bluuebunny/crossref_metadata_2025 huggingface dataset:
Post-thymic T cells undergo maturation in secondary lymphoid organs (82.11)
Authors: Houston Evan , Fink Pamela
Published: January 2023

T cell development was traditionally thought to occur in its entirety in the thymus, giving rise to fully developed naïve T cells ready to patrol the lymphoid periphery. However, characterization of T cells that have recently left the thymus was hampered by their lack of a unique cell surface marker. With the aid of new murine model in which those recent thymic emigrants (RTEs) are specifically labeled, RTEs been shown to undergo further changes in the lymphoid periphery prior to gaining full immunocompetence. With this model, our lab has demonstrated that RTEs are functionally dampened and phenotypically immature relative to the rest of the mature naïve T cell pool.

To aid in understanding the significance of this continued peripheral maturation, the mechanism driving this process is under investigation. We have found that exit from the thymus is required for full RTE maturation, and that this maturation process occurs progressively. We have also found that RTE maturation takes place within secondary lymphoid organs. In secondary lymphoid organs, IL-7 and MHC, homeostatic factors for naïve T cells, are dispensable for maturation, while dendritic cells do influence the maturation process. This work suggests that the process of RTE maturation is a dynamic process that is actively regulated by factors contained within the lymphoid periphery.

This research was supported by grants from the Cancer Research Institute and the National Institutes of Health.
Homeostasis of CD4+ memory and regulatory T cells occurs independently of NF-¿B (82.12)
Authors: Cornish Georgina , Ley Steve , Seddon Benedict
Published: January 2023

Deletion of I kappa B kinase (IKK2) under the control of cre-recombinant drivers lck-Cre and CD4-Cre, both expressed before thymic selection, interferes with normal naïve T cell activation and consequently mice fail to generate regulatory and memory CD4 T cells. However, it is not known whether the requirement for IKK2 expression extends beyond the initial inductive events required for generation of these populations. With this premise, the current study asks whether IKK2 signals are necessary for survival and/or maintenance of Treg and CD4 memory. We achieved this by specifically deleting IKK2 in mature peripheral T cells using an OX40cre driver, specifically deleting NF-κB signalling in regulatory and memory T cells post activation. Surprisingly, we find that IKK2 signalling is dispensable for normal homeostatic maintenance of both memory and regulatory T cells as mice with OX40 regulated deletion of IKK2 have normal numbers of Treg and CD4 memory. However, studies are ongoing to elucidate the mechanism by which of IKK2 mediates memory and regulatory T cell survival.
Toso expression during B cell development (82.3)
Authors: Choi Seung-Chul , Narayanan Sriram , Borrego Francisco , Coligan John E
Published: January 2023

Regulated apoptosis is essential for both the development and homeostasis of the immune system. Fas-mediated cell death has been implicated in apoptosis during the development and homeostasis of lymphocytes, and this apoptosis pathway is regulated by the interplay of BCL-2-family members, as well as other factors, such as cytokine withdrawal. However, Toso (also known as Fas Apoptosis Inhibitory Molecule-3) expression has been reported to inhibit Fas-mediated cell death. We isolated immune cells from the spleen and found that B cells expressed high levels of Toso mRNA compared to T cells, NK cells, granulocytes, macrophages and monocytes. After stimulation by LPS, anti-CD40 or anti-IgM, Toso expression is dramatically down-regulated in splenic B cells. Further analysis showed that bone marrow B cells express lower levels of Toso mRNA than splenic B cells. We sorted the bone marrow B cells into development stages and found that only immature B cells expressed high levels of Toso mRNA compared with pro-, pre- and mature B cells. We hypothesize that Toso has a role in normal B cell development and maintenance. To examine this, we are using Toso knockout and transgenic mice.

This work was supported by the NIAID intramural research program.
A role for CMV infection in expansion of dysfunctional IL-7 receptor alpha low memory CD8+ T cell with aging. (83.11)
Authors: Lee Won-Woo , Kang Seong-Wook , Lee Seung-Hyun , Shah Kamini , Kang Insoo
Published: January 2023

We recently reported an age-associated expansion of human memory CD8+ T cells expressing low levels of IL-7 receptor alpha chain (IL-7Rα) that is crucial for cell survival. These cells have limited T cell receptor (TCR) repertoire and impaired TCR-mediated proliferation. Chronic persistent viral infection is suggested as a factor that expands memory CD8+ T cells with aging and suppresses their function. Thus, we explored the role for cytomegalovirus (CMV) infection in expanding IL-7Rαlow memory CD8+ T cells and suppressing their TCR function via leukocyte Ig-like receptor-1 (LIR-1) with inhibitory function. CMV-infected donors have a higher frequency of IL-7Rαlow memory CD8+ T cells than CMV-uninfected donors in the elderly but not in the young. Most expanded IL-7Rαlow cells express LIR-1 and such expression correlates with replicative senescent marker CD57. The frequency of IL-7Rαlow memory CD8+ T cells is also higher in CMV-uninfected elderly donors than in CMV-infected and -uninfected young donors, suggesting that a factor(s) additional to CMV infection exists for expanding these cells. The expression of IL-2/15 receptor beta chain (IL-2/15Rβ) on IL-7Rαlow memory CD8+ T cells is higher in the elderly than in the young, suggesting IL-15-mediated expansion of them. Our data suggest that age-associated expansion of IL-7Rαlow memory CD8+ T cells is driven by both CMV infection and IL-15.
Basophils induce CD8 T cell IL-10 production (83.14)
Authors: Min Booki , Kim Sohee , Shen Tao
Published: January 2023

There is increasing evidence suggesting that basophils play a crucial role in developing Th2 type immunity. We previously reported that basophils cocultured with naive CD4 T cells stimulated with antigen promote differentiation of the T cells into IL-4-producing cells. In the present study, we examined whether basophils alter CD8 T cell differentiation pathway. Stimulation of OVA-specific OT-I CD8 T cells with peptide pulsed splenic DCs primarily induced T cell production of IFNγ. Addition of basophils into the coculture significantly inhibited IFNγ production. Unlike CD4 T cells, CD8 T cells cocultured with basophils did not produce IL-4. Instead, CD8 T cells activated in the presence of basophils produced significant levels of IL-10. The IL-10 production was greatly diminished when anti-IL-4 Ab or IL-4KO basophils were used in the culture, indicating that IL-4 is necessary for programming CD8 T cell IL-10 production. However, adding rIL-4 into CD8/DC coculture was not sufficient to induce IL-10 production. CD28-mediated costimulation dramatically enhanced T cell IL-10 production, yet neither ICOS nor CD86 was involved in IL-10 production. Finally, CD8 T cells producing IL-10 did not display regulatory cell phenotypes. Collectively, these results suggest a novel immunomodulatory function of basophils that efficiently induce CD8 T cell IL-10 production via IL-4-dependent and -independent mechanism.
The absence of Aire does not alter the CD8 T cell repertoire to infection (83.5)
Authors: Davenport Miles P , Valkenburg Sophie , Guillonneau Carole , Hubert Francois-Xavier , Cukalac Tania , Curtis Joan M , Stambas John , Scott Hamish , Venturi Vanessa , Kedzierski Lukasz , Kedzierska Katherine
Published: January 2023

Expression of the Aire molecule is thought to be important for self-tolerance through promoting ectopic expression of tissue-specific antigens in the thymus. In the absence of Aire, animals develop a variety of autoimmune conditions. It is unclear whether the absence of self-peptides in the thymus effects the selection of all T cells in the thymus (because they see a different mix of self-peptides), or only allows through a few self-specific cells, without perturbing selection of other cells. Here we study the CD8+ T cell responses to two well characterized influenza virus epitopes (DbNP366 and DbPA224) in AIRE +/+ and AIRE -/- mice using single cell sequencing of T cell receptors (TCR). Studying a total of approximately 1500 TCR Vbeta sequences from 25 mice, we find no differences between the responses in the presence or absence of Aire. This suggests that the absence of Aire does not perturb selection of the majority of T cells, but instead opens a small window allowing survival of a few self-specific cells.
Brief costimulation through ICAM-1 of human naïve CD4+ T cells induces differentiation to a memory phenotype days later (83.9)
Authors: Dotson Abby L. , Kohlmeier Jacob E. , Cool Kelli M. , Benedict Stephen H.
Published: January 2023

We previously demonstrated that costimulation through ICAM-1 induces differentiation of human naïve CD4+ T cells to a memory phenotype. Similar to results of costimulation through CD28, the phenotype includes expression of a cell surface protein array characteristic of T memory and a robust proliferative and cytokine response to low-level stimulation through the TCR complex alone. In the present work we describe the kinetics of differentiation in response to costimulation through ICAM-1 as compared with CD28 and define the minimal time that costimulatory signals are required for the naive T cells to achieve the memory phenotype. In addition, costimulation through ICAM-1 induces T cells with distinct homing properties.

This work was supported by the National Institute on Aging/NIH.
The expression level of autoantigen TRIM21 changes during lymphocyte development (84.10)
Authors: Yoshimi Ryusuke , Chang Tsung-Hsien , Wang Hongsheng , Atsumi Toru , Morse III Herbert C. , Ozato Keiko
Published: January 2023

The tripartite motif (TRIM) family member, TRIM21, is an E3 ubiquitin ligase that functions in both innate and acquired immunity. It is also an autoantigen known as Ro52/SS-A. While TRIM21 is thought to be ubiquitously expressed, its precise distribution patterns among various tissues and cells are largely unknown. To analyze detailed expression patterns of TRIM21, we generated mice with the Trim21 gene replaced by an EGFP reporter. Western blot data showed that EGFP is expressed particularly at high levels in lymph nodes, spleen and thymus. Flow cytometric analyses using splenocytes demonstrated that EGFP expression was generally high in T cells, macrophages and DCs, but lower in B cells and granulocytes. During B-cell development in bone marrow and spleen EGFP levels were high in pro-B cells, but fell in the subsequent stages until small pre-B cells and then rose again in later stages of development. During T-cell development in thymus EGFP expression was high in early CD4-CD8- double negative cells, and the expression gradually decreased as cells moved through mid to late stages of double negative cells and became much lower in double positive cells. Then EGFP expression rose again during the subsequent stages of single positive cells. These data suggest that TRIM21 may have a functional role during lymphocyte development.
Role of cFLIP in the initiation and maintenance of the germinal center response (84.18)
Authors: Coffey Francis J , Manser Tim
Published: January 2023

During a T cell-dependent immune response, B cells are activated and accumulate in germinal centers where they proliferate, undergo somatic hypermutation, and stringent clonal selection. These events are essential to the production of high affinity antibody responses and long-lived memory B cell populations. Apoptotic pathways are believed to play a pivotal role in regulating the expansion of activated B cells and negative selection events operative in germinal centers. Increased levels of the death receptor Fas on germinal center B cells should make them highly sensitive to Fas-induced cell death. However, previous studies have suggested a role for increased levels of cellular FLICE-inhibitory protein (cFLIP) in preventing Fas-mediated apoptosis in germinal center B cells. Therefore, regulation of cFLIP expression may important to the maturation of the immune response and the maintenance of peripheral tolerance. In the present study, we utilize mice with a B cell-specific deletion of cFLIP to assess its requirement during a T-dependent immune response. Analysis of bone marrow and splenic B cell populations in these mice indicates that there is no apparent maturation defect, despite a mild B cell-specific lymphopenia. However, data from immunization studies with various T-dependent antigens suggests an important role for cFLIP expression in the expansion of antigen-specific B cells and their participation in the germinal center response.

This work is supported by T32CA09683 to F.C. and AI038965 to T.M.
Mcl-1 promotes T cell survival independently of Bcl-2 through interaction with Bak (84.19)
Authors: Dunkle Alexis D , Dzhagalov Ivan , He You-Wen
Published: January 2023

T cells depend on the careful regulation of apoptosis for normal development, homeostasis and immune response. The Bcl-2 family of proteins has been shown to play critical roles regulating T cell apoptotic pathways. The anti-apoptotic proteins Bcl-2 and Mcl-1 each display a unique expression pattern throughout the lifespan of a T cell and both have been shown to be essential for T cell survival at multiple stages. However, it is unclear whether these proteins function in the same or separate molecular pathways. Here, we use genetic models to show that Mcl-1, through antagonism of Bak, has a unique function from Bcl-2 at multiple stages of thymocyte development. Loss of Bak rescues the death of Mcl-1-deficient thymocytes at the double negative and single positive stages. Loss of Bax, however, does not rescue Mcl-1-deficient thymocytes. Neither over-expression of Bcl-2 nor loss of the BH3-only protein Bim rescues the death of Mcl-1-deficient thymocytes. Interestingly, loss of Bak does not rescue activated T cells in an in vitro Mcl-1 deletion system, thus mechanisms may differ at different stages. These data indicate non-overlapping and distinct roles for Mcl-1 and Bcl-2 in T cells.

This work was supported by grants from the NIH.
Role of Itk in γδ T cell development and function (84.22)
Authors: Yin Catherine C , Felices Martin , Kosaka Yoko , Kang Joonsoo , Berg Leslie J
Published: January 2023

In conventional αβ T cells, the Tec family tyrosine kinase Itk is required for signaling downstream of the TCR. Itk also regulates αβ T cell development, lineage commitment, and effector function. A well established feature of Itk-/- mice is their inability to generate T helper type 2 (Th2) responses that produce IL-4, IL-5, and IL-13; yet these mice have spontaneously elevated levels of serum IgE and increased numbers of germinal center B cells. Here we show that the source of this phenotype is γδ T cells, as normal IgE levels are observed in Itk-/-Tcrd-/- mice. Further, we find that γδ T cells numbers are increased in Itk-/- mice, along with elevated proportions of γδ T cells expressing CD4 and NK1.1. When stimulated through the γδ TCR, Itk-/- γδ T cells produce high levels of the Th2 cytokines, but diminished IFNg. In addition, activated Itk-/- γδ T cells upregulate costimulatory molecules important for B cell help, suggesting that they may directly promote B cell activation and Ig class switching. Together, these data indicate that Itk signaling regulates γδ T cell lineage development and effector function, and is required to control IgE production in vivo.
Expression of CD226 Antagonizes Apoptotic Cell Death in Murine Thymocytes (84.4)
Authors: fang liang , Zhang Xinhai , Miao Jun , Zhao Fang , Yang Kun , Yang Angang , Chen Lihua , Jin Boquan
Published: January 2023

CD226 is known to be expressed on many types of peripheral lymphoid cells and involved in T cell differentiation, activation, and cytotoxicity. We found that CD226 was also expressed on mouse thymocytes at varying developmental stages, and its expression was associated with resistance of thymocytes against apoptosis. Expression of CD226 appeared to be closely coupled with thymocyte development, in that CD226 was preferentially expressed on CD4+CD8- and CD4-CD8+ thymocytes at all stages during mouse development. In DP, mainly expression of CD226 on CD69+ thymocytes showed that CD226 may be involved in positive selection. Inhibition of CD226 by shRNA in a fetal thymus organ culture (FTOC) model resulted in reduced thymus cellularity, which was associated with enhanced apoptotic cell death in the thymus as evidenced by the increased cell number with externalization of phosphatidylserine and increased caspase-3 activity. In contrast, CD226 transgenic mice displayed enlarged thymus and increased thymus cellularity. Expression of CD226 seemed to be associated with expression of survivin in that inhibition of CD226 down-regulated survivin expression and activition, and over-expression of CD226 rescued thymocytes from apoptosis through activition of survivin. Taken together, that CD226 is an anti-apoptotic molecule that may play an important role in murine thymus development.
NKAP, a novel modulator of Notch signaling, is required for T cell development (85.5)
Authors: Shapiro Virginia Smith , Pajerowski Anthony , Nguyen Chau , Aghajanian Haig , Shapiro Michael J.
Published: January 2023

T cell development depends on the coordinated interplay between receptor signaling and transcriptional activation/repression. We performed a genetic complementation screen, and identified a novel transcriptional repressor, NKAP. NKAP associates with HDAC3 and is part of a DNA-binding complex, as shown by chromatin immunoprecipitation. NKAP also associates with CIR, a part of the Notch corepressor complex. The expression of NKAP during T cell development inversely correlates with the expression of Notch target genes, implying that NKAP may modulate Notch-mediated transcription. To examine the function of NKAP in T cell development, Lck-cre NKAP conditional knockout mice were generated. Interestingly, loss of NKAP blocks development of alpha/beta but not gamma/delta T cells. In addition, lck-cre NKAP cKO DP T cells express 8- to 20-fold higher levels of Hes1, Deltex1 and CD25, providing in vivo evidence that NKAP functions as a transcriptional repressor, acting at least in part, by repression of the Notch pathway.
Cross-talk between IL-7Ra signaling and p53 pathway during thymopoiesis and lymphomagenesis (86.8)
Authors: Kibe Ryoko , Zhang Shu-zhong , Marrero Luis , Khan Shafin , Zieske Arthur , Huang Jiaqiang , Durum Scott K , Iwakuma Tomoo , Cui Yan
Published: January 2023

Thymopoiesis in IL-7Rα-/- mice is blocked at the CD4-CD8- double negative stage with failures in T cell receptor (TCR) γand β rearrangement. Early studies suggest that unsuccessful TCRβ rearrangement in mice defective in DNA damage repairs, incurring DNA double-strand breaks, results in p53 activation. However, it remains unknown whether p53 plays a role in the thymopoietic defect in IL-7Rα-/- mice. In this study, we demonstrated that p53 is highly activated in the thymocytes of IL-7Rα-/- mice. Considering the importance of p53 activity during TCR rearrangement and the observation of incomplete rescue of thymopoiesis in IL-7Rα-/- mice by modulating activities of Bcl-2 family members, we hypothesize that p53 activation may also contribute to the impaired thymopoiesis in IL-7Rα-/- mice and that temporal suppression of p53 activity by the IL-7Rα signaling facilitates thymopoiesis. With the generated IL-7Rα-/-p53-/- (DKO) mice, we found that p53 inactivation in IL-7Rα-/- background resulted in a 30-fold increase in thymic cellularity with increased TCRβ? cells. Furthermore, DKO mice developed thymic lymphoma at a higher incidence and died significantly earlier than p53-/- mice. Our study is the first to illustrate a functional interplay between IL-7Rα signaling and p53 pathways in facilitating TCRβ+ T cell development during thymopoiesis and lymphomagenesis.
Osteoclasts activation by receptor activator of NF-κB ligand (RANKL) enhanced the mobilization of hematopoietic progenitor cells from the bone marrow in acute injury (87.10)
Authors: CHO KYUNG-AH , Ju Sun-Young , Ryu Kyung-Ha , Woo So-Youn
Published: January 2023

Osteoclasts (OCLs) are specialized cells responsible for physiological bone resorption and as well as pathologic bone loss. In addition to unique ability to resorb bone, OCLs also play potential role in the mobilization of hematopoietic progenitor cells from bone marrow, particularly under various stress stimuli(e.g. hypoxia, injury or inflammation). We investigated effect of activated OCLs on the stem niche whether lead to mobilization of hematopoietic progenitors under liver specific injury, which was established in our previous acute liver disease study. We exposed mice to stress situation by inducing liver injury with CCl4 or combination of additional injecting of RANKL to activate OCLs, and compared the effect on the mobilization of hematopoietic progenitor cells from the BM. To investigate OCLs involved mechanism underlying mobilization, we induced activated OCLs from RAW 264.7 cell line through stimulation with RANKL and quantified the level of stem cell niche component, SDF-1 on the osteblasts and CXCR4 on the bone marrow cells(BMCs), after culturing with supernatants from activated OCLs. As a result, Under the stress situation in vivo, the mobilized hematopoietic progenitor cells were significantly increased after RANKL treatment under liver specific injury. Moreover, we found that functional OCLs cleaved SDF-1α on the osteoblasts and increased CXCR4 expression on the BMCs in vitro. These results suggest that OCLs might be involved in alteration of the interaction between SDF-1 and CXCR4 leading to mobilization of hematopoietic progenitor cells from the BM.
Disease stage-related changes in T cell receptor-VB profile in cutaneous T cell lymphoma (88.10)
Authors: Pomerantz Rebecca G , Patton, Jr Timothy J , Donnenberg Albert D , Mirvish Ezra D , Falo, Jr Louis D , Geskin Larisa J
Published: January 2023

The objective of this study was to evaluate the T cell receptor-Vβ (TCR-Vβ; Vβ) repertoire in patients with Mycosis Fungoides (MF) and Sezary Syndrome (SS) variants of cutaneous T cell lymphoma (CTCL) across all stages, in order to identify relationships between Vβ expression levels and disease stage. Blood was drawn from MF/SS patients (n=31) and the percentage of CD4+ T cells expressing each Vβ was determined by multiparameter flow cytometery and the change in expression levels with progressive disease stage was calculated with Spearman's rank correlation coefficients. We found that individual TCR-Vβ families were expanded in some patients with advanced MF/SS. Further, we detected significant decreases in the proportion of five TCR-Vβs with progressive stages of disease, while the proportion of all other tested TCR-Vβ families did not change with disease stage. These data show disease stage-related changes in the expression of certain TCR-Vβs known to be activated by viral superantigens. In particular, there was a highly significant decrease in expression of TCR-Vβ13, which has been found to be activated by an EBV-associated superantigen, in advanced CTCL. Based on these findings, we theorize that in early stages of CTCL, viral infection may play a role in the expansion of specific superantigen-associated T-cells; while in later stages, these same T-cells become eliminated by a yet unknown mechanism.

Funding Sources: National Institutes of Health, Dermatology Foundation, University of Pittsburgh Clinical & Translational Research Center
Preventive effects of Paeonol on cisplatin induced nephrotoxicity in mice (88.12)
Authors: Lee Hyojung , Yu Gun , Lee Hyoeun , Hong Moo-Chang , Shin Min-Kyu , Kim Yangseok , Bae Hyunsu
Published: January 2023

Paeonol, a major phenolic compound of Moutan Cortex, was known that to have antioxidant and anti-inflammatory activities. Cisplatin is a chemotherapeutic agent, however, its nephrotoxicity limits the use of this drug. In the present research, we examines whether paeonol has protective effects on cisplatin induced acute renal failure in mice. Animals were given 20mg/kg of paeonol intraperitoneally for 3 days. At 4 days, all groups of mice received a single dose of cisplatin at 30mg/kg intraperitoneally. Paeonol treated group marked attenuation in serum creatine, BUN, and survival. Paeonol treated group had a significantly reduced the levels of proinflammatory cytokines when compared to the control group. These results demonstrated that Paeonol pre-administration can prevent the renal toxic effects of cisplatin.
The role of transforming growth factor-beta signaling in a murine model of lung cancer (88.16)
Authors: Soó-Becker Kinga , Zhivkova Nataliya , Boross Ildikó , Koslowski Michael , Wiewrodt Rainer , Türeci Özlem , Lehr Hans A. , Finotto Susetta
Published: January 2023

The transforming growth factor beta TGFβ is known to be a potent tumour suppressor at early stages of cancer. However, cancer cells can lose responsiveness to TGFβ through mutations in the signaling pathway downstream of the TGFβ receptor.

TGFβ influences the development of regulatory T cells known to enhance cancer development. We analyzed TGFβ expression in the lung of subjects affected by lung adenocarcinoma. TGFβ mRNA level was increased in the lung of these patients and this value correlated with the progression of the disease. We thus asked whether targeting TGFβ signalling in T cells would inhibit regulatory T cells in a murine model of lung adenocarcinoma. Mice that overexpress a dominant negative form of TGFβ type II receptor lacking the cytoplasmic kinase domain in T cells were used. Histologically, we found a tumour regression in the abscence of TGFβ signalling in T cells. A hyperproliferation of the CD4+ T cells overproducing interferon gamma (IFNγ) was observed. Analysis of the regulatory T cells showed an intact supression function of these cells.

In summary, a defective TGFβ-signalling in lung T cells could lead to amelioration of lung adenocarcinoma in mice because inducing hyperproliferation of IFN-gamma producing CD4+ T cells thus limiting the T regulatory component in this disease.

This work is supported by the GK1043 in Mainz.
Genetic Approach to Study Complement Role in Ovarian Cancer (88.24)
Authors: Nunez-Cruz Selene , Connolly Denise , Roberts Amber , Markiewski Maciej , John Lambris D , Coukos George , Scholler Nathalie
Published: January 2023

The complement system plays a crucial role in inflammation, which is known to contribute to carcinogenesis and tumor progression. However, the exact role of complement in cancer development remains poorly understood. We investigated the contribution of complement to the development of ovarian carcinoma (OC). OC is the second most common gynecologic cancer and the fourth leading cause of cancer death in women. We assessed the influence of complement on angiogenesis and tumor microenvironment by crossing complement component 3 knockout (C3KO) with transgenic mice expressing the Simian Virus 40 TAg under the control of the Müllerian Inhibiting Substance type II Receptor promoter (MISIIR-TAg). MISIIR-TAg mice develop bilateral OC resembling human serous OC with ~100% penetrance.

We found that C3KO/MISIIR-TAg mice exhibited reduced tumor volume and microvessel formation compared to control wild-type C3 and C3 heterozygote (C3HET) littermates. In addition, we found that tumor-infiltrating macrophages, B cells and T lymphocytes from C3KO/MISIIR-TAg mice produced significantly less cytokines after ex-vivo culture. Furthermore, the number of CD3+/CD4+/FoxP3+ T-cells was reduced in these mice. These results support the hypothesis that C3 plays an important role in tumor progression and vascularization, and that complement deficiency might compromise tumor-infiltrating leukocyte functions.

This work was supported by P01 AI 068730 (NIH/NCI)
Neuropilins bind TGF-beta and its receptor components and promote Smad signaling (88.5)
Authors: Prud'homme Gerald J. , Stoilova Snejana , Glinka Yelena
Published: January 2023

Neuropilin-1 (Nrp1) and/or neuropilin-2 (Nrp2) are expressed by many tumors, correlating with a poor prognosis. Normally, Nrp1 is expressed by neurons, endothelial cells, dendritic cells (DCs) and Treg cells, while Nrp2 has a more limited distribution. Because neuropilins are coreceptors for VEGF, it has been thought they stimulate tumor angiogenesis, but they may also act otherwise. We recently reported that Nrp1 binds and activates LAP-TGF-beta1 (the latent form) and enhances Treg activity. Here, we report that both Nrp1 and Nrp2 interact with TGF-beta1, as well as TGF-beta receptor components, and induce internalization of the TGF-beta receptor complex. This enhances signaling by the Smad pathway. In accord with this, we found that both Nrp1 and Nrp2 activate latent TGF-beta1 on the membrane of breast cancer cells. The classical TGF-beta receptors (RI, RII and RIII) only bind active TGF-beta, but our data suggest that the neuropilins allow responsiveness to latent TGF-beta by its activation. We also find that TGF-beta competes with VEGF for binding to Nrp1/Nrp2, possibly altering angiogenesis. TGF-beta has been linked to metastasis and, thus, the neuropilins may promote metastasis by capturing and activating latent TGF-beta. Our novel findings are relevant to cancer biology, immune regulation and angiogenesis. This work was funded by the Ontario Institute for Cancer Research, Province of Ontario, Canada.
Signaling model of cell death shows cell-to-cell stochastic fluctuations are linked to apoptosis related diseases (88.8)
Authors: Raychaudhuri Subhadip
Published: January 2023

We have developed a detailed computational model of apoptotic cell death signaling that could explain some of the recent experimental observations on apoptosis (Goldkorn et al. Antioxid. Redox Signal, 2005). Specifically, our results show how apoptosis signaling can be slow (~ 10 hours) under weak stimulus due to large cell-to-cell stochastic fluctuations through the type 2 pathway of signaling. We use a novel set of stochastic differential equations to elucidate a robust network design and stochastic effects in apoptosis signaling in a cell-type independent manner. However, apoptosis signaling and diseases that arise from aberrant apoptosis signaling are often cell-type specific. For example, recent experiments have revealed neuroglobin protein as a novel inhibitor of the apoptotic pathway (T. Brittain et al., FEBS Letters, 2006) that has 1000 fold higher concentration level in neural and retinal cells (~ 100 micro-molar) compared to its 10-100 nanomolar concentration in most other cell types. We use our computational model to show that stochastic signaling through the type 2 pathway can explain the presence of such disparate levels of neuroglobin amount depending on the cell type. We also propose a molecular mechanism of apoptosis related diseases, such as degenerative disorders and cancer, due to aberrant signaling through the stochastic type 2 pathway and a faulty type 2 to type 1 switching of apoptotic signaling. A role of aberrant expression of apoptotic inhibitor Bcl2 in such apoptosis related diseases are also discussed.
Peptide-induced peripheral T cell deletion leads to tolerance mediated by TRAIL-expressing CD8+ regulatory T cells (89.16)
Authors: Gurung Prajwal , Ferguson Thomas A , Griffith Thomas S
Published: January 2023

Peripheral tolerance controls self-reactive T cells that escape thymic deletion. We previously showed that deletion of Ag-specific CD4+ T cells after soluble Ag injection leads to immune tolerance mediated by a CD8+ Treg population in response to the processing and presentation of the apoptotic T cells by the immune system. The CD8+ Treg maintain long-term tolerance to subsequent Ag challenge by deleting T cells that share the same Ag specificity. The present study explored the mechanism of action of these CD8+ Treg. Our data show OT-II T cells expand and contract equally in wildtype (WT) and TRAIL-/- mice after OVA323-339 injection. After contraction, WT mice were tolerant to subsequent OVA/CFA immunization, even when given fresh OT-II T cells, but TRAIL-/- mice displayed immunity. Analysis of the tolerant B6 mice found the transferred OT-II T cells did not undergo secondary expansion after OVA/CFA immunization, but secondary T cell expansion was evident in the non-tolerant TRAIL-/- mice. Subsequent investigation found direct killing of freshly transferred OT-II T cells in a TCR-specific manner in tolerant WT mice, but not in TRAIL-/- mice, and only CD8+ T cells from tolerant WT mice could transfer tolerance to naïve recipients. These data provide evidence for a novel mechanism of peripheral tolerance maintained through the cytotoxic activity of TRAIL-expressing CD8+ Treg against T cells of the same Ag specificity.
Characterization of mouse FoxP3+CD4+CD25+ regulatory T cells with an easy one-step flow cytometry analysis (89.17)
Authors: Qin Fenghua , Shin Jeffrey , Wei Bingyuan , Yang Xifeng , Kraft Brad , Lay Gene
Published: January 2023

Generally, CD4+CD25+FoxP3+ Tregs are characterized by cell surface staining with anti-CD25 and anti-CD4 antibodies, followed by intracellular staining with anti-FoxP3, which requires two steps. To simplify the characterization procedure, we developed a kit for labeling both surface and intracellular antigens of Tregs in one step. Mouse splenocytes were fixed and permeabilized at various fixing conditions: 1% paraformaldehyde (PFA), 0.5% PFA, 20% Ethanol, 70% Ethanol, BioLegend FoxP3 Permeabilization (Perm)/ Fixation buffer, or FoxP3 Perm/Fixation buffer with 0.01% Saponin. Then anti-mouse CD25 (mCD25), anti-mouse CD4 (mCD4), and anti-mouse FoxP3 (mFoxP3) antibodies were added to the splenocytes in one step. The cells were analyzed by flow cytometry after incubation for 20 minutes. Different fluorochrome combinations with anti-mCD25, anti-mCD4, and anti-mFoxP3 were tested. The results showed that anti-mFoxP3 Alexa Fluor 488 with the combinations of anti-mCD25-PE and anti-mCD4 APC produce comparable results to two-step staining in terms of percentage of CD4+CD25+FoxP3+ cells, absolute cell number, and fluorescence intensity. Among all these fixation conditions, the BioLegend FoxP3 Perm/ Fixation buffer yields the best results. Our development of a one step kit provides a fast, dependable new tool for evaluating and exploring effects of CD4+CD25+FoxP3+ Tregs in mice.
SHP-1 influences resistance of CD4+ T cells to regulatory T cell suppression (89.25)
Authors: Sankarshanan Mohan , Iype Tessy , Mauldin Ileana , Lorenz Ulrike M
Published: January 2023

The mechanistic and molecular understanding about factors that regulate the susceptibility and/or resistance of T cells to suppression by regulatory T cells (Treg) is very limited. The protein tyrosine phosphatase SHP-1 is a negative regulator of TCR-mediated signaling. SHP-1-deficient T cells hyper-proliferate in response to antigen stimulation. We therefore asked whether SHP-1 affects the susceptibility of CD4+ T cells to suppression by Treg cells. Our in vitro suppression assays showed that heterozygous (me/+) and homozygous (me/me) SHP-1-deficient CD4+ T cells were more resistant to Treg cell-mediated suppression than +/+ CD+ T cells, indicating that SHP-1 modulates the resistance to Treg cell suppression.

In order to gain a better understanding of the mechanism that controls the resistance to suppression, we examined the conjugate formation between CD4+ T cells and the antigen presenting cells (APCs). Using flow cytometric and ImageStream100 (Amnis) analyses, we observed that me/me and me/+ T cells form more conjugates with APCs than +/+ T cells both in the presence or absence of Treg cells. Moreover, +/+ T cells treated with an inhibitor of SHP-1 showed a comparable increased conjugate formation as SHP-1-deficient T cells indicating that the observed phenotype is T cell intrinsic and not due to developmental differences. Furthermore, SHP-1-deficient T cells exhibited elevated surface levels of ICAM-1. Ongoing studies are addressing whether ICAM-1 directly affects the resistance to suppression. Taken together, the increased resistance to suppression displayed by SHP-1-deficient T cells and the enhanced capability of forming conjugates with APCs compared to wild type T cells suggest that the ability of forming T cell-APC conjugates influences to the susceptibility and/or resistance of a T cells to suppression.

Source: NIH, RO1 AI 048672
Regulatory T cell enrichment by Adenosine A3 receptor agonist is mediated through PLD signal Inhibition (89.28)
Authors: CHANDRASEKARAN UMA , IWASHIMA MAKIO
Published: January 2023

Phospholipase D (PLD) is a phosphodiesterase that catalyzes the conversion of phosphatidyl choline (PC) to phosphatidic acid (PA). We have previously demonstrated that inhibition of PLD signaling leads to preferential expansion of naturally occurring Tregs. Here, we show that adenosine blocks PLD signaling in activated T cells and causes increased frequency of nTregs in vitro. Adenosine (Ado), a naturally arising nucleoside has been implicated to be immunosuppressive but the mechanisms involved in immune suppression are not clear.Ado is released from dead cells due to nucleotide degradation. It is also produced by hydrolysis of ATP by CD39/CD73. Ado elicits intracellular responses by activating G-protein coupled receptors (A1, A2a, A2b, and A3). Increased concentrations of Ado have been detected in the individuals with the defect in ecto-adenosine deaminase activity (ADA and CD26), organs with high level of cell death (e.g. thymus, placenta), or tissues under hypoxic conditions (e.g. large solid tumors). Increase of Ado concentration was associated with severely compromised immune responses.

T cells express two types of adenosine receptors, namely A2a and A3. Our data show that agonist specific to Adenosine A3 receptor blocks expansion of non-regulatory T cells by suppressing TCR mediated signaling events and reduction of cytokine production. However, expansion of CD4+CD25+ naturally arising regulatory T cells (nTregs) was not affected by A3 agonist. Thus, A3 mediated signaling may contribute to establish reduced immune responses in the Ado rich environment by changing the balance between effector and regulatory T cells.
Characteristics of CD8+CD25- cells induced to become suppressor cells ex-vivo with IL-2 and TGF-β (89.34)
Authors: Lu Ling , Wang Julie , Wang Xuehao , Zhang Feng , Lo Jiwen , Brand David , Horwitz David , Zheng Song Guo
Published: January 2023

CD8+ cells stimulated with IL-2 and TGF-β can be induced to develop suppressive activity. However, the phenotype and functional characteristics of these polyclonal regulatory T cells (iTregs) have not been well characterized. To distinguish expanded thymus-derived Foxp3+ CD8+ cells from conventional CD8+CD25- cells induced to become Foxp3+ Tregs, we used Foxp3 GFP transgenic mice. Splenic GFP- cells from these mice were stimulated with anti-CD3/28 coated beads. Following 4-day stimulation, 35% of CD8+ cells become CD25+GFP+ cells in the presence of IL-2 and TGF-β. Granzyme B expression was decreased compared to CD8+ cells activated without TGF-β. These cells were also anergic following restimulation. Although their Foxp3 expression is lower than TGF-β -induced CD4+ cells, they displayed similar suppressive activity in cell contact-dependent, non-cytotoxic suppressive activity manner in a standard in vitro assay. Neutralization of TGF-β, IL-10 or IL-10 receptor and the TGF-β receptor I (ALK5) inhibitor was unable to abolish the suppressive activity. Similar to CD4+ iTregs, adoptive transfer of CD8+ iTregs suppressed a chronic GVHD with a lupus-like syndrome. Thus, TCR stimulation with IL-2 and TGF-β can induce CD8+ cells as well as CD4+ cells to become Foxp3+ Treg cells and both of these subsets have protective effects against pathologic immune-mediated inflammation. (Supported by ACR REF Award and Outstanding youth Scientist Award from NSFC)
Characterization of Thymic Regulatory T cell development (89.6)
Authors: Hsieh Chyi S
Published: January 2023

The development of Foxp3+ CD4+ regulatory T (Treg) cells in the thymus is important for the preservation of self-tolerance and the prevention of spontaneous autoimmunity. It has been suggested that the CD4+CD8+ double positive (DP) stage is the earliest that Foxp3+ expression can be observed. We hypothesized that the antigen specificity of Foxp3+ DP cells may differ from Foxp3+ CD4+CD8- (CD4SP) cells due to the different antigenic repertoires presented by cortical thymic epithelial cells versus medullary APCs. However, purification of Foxp3+ DP cells proved difficult as many of those events on flow cytometry represented doublets of Foxp3+ CD4SP cells and Foxp3- DP cells. Our data therefore suggest that the frequency of Foxp3+ cells that can be found at the DP stage is around 1%, which is much smaller than previously reported. Rather, our current data suggests that thymic Treg cell development occurs primarily at the HSAhi CD4SP stage involving a two-step process involving both TCR-instructive and TCR-independent steps.
Tim-3 Regulation of T Cell Effector Function (90.13)
Authors: Su Ee W , Lee Judong , Kane Lawrence P
Published: January 2023

Tim-3 is a type I transmembrane glycoprotein first identified through a screen for TH1-specific cell surface markers. Since then, it has been detected on a wide variety of cell types. Galectin-9, a β-galactoside binding lectin is the only known ligand of Tim-3 and has been used to study the function of Tim-3 due to the lack of agonistic Tim-3 antibodies. Galectin-9 induces apoptosis and calcium flux in TH1 cells but promotes the maturation of immature monocyte-derived dendritic cells and induces IgE-sensitized mast cells to secrete higher levels of TH2 cytokines. To better understand the regulation of T cell effector function by Tim-3, we treated TH1 cells with varying doses of galectin-9 and found that at an optimal concentration, galectin-9 has the ability to enhance IFN-γ production by TH1 cells without affecting their viability. Then, using luciferase reporter assays, we found that overexpression of Tim-3 in Jurkat T cells can enhance NF-κB and NFAT/AP-1 activity downstream of the T-cell receptor and CD28. Therefore, these findings indicate that Tim-3 can positively affect T cell effector function.
Effect of ROR γt on regulation of IFN-γ secretion of T cell subpopulations (90.15)
Authors: LI Baiqing , XUE Zhuping , Ma Fei
Published: January 2023

It has been known that retinoid-related orphan receptor γt (RORγt) is a key transcription factor for development of IL-17 producing CD4 T cells (Th17), which are negatively regulated by interferon-γ (IFN-γ) produced by Th1 cells, however, whether the RORγt regulates IFN-γ production in T cell subsets has not been elucidated. In our study the peripheral blood mononuclear cells isolated from peripheral blood of healthy human donors were stimulated with CD3 mAB and cultured in IL-2 containing medium for 12 days. The activated T cells were transfected with three chemical synthesized small interfering RNA (siRNA) sequences for RORγt gene using liposomes. By using semi-quantity RT-PCR assay, the RORγt gene expression was inhibited in activated T cells that transfected with RORγt-siRNA-1026 and -1197, but was not inhibited in that with RORγt-siRNA-982. By using flowcytometry, the percentages of IFN-γ producing cells in total T cells, CD4+ T cells and CD8+ T cells that transfected with the sequence of RORγt-siRNA-1026, significantly decreased to 35.19, 32.78, and 41.84, respectively, in comparison of nontransfected group (43.39, 44.54, and 50.04, respectively) (p< 0.05). In conclusions, the transcription factor RORγt may also be involved in the positive regulation for IFN-γ production in human T cells, especially in CD4+ T cells.
Cytotoxicity of human CD8+ T-cells and NK-cells is suppressed by an alternative NKG2D splice variant (90.19)
Authors: Karimi Mobin  A , Bachmann Michael H , Cao Thai M , wang Chao , Kim Edward , Watts Tania H , Negrin Robert S , Zhang Li
Published: January 2023

NKG2D is an activating receptor crucial to diverse aspects of immune function. NKG2D-mediated cytotoxicity is utilized by human natural killer (NK) cells and CD8+ T-cells. Here, we functionally characterize an alternative splice variant of NKG2D in non-activated and activated human CD8+ T-cells, which is expressed as a membrane-anchored truncated protein that lacks the ligand-binding ectodomain. We found this truncated NKG2D (T/NKG2D) isoform to be unable to transmit an activating signal for cytolysis and its enforced expression to inhibit killing mediated by full-length NKG2D (F/NKG2D). Abundance of T/NKG2D transcripts is variable in non-activated and activated CD8+ T-cells, and inversely correlates with killing capacity. Co-immunoprecipitation showed that T/NKG2D pairs with DAP10 and heterodimerizes with F/NKG2D, suggesting that dominant negative regulation is due to formation of heterodimeric NKG2D receptor complexes incapable of binding to its ligands. Thus, competitive inhibition through the alternatively spliced NKG2D variant may constitute a novel mechanism that regulates NKG2D-mediated signaling and cytotoxicity in NK and CD8+ T-cells.
Distinct subsets of human skin dendritic cells differ on their ability to initiate Th17 responses (90.24)
Authors: Mathers Alicia R , Janelsins Brian M , Rubin Joseph P , Tkacheva Olga A , Shufesky William J , Morelli Adrian E , Larregina Adriana T
Published: January 2023

Activated human cutaneous dendritic cells (DCs) have the ability to initiate Th1 and Th2 immunity. However, whether human cutaneous DCs are capable of biasing pro-inflammatory Th17 responses remains little understood. Utilizing human skin explants composed by both epidermis and dermis or epidermal or dermal sheets to collect activated skin-migratory DCs (smiDCs), we demonstrated that smiDCs stimulate allogeneic naïve CD4+ T cells to differentiate simultaneously into two distinct effector Th17 and Th1 populations with ability of skin homing and inducing severe tissue damage. Of the two main myeloid DC populations resident in the skin, the subset of epidermal skin-migratory Langerhans cells (smiLCs) were capable of inducing Th17 responses. This effect depended on the combined effects of IL-15 and stabilized IL-6 trans-signaling of naïve CD4+ T cells. Purified skin-migratory DDCs did not synthesize IL-15 and were unable to bias Th17 responses however, they acquired the ability to bias Th17 cells in co-cultures with CD4+ T cells supplemented with IL-15 and stabilized IL-6. Overall, our data demonstrate that human cutaneous LCs induce Th17 responses by mechanisms different from those previously described for mouse DCs and human monocytes and highlight the need to target clinical treatments based on these variations.

Supported by NIH grants: R01 CA100893 (ATL)