showed that the increased ratio of Th1/Treg and Th1+Th17/Treg in every patient with ACS. The similar results were found in apolipoprotein E-deficient mice with atherosclerotic plaque. Our data suggests that imbalance of effective CD4 subsets (Th1 and Th17) and inhibitory T subset ( Tregs) was more important in the onset of acute coronary syndrome.
Tyrosine phosphatase SHP-1 negatively regulates the suppressive potential of CD4+CD25+Foxp3+ regulatory T cells (89.26)
Authors: Iype Tessy , Sankarshanan Mohan , Mauldin Ileana , Lorenz Ulrike M
Published: January 2023

CD4+CD25+Foxp3+ regulatory T (Treg) cells are well recognized as mediators of peripheral immune tolerance. However, their function remains poorly understood at the molecular and mechanistic level. We have previously shown that mice, which are deficient in the protein tyrosine phosphatase SHP-1 ('motheaten' or me/me mice), have increased numbers of Treg cells. Since SHP-1 is a negative regulator of TCR-mediated signaling in conventional T cell, we investigated whether SHP-1 also plays a regulatory role in Treg cells.

A comparison of the suppressive activities demonstrated that Treg cells derived from me/me or me/+ mice suppressed lymphocyte proliferation in vitro more efficiently than Treg cells from wild type mice indicating that SHP-1 is a negative regulator of Treg cell-mediated suppression. Since based on micro array analyses, the differences in suppressive activities are not due to developmental disparities, our data suggest that SHP-1 directly regulates the suppressive activity of Treg cells. To gain a better mechanistic understanding of Treg cell-mediated suppression, flow cytometric and in particular ImageStream100 (Amnis) analyses were performed. These studies showed that in the absence of SHP-1, Treg cells express different levels of a number of surface and intracellular molecules. Moreover, SHP-1 deficiency promoted increased conjugate formation between Treg cells and antigen presenting cells as visualized by ImageStream100. Conjugate formation with SHP-1-deficient Treg cells caused a more efficient down-modulation of the co-stimulatory molecules CD80/CD86 on the dendritic cells. Taken together, our data indicate that SHP-1 regulates the function and activation status of Treg cells and that under conditions of SHP-1 deficiency, Treg cells become more potent.

Source: NIH, RO1 AI 048672
Regulation of T cell specific IL-10 production by non-IL-10 expressing T cell subset (89.32)
Authors: Stoyanoff Korynn , Blum Arthur , Hang Long , Setiawan Tommy , Leung John , Weinstock Joel
Published: January 2023

Intro: IL10 is an anti-inflammatory cytokine, important for the regulation of immune responses including the modulation of intestinal inflammation. Induction of IL10 production is well studied, but little is known about inhibition of its production, which was the topic of this study. Method: Reporter mice, whose cells concurrently express IL10 and EGFP were used. Unfractionated Lamina propria mononuclear (LPMC) or Spleen (Sp) cells were cultured for 24hrs with aCD3 stimulation and then stained and sorted into IL10+Thy+, IL10-Thy+, and IL10-Thy- populations. IL10+Thy+ T cells were cultured alone or in combination with IL-10- cells for 48hrs. Supernatants were analyzed for IL10 production by ELISA. Results: IL10+Thy+ LPMC or Sp cells produce high levels of IL10 when cultured alone (0.614 + 0.156 ng/ml, 15K Sp cells, N=3). Coculture with IL10-Thy+ cells (45K) led to significant inhibition of IL10 production (0.129 + 0.05 ng/ml, p=0.018, N=3 for Sp, 79.06% reduction). Lesser inhibition was seen with IL10-Thy- coculture. Inhibition appears to be CD8+ T cell-mediated. Reduced IL10 production was not due to IL10+ T cell death. Discussion: We have identified a population of IL10- T cells in the gut and Sp with novel inhibitory effects on IL10 production by T cells. These cells may maintain the balance between pro- and anti-inflammatory immune responses through mechanisms previously not described . (DK38327, 058755, 034928)
A single scurfy T cell-derived TCR is sufficient to induce multi-organ autoimmune disease (89.37)
Authors: Killebrew Justin R , Perdue Nikole , Campbell Daniel J
Published: January 2023

While the majority of T cells expressing self-reactive T cell receptors (TCRs) are eliminated in the thymus during development, a fraction emerge into periphery. We have created the SFZ70 TCR transgenic mouse to better understand the in vivo behavior of these peripheral auto-reactive T cells. The SFZ70 TCR was cloned from the SFZ70 hybridoma, which was generated from a CD4+ T cell isolated from the skin-draining peripheral lymph nodes (PLN) of a scurfy mouse. The SFZ70 hybridoma responds specifically to dendritic cells (DC) isolated from the PLN of wild-type mice but not to DC isolated from other tissues, and therefore appears to recognize a cutaneous antigen. SFZ70+RAG1-/- mice contain both peripheral CD4+Foxp3+ regulatory T cells (Tregs) and CD4+Foxp3- T cells and do not exhibit inflammation in the skin or other organs. To examine whether SFZ70 Tregs are required for maintenance of peripheral tolerance, we generated SFZ70 mice lacking functional Tregs by crossing SFZ70 mice to scurfy mice (SFZ70+RAG1-/-Foxp3sf/Y). SFZ70+RAG1-/-Foxp3sf/Y mice develop severe autoimmune disease characterized by lymphocytic infiltration of the skin and liver. Additionally, CD4+Foxp3- T cells from these mice show increased proliferation and display a CD62LloCD44hi activated phenotype. Our results show that in the absence of Tregs, an individual scurfy T cell-derived TCR is capable of inducing multi-organ autoimmune disease.
Identification and characterization of macrophage regulatory cells (Mac-regs) with immunoregulatory properties (89.38)
Authors: Zorro Manrique Soraya , Zorro Manrique Soraya , Duque Maria Adelaida , Dominguez Ana Lucia , Mirza Noweeda N , Hoelzinger Dominique , Smith Shannon E. , Lustgarten Joseph
Published: January 2023

Macrophages with suppressive characteristics such as Tumor associated macrophages or Myeloid derived suppressor cells have been defined showing regulatory function under inflammatory or polarizing conditions. Until now, there are no reports indicating that under normal, non-inflammatory or non-polarizing conditions, macrophages could have immunoregulatory functions. Utilizing knock-in Foxp3-GFP mice, we identified a new population of Foxp3 positive cells that were CD11b+F4/80+ cells. These cells were observed in spleen, lymph nodes, bone marrow and thymus. Similar to Tregs, the CD11b+F4/80+Foxp3+ macrophages inhibited T cell proliferation, whereas Foxp3neg macrophages did not. The Foxp3neg macrophages could be converted to express Foxp3. These induced-CD11b+F4/80+Foxp3+ cells displayed inhibitory properties similar to natural Foxp3pos macrophages. Based on the immunoregulatory properties of these cells, we termed them Macrophage regulatory cells (Mac-regs). These Mac-regs express CD68hiF4/80hiCTLA4hiGITRhiIL-4Rhi. Interestingly, GR-1 is not a differentiation marker. The cytokine, chemokine, growth factor and genomic profiles of Mac-regs are different compared to CD11b+F4/80+Foxp3-cells. For the first time, we demonstrated the occurrence of a natural population of macrophages displaying regulatory function which may contribute to the homeostatic balance of the immune system.

This research was supported by Grants from the NIH and AFAR.
VEGFR1 as a New surface marker for CD4+ Regulatory T cells (89.41)
Authors: Shin Jin Young , Lim Jong Hyung , Park Chung Gyu
Published: January 2023

Regulatory T cells are a specialized subpopulation of T cells that control immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. The isolation and expansion of CD4+ Treg cells requires specific surface markers. Currently, CD25, CTLA-4 and GITR are used, but they are also expressed on the activated T cells. In this study, we identified vascular endothelial growth factor receptor 1 (VEGFR1) as a new surface marker for CD4+ regulatory T cells. We found that VEGFR1, a receptor for VEGF involved in angiogenesis, is constitutively expressed on the surface of T cells. CD4+ T cells that express high levels of VEGFR1 (0.5%-1% of CD4+ T cells) suppressed T cell activation as efficiently as CD4+CD25+ regulatory T cells (Treg cells). VEGFR1highCD4+ T cells are not overlapped with Foxp3+CD4+ or CD25+CD4+ T cells. We suggest that VEGFR1 can be a useful surface marker for CD4+ regulatory T cells and further characterization of VEGFR1highCD4+ T cells subset needs to be performed.
Developmental requirements of T follicular helper cells (90.10)
Authors: Nurieva Roza , Chung Yeonseok , Dong Chen
Published: January 2023

After activation, CD4+ helper T (Th) cells differentiate into distinct effector lineages. Th cell subset called T follicular helper (TFH) cells are recently found to be present in germinal centers and are characterized by their expression of chemokine (C-X-C motif) receptor 5 (CXCR5). Despite their potential importance in humoral immunity, the developmental regulation of TFH cells and their relationship with other Th subsets is unclear. We found that mouse TFH cells have a divergent gene expression profile from Th1, Th2 cells, and in vivo developed independently of these cell lineages. TFH cell generation was regulated by ICOS liagand expressed on B cells and was dependent on interleukin (IL)-21, IL-6 and signal transducer and activator of transcription 3 (STAT3). Although TFH cells shared IL-21 expression with Th17 cells, they did not express IL-17, IL-17F, IL-22. Moreover, differentiation of TFH cells did not require TGFβ or Th17-specific orphan nuclear receptors RORα?and RORγ in vivo. Most importantly, T cells activated in vitro in the presence of IL-21 but without TGFβ signaling preferentially acquired TFH gene expression and functioned to promote humoral immunity in vivo. Our data indicate that TFH cells are distinct in their gene expression, developmental regulation and immune function from Th1, Th2 or Th17 lineages.

The work is supported by a Scientist Development Grant from the American Heart Association.