bluuebunny/crossref_metadata_2025 huggingface dataset:
TIM-3 is a receptor for phosphatidylserine and allelic variants differentially mediate uptake of apoptotic cells (130.41)
Authors: Bu Xia , Ballesterous Angela , Chim Yee-Ling , Santiago Cesar , Lee Hyun-Hee , Umetsu Dale , Casasnovas Jose , DeKruyff Rosemarie , Freeman Gordon
Published: January 2023

TIM-3 is a member of the T cell/transmembrane, Ig and mucin (TIM) gene family. TIM-3 is expressed on Th1 cells and dendritic cells and generates an inhibitory signal resulting in apoptosis of Th1 cells. We found that TIM-3 is receptor for phosphatidylserine (PtdSer) exposed on apoptotic cells. A co-crystal structure showed a similar binding mode to TIM-4/PtdSer with a deep binding pocket coordinating calcium ion with PtdSer and flanked by two hydrophobic loops that can extend into a membrane. Fibroblasts transfected with BALB/c TIM-3 more effectively bound to and phagocytosed apoptotic cells than HBA TIM-3 transfected cells. Liposomes containing equal amounts of PtdSer and phosphatidylcholine reduced phagocytosis in a concentration dependent-manner. This suggests that functional differences in TIM-3 alleles may contribute to the Th1/Th2 differences between the strains. T or B cells expressing TIM-3 formed conjugates with but failed to phagocytose apoptotic cells, suggesting a role in signaling in lymphocytes without engulfment. These findings indicate that TIM-3 expressing cells can respond to apoptotic cells through TIM-3/PtdSer binding, but the consequence of TIM-3 engagement of PtdSer depends on the polymorphic variants of and type of cell expressing TIM-3. These findings establish a new paradigm for TIMs as PtdSer receptors and unify the function of the TIM gene family, which has been associated with ashma and autoimmunity and shown to modulate peripheral tolerance.
A vaccinia virus expression system to study antigen processing of multiple recombinant proteins (130.43)
Authors: Guterman Jacqueline , Wolf Benjamin , Kumagai Chiharu , Princiotta Michael
Published: January 2023

A major obstacle in designing effective vaccines is our limited knowledge of the mechanisms involved in eliciting a protective cell-mediated immune response. In recent years, research has focused on the development of recombinant vaccines, in which antigenic peptides derived from a specific pathogen are delivered to the cells via live vectors. For a vaccine to be effective in an out-bred population it must generate as many antigenic determinants as possible. One method that can be used to generate multiple peptide determinants is to express two or more recombinant proteins from a single live vaccine vector. To this end, we developed a recombinant vaccinia virus system that expresses two full-length influenza virus proteins; nucleoprotein (NP) and acidic polymerase (PA). In addition to the NP and PA proteins, our expression system is designed to produce yellow and red fluorescent proteins, which allow us to monitor, in a quantitative manner, recombinant protein expression both in vitro and in vivo. Using this novel expression system we have found that the route of immunization affects the immunodominance hierarchy of the host response to not only vector-derived peptides but also to recombinant peptides.
Immunization protocols towards shorter development time for monoclonal antibodies (130.44)
Authors: Hawksworth David , Brophy Susan
Published: January 2023

In the field of hybridoma development a wide variety of immunization regimens have been employed to develop fusion candidate animals in the shortest time possible. In studies presented here, we sought to discern the most immediate path to the highest affinity immune response. In the first study, we immunized two strains of mice using two distinct immunization regimens - 20 ug every six weeks or 5 ug bi-weekly, for 2 different proteins antigens. Results showed that, while additional boosts resulted in an improvement in average affinity, there was no difference in affinity between dosing groups at the end of the regimens. More importantly, it was discovered that some of the highest affinity animals had reached their maximum response without the need for an additional boost. Finally, for these two protein targets, BALB/c mice developed a higher affinity immune response than A/J mice. In a second study, mice were immunized three times with 20 ug protein using either a four or six week rest period between boosts. Results here showed no significant difference in relative affinity between mice rested 4 vs. 6 weeks between boosts. Overall, our results suggest that high affinity animals can be identified earlier in the boosting regimen and using shorter rest periods, thus allowing for the development of hybridoma cell lines in a shorter amount of time. Additionally, the more laborious bi-weekly dosing regimen showed no advantage over one with fewer boosts.
CD99 signal regulates the proliferation of multiple myeloma cell lines, RPMI8226. (131.10)
Authors: Gil Minchan , Park Seo-Jeong , Park Young Soo , Lee Hyangsin , Kim Hye-Mi , Park Chan-Sik
Published: January 2023

CD99 is a 32-kDa cell surface protein that is variously expressed on hematopoietic cells and hematopoietic-derived cancer cells. CD99 is highly expressed in some multiple myeloma cells including RPMI8226. In this study, we show that engagement of CD99 by agonistic anti-CD99 monoclonal antibody (mAb), YG32, inhibited proliferation of RPMI8226 multiple myeloma cells. However, YG32 mAb did not induce the cell death of RPMI 8226 cells. We carried out the microarray analysis to investigate the expression of genes involved in growth retardation by CD99 engagement. In microarray data, RPMI8226 cells engaged by anti-CD99 mAb had reduced expression of cyclin D1 and D2. Expression of CDKN2A gene, which codes cell cycle inhibitors p16INK4A and p19ARF, were induced by anti-CD99 mAb. These results suggest that CD99 signal regulates the proliferation of multiple myeloma cells by modulating proliferative and anti-prolifearative genes. The study was supported by National Research Foundation (NRF) grant of the Korean government A (R13-2008-023-01003)
Long-term cytokine therapy with IL-15/IL-15Rα complexes leads to impaired NK cell function (131.11)
Authors: Turley Shannon , Rubinstein Mark , Goldrath Ananda , Elpek Kutlu
Published: January 2023

Immunotherapies targeting effector lymphocytes are promising approaches for the treatment of cancer and infectious diseases. One such approach employs IL-15/IL-15Rα complexes which are considerably more potent than IL-15 in expanding cytotoxic lymphocytes including NK and CD8 T cells. Recent studies demonstrated that short-term treatment with IL-15/IL-15Rα complexes is highly effective in driving a transient but substantial reduction in tumor burden via immune-mediated mechanisms whereas continuous cytokine therapy improves survival only modestly and fails to control tumor outgrowth. Given the limited efficacy of long-term therapy, we sought to investigate the impact of continuous in vivo stimulation with IL-15/IL-15Rα complexes on NK and CD8 T cells in tumor-free mice. Long-term treatment with IL-15/IL-15Rα complexes triggered massive expansion of NK and CD8 T cells. Strikingly, however, NK cells exhibited marked defects in activation, IFNγ production, degranulation, and cytotoxicity whereas CD8 T cells showed enhanced functional capacity. Additionally, continuous treatment generated a suppressive environment for transferred naïve NK and CD8 T cells and resulted in accumulation of CD11b+Gr1+ cells. The mechanistic basis for NK cell dysfunction upon chronic stimualation with IL-15/IL-15Ra complexes will be discussed. Our results have important clinical implications for the design of immunotherapies and vaccines using multiple doses of immunostimulatory agents.
Abrogation of SHP-1 in tumor-specific effector T cells during adoptive immunotherapy of disseminated leukemia (131.15)
Authors: Stromnes Ingunn , Fowler Carla , Blattman Joseph , Tan Xiaoxia , Greenberg Philip
Published: January 2023

Adoptive therapy is a promising modality for treating patients with cancer. Currently, there are many obstacles to success, such as the low avidity of self/tumor-reactive T cells and the failure of transferred T cells to persist and to function in a tolerizing tumor environment. To overcome some of these obstacles, we asked if abrogating the Src homology region 2 domain-containing tyrosine phosphatase -1 (SHP-1), a negative regulator of lymphocyte activation, in tumor-reactive effector T cells would influence efficacy of adoptive therapy. A conditional knockout of SHP-1 in mature, tumor-specific transgenic T cells (TCRgag) was developed to analyze cell intrinsic effects of complete or partial ablation of SHP-1 on effector CD8 T cell function, persistence and efficacy in adoptive therapy of disseminated leukemia. In vitro-expanded TCRgag SHP-1-/- cells exhibited greater therapeutic efficacy than TCRgag SHP-1+/+ or SHP-1+/- cells, due to enhanced T cell expansion and survival in vivo, independent of providing exogenous IL-2. The increased expansion and cytokine production of SHP-1-/- effector cells was most pronounced in settings of lymphopenia. The role of SHP-1 in regulating signals received during lymphopenia-induced expansion and on the activation threshold of effector T cells will be discussed. Targeting SHP-1 in human T cells may provide a means to increase in vivo expansion following transfer and avidity for tumor targets during adoptive therapy of cancer.
Combination of CTLA-4 blockade with anti-CD40/IL-2 immunotherapy augments the CD8+ T cell response in resting and tumor-bearing mice (131.17)
Authors: Sckisel Gail , Nzigira Ngabo , Hurwitz Andrew , Blazar Bruce , Murphy William
Published: January 2023

Immunotherapy is a promising new option for the treatment of disseminated cancers. We have demonstrated that combined interleukin-2 (IL-2) and anti-CD40 (αCD40) therapy results in synergistic anti-tumor effects. While this strategy has shown success, it does not work well on less immunogenic tumors and can be extremely toxic. We sought to determine if removal of negative regulators such as cytotoxic T lymphocyte antigen-4 (CTLA-4) could result in augmentation of CD8+ T cell effects. We assessed total CD8+ T cell numbers and lytic ability after combination therapy in naïve and tumor-bearing mice. Here we present evidence that when the therapies are combined, there are increases in CD8+ T cells both systemically and intratumorally as well as a prolonged CD8+ memory expansion. We also saw increases in the lytic ability of CD8+ T cells against tumor cells as measured by chromium-51 release assay after combination with CTLA-4 blockade. We hypothesize that the amplification of T cell responses due to CTLA-4 blockade will enhance the anti-tumor effects of immunostimulatory therapies. It may also be possible to augment anti-tumor responses such that less immunotherapy will be needed to achieve equivalent anti-tumor responses.
SA-4-1BBL serves as an effective immunostimulatory component of HPV-16 E7 protein based therapeutic vaccine in a mouse model of cervical cancer (131.21)
Authors: Sharma Rajesh , Srivastava Abhishek , Yolcu Esma , Schabowsky Rich-Henry , Shirwan Haval
Published: January 2023

Cervical cancer is a major health problem in women worldwide. Current prophylactic vaccines are ineffective in therapeutic settings. Therefore, there is an acute need for the development of therapeutic vaccines. The HPV E7 oncoprotein is consistently expressed in cervical cancer, required for the maintenance of the transformed phenotype, and is not subjected to antigenic drift. However, the low antigenicity of this protein may require potent adjuvants for therapeutic efficacy. We have recently generated a novel form of the 4-1BBL costimulatory molecule chimeric with core streptavidin (SA-4-1BBL) and demonstrated its pleiotropic effects in soluble from on various cells of the immune system. We herein tested the utility of SA-4-1BBL as the immunomodulatory component of HPV-16 E7 recombinant protein based therapeutic vaccine in the E7 expressing TC-1 cervical cancer mouse model as a prelude to clinical trials. A single subcutaneous vaccination was effective in eradicating established TC-1 tumors in ~70% of mice. The therapeutic efficacy of the vaccine was associated with robust primary and memory CD4+ and CD8+ T cell responses, incresed CD8+ T cells and decreased T reg cells in tumor, enhanced NK cell killing, and generation of IFN-γ and IL-2 responses. Taken together, these data demonstrate the utility of SA-4-1BBL as a new class of immunomodulator for the development of therapeutic vaccines against cancer and chronic infections.
TNFRSF25 agonists as potent cancer vaccine adjuvants (131.25)
Authors: Schreiber Taylor , Podack Eckhard
Published: January 2023

Signaling through TNFRSF25 by its natural ligand, TNFSF15, or agonistic antibodies enhances cytokine production and effector function by antigen-activated T cells. Paradoxically, we recently discovered that TNFRSF25 signaling induces rapid and specific proliferation of CD4+FoxP3+ regulatory T cells (Treg) in vivo in the absence of exogenous antigen. When TNFRSF25 agonistic antibody, clone 4C12, was used in combination with heat shock protein (gp96-Ig) based vaccination, 4C12 treatment led to synergistic antigen-specific CTL expansion when given together with the vaccine, which occurred simultaneously with Treg expansion in vivo. The frequency of CTL and Treg reached 65% of total CD8+ cells and 45% of total CD4+ cells, respectively, within the same tissue following combined administration of 4C12 and gp96-Ig. TNFR25 driven synergism with gp96-Ig vaccination was dependent upon mTOR signaling. In a therapeutic setting, combined administration of 4C12 and gp96-Ig enhanced rejection of established EG7, LLC and 3-MCA induced sarcoma tumors. These data indicate that TNFRSF25 agonists enhance vaccine-induced anti-tumor immunity which correlates with increased magnitude and duration of tumor-specific CTL expansion, systemically. These findings illustrate that TNFRSF25 signaling may enhance either inflammatory or regulatory immunity depending on the temporal availability of its ligand and the inflammatory milieu.
Targeted delivery of interferon alpha via fusion to anti-CD20 results in potent antitumor activity against B-cell lymphoma (131.30)
Authors: Xuan Caiyun , Steward Kristopher , Timmerman John , Morrison Sherie
Published: January 2023

The anti-CD20 antibody rituximab has substantially improved outcomes in patients with B-cell non-Hodgkin lymphomas. However, many patients are not cured by rituximab-based therapies, and overcoming de novo or acquired rituximab resistance remains an important challenge to successful treatment of B-cell malignancies. Interferon alpha (IFNα) has potent immunostimulatory properties and anti-proliferative effects against some B-cell cancers, but its clinical utility is limited by systemic toxicity. To improve the efficacy of CD20-targeted therapy, we constructed fusion proteins consisting of anti-CD20 and murine or human IFNα. Fusion proteins had reduced IFNα activity in vitro compared with native IFNα, but CD20 targeting permitted efficient anti-proliferative and pro-apoptotic effects against an aggressive rituximab-insensitive human CD20+ murine lymphoma (38C13-huCD20) and a human B-cell lymphoma (Daudi). In vivo efficacy was demonstrated against established 38C13-huCD20 grown in syngeneic immunocompetent mice and large, established Daudi xenografts grown in nude mice. Optimal tumor eradication required CD20 targeting, with 87% of mice cured of rituximab-insensitive tumors. Gene knockdown studies revealed that tumor eradication required expression of type I IFN receptors on the tumor cell surface. Targeting type I IFNs to sites of B-cell lymphoma by fusion to anti-CD20 antibodies represents a potentially useful strategy for treatment of B-cell malignancies.
MUC1-specific Th1 immunotherapy induce differential levels and proportions of "inducible" and "natural" CD4 TReg cell subpopulations that results in enhanced tumor immunity and increased ovarian cancer patient survival (131.38)
Authors: Dobrzanski Mark , Rewers-Felkins Kathleen , Samad Khaliquzzaman , Robinson William , Wright Stephen
Published: January 2023

Adoptive T cell therapy is a viable treatment for cancer patients and optimally requires participation of CD4 T cells. Here, we assessed the therapeutic effects of adoptively transferred autologous MUC1 peptide-stimulated CD4+ effector cells in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4+/Th1 effector cell generation, we show that 3 monthly treatment cycles of T cell restimulation and intraperitoneal re-infusion modulated T cell mediated immune responses that correlated with diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease free, another patient survived long-term for nearly 18 months with recurrent disease and two patients expired within 3-5 months following final infusion. Long term survivors showed elevated levels of systemic CD3/CD4/CD25+ and CD3/CD4/CD25- T cells when compared to that of pre-treatment levels and similarly treated short-term survivors. Such cells co-expressed different levels of "Inducible" Tr1 (CD4/CD25-/FoxP3-/IL-10+) and "Natural" (CD4/CD25+/FoxP3+) TReg cell ratios that further correlated with disease-free survival. These patients showed elevated levels of Th1 cells and MUC1-specific T cells expressing chemokine receptors associated with T cell differentiation/memory. This suggests that MUC1-stimulated CD4 T cells induce differential levels and proportions of systemic TReg subpopulations that influence long term tumor immunity in such patients following treatment.
Evaluation of cellular therapies utilizing a fluorine-based magnetic resonance imaging (MRI) tracer agent (131.40)
Authors: Helfer Brooke , Balducci Anthony , Mailliard Robbie , Wesa Amy , Ahrens Eric
Published: January 2023

Natural killer cells (NK), T cells and dendritic cells (DC) are increasingly being used in the clinic to adjust patient immune function in response to cancer. One of the current limitations of such therapies is the inability to track the therapeutic cells once they are administered to a patient. Here we utilize a novel, non-radioactive fluorine-based (19F) MRI tracer agent to label clinically relevant cells ex-vivo and to non-invasively detect the transferred cells in vivo over time. We demonstrate that human NK, T cells and DC (immature and mature) can be effectively labeled without effect on their health, phenotype or function. Using a murine xenograft model as a pre-clinical “proof of principle” assay, we visualized and tracked the migration of human DC in-vivo. Following injection, the 19F labeled cells were detected and anatomically located by MRI. Mature DC but not immature DC were observed to migrate into the region of draining lymph nodes 18h post injection by MRI. Isolation and flow cytometric analysis of cells from draining lymph nodes confirmed the presence of human DC as well as the efficacy of this method. The application of this tracer agent across several cell types, along with non-invasive highly specific detection of 19F labeled cells by MRI, demonstrates the potential use of 19F in translational and clinical applications of cellular therapies. Ultimately, this technology may expedite a therapy’s transition from bench to bedside.
The role of Actin Binding Protein One (Abp1) in B cell development, activation, and B cell tolerance. (132.23)
Authors: Seeley Margaret , Onabajo Dr. Olusegun , Song Dr. Wenxia
Published: January 2023

The B cell receptor (BCR) serves as both signal-transducer and antigen-transporter. Optimal activation of B cells depends on coordinated BCR signaling, antigen processing, and actin cytoskeleton rearrangement events. Actin-binding protein 1 (Abp1/HIP-55/SH3P7) is a unique adaptor protein that has been shown to directly interact with components of signal transduction, endocytosis, and actin cytoskeleton apparatuses through its proline-rich, SH3, and actin binding domains. This suggests a role for Abp1 in mediating cross-talk among the three cell apparatuses during BCR activation. Here we study the role of Abp1 in B cell development, activation, and B cell tolerance. We discovered major defects in B cell development in Abp1 knockout mice, exhibited as reduced numbers of pre- and immature B cells in the bone marrow, and transitional B cells in the spleen. Furthermore, Abp1 knockout mice exhibit splenomegaly and have increased levels of anti-nuclear antibodies. At the cellular level, Abp1 is recruited to BCR surface signalosomes in response to antigenic stimulation. In Abp1 knockout mice, BCR-induced protein tyrosine phosphorylation of splenic B cells is elevated and phosphorylation of the MAP kinase JNK reduced compared to wild type B cells. These results suggest that Abp1 can regulate B cell development, B cell activation, and B cell tolerance via modulation of BCR signaling. This work supported by a NIH grant (AI059617) for W.S.
IL-2 signals promote the differentiation of effector CD8+ T cells (132.26)
Authors: Mitchell Diana , Williams Matthew
Published: January 2023

CD8+ T cells that do not receive IL-2 signals during the primary immune response to acute infection form stable memory populations but are unable to mount successful recall responses. In this study, we found a specific defect in the differentiation and persistence of effector and effector memory CD8+ T cells in the absence of IL-2 signals during the response to lymphocytic choriomeningitis virus (LCMV) infection. Although expression levels of the effector-associated transcription factors T-bet, Eomes, and Blimp-1 did not differ significantly, unbiased analysis of gene expression by WT or IL-2Rα-deficient CTLs revealed that optimal expression of CTL effector molecules, including Granzymes and Perforin, was IL-2-dependent. The end result of CD8+ T cell activation in the absence of IL-2 was therefore decreased effector CTL activity. The defect in effector differentiation was even more profound for secondary CTL responses and in models of localized tissue-specific infection. CD8+ T cells that homed to the lung following influenza infection failed to express Granzyme B. We conclude that IL-2 provides a differentiation signal to responding CD8+ T cells that enhances access to an effector gene expression program during the primary response to infection. Furthermore, our findings suggest that IL-2 permanently imparts a gene expression imprint to CD8+ memory T cells that enables them to re-access the CTL effector program upon subsequent encounter with antigen.
Characterization of intracellular signaling profiles induced by antigen receptor activation in murine B cell subsets (132.30)
Authors: Lyubchenko Taras , Liubchenko Ganna , Holers V. Michael
Published: January 2023

Protein phosphorylation and Ca2+ mobilization are among major signaling parameters characterizing the activation status of B lymphocytes. This study identifies quantitative differences between intracellular signaling profiles in subsets of follicular, marginal zone, transitional T1, T2, T3 murine B cells. Phosphorylation levels of signaling proteins and Ca2+ responses upon BCR engagement with anti-IgM were measured by multicolor flow cytometry. The results revealed previously uncharacterized diversity of signaling profiles in different B cells subsets that correlated with their developmental stage and previous antigen exposure. At least three important trends were identified: 1) B cells at earlier developmental stages had higher activation threshold, baseline Ca2+ and phosphorylation activity, and Ca2+ dynamics that in some instances did not correlate with sIgM expression levels; 2) phosphorylation levels within particular B cell subsets were heterogeneous which suggests the presence of various cell groups with different signaling properties within each subset; 3) analysis of phosphorylation profiles in individual signaling molecules revealed molecule-specific nonlinear characteristics of dose-response curves that are indicative of quantitative signaling differences between B cell subsets and subset-specific balance of BCR signaling cascades. These preliminary findings provide rationale for investigating roles of different B cell subsets in alterations of immune tolerance.
Activation of human naïve CD8 T cells by artificial antigen presenting cells (132.31)
Authors: Blair David , Dustin Michael
Published: January 2023

The precise mechanisms by which an activated T cell differentiates to a memory cell remain unclear. Recent evidence indicates that asymmetric cell division (ACD), an evolutionarily conserved process whereby multiple cell fates are derived from a common progenitor cell, may play an important role in the differentiation of T cells. However, these studies were conducted in mouse models and it is unknown whether parallel mechanisms occur in human T cells. To better understand these mechanisms, we employed the use of an established artificial antigen presenting cell (aAPC) system for the activation of human naïve CD8 T cells. Our efforts have focused on the early events following T cell activation up to the first division of T cells. We investigated atypical protein kinase C zeta (PKCζ), a protein involved in ACD, and its role in human CD8 T cell differentiation upon engagement of the T cell receptor. Anti-CD3 presentation to T cells by the aAPCs resulted in the partitioning of PKCζ distal to the immunological synapse in a temporal manner. This polarization was first observed at 12 hours following activation and was maintained for at least up to 42 hours of stimulation. However, in the absence of anti-CD3 stimulation by the aAPC, polarization of PKCζ did not occur in the T cells. These data indicate that partitioning of polarization complex proteins occurs in human T cells and may be important in the differentiation of CD8 T cell subsets following activation.
Human IL-7Rαlow memory CD8+ T cells have defect in the calcium signaling pathway in response to TCR triggering, leading to impaired IL-2 production and proliferation. (132.34)
Authors: Lee Ha Na , Yu Mi Na , Lee Dong-Sup , Kang Jae Seung , Hwang Young-il , Lee Wang Jae , Kang Insoo , Kim Hang-Rae
Published: January 2023

We have found two subsets of human CD8+ T cells expressing IL-7Rαhigh and IL-7Rαlow with different cell survival responses to IL-7. These IL-7Rαlow memory CD8+ T cells that produce effector cytokines and perforin have impaired survival in response to IL-7. Although these CD8+ T cell subsets fail to respond to TCR triggering, the mechanism for this is unknown. Thus we investigated the intracellular signaling pathways in differentially regulating proliferation and effector cytokine production in human IL-7Rαlow memory CD8+ T cells. Treating these CD8+ T cells with anti-CD3 Abs did not produce IL-2, but expressed IFN-γ and TNF-α. Although IL-7Rαlow memory CD8+ T cells showed anergic phenotypes in terms of cytokine production and proliferation, activation of MAPK pathway, which is known defects in anergic T cells, was intact. Importantly, human IL-7Rαlow memory CD8+ T cells had reduced calcium flux upon in vitro TCR stimulation, leading to impaired IL-2 production. Moreover, inhibition of IL-2 production was not recovered by PMA/ionomycin treatment. There findings provide new sight into that human IL-7Rαlow memory CD8+ T cells are rendered cellular characteristics of anergic with calcium signaling defects.
Antigen-specific precursor frequencies influence requirement of CD4+ T cell help and CD40-40L costimulation in primary and secondary responses (132.8)
Authors: Sokke Umeshappa Channakeshava , Xiang Jim
Published: January 2023

CD4+ T cells play a decisive role in generation of effective CD8+ T cell-mediated immunity against diseases associated with minor H antigens, including cancer, auto-immunity and allogenic tissue transplantation. The increased precursor frequency in reducing the need of CD4+ T cell help and costimulations in CD8+ T cell primary responses have been demonstrated. However, its similar roles in memory CD8+ T cell responses have not been fully understood. In a non-infectious vaccine model, here we show that, at endogenous precursor levels, CD4+ T cell help, and CD40-40L costimulation are required for both primary and memory responses. At increased precursor frequency, although CD4+ T cell help, and CD40-40L costimulation are not obligatory in primary responses, their requirement is still necessary for functional memory responses. Despite increased precursor frequency is ensured during priming, the memory CD8+ T cells developed from increased precursor frequency in the absence of CD4+ T cell help, and CD40-40L costimulation fail to induce complete protection following tumor challenge. These results further support the previous observations that efficient memory CD8+ T cell responses require CD4+ T cell help and CD40-40L costimulation.
The DRF motif in CXCR6 is important for selectivity among G-proteins in receptor signaling, which is cell-type specific (133.1)
Authors: Singh Satya , Foley John , Zhang Hongwei , Farber Joshua
Published: January 2023

CXCR6 is the receptor for CXCL16. Except for CXCR6, all human chemokine receptors contain the D3.49R3.50Y3.51 sequence, and all but two contain A3.53 at the cytoplasmic end of the third transmembrane helix (H3C). In contrast, the CXCR6 sequence is D3.49R3.50F3.51I3.52V3.53. We investigated the importance of canonical and non-canonical amino acids at these positions in CXCR6 in order to understand the role of this region in chemokine receptor function generally. We produced CXCR6 point mutants, including those where non-canonical amino acids were replaced with canonical amino acids singly and in combination, and analyzed the mutants in HEK-293T and Jurkat E6-1 cells. For the H3C of CXCR6, our data demonstrate that 1) non-canonical residues can function better than canonical residues, 2) sequences are matched, with changes having compensatory effects, 3) residues can affect both ligand binding and receptor signaling independently, 4) residues affect selectivity among G-proteins for receptor signaling, and 5) the affects of these residues on receptor function are cell-type specific. Our findings indicate that the H3C may not only serve as an on-off switch for GPCRs, as has been proposed, but also has broader effects on receptor function, which, surprisingly, depend on cell-type specific factors, and suggest that a GPCR structure may be constrained by a need to use G-proteins promiscuously in all the cells where a given receptors is expressed.
Kinetics and mechanisms of IL-4 suppression of Type I interferons’ responses in murine dendritic cells (134.22)
Authors: Sriram Uma , Bennett Heather , Xu Jun , Shivers Debra , Behrens Edward , Gallucci Stefania
Published: January 2023

Cytokines cross-talk and modulate immune responses. We have shown before that IL-4 suppresses the response of murine dendritic cells (DCs) to Type I interferons (IFN). In this study we analyzed the kinetics of Type I IFN and IL-4 interactions in myeloid bone marrow-derived DCs (BMDCs) for Type I IFN induced gene and protein expression. Pre-treatment with IL-4 suppressed IFN-a induced gene expression even at the sub-optimal dose of 0.25ng/ml of IL-4; suppressed phosphorylation of STAT1 and STAT2, suggesting that IL-4 acts at the level or upstream of STAT molecules in the Type I IFN signaling pathway. Post-treatment with IL-4 for a range of 2-24h after IFN-a stimulation, also suppressed IFN-b gene expression. IL-4 also suppressed the response to autocrine IFN-b that is induced upon IFN-a stimulation, acting as a positive feedback. Indeed, we found that BMDCs response to exogenous IFN-a is sustained for many hours - washing and replating BMDCs 30 min after IFN-a stimulation leads to a strong reduction of IFN-b expression that is stimulated only by autocrine IFN-b; post-treatment with IL-4 suppressed this second wave of response, suggesting that IL-4 can inhibit the response to paracrine IFNs and the secondary response to autocrine IFN. Cyclohexamide (protein synthesis inhibitor) abrogated the suppressive effects of IL-4 indicating that IL-4 induces the expression of a protein that possibly interacts with Type I IFN signaling and fades out the responses of DCs to Type I IFNs.
Interleukin-19 regulates the immune homeostasis to the airborne environment (134.5)
Authors: Grunig Gabriele , Sisco Maureen , Louten Jennifer , Daley Eleen , Hoffman Carol , Emson Claire , Gordon Terry , de Waal Malefyt Rene
Published: January 2023

Interleukin 19 (IL-19) is thought to have important roles in the immune control of pathogens, and in enhancing chronic inflammatory diseases of the skin (psoriasis), lung (asthma) and intestine (colitis). However, the biological function and the networked control of IL-19 are poorly understood. Naive mice deficient in IL-19 (IL-19-/-) on a 129xBL6 background had decreased alveolar macrophages and increased eosinophils in the broncho-alveolar lavage (BAL) and decreased B cells in the spleen and bone marrow. However, naïve IL-19-/- mice backcrossed to C57BL/6 or BALB/c, and mice housed in different locations exhibited an attenuated phenotype. Therefore, genetic backcross and challenge experiments with environmental stimuli (allergen, urban particulate matter) were conducted. The studies showed that abnormal composition of the cells in the BAL fluid, spleen and bone marrow in naïve IL-19-/- 129xBL6 mice was due to the deficiency in IL-19 combined with the activity of another gene inherited from the 129 strain. IL-19-/- mice on C57BL/6 or BALB/c backgrounds that were intranasally challenged with antigen or with antigen and urban particulate matter had decreased BAL eosinophils, alveolar macrophages and B cells when compared to wild type. Together our studies indicate that endogenous IL-19 is a critical component of immune homeostasis to the airborne environment and that mouse-strain specific genes control the function of IL-19.
Investigating Early Events in Sjogren’s Disease (135.8)
Authors: Shen Long , Wu Jing , Suresh Lakshmanan , Ambrus, Jr. Julian
Published: January 2023

Little is known about the early events in Sjogren’s disease. Previous studies have demonstrated that interleukin 14 alpha transgenic mice (IL14aTG) replicate the clinical and immunological features of Sjogren’s disease (Clin. Immunol. 130:304, 2009). Salivary gland function is lost before lymphocytic infiltration of the salivary glands. The current studies were designed to investigate the early events involved with salivary gland injury. Western blot studies demonstrated lymphotoxin (LTA) in the salivary gland secretions of IL14aTG mice, while salivary gland function was being lost. IL14aTG mice lacking LTA maintained normal salivary gland structure and function, although autoantibodies were deposited in the salivary glands. Because IL-14aTG mice produce high levels of interferon-a (INF-a) and INF-a can induce production of LTA, we investigated IL14aTG mice lacking the α/β interferon receptor. These mice retained normal salivary gland function, although mild lymphocytic infiltration was noted in the lachrymal glands. Because IL14aTG mice have increased numbers of B1 cells, we produced IL14aTG mice lacking btk. Interestingly, these mice maintained normal salivary gland function despite lymphocytic infiltration of the salivary glands. Thus cytokine injury involving LTA and IFN-a are necessary for the development of salivary gland injury in Sjogren’s disease. The roles of particular autoantibodies and lymphocytic infiltration of the salivary glands need to be elucidated.
Hemorrhagic shock primes for IL-1β processing: role of upregulated TLR2 (136.1)
Authors: Fan Jie , Li Yuehua , Xiang Meng , Billiar Timothy , Wilson Mark
Published: January 2023

IL-1β contributes to post-trauma sepsis, and its release requires processing in inflammasomes. Hemorrhagic shock (HS) primes for IL-1β release in response to LPS via yet unclear mechanisms. We have reported that HS-activated neutrophils (PMN) augment LPS/TLR4 signaling upregulation of TLR2 in alveolar macrophages (AM), thereby sensitizing the AM to TLR2 ligands. Here, we addressed the role of upregulated TLR2 in the HS-primed IL-1β processing. WT, TLR4-mutant and TLR2 KO mice were subjected to HS, followed by intratracheal LPS (30 µg/kg B.W.) and PGN (30 µg/kg B.W.) at 2h and 4h after HS, respectively. Bronchoalveolar lavage fluids (BALF) were collected at 2 h after PGN for detection of IL-1β. In vitro, AM were co-cultured with PMN isolated from HS mice, and treated with LPS, PMN was then removed and PGN was added at 2h. IL-1β and caspase-1 maturation in the AM were assessed. In vivo treatments of LPS and PGN caused a marked increase in IL-1β in BALF in the WT/HS mice, but not in the WT/sham, TLR4-mutant/HS or TLR2 KO/HS mice. HS-activated PMN increased TLR2 expression in AM in response to LPS, and subsequently increased caspase-1 and IL-1β maturation in the AM as well as IL-1β release in response to PGN. Moreover increasing extracellular [K+] failed to prevent the TLR2 signal-induced IL-1β maturation. In HS, amplified TLR2 signaling acts as a second signal in stimulating caspase-1 and IL-1β maturation via mechanisms independent of K+ efflux.
Differential IFN signature for TLR7 and 9 agonists (136.16)
Authors: Puig Montse , Tosh Kevin , Grajkowska Lucja , Verthelyi Daniela
Published: January 2023

Innate immune response modulators that stimulate TLR7 and 9 activate similar signaling pathways but their therapeutic range is distinct. Moreover, 3 structurally different types of stimulatory sequences for TLR9 have been identified (CpG ODN type D/A, B/K and C) that act on the same receptor but activate distinct immunostimulatory pathways and elicit different clinical effects. This study characterizes and compares gene expression in human and macaque cells treated with different TLR7 & 9 agonists. In vitro results showed that overall TLR7 and 9 agonists elicit transcription of a similar repertoire of immune-related genes; however the magnitude and kinetics of the gene expression differed significantly, particularly for IFN-related genes as well as cell mobilization and adhesion genes. Interestingly, while transcription level and kinetics of type I IFNs gene expression in cells treated with CpG ODN D, B and C differed significantly, the relative distribution of IFNα subtypes was almost identical. In contrast, the same cells stimulated with the TLR7 agonist elicited a different pattern of IFNα subtypes and IFNα-inducible genes. In vivo, only rhesus macaque monkeys treated with CpG ODN type D showed a robust IFN type I response both locally and systemically. The data suggest a higher mobilization and activation of the pDC with CpG ODN type D treatment. Understanding the nature of these differences has implications for the therapeutic application of TLR agonists in humans.
NOD2 mediates CNS inflammatory responses of primary murine glia in streptococcal meningitis (136.34)
Authors: Chauhan Vinita , Liu Xinjie , Marriott Ian
Published: January 2023

Resident cells of the central nervous system (CNS) such as microglia and astrocytes initiate and/or augment inflammation following trauma or infection. We have previously demonstrated that glia constitutively express nucleotide-binding oligomerization domain-2 (NOD2), a member of the novel nucleotide-binding domain leucine-rich repeat region-containing family of proteins (NLR) that functions as an intracellular receptor for a minimal motif present in all bacterial peptidoglycans. We have also shown that this NLR is essential for glial responses to Gram-negative pathogens and the subsequent in vivo CNS inflammation. In the present study, we have established that intact Streptococcus pneumoniae, the major causative agent for Gram-positive bacterial meningitis in adults, is a potent stimulus for the activation of the pivotal inflammatory transcription factor NF-kB and production of inflammatory cytokines in primary murine microglia and astrocytes. We demonstrate that NOD2 is essential for the maximal responses of these cells to intact S. pneumoniae but not cellular lysates. Finally, we have shown that NOD2 is required for the elevated inflammatory mediator levels, astrogliosis, and demyelination, following in vivo administration of this Gram-positive CNS pathogen. As such, we suggest that NOD2 plays a critical role in the establishment of the lethal inflammation associated with streptococcal meningitis.
Cross-talk between innate and adaptive immunity through pentraxins and Fc receptors (136.42)
Authors: Lu Jinghua , Marjon Kristopher , Marnell Lorraine , Mold Carolyn , Du Clos Terry , Sun Peter
Published: January 2023

The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein (CRP), which are two of the acute-phase proteins and able to opsonize microbial pathogens. In 2008, we reported the structural mechanism for pentraxin’s binding to FcγRIIa and its functional activation of FcγR-mediated phagocytosis and cytokine secretion. We further extend these findings in two aspects: 1) High-affinity IgG receptor, FcγRI, is also recognized by pentraxins. The crystal structure of human FcγRI reveals a unique architecture of the three-Ig domains of FcγRI, which is reminiscent of the head of a seahorse. Despite the additional third Ig domain (D3), the first two Ig domains (D1 and D2) of FcγRI adopt a similar structure to that of FcγRIIa and also confer the high affinity for the binding of IgG. Therefore, like FcγRIIa, FcγRI binds to two diagonally-spanned subunits of each CRP or SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. 2) We show that pentraxins also recognizes FcαRI, both in solution and on cells. FcαRI bound to the effector face of CRP and SAP in a region close to but not identical with that of FcγRs. The binding of CRP to FcαRI transfected RBL cells induced degranulation and the phosphorylation of Syk kinase. Taken together, these results establish antibody-like functions for pentraxins in the Fc receptor pathway and suggest a new crosstalk between the innate and adaptive immune systems.
Proteomics decode RLR and TLR signaling circuitry (136.5)
Authors: Li Shitao , Wang Lingyan , Dorf Martin
Published: January 2023

RIG-I-like receptors (RLRs) and Toll-Like Receptors (TLRs) play a critical role in sensing microorganisms and activating the early innate immune response. The signaling circuit of innate immunity remains incompletely understood. To systematically investigate the network, we initiated a global proteomic analysis of protein complexes found in the innate immunity pathway. Fifty-five genes involved in TLR and RLR pathways were tagged with FLAG epitopes and stably transfected into 293 cells engineered to express TLR3 or TLR4. After treatment with or without poly(I:C), LPS, or poly(dA:dT), cells were collected to purify the tagged protein complexes. Proteins in each complex were identified by mass spectrometry and data were statistically analyzed. Protein interaction networks were established using Cytoscape software. Sixty-five known interactions were confirmed and seventy-nine new interactions were detected.
Crucial role of effector T cell-derived IL-10 in regulating lung inflammation and pulmonary function during primary RSV infection (137.13)
Authors: Sun Jie , Cardani Amber , Braciale Thomas
Published: January 2023

Respiratory syncytial virus (RSV) infection is the leading viral cause of severe lower respiratory tract illness in young infants. Clinical studies have documented that genetic polymorphism of a regulatory cytokine IL-10 is associated with the development of severe bronchiotis in RSV infected infants. However the tempo, source and function of IL-10 during the RSV infection remain unclear. Here, we have examined the role of IL-10 in a murine model of primary RSV infection. We found that high levels of IL-10 are produced in the respiratory tract at the onset of adaptive immune responses. The main cellular sources of the IL-10 are infiltrating anti-viral effector CD4+ and CD8+ T cells (Te). We showed that the function of the Te-derived IL-10 in vivo is mainly to limit the pro-inflammatory activity of the anti-viral Te themselves and thus to control the exaggerated pulmonary inflammation induced by the infiltrating Te. Furthermore, we demonstrated that the inhibition of the exaggerated inflammation by IL-10 is essential in maintaining normal pulmonary function of the host. Our data not only discovered a novel function of IL-10 (i.e., autocrine inhibition of the inflammatory activity of Te) during infection but provided potential explanation and cellular mechanism for the early clinical studies demonstrating a link between IL-10 gene polymorphism and the development of pulmonary diseases during RSV infection.
HCV+ hepatocytes induce human CD4+ regulatory T cells through the production of TGF-β (137.19)
Authors: Hall Caroline , Hahn Young
Published: January 2023

Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and impaired T cell responses are reported to be responsible for facilitating and maintaining this infection. CD4+ regulatory T cells detected in chronic HCV patients have been implicated in dampening antiviral T cell responses to HCV infection. However, the underlying mechanism for induction and expansion of regulatory T cell responses has yet to be determined. To this end, we examined whether HCV-expressing hepatocytes, as the primary target for HCV infection and replication, are able to induce regulatory T cells. These studies were accomplished by establishing the co-culture of Huh7.5 containing the full-length genome of HCV genotype 1a (Huh7.5-FL) with activated CD4+ T cells, using Huh7.5 with no HCV (Huh7.5) and a subgenomic portion (Huh7.5-SG) as controls. The production of IFN-γ and proliferative response of antigen-independent CD4+ T cells was diminished following co-culture with Huh7.5-FL compared to controls. Notably, these CD4+ T cells in contact with Huh7.5-FL express an increased level of regulatory T cell markers (FoxP3, CTLA-4, LAP) and are able to suppress the proliferation of effector T cells. Intriguingly, Huh7.5-FL upregulate the production of TGF-β and blockade of TGF-β abrogates FoxP3 expression and IFN-γ production. These results suggest that hepatocytes may play a pivotal role in impairing antiviral T cell response by the induction of regulatory T cells.
TAT-CD137 Ligand cytoplasmic domain as the effective activator for reverse signaling (138.19)
Authors: Kim Jung , Lee Eun , Quang Nguyen , Kwon Byungsuk
Published: January 2023

The CD137 (also called 4-1BB), a T-cell co-stimulatory receptor induced by TCR activation, is a member of tumor necrosis factor (TNF) receptor superfamily. The ligand of CD137 (4-1BBL) , also a TNF superfamily member, is known to be expressed on activated antigen-presenting cells such as B cells, macrophages and dendritic cells (DCs). Bi-directional transduction of signals exists for the CD137 receptor/ligand system. Here we developed the TAT fused CD137 Ligand cytoplasmic domain (TAT-CD137Lcy), which has cell-penetrating ability, to regulate the reverse signaling. The TAT-CD137Lcy protein induced the production of IL6 and TNF-a on peritoneal macrophages and also increased the level of mRNA transcripts of IL-6 and TNF-a. The TAT-CD137Lcy protein increased phosphorylation of p38, JNK and Erk but not the degradation of the inhibitor of NF-kB (IkBa). The TAT-CD137Lcy protein potentially functions as activator for reverse signaling. To confirm the positive effect of TAT-CD137Lcy protein, B cells proliferation assay was performed. The TAT-CD137Lcy protein enhanced the proliferation of splenic B cells stimulated with anti-CD40 monoclonal antibody (mAb) and anti-IgM mAb. Our results indicated that the TAT-CD137Lcy protein is the effective activator for reverse signaling. For activation of reverse signaling, The TAT-CD137Lcy protein is more effective than 4-1BBFc and TKS-1 mAb because the TAT-CD137Lcy protein does not interrupt the interaction between receptor and ligand.