bluuebunny/crossref_metadata_2025 huggingface dataset:
BCR Signaling Generates NF-kB2 (p100) For BR3-Mediated Processing And Survival (83.2)
Authors: Stadanlick Jason E , Scholz Jean L. , Miller Juli P. , Cancro Michael P.
Published: January 2023

Signaling through both the B cell receptor (BCR) and the BLyS receptor, BR3, are required for primary B cell survival. Therefore, understanding how the downstream signaling pathways from these receptors interact should help reveal the molecular mechanisms underlying primary B cell selection and survival. The classical and non-classical NF-kB pathways are an attractive starting point for interrogation, since its members operate downstream of several key B cell signaling systems. Accordingly we have examined BLyS-mediated signaling and survival both in isolation as well as in the context of signals derived form the BCR. In NH3T3 cells expressing BR3, as well as in primary splenic B cells, we find that BLyS signaling via BR3 predominantly activates the non-classical pathway. This is evidenced by an increase in p52 levels and the corresponding diminution of the p100 pool upon BLyS treatment. In contrast, while BCR signaling potently upregulates p100 levels, it does not result in processing to the active p52 form. However, simultaneous BCR and BR3 signaling yields both p52 accumulation and sustained p100 availability. These findings suggest a model whereby BCR signaling is required to maintain available substrate for the viability-promoting BR3 pathway. In support of this model, blockade of proximal BCR signaling with a Syk kinase inhibitor leads to the depletion of p100 stores and the failure of BLyS-mediated survival. Together, these findings lead us to propose a model whereby BCR signaling rations a key survival pathway substrate, thus governing selection and homeostasis.
Regulation of B-lineage specification by the IL-7/JAK3/STAT5 pathway (83.6)
Authors: Gerstein Rachel Maurie , Tian Linhua , Wallace Morgan , De Pallabi , Prince Amanda , Berg Leslie , Allman David
Published: January 2023

The cytokine IL-7 is essential for early B cell development in adult bone marrow. We previously showed that B cell genesis arrests at the common lymphoid progenitor (CLP) stage in mice lacking the common gamma chain or the alpha-chain of the IL-7 receptor (IL-7R). Other studies suggest that CLPs are enriched for B-lineage specified (but uncommitted) precursors, as CLPs are not requisite for T cell development, and V(D)J recombinase activity in CLPs requires a B-specific enhancer element in the RAG locus. Here we probe events downstream of the IL-7R that promote B-lineage specification. We find that JAK3, the principal kinase downstream of the IL-7R, is essential for B cell development beyond the CLP stage. We further demonstrate that JAK3-deficient CLPs exhibit a dramatic loss in RAG1/2 expression and V(D)J recombinase activity. JAK3-null CLPs also lacked transcripts for EBF1, a B-lineage restricted transcription factor previously suggested to promote RAG expression. We also find that conditional inactivation of STAT5, the key transcription factor activated by JAK3, results in loss of B-lineage precursors beyond the CLP and diminished V(D)J recombinase activity. Together these data suggest that IL-7R activation of the JAK3/STAT5 pathway plays a key role in initiating several molecular events required for early B cell development in CLPs.
Novel aspects of the FADD function in lymphocytes revealed using hypomorphic FADD mutant and conditinal FADD-deficient mice (84.4)
Authors: Zhang Jianke , Imtiyaz Hongxia , Rosenberg Stephen
Published: January 2023

FADD was initially isolated as a death domain-containing protein interacting with the death receptor Fas. In the context of apoptotic signal transduction, the death domain of FADD interacts directly with the death domain of Fas and the death effector domain of FADD interacts with the death effector domain of pro-caspase-8. This leads to activation of the apical caspase-8 and subsequently downstream caspases such as caspase-3, -6 and -7. Fas-induced apoptosis is essential for maintaining lymphoid homeostasis and suppressing autoimmunity. Mutations in the Fas gene result in massive accumulation of lymphocytes of potential auto-reactivity, and lead to lymphadenopathy and autoimmune diseases in the mouse and human. However, deletion of FADD resulted in early embryonic lethality. To further analyze the function of FADD, we generated several lines of mutant mice carrying point mutations of FADD. Analysis these hypomorphic mutant mice as well as the previously generated conditional FADD-deficient mice revealed novel aspects of the mechanism involved in the regulation of innate and adaptive immune responses mediated by FADD.
T CELLS UNDERGO ASYMMETRIC CELL DIVISION – A NEW MECHANISM TO DICTATE T CELL FATE (84.5)
Authors: Russell Sarah May , Oliaro Jane , Pasam Anupama , Sacirbegovic Faruk , Pham Kim
Published: January 2023

We have previously shown that T cells utilize an evolutionarily conserved network of polarity proteins to orchestrate cell shape and polarity, and that these proteins are required for migration and immunological synapse formation in T cells (1). We describe here in vitro evidence using the OT-1 model system that T cells utilize this polarity network to conduct a physiological process not previously ascribed to lymphocytes, that of asymmetric cell division. We demonstrate that naive T cells remain attached to antigen presenting cells (dendritic cells pulsed with ovalbumin peptide) throughout cell division, and utilize this attachment to orient their axis of cell division. By maintaining the asymmetry originally associated with immunological synapse formation, the daughters of the T cell division inherit different molecular characteristics, which provide the capacity to dictate different subsequent fates.

A network of PDZ-containing proteins regulates T cell polarity and morphology in motility and immunological synapse formation
Runx1/AML1 and hematopoietic dysplasia (84.6)
Authors: Telfer Janice C , Malcuit Nancy , Hedblom Emmett , Kane Christyne
Published: January 2023

We expressed several mutated forms of the Runx1 transcription factor in hematopoietic stem cells (HSC) and used them to reconstitute lethally irradiated mice. We found that the Runx1 mutant truncated shortly after the DNA-binding runt domain (Runx1.d190) transported appropriately to the nucleus and had strong dominant negative activity. Surprisingly, Runx1 mutants truncated before the runt DNA-binding site (Runx1.d49) also reiterated some of the effects of the Runx1.d190 dominant negative mutant, despite the lack of a nuclear localization site and DNA-binding capacity, suggesting that the N-terminus plays an active role in some Runx functions and that the N-terminal activity may be significant in Runx mutants lacking DNA-binding activity. The Runx1.d190 mutant, when expressed in HSC, results in the expansion of the cellular compartment containing HSC. In a competitive reconstitution in a lethally irradiated mouse, these Runx1.d190-positive cells and their progeny almost totally out-compete untransduced cells and their progeny. Some of the Runx1.d190-positive hematopoietic stem cells are able to reconstitute a sub-lethally irradiated mouse in all hematopoietic lineages and maintain that reconstitution for over a year, indicating transformation of a hematopoietic stem cell or its immediate progeny. The progeny of the Runx1.d190-positive hematopoietic stem cells are defective in several lineages, such as B cells, T cells, neutrophils, megakaryocytes, and erythrocytes. There is a significant loss of erythropoiesis in the bone marrow and the occurrence of splenic extramedullary hematopoiesis.
Impaired GATA3-dependent chromatin remodeling and Th2 cell differentiation leading to attenuated allergic airway inflammation in aging mice (85.1)
Authors: Hasegawa Akihiro , Nakayama Toshinori
Published: January 2023

Age-related changes in lymphocytes are most prominent in the T cell compartment. In the present study, we used 8–12 month-old aging mice and a well-established in vitro Th1/Th2 cell differentiation culture system to identify molecular defects in Th1/Th2 cell differentiation that can be detected in the relatively early stages of aging. The capability to differentiate into Th2 cells is reduced in aging mouse CD4+ T cells. Decreased activation of the ERK MAPK cascade upon TCR stimulation, but normal [Ca2+]i mobilization and normal IL-4-induced STAT6 activation were observed in aging mouse CD4+ T cells. In addition, reduced expression of GATA3 was detected in developing Th2 cells. Chromatin remodeling of the Th2 cytokine gene locus was found to be impaired. Th2-dependent allergic airway inflammation was milder in aging mice compared with in young adult mice. These results suggest that the levels of Th2 cell differentiation and resulting Th2-dependent immune responses, including allergic airway inflammation, decline during aging through defects in the activation of the ERK MAPK cascade, expression of GATA3 protein and GATA3-dependent chromatin remodeling of the Th2 cytokine gene locus. In the present study, we provide the first evidence indicating that a chromatin-remodeling event in T cells is impaired by aging.
Neonatal recent thymic emigrants (RTE): phenotypic and functional comparison to adult RTE (85.16)
Authors: Opiela Shannon Jacqueline , Adkins Becky
Published: January 2023

Neonatal CD4+ T cells typically produce high levels of IL-4, resulting in Th2-biased responses. The neonatal CD4+ compartment contains a high frequency of recent thymic emigrants (RTE), in contrast to the small proportion found in adults. Using intrathymic labeling to identify RTE, it has been described that adult RTE produce high levels of IL-4. Thus, neonatal Th2 biased responses may result from their over-represented RTE population. Here we have compared neonatal and adult CD4+ RTE for the first time, using a mouse strain that allows for the identification and purification of RTE from the periphery. In contrast to the intrathymic labeling system, adult RTE from these mice produced less IL-4 and IFNγ than the resident cells. Similarly, neonatal RTE produced less of both IL-4 and IFNγ than resident neonatal cells. The phenotypic changes accompanying post-thymic maturation of RTE were also similar in neonates and adults. Thus, both neonatal and adult RTE appear to require a period of post-thymic maturation to acquire mature Th2 and Th1 function and phenotype. Importantly, despite these similarities, neonatal RTE produced more IL-4 than adult RTE. Together, these results indicate that neonatal and adult RTE differ functionally but the subsequent processes of post-thymic maturation are similar in early development and adult life.

This work was supported by NIAID grant number R01 AI44923-02 (B.A.)
Effector CD8 T cells transit to memory cells in the presence of tumor-associated antigen (85.17)
Authors: Rolle Cleo E. , Carrio Roberto , Malek Thomas R.
Published: January 2023

It is difficult to study the time frame and requirements for antigen removal as CD8 T effector cells transit to memory cells during an immune response because memory cells only readily appear after contraction of the effector response and clearance of antigen. To overcome these limitations, we have modeled the effector to memory transition in vitro and by the adoptive transfer of effector CTL to normal mice. When OVA-specific OT-I CTL were cultured in IL-15 without antigen, they acquired phenotypic and functional properties of memory cells 48 hr later. Such memory conversion occurred in the presence of 0.01 nM OVA peptide and APC, a dose that only induces a strong recall response, but not at a higher dose (0.1 nM) that readily activates naïve OT-I T cells. Following adoptive transfer of OT-I CTL to antigen-free normal mice, OT-I effector cells developed into central memory-like cells by 3 days post-transfer. Upon adoptive transfer to mice bearing the OVA-expressing E.G7 tumor, the OT-I CTL effector cells also readily converted to memory-like cells by day 3 post-transfer while the effector cells rejected the tumor. However, under conditions where the tumor was not cleared, memory production was impaired. Collectively, these data suggest that some CTL rapidly convert to memory cells concurrent with an effector response, without complete clearance of antigen, provided that antigen level is not too high.

(Supported by the NIH)
Epigenetic patterns at the neonatal Th2 cytokine locus are developmentally regulated (85.2)
Authors: Foote Monica Ruth , Rose Shawn , Lichtenheld Mathias , Adkins Becky
Published: January 2023

Murine neonatal immune responses are commonly Th2 biased, characterized by rapid and high level Th2 cytokine production. These Th2-biased responses entail cell-intrinsic properties whose molecular basis is now beginning to become unveiled. We have recently found that the Th2 regulatory region CNS-1 is nearly 25% more demethylated in naïve neonatal CD4+ lymph node T cells and in CD4+ single positive (SP) thymocytes than in the same adult populations. Importantly, IL-4-expressing neonatal thymocytes exhibit substantially greater levels of hypomethylation at CNS-1 compared to unactivated cells. These results suggest that a hypomethylated state at CNS-1 is permissive for IL-4 expression in neonatal cells. Additional studies have revealed that CNS-1 exists in this highly demethylated state in freshly isolated fetal (d15 gestation) thymocytes, which are predominantly double negative cells and are the precursors of CD4+ SP thymocytes found in the neonate. These new findings suggest that epigenetic patterns at the neonatal Th2 cytokine locus are established within the fetal thymus, or even earlier, possibly in fetal liver precursor cells. Together, these observations lead to the intriguing idea that epigenetic modifications may be used as a developmental strategy within the CD4+ T helper cell lineage to achieve differential expression of cytokine genes at different times of life.

This work was supported by NIAID grant number R01 AI44923-02 (B.A.).
Effects of aging on the ability of B lymphocytes to undergo early activation events and their proliferative capability (85.26)
Authors: Blaeser Tony Ryan , McGlauchlen Kiley S , Vogel Laura A
Published: January 2023

Dysfunction of the humoral immune response is associated with aging. It is uncertain, however, if this dysfunction is due to intrinsic B cell defects or defects of the aged microenvironment, such as lack of appropriate T cell help. Previous in vivo studies by our lab found decreased antigen-induced expansion by aged B cells but could not rule out the possibility of decreased migration. Here we examine early activation events in vitro in response to anti-CD40 and LPS stimulation. Aged B cells showed similar resting levels of CD40 to that of their young counterparts but were less able to respond to CD40 induced signals. Aged cells were defective in their ability to down-regulate CD38 and up-regulate CD69 over a 48 hour period. Aged cells, however, were able to up-regulate CD86 at similar levels to that of young cells. There also was a decrease in blast formation with aged B-lymphocytes as well as slightly decreased proliferative response. Hence, aged B cells appear to be defective in their response to some early activation events but appear to always show a partial response.
Costimulation through ICAM-1 induces differentiation of naive human CD4+ T cells from aging individuals (85.27)
Authors: Dotson Abby L. , Kohlmeier Jacob E. , Cool Kelli M. , Benedict Stephen H.
Published: January 2023

With age, the immune response is less effective at protection and less responsive to vaccines. The events leading to immunosenescence have not been fully described. We have been investigating the hypothesis that the nature of the T cell costimulatory signal governs in part the phenotype of the post thymic developing naïve T cell as it responds to cognate antigen. The costimulatory molecules, CD28 and LFA-1 are the best characterized; however we have demonstrated that ICAM-1, resident on a T cell, can receive signals that drive naïve human CD4+ T cells taken from young individuals to effector and memory phenotypes that differ from those induced by CD28 or LFA-1. For the present study, we are investigating the participation of these three costimulatory signals in activation and differentiation of human naive CD4+ T cells from aging individuals. We will describe differential effects on development in response to costimulation of naïve T cells taken from older as compared with young human subjects.
TRAF3 is a critical regulator of B cell homeostasis in secondary lymphoid organs (86.2)
Authors: Xie Ping , Stunz Laura L , Larison Karen D , Yang Baoli , Bishop Gail A
Published: January 2023

TRAF3, a cytoplasmic adaptor protein, is utilized by receptors of the tumor necrosis factor receptor (TNF-R) superfamily and Toll-like receptors for signaling. Despite in vitro evidence that TRAF3 plays diverse roles in different cell types, little is known about the in vivo functions of TRAF3. To address this gap in knowledge and to circumvent the early lethal effect of TRAF3 null mutations, we generated conditional TRAF3-deficient mice. We found that specific ablation of TRAF3 in the B cell lineage led to remarkably prolonged B cell survival and greatly expanded B cell compartments in secondary lymphoid organs. This culminated in splenomegaly and lymphadenopathy, hyperimmunoglobulinemia, and autoimmune reactivity. These findings identify a novel critical role for TRAF3 in regulating B cell homeostasis in secondary lymphoid organs, and implicate the involvement of TRAF3 in peripheral self-tolerance induction.

This study is supported by a grant from the American Heart Association (PX), NIH grants AI28847, AI49993, CA099997, and a VA Career Award (GAB).
Class IA PI3K is involved in the pre-B cell antigen receptor-mediated signaling (86.6)
Authors: Koyasu Shigeo , Fujiwara Mari , Matsuda Satoshi , Kadowaki Takashi , Kawano Yohei , Maki Kazushige , Karasuyama Hajime
Published: January 2023

We have previously generated p85α−/− mice deficient for the most abundantly and ubiquitously expressed regulatory subunit of class IA PI3Ks. In p85α−/− mice, B cell development from pro-B cells to pre-B cells in the bone marrow is impaired and the number of mature B cells in the periphery is decreased. Here we further investigated the role of PI3K in early B cell development. First, we crossed p85α−/− mice with a transgenic mouse line expressing rearranged IgH and IgL chains Ig(H+L) specific for hen egg lysozyme (Hel-Tg). Introduction of functional Ig(H+L) chains did not restore the number of peripheral B cells in mice on a p85α−/− background. We next crossed p85α−/− mice with Rag-2−/− mice to generate p85α−/−Rag-2−/− double deficient mice. We compared the transition from B200+c-kit+CD25− pro-B cell stage to the B200+c-kit−CD25+ pre-B cell stage upon anti-Igβ antibody injection between Rag-2−/− mice and p85α−/−Rag-2−/− double deficient mice. While B200+c-kit−CD25+ cells were readily observed in Rag-2−/− mice after administration of anti-Igβ antibody, such transition was impaired in p85α−/−Rag-2−/− double deficient mice, indicating that the class IA PI3K is involved in the pre-BCR signaling. Our results indicate that class IA PI3K is important in pre-B cell antigen receptor-mediated signaling to induce pre-B cell expansion rather than the Ig gene rearrangement.
NON-APOPTOTIC INDUCTION OF CASPASE-8 IN T-CELLS DEPENDS ON FADD AND CELL CYCLE PROGRESSION (87.1)
Authors: Leverrier Sabrina , Weist Brian M , Walsh Craig M
Published: January 2023

Upon triggering of apoptosis by tumor necrosis factor (TNF) receptor family member engagement, FADD (Fas-Associated Death Domain-containing protein) acts as an adaptor protein by recruiting caspase-8 and promoting its autocatalytic activation. Surprisingly, it was found that FADD and caspase-8 are also critical for T lymphocytes proliferation. Indeed, T cell specific overexpression of a FADD dominant-negative mutant (FADDdd) or conditional deletion of caspase-8 or FADD in T cells leads to profound defects in proliferation. While it is known that FADD transmits an apoptotic signal by recruiting caspase-8 within the cytoplasm, it is unclear how FADD and caspase-8 promote T cell proliferation. Since it has been shown that both proteins can localize within the nucleus, we investigated caspase-8 catalytic activity in the nucleus of proliferating T cells and detected an increasing level of caspase-8 activity upon stimulation with anti-CD3 and anti-CD28. Using FADDdd T cells, we determined that this nuclear catalytic activity was dependent on normal FADD function. Blocking cell cycle progression at different stages also drastically reduced caspase-8 activation in non-apoptotic T cells, whereas inhibiting TNF receptor activation did not appreciably diminish caspase-8 activity. We are currently attempting to identify caspase-8 nuclear substrates involved in T-cell proliferation. Taken together, our results suggest that specific subcellular compartmentalization is associated with a distinct magnitude in caspase-8 activity, possibly leading to new substrate selectivity during cell cycle progression. These findings may explain how two apoptotic regulatory proteins, FADD and caspase-8, alternatively promote cell death or proliferation in T cells.
IFN-gamma production by CD4 T cells requires Histamine H1 receptor during their initial phase of activation (87.25)
Authors: Noubade Rajkumar , Rincon Mercedes , Teuscher Cory
Published: January 2023

Histamine H1 receptor (H1R) signaling in T cells plays an important role Th1 effector functions. H1R is an autoimmune susceptibility gene controlling experimental autoimmune encephalomyelitis (EAE). H1R knockout (H1RKO) mice also develop significantly less severe EAE compared to C57BL/6J wild-type mice in association with immune deviation of the CD4+ T-cell response from a Th1- to a Th2-like response. Little is known about the role of direct H1R signaling in the regulation of T-cell effector responses. In this study, T-cells from C57BL/6J and H1RKO mice were used to study H1R signaling in directly regulating such responses. Attempts to restore IFN-gammaproduction in H1RKO T-cells following retroviral transduction of the H1R were unsuccessful. In contrast, H1RKO transgenic mice selectively expressing the H1R in peripheral CD4+ T-cells under the control of the distal-lck promoter restored the IFN-gamma response and susceptibility to EAE in an H1R gene dose-dependent fashion. Moreover, activated H1RKO CD4+ T-cells show reduced phosphorylation of p38 MAP kinase compared to T-cells from wild-type mice which was again restored in activated T-cells from transgenic mice. These results show that H1R signaling regulates IFN-gamma production in CD4+ effector T-cells by direct signaling during initial activation of naïve T-cells through phosphorylation of p38 MAP kinase.
Upregulation of Th1-specific transcription factors and inflammatory responses by IFNg and IL27 in splenocytes from estrogen treated mice (87.33)
Authors: Karpuzoglu Ebru , Phillips Rebecca A , Dai Rujuan , Lengi Andrea J , Ahmed S. Ansar
Published: January 2023

Estrogen, an immunomodulator, is believed to be involved in many autoimmune inflammatory conditions. We reported that estrogen treatment of C57BL/6 mice favors the induction of Th1 responses such as secretion of IFNγ. However, the mechanisms involved in response to IFNγ are not well known. Therefore, we investigated the molecular pathways in splenocytes from estrogen-treated mice following in vitro exposure to Th1(IFNγ)-inducing cytokines: IFNγ, IL12 and IL27. ConA activated splenocytes from estrogen treated mice induced an IFNγ-dependent-increase in iNOS, nitric oxide, COX2 and MCP1. In vitro addition of IFNγto ConA activated splenocytes from IFNγ −/− mice induced iNOS and MCP1 primarily in estrogen-treated mice. Since IFNγ is a potent inducer of IRF1, interestingly that neither direct addition of IFNγ to splenocytes from wildtype or IFNγ −/− mice nor the addition of IFNγ to T cells, induced IRF1 in cells from estrogen-treated mice. Activated splenocytes from estrogen-treated mice had noticeably increased levels of the IFNγ-specific transcription factor T-bet, although there were no marked changes in phosphorylated STAT1. Estrogen-induced upregulation of T-bet was largely due to IFNγ and IL27, but not IL12. Thus, these findings contribute novel insight regarding the pro-inflammatory effects of estrogen on the immune system and may lead to novel therapeutic approaches for autoimmune diseases.

Supported by NIH grant 5RO1 AI51880.
Granzyme B Is Critical for T-Cell Receptor Activation Induced Cell Death of Type 2 Helper T Cells (87.34)
Authors: Devadas Satish , Das Jyoti , Liu Catherine , Zhang Liying , Roberts Arthur I. , Pan Zui , Moore Paul A. , Das Gobardhan , Shi Yufang
Published: January 2023

Although CD95L is required for T cell receptor activation induced cell death (AICD) in T helper 1 (Th1) cells, the molecular mechanisms mediating AICD in Th2 cells are unknown. We found that death receptor medicated apoptosis was not involved in AICD of Th2 cells because blocking their cognate ligands with recombinant soluble receptors of specific antibodies had no effect on apoptosis of activated Th2 cells. Furthermore, we showed that the major caspases were not actively involved in AICD of Th2 cells. However, inhibition of granzyme B (Gzmb) specifically abolished AICD in Th2 cells but not Th1 cells. Likewise, Th2 cells derived from Gzmb-deficient mice were resistant to AICD, and Gzmb deficiency or inhibition of Gzmb activity consequently enhanced the production of Th2 cytokines. Most interestingly, Gzmb-deficient mice exhibited increased susceptibility to allergen-induced asthma. Thus, Gzmb plays a critical role in the AICD of Th2 cells. This finding provides a novel mechanism for the balance between Th1 and Th2 cells during immune responses.
LAT Plays Important Role in Thymocyte Survival and Mature T Cell Activation (87.35)
Authors: Shen Shudan , Zhu Minghua , Zhang Weiguo
Published: January 2023

LAT (Linker for Activation of T-cells) is a membrane-associated adaptor protein that plays a critical role in linking T-cell receptor engagement to activation of its downstream signaling events. Upon tyrosine phosphorylation, LAT binds Grb2, Gads, and PLC-gamma1. Previous studies show that LAT is essential in thymocyte development as LAT-deficient mice completely lack mature T cells. In addition, LAT-deficient Jurkat cells have a severe defect in TCR-mediated signaling. In order to investigate the role of LAT in T cell activation, survival, and homeostasis, mice in which the LAT gene can be inducibly deleted from thymocytes and mature T cells by injection of tamoxifen were generated. Our data showed that tamoxifen treatment of these mice led to a dramatic loss of thymocytes, especially DP thymocytes. Deletion of LAT in mature T cells affected TCR-mediated signaling, IL-2 production, and proliferation. These data suggested that LAT plays an important role in thymocyte survival and TCR-mediated signaling in mature T cells.
Participation of nitric oxide synthase (NOS) and atypical protein kinase C (PKC) ζ in thyroid hormone-mediated apoptosis of murine T lymphocytes (87.39)
Authors: Cremaschi Graciela Alicia , Klecha Alicia Juana , Vercelli Claudia , Franchi Ana María , Genaro Ana María , Arcos María Laura Barreiro
Published: January 2023

Thyroid hormones (TH) regulate immune responses and are able to influence proliferation and differentiation in several cell types. However, their direct actions on lymphocytes have not been elucidated. We have demonstrated that short time culture (up to 72 h) of quiescent BW5147 (BW) T cells in the presence of TH induced cell division. Here, prolonged actions of TH on BW cells and the intracellular signals involved were studied. After 5 days of culture, TH inhibited BW growth inducing apoptosis, as shown by Hoescht staining and DNA ladder assay. These effects were accompanied by an increase in NOS activity measured by [14C]-citrulline formation. Also, an increase in iNOS and a decrease in the PKC ζ isoform, both at the protein (by western blot analysis) and mRNA levels (by RT-PCR evaluation) were seen An increase in reactive oxygen species (determined by conversion of dichlorofluorescin-diacetate to the fluorescent DCF) was found. Additionally, TH induced an increment in protein nitrosylation.

These results show that prolonged TH exposition lead to an exacerbate increase in iNOS activity, that would in turn result in PKC ζ decrease, probably through protein nitrosylation. These intracellular signals would trigger apoptotic mechanisms involved in TH regulation of lymphocyte activity.
Importance of siRNA negative control selection: Evaluation of non-specific protein knock-down by negative control siRNA (87.42)
Authors: Sullivan Yvonne B. , Hughes Douglas E. , Narahari Janaki , Webb Brian L.
Published: January 2023

Small interfering RNA (siRNA) are double-stranded RNA consisting of 19–22 base-pairs that direct the cleavage of complementary mRNA targets. The utility of siRNA as a research tool depends on the specificity of the gene silencing, as knockdown of unintended gene targets (off-target effects) can potentially complicate the interpretation of siRNA data. To investigate the extent of off-target effects at the protein level, we evaluated RNAi negative control products from three commercial vendors for non-specific protein knockdown utilizing the SuperSignal® siRNA/Antibody Modules and SuperSignal siRNA Chemiluminescent Detection Module. Using Western blotting we found that the expression level of a number of different proteins was reduced when A549 cells were transfected with negative control siRNAs from two different vendors. Minimal non-specific protein knockdown was detected with the ON-TARGETplus™ siCONTROL® Non-Targeting Pool. These data suggest that the selection of an optimal negative control is critical to correct interpretation of siRNA experiments and that non-specific knockdown of protein should be evaluated.
Synergy Between Molecular targets of Costimulation in Promoting Persistence of CD8 T cells (87.5)
Authors: Song Jianxun , Song Ava , Croft Michael
Published: January 2023

Costimulation regulates multiple cellular processes including proliferation, expansion and survival of T cells, and molecular targets of costimulation might be useful to augment T cell activities. Two defined targets of costimulatory signals in CD4 T cells are the anti-apoptotic Bcl-2 family molecule Bcl-xL, and survivin, an Inhibitor of Apoptosis family member that might regulate cell division and survival. However, the relative importance and relationship between these molecules in CD8 T cells is not clear. To understand whether they have overlapping or synergistic functions, we used retroviral transduction to introduce Bcl-xL and survivin separately, or together linked by a 2A picornavirus self-cleaving peptide, into antigen-responding CD8 cells. In vitro, we found that CD8 effector T cells expressing both Bcl-xL and survivin strongly expanded at an early stage and had a long-term survival advantage. In vivo, with response to tumor-expressed Ag following adoptive T cell transfer, antigen-reactive CD8 T cells expressing both Bcl-xL and survivin displayed a greatly enhanced tumor protective activity compared CD8 cells expressing either molecule introduced separately. These results indicate that Bcl-xL and survivin can critically contribute in a synergistic, non-redundant, manner to augment the accumulation and persistence of CD8 T cells following encounter with antigen. The data provide new insights into why costimulatory signals might need to be sustained over time or provided periodically via ligation of multiple receptors.
Counting agonist in functional T cell receptor clusters using nanopatterned corrals (87.51)
Authors: Rossenova Boryana , Jackson Bryan , Dustin Michael , Groves Jay T
Published: January 2023

T lymphocytes, the initiators of the adaptive immune response, are remarkable for their ability to recognize very few agonist peptides presented on APC, using their T cell receptors (TCR). Recent studies emphasize the role of spatial organization of TCR at the interface between the two cells in regulating T cell activation - a central cluster and peripheral microclusters. The microclusters are thought to initiate, amplify and maintain signaling, however their mechanism of function is largely unknown. Using nanopatterned square corrals, 1 micrometer in length, we can regulate the maximum number of agonist-MHC molecules per cluster presented by a fluid supported lipid bilayer containing MHC and ICAM-1 molecules. We find that at activating mean concentrations of agonist, T lymphocytes are unable to initiate signaling if the maximal number of agonist molecules per corral is 1–2. Surprisingly, agonist number as little as twice this amount per corral, results in full cell activation, as measured by the calcium flux inside the cell. These observations suggest that microclusters are not necessary to amplify signaling beyond what 3–4 agonist molecules are sufficient to do.
Protracted Contraction and Memory T Cell Differentiation in Mice Deficient in TNF and Perforin (87.6)
Authors: Schmidt Nathan W. , Harty John T.
Published: January 2023

Following infection, pathogen-specific T cells undergo an expansion in numbers followed by contraction to relatively stable memory numbers. Work from others suggests that CD8 T cell responses to infection are altered in mice lacking TNF receptors or perforin. To understand how TNF regulates T cell responses we infected perforin/TNF-double deficient (DKO) mice, perforin-deficient (PKO) mice, and TNF-deficient (TKO) mice with Listeria monocytogenes (LM) and analyzed the MHC Ia-, MHC Ib-, and MHC II-restricted T cell responses. DKO mice exhibited a delay in contraction of MHC Ia- and MHC II-restricted T cells compared to WT mice. All three T cell responses exhibited delayed acquisition of CD62L, a central memory marker, in both DKO and TKO mice. DKO mice have a prolonged infection of LM, however, this did not account for the delay in contraction of the T cell populations, which still occurred in mice treated with antibiotics to eliminate the prolonged infection. These data suggest that TNF and perforin regulate antigen-specific T cell homeostasis, while TNF regulates the phenotype of antigen-specific T cells. Ongoing studies are addressing the mechanisms underlying delayed contraction and whether additional memory characteristics are altered by TNF deficiency.
Pretreatment of CD138− B Lymphocytes with a Covalent Reactive Antigen Analog to Factor VIII Reduces Antibody Production in response to FVIII (88.14)
Authors: Smith Keri Csencsits , Planque Stephanie , Nishiyama Yasuhiro , Taguchi Hiroaki , Escobar Miguel , Paul Sudhir
Published: January 2023

B lymphocytes producing high titer antibodies to factor VIII (FVIII) are of major concern in the treatment of bleeding episodes in a subpopulation of patients with hemophilia A. We hypothesize that an electrophilic, covalent reactive analog of FVIII (FVIII-CRA) can induce antigen-specific tolerance by permanent engagement of B cell receptors. FVIII-CRA was prepared by derivitizing lysine side chains with phosphonate diester groups. These groups are predicted to bind enzyme-like nucleophilic residues located within the BCR. To test the effects of FVIII-CRA binding on B lymphocytes, we isolated CD138− splenocytes from FVIII-deficient mice (B6;129S4-F8tm1Kaz/J) immunized with FVIII. These putative memory B lymphocytes were then re-stimulated for 6 days with identical doses of FVIII or FVIII-CRA. The number of antibody secreting cells (ASC) produced in response to FVIII-CRA re-stimulation was significantly lower than FVIII re-stimulation (avg of 36 ASC vs 111 ASC, p = 0.03). In addition, cell viability following low dose FVIII-CRA treatment was decreased by ~ 22% when compared with identical doses of FVIII. These results suggested that FVIII-CRA may tolerize memory B lymphocytes. To confirm this, we pretreated CD138− splenocytes with FVIII-CRA and then added a stimulatory dose of FVIII to the cultures. Pretreatment with FVIII-CRA resulted in a significant decrease in the number of ASC produced in response to FVIII (by 17-fold compared to control pretreatment). These results suggest that FVIII-CRA may represent a viable means to induce antigen-specific B cell tolerance in hemophilia.
Costimulatory molecule-targeted immunotherapy of chronic graft-versus-host disease (88.17)
Authors: Kim Juyang , Kim Hye J. , Park Keunhee , Kim Jiyoung , Choi Hye Jeong , Yagita Hideo , Nam Seok H. , Cho Hong R. , Kwon Byungsuk
Published: January 2023

Chronic graft-versus-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Current therapies for cGVHD reduce symptoms but are not cures. Using the B10.D2„³Balb/c (H-2d) minor histocompatibility antigen-mismatched model which reflects clinical and pathological symptoms of human cGVHD, we show that a single injection of an agonistic monoclonal antibody (mAb) to CD137, a member of tumor necrosis factor receptor superfamily, reverses skin fibrosis, ulceration and alopecia, ultimately improving general health conditions. The reversal is associated with markedly reduced CD4+ T cell cytokines and increased apoptosis of donor CD4+ T cells. The Fas pathway is required for the therapeutic effect of anti-CD137 mAb on cGVHD. Taken together, these data indicate that anti-CD137 mAb has a therapeutic effect on cGVHD by removing donor CD4+ T cells mediating cGVHD. Our study demonstrates an agonistic mAb specific for a costimulatory molecule as a possible target for therapeutic intervention in cGVHD.
Bovine apolipoprotein B-100 is a dominant immunogen in therapeutic cell populations cultured in FCS in mice and humans (88.29)
Authors: Sakamoto Norihisa , Tuji Kuzuhide , Muul Linda M , Lawler Ann M , Candotti Fabio , Metcalf Julia A , Tavel Jorge A , Lane H.Clifford , Urba Walter J , Fox Bernard A , Varki Ajit P , Lunney Joan K , Rosenberg Amy S
Published: January 2023

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine ES cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100 which binds to abundant low density lipoprotein receptor on the cell surface and is internalized. Critically, we have shown that in the majority of patients administered three different types of cell-based therapies utilizing cells grown in FCS containing media, an antibody response to bovine apoB-100 develops following the second infusion and is the dominant specificity. The low level of engraftment of genetically modified autologous cells in an ADA-SCID patient with a strong bovine apoB response suggests that such antibodies may influence cell engraftment.
Regulation of Airway Tolerance and Antigen-specific Regulatory T cells by the Costimulatory Molecule OX40 (88.39)
Authors: Duan Wei , Croft Michael
Published: January 2023

Respiratory exposure to soluble antigen leads to airway tolerance and antigen-specific suppression of airway inflammation. There is increasing evidence that this involves inducible regulatory T cells (Treg) that suppress pathogenic Th2 cells. What controls Treg activity is not clear. We investigated the role of the costimulatory molecule OX40 in the generation and function of Foxp3+ Treg induced by soluble OVA, given either intravenously or intranasally. By tracking adoptively transferred OVA-specific CD4 cells, we observed that markedly fewer Foxp3+ cells were induced in the absence of OX40, suggesting OX40 can control their expansion and/or survival. Also, already generated OVA-specific CD4 Foxp3+ cells when adoptively transferred into sensitized mice, suppressed lung inflammation. This suppression was impaired if the Treg lacked OX40, further demonstrating that OX40 can positively control the function of this Treg subset. In contrast, we found that an OX40 agonist antibody reversed established airway tolerance when given with antigen immunization, implying that anti-OX40 either augmented Th2 cell expansion or inhibited Treg. Thus, OX40 signals can positively affect inducible Treg but they might equally control pathogenic T cell activity. These studies further substantiate the idea that OX40 is central to the activities of several subsets of T cells that control airway inflammation.
T cell response after TCR antagonism: A unique unresponsive CD4+ T cell phenotype (88.41)
Authors: Edwards Lindsay J , Evavold Brian D
Published: January 2023

The outcomes of T cell interaction with ligands of varying potency include the induction of clonal unresponsiveness (anergy) and inhibition of T cell responses (antagonism), among others. The phenotype of peptide-induced anergy has been extensively described, and a number of groups have demonstrated antagonism. However, the phenotype of CD4+ T cells after encountering an antagonistic stimulus has not been previously characterized, despite the fact that several pathogens, including HIV, HCV and malaria, include epitopes that have been defined as T cell antagonists. We demonstrate that post-antagonism, T cells fail to proliferate. However, these cells produce large quantities of IL-2 and express CD25 levels similar to wild type stimulated cells. Our data indicate that antagonized CD4+ T cells fail to respond to IL-2 stimulation, despite the production of this cytokine, as well as expression of its receptor. This response represents a novel phenotype, distinct from all previously described forms of anergy.

This research was supported by NIAID (R01 AI056017).
Class II MHC Deficient Mice Have Contact Hypersensitivity Responses that Are Regulated by CD4+CD25+ T Cells (88.5)
Authors: Kish Danielle D. , Gorbachev Anton V , Fairchild Robert L
Published: January 2023

CD4+CD25+ T cells limit the expansion of effector CD8 T cells in skin draining lymph nodes (LN) during hapten sensitization for contact hypersensitivity (CHS) responses. This regulatory function results in decreased swelling responses to hapten challenge of the skin. Depletion of CD4 T cells prior to hapten sensitization results in increased numbers of hapten-primed effector CD8 T cells and CHS responses that are elevated in magnitude and extended in duration. These increased CHS responses are similar to those observed in sensitized and challenged class II MHC−/− mice. However, depletion of CD4 T cells in class II MHC−/− mice increased CHS responses to hapten sensitization and challenge when compared to untreated class II MHC−/− mice. ELISPOT analyses indicated increased hapten-specific CD8 T cell priming in LN of class II MHC−/− mice compared to the wild-type and these numbers increased significantly with anti-CD4 or anti-CD25 mAb treatment prior to sensitization. Flow cytometry analyses of LN cells from class II MHC−/− mice revealed a small but distinct population of CD4T cells and ≥50% expressed CD25. qRT-PCR analyses of RNA prepared from LN cells of class II MHC−/− and wild-type mice indicated the presence of FoxP3 expression that was absent when the animals were depleted of CD4 or CD25, but not NK1.1, cells. Purified CD4+ CD25+ T cells from class II MHC−/− and wild-type mice were equivalent in the ability to limit CD4+CD25− T cell proliferation in vitro. These results identify a CD4+ CD25+ regulatory T cell population in class II MHC−/− mice.
CD11c is Critical in Modulating Cytokine Production by Activated T cells (88.6)
Authors: Adams Jillian E , Barnum Scott
Published: January 2023

It is well established that CD11a (a.k.a. LFA-1) functions in T cell activation by providing a co-stimulatory signal and by participating in the formation of the immunological synapse. The role of other β2-integrins in T cell activation remains unknown. Recent EAE studies have suggested a role for CD11b and CD11c in T cell biology. These findings prompted us to simultaneously stimulate naïve T cells in vitro, using anti-CD3ε and anti-CD11c antibodies. We found that treatment of T cells with anti-CD3ε and anti-CD11c (clones N418 or HL3) antibodies substantially suppressed IFN-γ production at 72 hours. T cells treated with anti-CD3ε and anti-CD11c antibodies do not produce detectable amounts of IL-4 and IL-10 at 12, 24, and 48 hours. In these same cultures, IFN-γ production was only detected at 12h post-stimulation, but at a concentration 3 logs lower than that observed with anti-CD3ε treatment alone. Interestingly, TGF-β1 production was increased 3 fold on treatment with anti-CD11c and anti-CD3ε compared to anti-CD3ε treatment alone. The suppression of IL-4, IL-10, and IFN-γ production could not be rescued by co-stimulation with anti-CD28 antibodies. Our data suggest that CD11c may be a novel negative regulator in T cell activation.

Supported by NH46032 from the NIH.
SHPS-1 positively regulates T cell priming and cytokine production by dendritic cells (DCs). (89.15)
Authors: Tomizawa Takeshi , Kaneko Yoriaki , Okajo Jun , Okuzawa Chie , Saito Yasuyuki , Sekigami Tomomi , Kaneko Yuka , Matozaki Takashi , Nojima Yoshihisa
Published: January 2023

[Background] SHPS-1, a transmembrane protein that binds protein tyrosine phosphatases in its cytoplasmic region, is predominantly expressed on DCs, but the role of SHPS-1 signals during T cell priming by DCs is largely unknown. Here, we investigated the physiological roles of SHPS-1 in the functions of DCs by the use of SHPS-1 mutant mice, which lack most of the cytoplasmic region of this protein.

[Methods] DCs were prepared from spleens by Auto MACS. The expression of SHPS-1 or costimulatory molecules on DCs were examined by FCM. IL-12-induced IFN-γ production by DCs was determined by ELISA. The ability of DCs for antigen presentation was evaluated by the incubation of OT-II cells with OVA-loaded DCs.

[Results] FCM analysis showed that SHPS-1 was predominantily expressed on CD8α− DCs. The expression level of SHPS-1 was reduced in DCs from SHPS-1 mutant mice, but the expression levels of costimulatory molecules on DCs from SHPS-1 mutant mice were almost comparable to that on WT DCs. On the contrary, IL-12-induced production of IFN-γ was reduced in DCs from SHPS-1 mutant mice. The ability of DCs to stimulate proliferation of OVA-specific CD4+ T cells was also impaired in DCs from SHPS-1 mutant mice.

[Conclusion] SHPS-1 positively regulates DC functions including CD4+ T cell priming and cytokine production.
Cell Specific Inactivation of Multiple TLRs via Conditional Deletion of Their Master Chaperone gp96/grp94 (89.16)
Authors: Yang Yi , Li Zihai
Published: January 2023

gp96/grp94 is the endoplasmic reticulum (ER) resident protein chaperone of the HSP90 family. In contrast to its cytosolic counterpart HSP90, very few client proteins and yet no cochaperones of gp96 have been uncovered. To study the function of gp96 in vivo, we generated a gp96 conditional knockout mouse model. By crossing with a macrophage-specific Cre recombinase transgenic mouse, we successfully deleted gp96 in macrophages, but not in any other cell types, including dendritic cells (DCs). We found that the mutant macrophages failed to respond to multiple Toll-like receptor (TLR) ligands, including TLR2, TLR4, TLR5, TLR7 and TLR9. Since gp96 is indispensable for TLRs, the gp96 conditional knock-out mouse model provides a unique system to examine the contribution by TLR in inflammation and immune response in vivo. We found that macrophage-specific gp96 knockout mice were resistant to endotoxin shock, but highly susceptible to Listeria monocytogenes infection, demonstrating that macrophages but not other myeloid cells are the dominant source of proinflammatory cytokines in these conditions. Our finding is significant in that it not only confirmed a previous report that gp96 is required for cell surface expression of certain TLRs, but also for the first time revealed that gp96 is required by those TLRs that normally reside in the endocytic compartments. We conclude that the Toll-like receptor family members are delicately regulated by gp96 as their master chaperone.