bluuebunny/crossref_metadata_2025 huggingface dataset:
Towards site-directed covalent vaccination: HIV neutralization by antibodies to an electrophilic gp120 V3 peptide (47.38)
Authors: Nishiyama Yasuhiro , Mitsuda Yukie , Planque Stephanie , Hara Mariko , Taguchi Hiroaki , Salas Maria , Hanson Carl V , Paul Sudhir
Published: January 2023

We observed recently that antibodies (Abs) to an analog of full-length HIV gp120 analog containing electrophilic phosphonate groups located at Lys (E-gp120) form stable immune complexes characterized by very slow dissociation and resistance to denaturants. We hypothesize that the unusual stability is due to the partial covalent character of the complexes, mediated by enhanced Ab nucleophilic reactivity developing in response to electrophilic immunization. Here we tested the feasibility of inducing an epitope-specific nucleophilic Ab response on-demand using as immunogen the electrophilic analog of the principal neutralizing determinant of HIV gp120 (E-PND). Murine IgG Abs raised to E-PND and non-electrophilic PND displayed binding to intact HIV particles. Complexes of HIV with anti-E-PND IgG dissociated ~25-times slower than the complexes with anti-PND IgG. Anti-E-PND IgG neutralized HIV more potently than anti-PND IgG, determined using a T cell line assay (difference in IC50, ~50-fold). The slower dissociation kinetics can be expected to maintain long-lasting blockade of gp120 recognition by host receptors, resulting in superior HIV neutralization. These results suggest the utility of electrophilic immunization to enhance the protective efficacy of site-specific humoral immunity against peptide antigens.
Anti-proliferative effect of interleukin-27 on melanoma cells (48.23)
Authors: Yoshimoto Takayuki , Morishima Noriko , Mizuguchi Junichiro
Published: January 2023

Interleukin (IL)-27 is a member of the IL-6/IL-12 family and activates both signal transducer and activator of transcription (STAT)1 and STAT3 through its receptor, which consists of WSX-1 and gp130. We previously demonstrated that IL-27 has potent antitumor activities, which are mediated through CD8+ T cells, natural killer (NK) cells or its anti-angiogenic activity. Here, we demonstrate that IL-27 also possesses a direct anti-proliferative activity on melanoma cells. Although WSX-1 expression was hardly detected in parental mouse melanoma B16F10 cells, IL-27 activated STAT1 and STAT3 and up-regulated major histocompatibility complex (MHC) class I in B16F10 transfectants expressing wild-type WSX-1. In contrast, IL-27 failed to activate STAT1 and up-regulate MHC class I in those expressing mutant WSX-1, in which the putative STAT1-binding Tyr-609 of the cytoplasmic region was replaced by Phe. IL-27 greatly inhibited the cell growth of transfectants expressing wild-type WSX-1 but not the mutant, and induced apoptosis and G1 arrest. Several human melanoma cells were also reveled to express both IL-27 receptor subunits, and activation of STAT1 and STAT3 and growth inhibition by IL-27 were observed. These results suggest that IL-27 has an anti-proliferative activity on mouse and human melanoma cells through WSX-1/STAT1.
Cell-based Mechanism of Action of a TCR/IL-2 Fusion Protein against Established Tumors (48.36)
Authors: Wen Jinghai , Zhu Xiaoyun , Liu Bai , You Lijing , Lee Hyung , Han Kaiping , Sheehan John , Chavaillaz Pierre-Andre , Rhode Peter , Wong Hing
Published: January 2023

We have previously reported that a single-chain T cell receptor/IL-2 fusion protein, recognizing aa264–272 peptide of human p53, exhibits potent antitumor activities in mouse xenograft models. In this study, we compare the biological activities of 264scTCR/IL-2 with that of Mart-1scTCR/IL-2, a TCR/IL-2 fusion lacking p53 recognition. Although 264scTCR/IL-2 and Mart-1scTCR/IL-2 exhibit equivalent activities for NK cells activation, 264scTCR/IL-2 shows significantly better antitumor activity than Mart-1scTCR/IL-2 against p53+/HLA-A2+ human tumors in mouse xenograft models. We also observed a dose-dependent increase in activated NK cells infiltration into tumors following 264scTCR/IL-2 treatment and significantly more cell infiltration in tumors of 264scTCR/IL-2 treated mice than in those treated with Mart1scTCR/IL-2. The data are consistent with our hypothesis that 264scTCR/IL-2 activates immune cells, such as NK cells, to express high-affinity IL-2 receptors. Following stable interactions between the IL-2 domain and these receptors, the scTCR domain of the fusion molecule guides the immune cells to tumors displaying target peptide/HLA complexes where they mediate antitumor effects.

NIH-2R44CA097550
Inhibition of metastatic B16-F10 melanoma tumor growth by murine dialyzable spleen extracts (DSE) (48.39)
Authors: PEREZ-TAPIA SONIA MAYRA , MARTINEZ/RUIZ GUSTAVO , SALINAS NOEMI , ESTRADA-GARCIA IRIS CITLALLI , ESTRADA-PARRA SERGIO ANTONIO , VELASCO-VELAZQUEZ MARCO ANTONIO
Published: January 2023

Surgery, radiotherapy and chemotherapy are the most widely used and well-established modalities for treating malignant diseases. However, all modalities have major drawbacks. An alternative approach is to expand and activate tumour-specific immune cells in vivo. Dialyzable spleen extracts (DSE) are obtained from freeze-thawed murine spleen cell suspensions and subsequent dialysis (<12 KDa), they have immunomodulatory activity and have shown to cure experimental murine tuberculosis. In this work a BALB/c DSE was used to examine in mice its effectiveness as a prophylactic anti-metastatic therapy, using the mouse B16-F10 melanoma model. 1 Unit DSE (equiv. 1x105 spleen cells) or 0.1 U, were orally given daily for 7 days to 6 and 4 mice respectively. A third group of mice (N=6) was treated with placebo. On day 8, all mice were bleed and each animal inoculated with 1x106 B16-F10 cells. On day 28 all animals were sacrificed, lungs were dissected and macroscopic metastatic nodules counted. An statistically decrease in metastatic nodules (P¡Ü0.01) was observed in both groups that received DSE when compared with the control group. These results indicate that DSE is a promising approach for inhibiting metastatic tumor growth. Further work will focus on the mechanism which elicit this antimetastatic activity.

This work was supported in part by SIP/IPN. 1Are fellows of COFAA, EDI and SNI.
CDC2-related kinase and metabolic regulation in B cell malignancies (49.19)
Authors: Saltis Mark , Davidson Wendy
Published: January 2023

Microarray and qPCR analyses revealed that pre-transformed murine B cells and a variety of human and murine B cell lymphomas constitutively express high levels of Pftk1, a CDC2-related kinase. Expression of Pftk1 also was induced in normal b cells following activation. These findings suggest that Pftk1 is associated with B cell activation and that dysregulation of expression may be an early event in B cell transformation. While the function of Pftk1 in B cells is unknown, published studies on adipocytes suggest that it may have a regulatory role in glycolysis. Concurrent with the increased expression of Pftk1 in B cell tumors, there is a 2–6 fold increase in the levels of transcripts for genes encoding the glycolytic pathway enzymes, including Hk2, Pgam1, Ldh1, and Pkm2. Currently, we are using a variety of molecular approaches to further investigate the relationship between enhanced Pftk1 expression and increased glycolysis in malignant B cells and to determine whether Pftk1 functions as an oncogene.

This work was supported by Arthritis Foundation Chapter Research Grant and NIH grant CA82872-02 awarded to W.F.D.
Vitamin C induces apoptosis in human colon cancer cells through endoplasmic reticulum stress and mitochondrial pathway (49.25)
Authors: Kim Jee Eun , Kang Jae Seung , Jung Da Jung , Hahm Eunsil , Lee Seung Koo , Bae Se Yeon , Hwang Young Il , Shin Dong Hoon , Lee Wang Jae
Published: January 2023

It has been reported that vitamin C plays an effective role in the treatment and prevention of cancer, but its specific mechanism is still not fully understood. In particular, the effect of vitamin C on the treatment and prevention of colorectal cancer, which has been increasingly a common disease in South Korea due to westernized diet of many Koreans, is still largely unknown. Therefore, we have examined the effects of vitamin C on the induction of apoptosis on HCT-8, human colon cancer cell line. We have first confirmed that the optimal concentrations of vitamin C for the induction of apoptosis on human colon cancer cell line, HCT-8 is 2 mM of vitamin C. Here we demonstrated that vitamin C induced ER stress through redox imbalance as antioxidant and, increased cytosol calcium level. Then, we investigated the changes of ER-stress induced molecules. As a result, the phosphorylation of pro-apoptotic protein, Bad decreased and Bad, which was subsequently released from 14-3-3, was translocated to the mitochondria. In addition, we also found that the vitamin C increased expression of pro-apoptotic protein, Bax and tumor suppressor gene product, p53, which usually resulted in apoptosis, in a time dependent manner. Moreover, we have witnessed that vitamin C treatment disrupts mitochondrial membrane potential in HCT-8, human colon cancer cell line. Taken together, vitamin C induced redox imbalance, resulting in apoptosis in human colon cancer cells by both endoplasmic reticulum stress and Bad/p53/Bax-mediated activation of mitochondrial pathway.
TRP-2 TCR Transgenic Mice as a Model to Study CD8+ T cell Responses to Melanoma Antigens (50.15)
Authors: Singh Vinod , Ji Qingyong , Hurwitz Arthur A.
Published: January 2023

To study the effects of T cell avidity on tumor immunity and autoimmunity, we have generated mice that bear T cell receptor (TcR) transgenes specific for a peptide from tyrosinase-related protein 2 (TRP-2), a melanosomal enzyme expressed by both normal melanocytes and melanoma cells. We have begun characterizing a founder mouse line derived from an intermediate avidity T cell line, designated clone 37. Interestingly, the clone 37 TcR transgenic (Tg) mice did not develop autoimmune depigmentation despite the presence of TRP-2-reactive T cells in the lymph nodes and systemic expression of TRP-2. These Tg T cells displayed a naïve phenotype (CD44low, CD62hi, CD69neg, and CD25neg) and proliferated upon stimulation in vitro with cognate antigen. No difference in T cell responsiveness or frequency was observed in the development of Clone 37 T cells following bone marrow reconstitution of wild type C57Bl/6 or TRP-2−/− mice. Adoptive transfer of clone 37 TCR Tg cells into C57BL/6 showed that these T cells do not encounter the endogenous TRP-2 antigen as well as a subcutaneous B16/BL6 melanoma tumor (i.e., they are “ignorant”). However, vaccination with TRP-2 peptide-pulsed dendritic cells induced proliferation of these adoptively transferred CD8+ T cells and subsequent migration into subcutaneous B16/BL6 tumors, but did not inhibit progressive tumor growth. Similarly, B16/BL6 tumors grew progressively in clone 37 TCR Tg mice despite the large number of tumor-associated antigen-specific CD8+ T cells. These findings demonstrate the ability of tumors to evade an anti-tumor immune response despite the presence of tumor-specific T cells. Our on-going studies will characterize two additional mouse lines bearing TcR transgenes from high and low avidity T cell clones and study the impact of different T cell avidities on tumor immunity and autoimmunity.
Role of Type I and Type II NKT cells in the host’s innate antitumor immune response to a B-cell lymphoma (50.26)
Authors: Gourapura Renukaradhya Jayamurthy , Khan Masood A , Vieira Marcus , Brutkiewicz Randy R
Published: January 2023

Natural killer T (NKT) cells are a T cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant TCR [alpha] chain rearrangement and are called Type I NKT cells; all other NKT cells are Type II. In the current study, we have analyzed the roles for NKT cell subsets in the host’s innate antitumor response against the murine NS0 B-cell lymphoma model in vivo. As the NS0 cell line lacks detectable cell surface CD1d expression, murine CD1d1 cDNA or empty vector was transfected into NS0 cells to generate CD1d+ and control cell lines. In tumor-bearing mice, we found that Type I NKT cells conferred protection in a CD1d-dependent manner, whereas Type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice were predictive of tumor progression. Myeloid suppressor cells (CD11b+Gr1+) were present in larger numbers at the tumor site and in the spleen of tumor-bearing Type I NKT-deficient mice, suggesting a possible inhibition of antitumor immunosurveillance mediated by these cells. Therefore, there are distinct roles for NKT cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in the immunotherapy of blood cancers.

This study was supported by NIH grants RO1 CA89029 and AI46455 to RRB.[alpha]
Regulatory T cells (Tregs) contribute to suppression of anti-malarial immunity by concurrent nematode infection (51.1)
Authors: Stevenson Mary M. , Su Zhong , Segura Mariela
Published: January 2023

Here, we investigated the mechanisms involved in modulation of anti-malarial immunity by concurrent nematode infection. Both WT C57BL/6 (B6) and STAT-6 deficient (KO) mice co-infected with the gastrointestinal nematode Heligmosomoides polygyrus (Hp) 2 wk prior to blood-stage Plasmodium chabaudi AS (Pc) infection had significantly higher parasitemias and significantly lower serum IFN-γ levels compared to their respective controls infected with Pc alone. Hp-infected STAT-6 KO compared to WT mice produced significantly lower levels of Th2 cytokines (IL-4, IL-13) but Tregs (CD4+CD25+Foxp3− and CD4+CD25−Foxp3+) were significantly and similarly increased within 2 wk after Hp infection in both strains. Transfer of CD4+CD25+ Tregs from uninfected and Hp-infected B6 mice significantly increased malaria parasitemia in recipient mice while depletion of CD25+ cells suppressed parasitemia in Pc-infected mice but not in co-infected mice. FACS analysis revealed that anti-CD25 mAb (PC61) treatment reduced both CD4+CD25+ Tregs and CD4+Foxp3+ Tregs in Pc-infected but not in co-infected mice. Co-infection with Hp significantly enhanced Pc-induced TGF-β1 and IL-10 production. In vivo neutralization of TGF-β1 and blocking IL-10R with mAbs significantly reduced parasitemia in both Pc-infected and co-infected B6 mice; anti-IL-10R mAb treatment resulted in severe mortality. These results suggest that Tregs, and possibly immunoregulatory but not Th2 cytokines, contribute to suppression of anti-malarial immunity in nematode and malaria co-infected mice.
FcγRIII mediates IgG-induced IL-10 and is required for chronic Leishmania lesions (51.2)
Authors: Buxbaum Laurence U. , Thomas Bolaji N.
Published: January 2023

Chronic disease caused by the protozoan parasite Leishmania mexicana, requires IL-10 and the common γ chain (FcRγ) shared by the activating IgG receptors FcγRI, III, and IV and by FcαR and FcεR. FcRγ can also take the place of CD3ζ in some T cells. Which pathway is responsible for IL-10 production and suppression of a protective immune response is unknown. We recently showed that FcγRIII KO mice, like IL-10 and FcRγ KO mice, resolve L. mexicana lesions with a stronger Th1 response and much lower parasite burdens (by 4–5 logs) than WT mice. We now show that FcγRIII KO macrophages have a severe IL-10 defect, when induced by IgG-opsonized parasites, and that this defect is comparable to FcRγ KO macrophages. Thus FcγRIII appears to contribute most of the IgG-induced IL-10, supporting the in vivo findings. We also found that anti-Leishmania IgG responses (IgG1 and IgG2a) were not altered in FcγRIII KO mice compared to WT mice, consistent with the model that parasite-bound IgG alters T cell development and thus may determine the strength of the Th1 response rather than the reverse. Furthermore, depletion of CD25+ T cells did not change the chronic nature of L. mexicana lesions, nor was T cell IL-10 production from FcγRIII KO mice lower than from B6 mice. When taken together these findings support a model in which macrophage FcγRIII, rather than Treg cells, provides the IL-10 that suppresses a protective immune response to L. mexicana.
Macrophages produce a soluble mediator which enables neutrophils to kill the parasitic nematode Strongyloides stercoralis (51.4)
Authors: O’Connell Amy E , Abraham David
Published: January 2023

Neutrophil recruitment to the parasite microenvironment via CXCR2 is required for killing of Strongyloides stercoralis infective larvae during the primary and secondary immune responses. Neutrophils isolated from other cell types within diffusion chambers are capable of killing L3 in vivo, however, isolated neutrophils are unable to kill larvae in vitro. Macrophages have been shown to be required for in vivo killing of other nematode species. We hypothesized that neutrophils require soluble mediators that can be supplied by macrophages in vivo, explaining their inability to kill in isolation in vitro. Combining neutrophils with macrophages at a 4:1 ratio in vitro led to a robust larvicidal response, while neither cell type was able to facilitate larval killing independently. The killing response occurred whether naïve or immune mouse serum was used, which suggests that neutrophil-macrophage cooperation is a component of the primary immune response. Furthermore, the killing process was complement-dependent, as killing was ablated when serum from C3−/− mice was used. Combining cell free supernatant from cultured macrophages with neutrophils also led to larval killing, indicating that the cooperation between neutrophils and macrophages is dependent on a soluble factor from macrophages inducing larval killing by neutrophils.
Defensin α-1 expression is up-regulated in human cells in response to early Trypanosoma cruzi infection as an innate immune mechanism to decrease cellular infection via membrane pore formation leading to apoptosis (51.6)
Authors: Madison Marisa Nia , Kleshchenko Yuliya , Nde Pius , Simmons Kaneatra , Lima Maria F , Villalta Fernando
Published: January 2023

Defensins play a fundamental role in the initiation of immune responses to selected human pathogens. Here, we show that early cellular infection by T. cruzi up-regulates the expression and secretion of defensin α-1, which displayed a trypanocidal role mediated by pore-formation resulting in apoptosis of the parasite. Analysis of the whole human epithelial cell transcriptome upon short infection of cells by T. cruzi indicates that the parasite up-regulates the levels of transcripts of defensin α-1. Defensin α-1, at concentrations not toxic for host cells, significantly reduced the viability of trypomastigote forms of T. cruzi. Defensin α-1 integrates into the trypanosome membrane to cause pore formation, cellular and mitochondrial membrane depolarization, membrane disorganization and blebbing, DNA fragmentation, and cytoplasmic vacuolization leading to damage and death. Pre-incubation of trypomastigotes with exogenous defensin α-1 followed by exposure to human epithelial cells significantly reduced trypanosome infection. Membrane depolarization of trypanosomes abolished the trypanocidal activity of defensin α-1, indicating that the mechanism of defensin α-1 mediated trypanosome killing is membrane-voltage dependent. We conclude that defensin α-1 gene expression and peptide secretion is an effective host innate immune response to control T. cruzi infection.

(Supported by NIH grants)
Heat shock protein gp96 is a critical chaperone for Toll-like receptor 9 (52.2)
Authors: Liu Bei , Yang Yi , Li Zihai
Published: January 2023

Toll-like receptors (TLR) are important pattern-recognition receptors and play significant roles in both innate and adaptive immunity. As a ubiquitous ER luminal protein, gp96 has been previously implicated in the folding and surface expression of TLR1, TLR2 and TLR4. However, whether gp96 chaperones other TLRs, especially intracellular TLR9, is unknown. We herein addressed this question using a genetic approach. We found that gp96 mutant cells, via insertional mutagenesis or homologous recombination, were unresponsive to TLR9 ligand, which was completely rescued by restoration of gp96. Furthermore, we demonstrated the direct interaction between gp96 and TLR9; such an interaction is dependent on N-linked glycosylation and normal ER calcium store. These findings illustrate that gp96 is an essential chaperone for the folding of TLR9, and provide a new potential target to regulate TLR function in the treatment of a variety of diseases.
Complement C5 regulates insulin-like growth factors binding proteins in chronic Experimental Autoimmune Encephalomyelitis (EAE) (53.8)
Authors: Cudrici Cornelia Diana , Zafranskaia Ekaterina , Weerth Susanna , Chen Hegang , Gherman Adrian , Ito Takahiro , Rus Violeta , Raine Cedric , Rus Horea
Published: January 2023

Complement activation plays a central role in autoimmune demyelination. We have analyzed the role of complement C5 in EAE using C5-deficient (C5-d) and C5-sufficient (C5-s) mice and found that the absence of C5 resulted in fiber loss and extensive scarring. In this study, we investigated thetranscriptional profile induced by C5 in chronic EAE using oligonucleotide array.During chronic EAE (day 90, post-immunization), we found that 462 genes were differentially regulated in C5-s when compared with C5-d mice. The majority of these genes were related to cell growth and signal transduction; agroup of genes belonging to the insulin-like growth factors binding proteins family was also significantly regulated by C5. This category include insulin like growth factors (IGFBP) 3, 4 and 6, connective tissue growth factor (CTGF), WNT1 inducible signaling pathway protein 2, cysteine rich protein 61 and endothelial cell specific molecule-1. Using Real-time PCR we confirmed the down-regulation of these genes in C5-d mice in chronic EAE. When IGFBP3 and CTGF levels in chronic EAE were compared with those in control mice (day 0, post-immunization), significantly lower levels of mRNA expression were found only in C5-d mice. The down-regulation of genes belonging to the insulin-like growth factor binding family suggests that they might be responsible for the axonal loss and gliosis seen in C5-d during chronic EAE.
Cellular interactions during GALT development (81.10)
Authors: Yeramilli Venkata , Knight Katherine L.
Published: January 2023

In rabbits, B cells develop in the bone marrow and migrate to the GALT where they proliferate, form organized follicles, and develop the primary antibody repertoire by Ig gene diversification. Our goal is to determine the mechanism by which these processes are induced in GALT. We hypothesize that B cells in GALT are polyclonally stimulated in an antigen and T cell independent manner. To test this idea, we injected newborn rabbits with adenoviruses (Ad) expressing soluble receptors, CTLA4Ig and CD40Ig, to inhibit activation of T cells and T-B cell interactions, respectively. We found that GALT developed normally in rabbits injected with AdCTLA4Ig, as evidenced by the presence of follicles with proliferating B cells. In contrast, we found no follicles with proliferating B cells in rabbits injected with AdCD40Ig. These results indicate that activated T cells are not required for GALT development, but that CD40:CD40L interactions are required. Using a similar approach, we found that B cells require signaling through the BAFF receptor and complement receptor 2 (CR2) for proliferation in GALT. The source of CD40L, BAFF and CR2 ligand, molecules required for B cell activation in GALT, are under investigation.

Supported by NIH AI50260
Functional deficiency in IL-7 caused by an ENU-induced point mutation (82.11)
Authors: Wang Hongsheng , Feng Jianxun , Morse Herbert C.
Published: January 2023

N-ethyl-N-nitrosourea (ENU)-induced mutagenesis provides a powerful approach to identifying genes involved in immune regulation and diseases. Here we describe a new mutant strain, HLB368, with hereditary leukopenia. At necropsy, the mutant mice had very small thymuses and spleens. All but the inguinal nodes were absent and there were no Peyer’s patches. By flow cytometry, the ratios of T cell subsets were normal, but B cell development was blocked at the pre-pro-B cell stage. The development of B1 and marginal zone B cells was relatively normal. The mutation was mapped to chromosome 3 between D3Mit221 and D3Mit224, a region that contains the Il7 gene. cDNA and genomic DNA sequences of Il7 revealed a T-to-C missense transition resulting in a change of Leu to Pro within the leader peptide that would be predicted to inhibit secretion. In keeping with this concept, we found that in vitro treatment of B cell progenitors from mutant mice with IL-7 induced them to differentiate into pre-BII cells. Phenotypic comparisons of HLB368 with genetically targeted Il7 null mice showed many similarities along with a few differences, indicating that this ENU-induced mutant carries a novel allele. This new strain thus provides a new model for studying the functions of IL-7 on a pure C57BL/6 background.
Exposure to bead immobilized antigen dramatically increases the survival and expansion of anti-CD40 stimulated HEL transgenic B cells. (83.9)
Authors: Marran-Nichol Kathleen
Published: January 2023

Since B cells primarily respond to conformational determinants produced by the three-dimensional assembly of proteins, the assumption has remained that B cells respond mainly to soluble antigens. Recently data clearly show that dendritic cells derived from inflammatory sites exhibit the capability of presenting non-processed and cell surface associated antigens to resting B cells. We show that HEL B cells stimulated with bead associated HEL plus anti-CD40 proliferate and survive to a much greater extant than similar B cells stimulated in culture with soluble HEL plus anti-CD40. ACS analysis of phenotypic characteristics and CSFE staining patterns demonstrate that although similar numbers of B cells initially enter cell cycle in both experimental groups, HEL B cells stimulated by immobilized antigen plus anti-CD40 are found to complete up to twice as many division cycles. In addition, a subset of these B cells appears to enter the plasmocyte differentiation pathway in the absence of exogenous cytokine supplementation. These findings indicate that presentation of bound antigen to resting HEL B cells impacts on the ability of the B cells to effectively respond to T helper cell-derived signals and support the possibility that direct physical presentation of intact antigen by APC emigrating from inflammatory sites represents an additional means for communication between the innate and adaptive immune systems.
Thymopoiesis is Enhanced by Inhibition of gp130 Signaling in Young and Aged Mice (85.15)
Authors: Kendall Shellie Marie , Ventevogel Melissa S. , Sempowski Gregory D.
Published: January 2023

Normal aging is characterized by attenuation of cellular immune function and progressive decline in ability to respond to pathogens. Understanding mechanisms of immunosenescence with aging will enable development of targeted therapies for enhancing immune response. We previously reported that both human and mouse thymus aging is characterized by increased expression of IL-6 family cytokines. Further we demonstrated that these cytokines are directly suppressive and involute the thymus. IL-6 family cytokines activate the gp130 cell surface receptor on thymocytes, thus we hypothesized inhibition of gp130 receptor signaling will stimulate thymopoiesis in aged mice. To test this hypothesis we infused aged BALB/c mice (20 mo) or young mice (2 mo) with neutralizing gp130 Ab and monitored thymus function over 8 weeks. Inhibition of gp130 signaling significantly elevated thymus weight and TCR gene rearrangement in the aged thymus. In young mice we also observed with gp130 inhibition significant increases in thymus weight, cellularity, TCR gene rearrangement, and naïve export to the periphery. Overall, these data demonstrate release of thymosuppressive cytokine signaling stimulates thymopoiesis by both young and aged thymus tissue. Furthermore, gp130 signaling is an important thymoregulatory pathway to explore in the pursuit of human T cell immune reconstitution therapies.

Supported by NIH HL67314, AG025150.
Aged mice exhibit increased frequencies of CD4+ T cells with a regulatory phenotype (85.20)
Authors: Maue Alexander C , Eaton Sheri , Haynes Laura
Published: January 2023

In vivo, age-related defects in CD4+ T cell function lead to reduced expansion, IL-2 production, NF-κB activation and B cell helper activity. These defects are significant as they result in reduced vaccine efficacy in the elderly. To date, relatively little is known about what factors contribute to these age-related defects in vivo. By using real time-PCR (RT-PCR) and intracellular flow cytometry, we have identified candidate cell subsets and molecules that may contribute to the observed immunosuppression. Spleens of young (4 months) and aged (24 months) mice displayed significantly higher expression of interleukin-10 (IL-10) as determined by RT-PCR. Analysis of cell subsets isolated by sorting revealed a significant increase in IL-10 expression in aged CD4+ T cells compared to young. Following intracellular cytokine staining, a statistically significant increase in CD4+IL-10+ cells (~15 fold) was observed in aged (26 months) compared to young (2 months) mice. Aged CD4+ T cells exhibited significantly higher expression of CD25 (~3 fold), GITR (~5 fold) and CTLA-4 (~4 fold) compared to young CD4+ T cells, as well as greater overall numbers of cells expressing each marker. Following intracellular staining, both intact and transgenic CD4+ T cell populations in aged mice displayed greater numbers of Foxp3-expressing cells compared to young populations. Our results demonstrate that in aged mice, several molecules and cells associated with immune regulation are present in significantly higher amounts than in young mice and that future experiments are required to determine the effects of these elements on naïve CD4+ T cell and aged immune responses.

This research was supported by NIH grants AG02160 and AG21054.
Inflammation regulates memory CD8 T cell development (85.3)
Authors: Cui Weiguo , Joshi Nikhil , Kaech Susan
Published: January 2023

During viral or bacterial infection, the distinct effects of inflammation and antigenic stimulation on effector and memory cell differentiation in CD8 T cells remain to be better defined. Our recent findings show that two subsets of effector CD8 T cells that have distinct cell fates are formed during acute infection -- the KLRG1hi IL-7Rlo short-lived effector cells (SLECs) and the KLRG1lo IL-7Rhi memory precursor cells (MPCs). SLECs will undergo apoptosis after infection subsides whereas MPCs survive and become memory CD8 T cells. Here we show that inflammation plays an important role in generating SLECs during infection. Specifically, we found that priming CD8 T cells in the absence of overt inflammation using peptide-coated dendritic cells (DCs) induced primarily MPCs, but SLECs did not form. However, if CD8 T cell activation occurred in the presence of CpG then SLECs formed, and this was TLR9-dependent. In addition, the two signals (antigen + inflammation) needed to act in concert to induce formation of the SLECs. Next, we found that IL-12 alone, but not IFNγ, was sufficient to induce SLECs. However, IFNγ appears to be permissive in this process since IFN γR−/− CD8 T cells failed to form SLECs with peptide-coated DCs + CpG. Together, these results provide new insight into how inflammatory signals control effector cell differentiation and longevity and thus, provides a novel link between inflammation and T cell homeostasis.
Impaired Peripheral B cell Development and Function in RasGRP3-deficient Mice (86.17)
Authors: Hoek Kristen L , Carlesso Gianluca , Castro Iris , Rapoport Rebecca , Bautch Victoria L , Khan Wasif N
Published: January 2023

Signaling through the B cell receptor (BCR) is critical for peripheral B cell maturation. We have shown that diacylglycerol (DAG) is preferentially produced in late (T2) compared to early (T1) transitional immature splenic B cells upon BCR engagement. Two major downstream effectors of DAG in B cells are PKCs and RasGRP3. BCR-mediated activation of RasGRP3 leads to activation of the Ras/ERK pathway, which is important for B cell proliferation and differentiation. To determine the role of RasGRP3 in B cell development, we analyzed the B cell compartment of RasGRP3-deficient mice obtained by gene trap (Mol Cell Biol 24:101515–28). Here we show that loss of RasGRP3 results in a decrease in splenic B cells compared to wt controls (25.6±16.6 vs 69.3±23.1, mean B cell number (x106) ± SD), with a block in peripheral B cell development between the T2 and mature follicular (FoB) stages. Additionally, peritoneal B2 B cells are severely reduced in RasGRP3−/− animals. However, development of marginal zone (MZ) and B1 B cells appears unaffected. In agreement with previous reports, we found that RasGRP3−/− B cells are impaired in BCR-mediated functions in vitro; B cell proliferation, induction of activation markers, and upregulation/maintenance of survival proteins are severely impaired in response to anti-IgM stimulation. These results reveal that RasGRP3 is necessary for BCR-mediated B cell differentiation/survival.
Zinc functions as ionic signaling molecule in primary T cell responses (87.30)
Authors: Lee Won-Woo , Pryshchep Sergey , Cui Dapeng , Weyand Cornelia M. , Goronzy Jorg J.
Published: January 2023

Zn is an important catalytic cofactor for enzymes and transcription factors, however, it is unknown whether these mechanisms account for the compromised immune competence with Zn deficiency or immune-enhancing function of Zn. To examine the effect of Zn on T cell priming, we stimulated naive CFSE-labeled CD4 T cells with DCs and the superantigen TSST. The system allows for separately examining high- (Vß2+) and low-avidity (Vß2-) T-cell responses. External Zn2+ concentrations inversely correlated with the activation threshold of low-avidity T-cells, pointing to an early signaling event. FACS showed that external Zn concentrations only influenced cytoplasmic Zn in activated and not in resting cells. Confocal microscopy was used to monitor Zn fluxes in FluoZin3-labeled T cells after activation with TSST-loaded DC. TCR synapse formation was visualized with anti-CD3. Zn2+ accumulated in the subsynaptic region of the T-cell activation platform within 5 min after activation, likely due to relocation and activation of zinc importers. The local Zn accumulation correlated with external Zn2+ concentration, peaked early, and started to decline again within ten min. Free Zn2+ did not show the global cytoplasmic spreading seen with Ca2+. Our data suggest that Zn functions as an ionic signal. TCR-induced Zn influx may modulate early signaling events which increases sensitivity of T-cell responses to subthreshold stimuli.
Galectin-3 translocates to the immunological synapse and inhibits cytokine production in CD4+ T cells activated by engagement of TCR (88.12)
Authors: Chen Huan-Yuan , Weng I-Chun , Maverakis Emanual , Yang Ri-Yao , Hsu Daniel K , Liu Fu-Tong
Published: January 2023

Galectin-3 is induced in CD4+ T cells upon activation by mitogens or TCR engagement, but its function in this cell type has not been determined. We have compared the cytokine responses of gal3−/− and gal3+/+ CD4+ T cells upon activation induced by TCR engagement and found that gal3−/− CD4+ T cells secreted more IFNγ and IL-4 compared to gal3+/+ cells. We also found that serum levels of IFNγ were significantly higher in gal3−/− than gal3+/+ mice after exposure to Staphylococcus enterotoxin B. In addition, gal3−/− CD4+ T cells exhibited higher levels of tyrosine-phosphorylated proteins, as well as enhanced phosphorylation of early signaling molecules, including LAT and Zap70, upon anti-CD3 stimulation. These results suggest that endogenous galectin-3 suppresses T cell responses. Immunofluorescence analysis revealed that galectin-3 was recruited to the immunological synapse in CD4+ T cells after TCR engagement. Our preliminary data suggest that galectin-3 expression in T cells correlates with an enhanced down-regulation of TCR when the cells are activated by engagement of the receptor, in a manner similar to that described for other intracellular proteins that negatively regulate T cell activation. We conclude that galectin-3 is an inhibitory regulator of T cell activation that down-regulates signal transduction, and functions by recruitment to the immunological synapse following activation by TCR engagement.
CD4+ T cells promoting humoral responses in mice (88.22)
Authors: Eddahri Fouad , Denanglaire Sébastien , Leo Oberdan , Andris Fabienne
Published: January 2023

Following antigen inoculation in naïve transgenic mice, we identified an antigen-specific CD4+ T cell subset expressing high level of both CD62L and CD44 that differ from the well characterized inflammatory cytokine producer CD62Llo CD44hi T cells. Antigen-primed CD62LhiCD44hi T cells induced extensive antibody secretion in B cell cooperation tests despite defective IL-4 and IFN-γ production, while CD62Llo CD44hi T cells secreted high amount of IL-4 and IFN-γ but did not induce optimal IgG secretion by cocultured B cells, suggesting that T cell-dependent antibody secretion by B lymphocytes (Th-B function) could be dissociated from elevated Th1 and Th2 cytokine production.

We therefore studied the stimulation requirements leading to humoral and inflammatory immune responses following a dendritic cell-based immunisation protocol. The data obtained suggested that injection of antigen-pulsed mature DC primed naïve T cells for IFN-γ secretion in vivo whereas injection of antigen-pulsed immature DC did not. However, immature DC were fully capable of promoting antigen specific antibody secretion, suggesting that activation protocols described as “suboptimal” for Th cell cytokine secretion induced optimal Th-B capacities.

Together, these studies should help in the development of novel vaccination strategies inducing optimal antibody responses while minimizing local inflammatory reactions.
FOXP3 is a homo-oligomer, and it interactions with histone acetyltransferase and class II histone deacetylases are required for repression (88.30)
Authors: Li Bin , Samanta Arabinda , Song Xiaomin , Iacono Kathryn T , Brennan Patrick , Riley James , Chatila Talal A , Banham Alison , Shen Yuan , Saouaf Sandra J , Greene Mark I
Published: January 2023

We have found that FOXP3 is an oligomeric component of a large supramolecular complex. The Zinc-Leuzip region of FOXP3 is sufficient to mediate its homotetramerization. Wild type FOXP3 directly binds to the human IL-2 promoter, but the E251 deletion in FOXP3 in XLAAD/IPEX patient’s T cells disrupts its homo-oligomerization, heteromerization with FOXP1, its association with the IL-2 promoter, and limits repression of IL-2 transcription after T cell activation. Additionally we report that transcriptional repression by FOXP3 involves a histone acetylase-deacetylase complex that includes TIP60 and HDAC7 and HDAC9. FOXP3 can be acetylated, and TIP60 promotes FOXP3 acetylation in vivo. Knockdown of endogenous TIP60 relieved FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation, and can be restored by the Trichostatin A indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a novel complex based mechanism by which FOXP3 actively mediates transcriptional repression.
Immune modulation through silencing CD80 and CD86 in dendritic cells using siRNA (88.4)
Authors: Suzuki Motohiko , Zhang Xusheng , Zheng Xiufen , Vladau Costin , Li Mu , Chen Dong , Gracia Bertha , Min Wei-Ping
Published: January 2023

Dendritic cells (DC) are central cells of the immune system that can stimulate or suppress immune responses, partially depending on their expression of costimulatory molecules. CD80 and CD86 are critical costimulatory molecules for both T cell responses and Th differentiation. Thus, silencing DC expression of CD80 and CD86, using siRNA, could potentially enable manipulation of immune responses. Since silencing of CD80 or CD86 in DC has not been reported, in this study we investigated the tolerogenesis and Th differentiation capacity of CD80/CD86 in DC after siRNA-mediated gene silencing. Gene silencing was performed by transfecting mouse bone marrow-derived DC with CD80 or CD86 siRNA. The gene silencing efficacy was measured by flow cytometry, RT-PCR, and real time PCR. DC that were transfected with CD80 or CD86 siRNA induced inhibition of T cell responses in MLR and generated T regulatory cells. The CD4+CD25+FoxP+ cell subset was significantly increased in a co-culture system that contained CD80- or CD86-silenced DC and allogeneic T cells. Furthermore, CD80 siRNA significantly decreased IFN-gamma, although a reduction in IL-4 was not observed. Conversely, CD86 siRNA significantly reduced IL-4 but did not reduce IFN-gamma. This study suggested, for the first time, that CD80 siRNA and CD86 siRNA can be useful tools for generating tolerogenic DC with a differential effect on modulating cytokine production.
The Importance of IL-2 for FOXP3 expression (88.8)
Authors: Popmihajlov Zoran , Smith Kendall A
Published: January 2023

To examine the role of TCR-induced Interleukin-2 (IL2) in TCR-regulated FOXP3 expression, PBMC from normals were stimulated in vitro with anti-CD3 monoclonal antibody (OKT3) +/− anti-CD25 or anti-IL2, and/or human rIL-2. 4-color flow cytometry was used for the expression of FOXP3. OKT3 increased FOXP3 expression in both CD4+ and CD8+ T cells. Peak occurred at 24h (CD4+ 13.63% ± 0.45 (SEM)) vs. (CD8+ 5.62% ± 0.27 (SEM) (n=26), & by 96h was back to baseline levels (CD4+ 1.82% ± 0.24 (SEM) vs. CD8+ 0.13% ± 0.05 (SEM))(n=18). Both anti-CD25 and anti-IL2 inhibited FOXP3 expression, to a maximal suppression of ~ 60% in both CD4+ and CD8+ T cells. Human rIL-2 restored and prolonged FOXP3 expression. By comparison, anti CD28/CD3 stimulation +/− rIL2 of murine T cells does not activate FOXP3 expression by CD4+ cells and CD8+ cells do not express FOXP3 +/− stimulation. T cells from IL2 KO mice expressed ~ 50% FOXP3 vs. WT mice. Therefore, TCR-activated FOXP3 expression by mature human T cells is regulated in part by IL-2 signaling, and only occurs transiently in a subset of both CD4+ and CD8+ cells. In contrast, murine T cell FOXP3 expression cannot be activated via the TCR and IL2 deficiency reduces, but does not fully abrogate FOXP3 expression.

Supported by NIAID grant # RO1-AI-51181 and the Belfer Foundation.
Identification of a Non-Receptor Form of OSCAR Gene (89.39)
Authors: Khan Emad A , Cui Cai-Bin , Aukhil Ikramuddin , Flood Patrick M
Published: January 2023

Since its discovery, osteoclast-associated receptor (OSCAR) gene has been studied as a receptor protein. Our research is the first to identify and characterize a non-receptor form of OSCAR. While searching for proteins differentially expressed during bone remodeling, a clone of a non-receptor form of OSCAR gene (3D10) was isolated using representational difference analysis. 3D10 was found to be one of a non-receptor group of OSCAR. This group was found to have a non-spliced intron resulting in larger transcripts and proteins lacking a transmembrane domain. Comparisons were made in tissue distribution between the non-receptor and the membrane receptor forms of OSCAR using specific PCR primers and a rabbit polyclonal antibody that we synthesized. The non-receptor forms were found to be expressed in the lung, pancreas, spleen, kidneys and bone marrow, as well as in multinucleated cells and peripheral blood leukocytes. To determine potential binding partners, yeast two-hybrid screening was performed, which showed several potential binding proteins with multiple hits like URP2, PFAAP5 and BAT3. Interestingly, while 3D10 does not have a nuclear localization signal, two of its binding partners were nuclear proteins one of which is an apoptotic regulator (BAT3). 3D10 and some of its binding partners were studied using transfected cell lines with various constructs. Transfection studies using luciferase reporter plasmid for NF-κB binding sites and HEK 293 cells suggest that 3D10 down-regulates NF-κB-dependant inflammatory responses. In conclusion, 3D10 represents a novel non-receptor form of OSCAR which may be involved in the regulation of NF-κB-mediated inflammatory responses and apoptosis regulation.
Alternative Signaling of TH17 Cell Development by Sphingosine 1-Phosphate (S1P) (90.2)
Authors: Goetzl Edward J , Liao Jia-Jun , Huang Mei-Chuan
Published: January 2023

S1P from diverse blood and lymphoid cells signals T cell migration and other functions through its type 1 G protein-coupled receptor (S1P1). Our recent observation of increased levels of Th17 cells in mice with T cell-selective overexpression of transgenic S1P1 prompted studies of mechanisms of stimulation of Th17 cell development by the S1P- S1P1 axis. Incubation of C57BL\6 mouse splenic CD4 T cells on adherent anti-CD3 + anti-CD28 antibodies with IL-1beta, IL-6, and TGF-beta1 led to development of 26+/−9% Th17 cells (mean+/-SD, n=4), quantified by an ELISpot assay, that increased to 59+/−5% with 10-6 M S1P and 61+/−4% with IL-23 after 5 days. On day 5, secretion of IL-17 quantified by ELISA increased from 52+/−30 to 261+/−117, 470+/−98, and 438+/−61 pg/ml/24 hr/106 T cells with S1P, IL-23, and the S1P1–selective agonist SEW2871, respectively. CD4 T cell secretion of IL-4 and IFN-gamma were decreased significantly as IL-17 increased, and addition of recombinant IL-4 + IFN-gamma blunted IL-17 responses significantly. S1P-S1P1 enhancement of Th17 development was suppressed as well by IL-27 and NF-kappaB inhibition. The importance of stimulation of Th17 development and activity by the S1P- S1P1 axis is supported by the similarity of magnitude of effect and susceptibility to immunoregulation to that of IL-23.
Alpha-2-Macroglogulin (a2M) Mediates T Cell Suppression via a Peptide Sequence in its Cytokine-Binding Site but not by Cytokine Neutralization (90.6)
Authors: Koo Peter H. , Khol Matthew
Published: January 2023

a2M family proteins are protease inhibitors, but can also neutralize certain microbial virulence factors and influenza viruses, carry diverse types of cytokines, and inhibit T cell growth. Monoamine-reacted a2M (MA-a2M) is much more inhibitory to T cells than native a2M (N-a2M), but its mechanism of inhibition is unknown. We demonstrated that a2M family proteins form stable complexes with placental protein-14 (PP14; a potent immunosuppressive cytokine) besides TGF-β1; in addition, they enhance PP14’s ability to inhibit T-cell growth and IL2 production while not affecting IL4. Hence we postulate that a2M family proteins belong to a novel class of regulators for T cells. To investigate the role of a2M in T cell regulation and its mechanism of action, we employed a cloned Th cell line (HT), which requires IL2 or IL4 for growth and responds to TGF or PP14. TGF-β1 dose-dependently inhibits IL4-promoted but stimulates IL2-promoted HT-growth. While MA-a2M is consistently much more inhibitory to IL4-promoted HT growth than N-a2M, it dose-dependently neutralizes the immunosuppressive activity of TGF-β1; whereas N-a2M produces little effect. To study the mechanism of T cell regulation by a2M, we tested two recombinant peptides in and around a2M’s cytokine-binding site: the GST-a2M-peptide fusion proteins respectively encoding N591-N774 (FP3, which binds cytokines) and N775-N1082 (FP4, as negative control). Neither FP3 nor FP4 could block the immunosuppressive activity of MA-a2M or TGF-β1. While FP3 is potently inhibitory and adds to the inhibitory activity of TGF-β1 on HT cell growth, FP4 is not. Our data implicates an 184-a.a. sequence in a2M’s cytokine-binding site as the site for mediating T cell suppression but not for neutralization of cytokines.