bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety, Efficacy and pharmacokinetics of T1h, a humanized anti-CD6 monoclonal antibody, in moderate to severe chronic plaque psoriasis - Results from a randomized phase II trial. (96.13)
Authors: Anand Arun , Assudani Deepak , Nair Pradip , Krishnamurthy Sharmitha , Deodhar Sarika , Arumugam Muruganandam , Iyer Harish , Melarkode Ramakrishnan
Published: January 2023

T1h, a novel humanized monoclonal antibody targeting CD6, a co-stimulatory receptor present on T cells, was evaluated as a potential therapy for psoriasis. In this multi-centric phase II, dose finding, single-blind study, 40 patients with moderate-to-severe psoriasis were randomized to receive T1h intravenously at 3 different doses across weekly, two-weekly and four-weekly schedules (n=5 per dose cohort) for 8 weeks, and followed for further 24 weeks. Safety and PK were the primary endpoints. Efficacy of T1h was evaluated using PASI and PGA. SF-36 and DLQI questionnaires were used to assess changes in quality of life. T1h was well tolerated. The most common adverse events were chills and pyrexia. Only 2 serious adverse events (both related to psoriasis) were ascribed to T1h therapy. T1h was weakly immunogenic with one patient developing an immune response. T1/2 of T1h ranged from 11.72 days to 18.51 days across different dose-schedules. Significant reductions in mean PASI score from baseline were observed at week 12 (22.32 to 6.23, P< 0.0001) with the best response observed in cohort receiving 1.6 mg/kg T1h administered every 2 weeks. Overall, the proportion of patients with PASI50 and 75 responses were 72.5% and 45% respectively. 26 patients (65%) achieved a PGA score of clear or minimum at 12 weeks. A significant improvement in DLQI, SF36 and epidermal thickness was also noted. T1h exhibited a favourable safety profile in the treatment of moderate to severe psoriasis.
Negative modulation of B-cell function via inhibitory receptor signaling by using novel diabody-based Dual-Affinity Re-Targeting (DART) molecules (96.14)
Authors: Veri Maria-Concetta , Burke Steve , Huang Ling , Li Hua , Gorlatov Sergey , Tuaillon Nadine , Alderson Ralph , Rainey Jonah , Ciccarone Valentina , Zhang TengFei , Shah Kay , Jin Linda , Moore Paul , Koenig Scott , Johnson Syd , Bonvini Ezio
Published: January 2023

The Fc receptor, CD32B, functions as an inhibitory receptor and, when co-ligated with the B-cell antigen receptor (BCR) complex, dampens signaling and immune activation. Ligation of activating and inhibitory receptors is an obligatory event for CD32B inhibition coupling in B cells, which heightens the need for a bispecific molecule for interventional purposes. To exploit this mechanism, we have engineered a novel and stable bispecific diabody scaffold that facilitates heterodimeric assembly with high predictability. This Dual-Affinity Re-Targeting (DART) scaffold was used to couple an Fv specific for the inhibitory receptor, CD32B, with an Fv specific for the Ig-beta component (CD79B) of the BCR. The resulting CD32BxCD79B DART molecule was capable of co-ligating the activating and the inhibitory receptors on the B-cell surface, resulting in reduced BCR-induced calcium mobilization and phosphorylation of selected signaling molecules. These biochemical changes correlated with inhibition of B-cell proliferation and Ig secretion upon B-cell activation. In addition, a DART protein specific for the mouse orthologs of CD32B and CD79B reduced disease severity in a collagen-induced arthritis model. This novel approach to B-cell function modulation forms the basis for translating inhibitory signaling into therapeutic tools for managing autoimmune and inflammatory diseases.
Anti-inflammatory curcumin inhibits AID expression within cycling human B cells (96.21)
Authors: Haque Shabirul , Lee Hyunjoo , Waqas Bukhtawar , Chiorazzi Nicholas , Mongini Patricia
Published: January 2023

Curcumin is a natural phenolic compound extracted from the spice, Curcuma longa. It has noted anti-inflammatory effects, in part due to its suppression of NF-kB. Activation-induced cytosine deaminase (AID) is a NF-kB-regulated enzyme essential for Ig class switch recombination and somatic hyper-mutation with a demonstrated role in lymphomagenesis. This study has examined the effect of curcumin on the division-dependent upregulation of AID protein and mRNA within in vitro-activated normal human B lymphocytes and the AID-expressing B cell line, CL-01. CFSE-labeled, IgM+ human B2 cells isolated from spleen/tonsil were pre-activated for 4-5 days with stimuli likely encountered in sites of inflammation, i.e. limiting surrogate C3dg-coated antigen (anti-IgM: anti-CD21: dextran) + IL-4 + BAFF. Curcumin at doses from 6 to 50 µM, or parallel vehicle control, was pulsed into dividing B cell cultures and AID mRNA and protein were assessed after 1 or 2 d, respectively. Expression of mRNA was monitored by both quantitative and qualitative RT-PCR. AID protein was assessed, by two-color flow cytometry of CFSE-labeled cells and immunoblotting. Curcumin significantly down-regulated AID mRNA and protein, in dose dependent fashion. Accompanying this decline was a diminished recovery of IgG+ class-switched B cells within the divided population. This study suggests that curcumin could have a role in treating B cell autoimmune disease and reducing the risk of malignant transformation.
B-cell Delivered Gene Therapy for Tolerance in EAE: Role of Antigen-specific B Cells (96.27)
Authors: Zhang Ai-Hong , Onabajo Olesegun , Su Yan , Skupsky Jonathan , Scott David
Published: January 2023

B-cell delivered gene therapy has been useful for tolerance induction in several mouse models for autoimmune diseases. However, it is not known whether Ag specific B-cells can be transduced to serve as tolerogenic antigen-presenting cells (APC), since such cells would be present in primed hosts. To test this, LPS activated splenic B cells were transduced with a retroviral vector encoding the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG35-55) in frame with IgG (MOG-Ig). Unlike transduced B-cells from wild type C57Bl/6 (B6) donors, adoptive transfer of MOG-Ig transduced B-cells from MOG-specific BCR transgenic mice (αMOG B-cells) failed to ameliorate MOG35-55 induced EAE in either pretreatment or therapeutic protocols. However, αMOG B-cells transduced with control vector encoding OVA323-339 (OVA-Ig) were highly tolerogenic in terms of T-cell recall response to OVA323-339. Interestingly, significant protection in mice from EAE was observed when ~1% transduced αMOG B-cells were used in the EAE model (+99% MOG-Ig transduced B6 B-cells). These data suggested that Ag specific B-cells in nature can be transduced as tolerogenic APC when a non-relevant Ag was targeted, and Ag specific tolerance induction can still be achieved in the presence of low number of pathogenic antigen specific B-cells. (Supported by NIH grant RO1 AI035622 and a Pre-doctoral fellowship from the AHA)
Experimental extracorporeal photopheresis therapy prevents type 1 diabetes in non-obese diabetic mice (96.3)
Authors: Xia ChangQing , Chernatynskaya Anna , Campbell Kim , Clare-Salzler Michael
Published: January 2023

In our previous studies, we demonstrated that infusion of apoptotic cells significantly prevented type 1 diabetes (T1D) in non-obese diabetic (NOD) mouse. Extracorporeal photopheresis (ECP) is an apoptotic cell-based therapy in clinic for immune-mediated disorders. In this study we examined experimental ECP therapy in T1D prevention in NOD mice. We discovered that five weekly intravenous injections of ECP-treated NOD spleen cells significantly delayed diabetes onset when NOD mice started to receive treatment at 8 weeks of age. Further cell dose studies demonstrated that low dose ECP-treated spleen cells as low as 2x105/injection/mouse had similar diabetes protection compared to high dose (5x106). In contrast to ECP therapy alone, ECP therapy combined with β cell antigens appeared to improve the protective effect with markedly reduced insulitis in pancreas. ECP and ECP plus β cell antigen therapies increased Foxp3+ Tregs in spleen, and β cell antigen-specific T cell proliferation was significantly suppressed in vivo in these two groups. In addition, we found that ECP therapy did not induce global immunosuppression as well as autoimmunity against nuclear materials. In conclusion, ECP therapy is a safe and effective approach in T1D prevention and has great potential for clinical translation to treat human T1D.
Efalizumab down-regulates CD25 expression on FOXP3+ regulatory T cells and exacerbates the autoimmunity in primary Sjogren’s syndrome (96.4)
Authors: Tran Dat , Bebris Lolita , Shevach Ethan , Illei Gabor
Published: January 2023

Rationale: FOXP3+ regulatory T cells (Tregs) are central to prevention of autoimmunity. Tregs expressed high level of CD25 and require IL-2 signaling for their function. Since CD25 is up-regulated with activation and LFA-1 is required, we hypothesize that efalizumab treatment would result in down-regulation of CD25 on Tregs and potential exacerbation of autoimmunity. Methods: Subjects with Sjogren’s syndrome were enrolled in a phase II clinical trial for weekly injection of efalizumab (1mg/kg) up to 25 weeks. Peripheral bloods were analyzed for Tregs by flow cytometry. Clinical conditions were measured by objective improvement in salivary and lacrimal flows and minor salivary gland (MSG) inflammation. Results: All three subjects had progressive down-regulation of CD11a and CD18 on Tregs (CD4+FOXP3+), which also correlated with dramatic decrease in CD25. There was no clinical improvement but instead repeat MSG biopsy showed significant increase in inflammation. Two subjects developed anti-dsDNA; one of whom developed multiple autoantibodies and lupus-like syndrome which resolved after efalizumab discontinuation. Conclusions: Efalizumab exacerbates Sjogren’s syndrome and induces significant increase in local inflammation and systemic autoimmunity. Inhibition of LFA-1 interaction on Tregs negatively affected their CD25 without altering their FOXP3 level. This study cautions the potential detrimental effect of efalizumab on Tregs and autoimmunity.
Anti-inflammatory effects of glucosamine in regulating the development of experimental autoimmune encephalomyelitis (EAE) (96.6)
Authors: Zhou Juhua , Nagarkatti Prakash , Zhong Yin , Nagarkatti Mitzi
Published: January 2023

We attempted to understand the anti-inflammatory regulation of glucosamine-6-phosphate (GS-6-P) and glucosamine-6-sulfate (GS-6-S) in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Clinical symptoms in MOG35-55-induced EAE mice significantly decreased following either oral administration or intravenous injection of GS-6-P, whereas only intravenous injection of GS-6-S inhibited EAE development. After clinical symptoms of EAE were induced, intravenous injection of both GS-6-P and GS-6-S could alleviate EAE in mice although GS-6-P was more potent. Further analysis showed that both GS-6-P and GS-6-S dramatically inhibited lymphocytic infiltration in the brain and spinal cord, recovered IL-10 producing cells to a normal level, and significantly decreased total number of splenocytes, CD4 T cell populations, IFN-γ producing cells and Th17 cells in mice. Besides, both GS-6-P and GS-6-S significantly inhibited the gene expression of IL-12, T-bet and IFN-γ, suggesting that glucosamine suppressed Th1 CD4 T cell differentiation and thus blocked EAE. GS-6-P also significantly increased IL-4 producing cells in spleen, but GS-6-S did not, suggesting GS-6-P could also promote Th2 CD4 T cell differentiation and thus further inhibited EAE. Our results suggested that GS-6-P could be a new therapeutic drug for MS treatment. (Supported in part by NIH grants R01AI053703, R01ES09098, R01 AI058300, R01DA016545, R01HL058641, ES 019313 and P01AT003961).
Tumor derived gangliosides promote regulatory cell activity (98.10)
Authors: Jales Alessandra , Falahati Rustom , Herzog Dallen , Ladisch Stephan , Leitenberg David
Published: January 2023

Gangliosides, shed by tumor cells, have been shown to have immunomodulatory properties, suggesting that gangliosides may be a critical factor in initiating an immunosuppressive tumor microenvironment. To better define the immunomodulatory properties of gangliosides on antigen-specific T cell activation and development, independent of other tumor associated factors, we have developed an in vitro system utilizing ganglioside-treated murine bone marrow derived dendritic cells to prime and activate antigen-specific CD4+ T cells from AND T cell receptor transgenic mice. Ganglioside treatment promotes the development of a regulatory DC population characterized by decreased CD86 expression, and decreased IL-12 and IL-6 production. CD4 T cells primed with ganglioside-treated DCs proliferate normally, but have a defect in Th1 effector cell development and IL-2 production. This defect in Th1 effector cell responses is due in part to the development of regulatory T cell activity that can suppress the activation of previously primed Th effector cells in a contact-dependent manner. Depletion of CD25+FoxP3+cells, and addition of exogenous IL-2 partially rescues Th1 responses in effector T cells co-cultured with ganglioside primed T cells. Collectively, these data suggest that ganglioside-exposed dendritic cells promote regulatory T cell activity that may have long lasting effects on the development of tumor specific immune responses.
Developing regulatory dendritic cells with the pregnancy estrogen estriol (98.11)
Authors: Muth Dillon , Taylor Cassandra , Whitacre Caroline , Boyaka Prosper , Papenfuss Tracey
Published: January 2023

In addition to inflammatory (immunogenic) capabilities, DCs have a potent role in regulating inflammation and immunity. We have found that DCs from estriol (E3) treated mice are activated regulatory DCs (regDCs). Specifically, these splenic E3 regDCs have elevated CD80, CD86, PD-L1, PD-L2, B7-H3 and B7-H4, hypo-produce IL-12, inhibit T cell proliferation and, upon adoptive transfer, protect mice from EAE through promoting Th2 responses. In these studies, we generated regDCs in vivo and in vitro and developed an in vitro correlate to splenic E3 regDCs. Mice given murine Flt3L (mFL)-secreting cells expanded both CD11b-CD11c+ and CD11b+CD11c+ cells, demonstrated mononuclear hyperplasia within bone marrow, lymph nodes and spleens and E3-exposed DCs were phenotypically similar to human Flt3L (hFL) E3 regDCs. Bone marrow DCs (BM-DCs) were generated with mFL, hFL or GM-CSF with or without E3. E3 inhibited FL BM-DC differentiation while GM-CSF BM-DCs were less affected. Phenotypic evaluation showed elevated CD80, CD86, PD-L1 and PD-L2 and CD4+ T cells proliferated less when cultured with E3 BM-DCs. These results demonstrate that E3 generates regDCs in vitro and in vivo and demonstrate an in vitro correlate to in vivo generated E3 regDCs. Generation of regDCs has important therapeutic applications in autoimmunity and inflammatory diseases.
Maturation of dendritic cell by edible mushroom Sparassis crispa (98.15)
Authors: Kim Hyung Sook , Kim Jee Youn , Roh Eunmiri , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Dendritic cells (DCs) play a pivotal role in the initiation of T cell-mediated immune response, making them an attractive cellular adjuvant for use in cancer vaccines. During the evolution of neoplastic diseases, dendritic cell (DC) functions are usually attenuated, and this presents a problem to DC-based immunotherapies against cancer. Here, we investigated the effects of beta-glucan (SCG), purified 6-branched 1, 3-beta-D- glucan obtained from Sparassis crispa, on Dc maturation. SCG efficiently increased the maturation of tlr4+/+ DCs from C57BL/6 and C3H/HeN mice, but not tlr4-/- DCs from C3H/HeJ mice. Phenotypic maturation was confirmed by the elevated expressions of CD40, CD80, CD86, MHC-I, and MHC-II molecules, and functional maturation by increased in the gene expression of IL-12, IL-1beta, TNF-alpha, IFN-alpha, IFN-beta production and decreased endocytosis. Also, SCG made DCs to activate allogenic T cells to proliferate and produce cytokines such as IL-2 and IFN-gamma. As a mechanism of action, SCG activated mitogen-activated protein kinases, proved by enhanced phosphorylation of ERK, p38 and JNK. In addition, SCG increased nuclear translocation of NF-κB p65/p50, which are signaling molecules down-stream of toll-like receptor 4 (TLR4). These results indicate that SCG induces DC maturation through TLR4.
Association of TREM2-DAP12 with DAP10 is required for the regulation of PI3K in macrophages (98.18)
Authors: Peng Qisheng , Malhotra Shikha , Humphrey Mary Beth
Published: January 2023

The regulation of PI3K following TREM2 ligation in macrophages is unclear and was the focus of this study. Immunoprecipitation studies show that DAP10 signaling adapter protein is required for recruitment of PI3K (p85 subunit) to a TREM2-DAP12 complex following TREM2 ligation. Once recruited to the complex, PI3K mediates TREM2-DAP12 dependent activation of AKT and ERK1/2. Compared to wild type macrophages, TREM2 stimulation failed to induce p85 recruitment to DAP12 in DAP10-deficient macrophages leading to a significant reduction in activated AKT and ERK1/2. Calcium flux induced by TREM2 ligation remained intact in DAP10-deficient macrophages, indicating that it is DAP10-independent. Both DAP10 and DAP12 could associate with TREM2 and each other in macrophages suggesting a tri-molecular complex of TREM2-DAP12-DAP10. We further show that the recruitment of PI3K to TREM2-DAP12 complexes is negatively regulated by the lipid phosphatase, SHIP1, in a DAP12 ITAM- dependent manner. Confocal study showed that DAP10 is not required for the translocation of SHIP1 or DAP12 to the membrane. Thus, our data suggests that DAP10 is required for the recruitment and activation of PI3K induced by TREM2-DAP12 signaling but not for calcium flux. Experiments are now being done to further understand the role of this tri- molecular complex (TREM2-DAP12-DAP10) leading to optimal functioning of bone marrow- derived macrophages.
PU.1 is required for generating the IL-9-producing Th9 phenotype (99.10)
Authors: Yao Weiguo , Tepper Robert , Kaplan Mark
Published: January 2023

IL-9 secreting Th9 cells are a newly defined subset of CD4 T cells that differentiate in the presence of TGF-β and IL-4, and that we have found have increased expression of the transcription factor PU.1. In a cohort study on infants with dermatitis, we found cultured PBMCs from atopic patients secreted higher level of IL-9 than non-atopic patients following anti-CD3 stimulation. In addition, specific cat or mite allergen stimulation of these PBMCs resulted in higher IL-9 production in cultures from atopic patients than from non-atopic patients. To investigate the underlying mechanism of IL-9 production and the development of Th9 cells in human allergic disease, we differentiated Th9 cells from naïve CD4 T cells isolated from atopic and non-atopic groups. We found that, after differentiation, Th9 cells from atopic patients have higher expression of PU.1 and IL-9 than those from non-atopic patients. Moreover, atopic samples showed increased pSTAT6 levels during differentiation, compared to samples from non-atopic patients. To determine if PU.1 is a regulator of the Th9 phenotype, we transfected Th9 cultures from normal human PBMCs with PU.1-specific siRNA. Our data showed that decreasing PU.1 expression impaired IL-9 production. In conclusion, these data demonstrate a critical role of PU.1 in the regulation of Th9 differentiation and suggest that PU.1-expressing Th9 cells may be involved in human allergic disease.
Dec2 regulates initial TH2 lineage commitment (99.12)
Authors: Yang Xuexian , Angkasekwinai Pornpimon , Zhu Jinfang , Peng Juan , Liu Zhiduo , Nurieva Roza , Liu Xikui , Chung Yeonseok , Chang Seon Hee , Sun Bing , Dong Chen
Published: January 2023

How naive CD4+ T cells commit to the T helper type 2 (TH2) lineage is poorly understood. Here we show that the basic helix-loop-helix transcription factor Dec2 is selectively expressed in TH2 cells. CD4+ T cells from Dec2-deficient mice exhibits defective TH2 differentiation in vitro and in vivo in an asthma model and in response to challenge with a parasite antigen. During early TH2 differentiation, Dec2 directly binds to and activates transcription of the Junb and Gata3 genes and promotes interleukin 4 expression. In addition, over-expression of GATA3 or JunB rescues TH2 cytokine expression in Dec2-deficient CD4+ T cells while optimal expression of these cytokines requires Dec2. As GATA3 induces Dec2 expression, these findings also indicate a feed-forward regulatory circuit during TH2 differentiation.
Cutaneous delivery of Poly(dG) conjugated protein antigens targets and activates DCs in vivo.(100.32)
Authors: Toussaint Helene , Erdos Geza , Donahue Cara , Falo, Jr. Louis
Published: January 2023

Efficient and specific targeting of exogenous antigen to antigen presenting cells (APCs) in vivo is a critical challenge for vaccine design. APCs acquire antigens from a variety of sources and endocytic mechanisms can predetermine intracellular antigen routing, processing and presentation to CD4+ or CD8+ T cells. We have designed an APC targeting strategy based on simple and direct coupling of Poly(dG) oligodeoxynucleotides to protein antigens. In comparison to soluble OVA, we find that conjugates of Poly(dG) and OVA (OVA-Poly(dG)) are rapidly internalized by dendritic cells (DCs) and follow distinct intracellular processing pathways. Functionally, OVA-Poly(dG) is both presented to OVA-specific CD4+ T cells (OT-II cells) and efficiently cross-presented to OVA-specific CD8+ T cells (OT-I cells) by DCs in vitro. Importantly, immunization of naïve animals by direct injection of OVA-Poly(dG) results in efficient priming of potent antigen specific CTL responses. Further, OVA-Poly(dG) generate a specific memory CD8+ T cells response and promote OVA-specific antibody responses. Extending our findings to human skin, we show that I.D. injection of antigen-Poly(dG) conjugates results in efficient uptake by human skin migratory DCs, preferentially dermal CD14+DCs that also acquire potent T cell allostimulatory function. Our data suggests that Poly(dG) can both target antigens to human skin immune APCs and function as an adjuvant to induce potent CD4 and CD8 mediated T cell immunity.
Regulation of expression of “pancreatic” proteases in dendritic cells: a novel mechanism of peripheral tolerance (100.34)
Authors: Farkas Adam , Finn Olivera
Published: January 2023

MUC1 is a tumor-associated antigen. MUC1-specific TCR transgenic CD4+ T cells transferred into MUC1 Tg mice show less proliferation in response to vaccination with DC loaded with MUC1 peptide (DC/MUC1p) compared to WT. To query the underlying regulatory mechanisms, we vaccinated WT and Tg mice with DC/MUC1p and conducted transciptome analysis of splenic RNA. Proteases previously characterized as pancreas-restricted (Trypsin, Elastase and Carboxypeptidase B1) were significantly down-regulated in splenic DC from Tg mice relative to WT. Vaccination with soluble MUC1p gave the same result. Protease mRNA down-regulation in DC correlated with the reduction at the protein level. In related work we have shown that these enzymes are regulated by DC/T effector versus T reg interactions. Inhibition of Trypsin or CPB1 decreases the ability of DC to elicit MUC1-specific CD4 T cell response in vitro, supporting a correlation between deficient protease expression in DC and reduced T cell response in vivo. DC from MUC1p-vaccinated Tg mice express lower levels of MHC II and costimulatory molecules concurrent with decreased protease levels. These proteases are found in MHC II+ and lysosomal compartments, sites of antigen processing. We suggest that one mechanism of peripheral tolerance that regulates anti-MUC1 immunity is inhibition of antigen processing. We propose that pharmacologic modulation of these enzymes could be one approach to improving the potency of TAA-based cancer vaccines.
A new dynamic model of three cell interactions by CD4+ T-licensed DCs and DC-primed CD4+ T cells (100.38)
Authors: Xiang Jim
Published: January 2023

CD8 cytotoxic T lymphocytes (CTLs) kill pathogen-infected cells. Recent in vivo imaging study suggested that presence of DC-CD4 T clusters during CTL priming are essential for T-cell memory, challenging independent roles for the DCs or the CD4 T-cells that dissociate following DC-CD4 T interactions. We have developed a novel in vivo experimental system based on the rapid inducible ablation of diphtheria toxin receptor (DTR) transgenic-DCs and antibody-mediated depletion of CD4 and CD8+ T cells in mice to create DC-CD4 T interactions in vivo first, followed by killing of either CD4 T-licensed DCs or DC-primed CD4 T cells. Later, adoptively transferring naive polyclonal CD8 T cells facilitated their interactions with either licensed-DCs or primed-CD4 T cells, thereby revealing their respective roles in CTL immunity. Our results provide direct in vivo evidence that CD4 T-cells and DCs, once activated by DC-CD4 T interactions, can stimulate CTL responses and programme memory on CTLs independent of DC-CD4 T clusters, uncovering the co-existence of direct and indirect mechanisms of T-cell help for CTLs by licensed DCs and primed CD4 T cells in non-inflammatory situations. In situations in which CTL immunity is heavily dependent on CD4 T-cell help, our findings suggest a new dynamic model of three cell interactions in CTL responses, that dissociated stage of primed CD4 T-cells and licensed DCs represent crucial “immune-intermediates” for CTL responses and memory development.
MHC class I-related FcRn-mediated presentation of immune complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells (100.56)
Authors: Zhu Xiaoping , Liu Xindong , Lu Li , Yang Ziyan , Palaniyandi Senthilkumar , Zeng Rongyu , Gao Lian-Yong , Mosser David , Roopenian Derry
Published: January 2023

The Fc receptors for the immunoglobulin (Ig) G antibody (FcgammaRs) found on macrophages and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed (IC)-antigens to T cells. We found that the neonatal Fc receptor for IgG (FcRn) in both macrophages and DCs failed to have a strong effect on the cross-presentation of IC ovalbumin (OVA) antigen to CD8+ T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to OT-II CD4+ T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on antigen presentation between macrophages and DCs. The presentation of phagocytosed latex-OVA-ICs to CD4+ T cells was also considerably enhanced on wild-type versus FcRn-deficient macrophages, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to below 6.5 in macrophages but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on antigen processing and presentation of ICs to CD4+ T cells in the endosomal versus phagosomal compartments of macrophages versus DCs.
Foxo1 is essential for germinal center B cell functions and the development of collagen-induced arthritis (101.15)
Authors: Chang Shih-En , Zheng Biao
Published: January 2023

Foxo1 has been shown to be critical for B-cell lymphopoiesis by regulating several genes involved in immunoglobulin (Ig) rearrangement. However, the role of Foxo1 in germinal center (GC) B cell functions remains unclear. Unlike being downregulated in activated B cells, Foxo1 expression is elevated in GC B cells, suggesting a distinctive regulatory role. In Foxo1 GC B-cell conditional knockout (CKO) mice, both primary and memory antibody responses are severely impaired after immunization with T-dependent antigen NP-CGG. The numbers of NP-specific IgG1-secreting B-cells, as well as the NP-specific IgG1 antibody titer in the serum, are dramatically reduced in the mutant mice. The results indicate an inability to undergo Ig class-switch recombination (CSR) in Foxo1-deficient GC B cells, possibly due to the abolished expression of activation-induced cytidine deaminase. In addition, analysis of mutation rate of Ig genes in the NP-specific B cells reveals that Ig somatic hypermutation (SHM) is impaired in the Foxo1-deficient GC B cells. The clonotypes of B cells responding to NP are also altered in the mutant mice. Remarkably, using collagen-induced arthritis model, we have found that Foxo1 GC B-cell CKO mice are resistant to arthritis induction. Taken together, our data indicate that Foxo1 is essential for normal antibody responses, CSR and SHM of GC B cells. The absence of Foxo1 in GC B cells prevents the development of autoimmune arthritis.
Extra-telomeric function of telomerase in rheumatoid arthritis (101.2)
Authors: Shao Lan , Fujii Hiroshi , Grisar Johannes , Jörg Goronzy , Weyand Cornelia
Published: January 2023

Telomerase, an enzyme that adds DNA onto telomeric ends is upregulated in T lymphocytes upon antigen recognition. How telomerase affects T-cell function is unknown. In patients with the autoimmune syndrome rheumatoid arthritis (RA) induction of T-cell telomerase is impaired and associated with premature immunosenescence. To suppress telomerase activity healthy naïve CD4 T cells were stimulated through the T-cell receptor and transfected with control or hTERT-specific siRNA. Telomerase inhibition significantly increased p53 mRNA (p=0.05) and p53 protein (p=0.002). Pro-apoptotic BH3-only proteins were selectively upregulated; PUMA was increased (p=0.04), Noxa and Bim remained unaffected. hTERT silencing doubled frequencies of apoptotic cells. In RA T cells hTERT expression was spontaneously reduced (p<0.0001); immunostaining localized most of the protein to the cytoplasm (p=0.002). Upon TCR ligation RA T cells had higher levels of p53, phosp53 and PUMA. p53 or PUMA knockdown effectively rescued RA T cells from apoptosis. hTERT over-expression suppressed p53 and PUMA and normalized cellular yield. p53 and PUMA silencing did not correct hTERT expression, placing telomerase upstream of the p53-pathway. In conclusion, T-cell telomerase has extra-telomeric functions and regulates life-death decisions after antigenic stimulation. In patients with rheumatoid arthritis, the survival-protective function of telomerase is defective, causing T cell loss and proliferative stress.
A unique T cell subset, Th40, are pathogenic and diagnostic in mulitple sclerosis (101.30)
Authors: Waid Dan , Carter Jessica , Vaitaitis Gisela , Wagner Rebecca , Gottlieb Peter , Corboy John , Fitzgerald-Miller Lisa , Wagner David
Published: January 2023

Auto-aggressive T cells have been implicated in the pathogenesis and maintenance of MS, including mouse models and human disease. We described a T cell subset, Th40 cells, which proved highly pathogenic and auto-aggressive in autoimmune diabetes. Using flow cytometry, we compared Th40 levels in 90 MS subjects of all subtypes, 238 T1DM and 208 control patients. A subset of MS patients were followed during and after pregnancy as well. RRMS subjects have significantly increased percentages of Th40 cells in PBL compared to non-autoimmune controls. MS subjects (75) had Th40 cell levels equal to or greater than autoimmune T1D (238) subjects. The majority of Th40 cells from MS subjects' are T helpers producing pro-inflammatory cytokines IFNg, TNFa and IL-17. Th40 cells produce high levels of IL-6, an acute phase response cytokine that shunts regulatory T cells into Th17 pathway. During RRMS Th40 cell expansions occur regardless of age or gender and are responsive to known MS associated self antigens including MOG, MBP and PLP. A clonal expansion of TRAV 8.1.3+ cells within the Th40 population was evident. During pregnancy Th40 cell levels dropped dramatically corresponding with remission returning to pathogenic levels postpartum. In the mouse EAE model of MS Th40 cells are highly expanded. Th40 cells could prove diagnostic in RRMS and prove diagnostic to predict relapse. Th40 cells are pathogenic in MS and identification of a clonal expansion in MS further suggests autoimmunity.
Identification of a second mimicry epitope from Acanthamoeba castellanii that induces CNS autoimmunity through the generation of cross-reactive T cells for myelin basic protein 89-101 (101.35)
Authors: Massilamany Chandirasegaran , Steffen David , Reddy Jay
Published: January 2023

Autoimmune responses to myelin antigens have been implicated in the pathogenesis of multiple sclerosis and this requires the mediation of autoreactive T cells and B cells, but the mechanisms by which the disease is initiated are unknown. Environmental microbes are commonly suspected in the initiation of the disease and emerging evidence suggests that exposure to multiple organisms might be a critical requirement to trigger central nervous system (CNS) autoimmunity. We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139-151 capable of inducing autoimmune encephalomyelitis in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89-101 namely, NAD 108-120 and the mimicry sequence is derived from NADH dehydrogenase subunit 2 of Acanthamoeba. NAD 108-120 shares 46% sequence identity with MBP 89-101 and it induces clinical and histological EAE similar to that of cognate peptide through the generation of cross-reactive T cells and proinflammatory cytokines that favor CNS autoimmunity. This is the first report to demonstrate that a solitary microbe can predispose to CNS autoimmunity by generating cross-reactive T cells for multiple myelin antigens.
T cell specific deletion of the phosphatase SHP1 decreases incidence and severity of experimental autoimmune encephalomyelitis (101.41)
Authors: Edwards Lindsay , Mauldin Ileana , Lorenz Ulrike , Evavold Brian
Published: January 2023

T cell activation is regulated by a dynamic balance between kinase and phosphatase activity. The tyrosine phosphatase SHP1 is a critical negative regulator of the immune system, and a proximal regulator of T cell receptor signaling. Here, we have utilized two novel models to study the deletion of SHP1 specifically in mature T cells. We have bred mice bearing a floxed SHP1 allele that deletes the majority of the gene with mice bearing an inducible Cre (Mx1 promoter). We also utilized a dominant negative allele of SHP1 whose expression requires Cre-driven recombination. By transferring mature T cells from these mice into T cell deficient hosts and subsequently inducing Cre expression we have isolated the effect of loss of SHP1 activity to T cells. We have determined that selective loss of this phosphatase in mature T cells dramatically attenuates experimental autoimmune encephalomyelitis (EAE). Both severity and incidence were decreased significantly in the conditional knockout and dominant negative mice relative to controls (mean max score 1.0, 1.7, and 2.7, respectively and mean incidence 29, 37 and 79%). These data differ from results in mice heterozygous for the motheaten allele, a naturally occurring SHP1 mutant, which exhibit EAE severity that is similar to or slightly worse than controls. Collectively, this suggests that lack of SHP1 activity in T cells may protect from disease, while deletion in other cell types may exacerbate susceptibility or symptoms.
Memory phenotype CD8 T cells generated in response to inducible self antigen (101.42)
Authors: Jellison Evan , Turner Michael , Blair David , Lefrançois Leo
Published: January 2023

Chronic antigenic stimulation of CD8 T cells by self antigen or chronic infection results in an altered CD8 T cell response and may lead to antigenic tolerance. To study these processes we generated transgenic mice in which antigen (Ag) expression is tightly controlled in DCs by doxycycline (dox). Our system provides the unique ability to examine endogenous CD8 T cell responses to neo-self Ag in the absence of overt inflammation. Dox can be given both chronically and acutely, and Ag expression ceases upon dox withdrawal. Importantly, the transgenic mice are ignorant of Ag prior to induction. Chronic antigen induction (dox on) in these mice resulted in a massive expansion of endogenous Ag-specific CD8 T cells. They upregulated CD44 and LFA-1, and expressed CD69 and PD1, but predominantly did not express KLRG1 or CD127, nor did they generate effector cytokines. Upon Ag removal (dox on, then dox off), the Ag-specific CD8 T cell population waned but the surviving population was enriched for CD127+ memory phenotype cells. The remaining CD8 T cells recalled to subsequent pathogen challenge but did not recall to self Ag challenge (dox back on). These studies indicate that chronic self Ag exposure generates a CD8 T cell population that is non functional, but persists through an addiction to Ag. However, when self Ag is removed, a functional memory phenotype CD8 T cell population emerges, which is regulated depending on the context of subsequent Ag presentation.
Activation of TLR4 on endothelium alone initiates neutrophil adhesion within the liver microcirulation during endotoxemia (102.22)
Authors: McDonald Braedon , Zhuo Lisheng , Kimata Koji , Kubes Paul
Published: January 2023

During sepsis and endotoxemia, neutrophils are recruited to the liver where they cause tissue damage and vascular dysfunction. We have previously reported that neutrophils adhere within sinusoids of the endotoxemic liver via interactions between neutrophil CD44 and endothelial hyaluronan. In this study, we aimed to characterize the cellular sentinels that detect circulating LPS via TLR4 and the pathways leading to initiation of neutrophil adhesion via CD44-hyaluronan interactions. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow derived cells was required for neutrophil recruitment into the liver during endotoxemia (1 mg/kg LPS, i.v.). Furthermore, LPS-induced neutrophil adhesion in sinusoids was equivalent between wild-type mice and transgenic mice that express TLR4 only on endothelium (tlr4-/-Tie2tlr4, named endoTLR4), but surprisingly, sinusoidal occlusion (vascular damage) was attenuated in endoTLR4 mice. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 induced the deposition of serum-derived hyaluronan associated protein (SHAP) within liver sinsusoids, which was required for activation of endothelial hyaluronan to bind neutrophil CD44. These data reveal that endothelial cells are the key sentinels that recognize circulating LPS and initiate neutrophil adhesion in the liver during endotoxemia, but the development of liver pathology requires TLR4 activation on additional cell types.
TLT2 signaling regulates a positive feedback loop that promotes neutrophil activation and chemotaxis. (102.24)
Authors: Halpert Matthew , King R. Glenn , Justement Louis
Published: January 2023

TLT2 is one of four receptors conserved between mouse and human within the TREM locus, and is expressed on B cells, macrophages, and neutrophils. TLT2 ligation on murine macrophages induces the production of chemokines and growth factors, as evidenced by ex vivo treatment with αTLT2 mAbs. This is recapitulated in vivo following injection of αTLT2 mAbs resulting in the production of chemokines and growth factors, which ultimately lead to enhanced neutrophil recruitment. Ligation of TLT2 on murine neutrophils results in an enhanced respiratory burst, as well as potentiation of degranulation and chemotaxis in response to agonists that bind G protein coupled receptors (GPCR), including FMLP, KC, MIP-2, IL-8, and C5a. However, the neutrophil responses to GM-CSF, LPS, and FcR ligation were unaltered, suggesting that TLT2 specifically potentiates the neutrophil response to signals derived from GPCRs. Administration of αTLT2 mAb results in enhanced neutrophil accumulation in response to nonspecific inflammatory mediators in vivo, and competitive adoptive transfer experiments demonstrate enhanced recruitment of αTLT2 mAb treated neutrophils compared to control neutrophils in response to lung inflammation. These results demonstrate that TLT2 regulates an intricate feed-forward loop wherein it is important for driving the production of factors that recruit neutrophils, as well as enhancing the response of neutrophils towards chemokines and bacterial products that signal via GPCRs.
Langerhans cells are critical in the pathogenesis of atopic dermatitis via TSLP receptor signaling (103.1)
Authors: Nakajima Saeko , Miyachi Yoshiki , Kabashima Kenji
Published: January 2023

In the skin, there exist at least two populations of dendritic cells (DCs), epidermal Langerhans cells (LCs) and dermal DCs. It has been reported that cutaneous DCs mediate the pathogenesis of atopic dermatitis (AD), but it remain unknown which subset is directly involved. Using mice with Langerin-diphtheria toxin receptor, we found that LC depletion showed much milder clinical manifestations together with lower serum IgE levels in a topical OVA application-induced AD-like mouse model. In addition, LC depletion impaired OVA-specific T cell proliferation and IL-4 production in the draining lymph nodes. Recent work has identified TSLP as promoting Th2-dominant inflammations. Since TSLP is expressed in the epidermis, we hypothesize that TSLP secreted by keratinocytes acts on epidermal LCs to prime naïve T cells into Th2 cells. To this end, we used TSLP receptor-deficient mice and newly generated LC-specific TSLP receptor-deficient mice by bone marrow reconstitution of wild-type mice into irradiated TSLP-receptor deficient mice by taking advantage of radio-resistance of LCs. We induced AD-like skin conditions to these mice by sensitization with OVA percutaneously. We found that LC-specific TSLP receptor-deficient mice showed much milder clinical and histological findings together with lower IL-4 and IgE levels. Taken together, these results suggest that epidermal LCs play a critical role in the development of AD via TSLP-TSLP receptor signaling.
Lung surveillance revealed by two-photon live imaging (103.3)
Authors: Thornton Emily , Looney Mark , Sen Debasish , Sheppard Dean , Huang Xiaozhu , Krummel Matthew
Published: January 2023

The lung is a major mucosal site of allergen and pathogen encounter and is also the site of damage resulting in immune-based repair. Despite the interest in how immune cells enter and function in this organ, direct observation of these events has not been possible due to obligate movements made during respiration and blood flow. Real-time imaging of cellular and sub-cellular dynamics in the lung requires image resolution, image-registration, and intact physiology to be simultaneously optimized. We have developed both in vitro slice and intravital stabilized imaging systems. We have applied these methods to visualize how phagocytes function along this mucosal surface. Imaging under basal conditions reveals constitutive surveillance of the alveolar surface by resident dendritic cells (DC). These DCs actively sweep the epithelium, taking up model antigens, whereas alveolar macrophages (AMs) lie immotile within the alveolar space. DC number dramatically increases after allergen challenge; however, sampling behavior is not affected, leading to increased antigen uptake by these proinflammatory cells. These antigen-positive DCs then play a critical role in the activation of T cells visualized in this system. We will discuss the integration of innate and adaptive cells as it occurs in the lung.
A critical functional role for neuroimmune semaphorin 4A in asthma severity (103.7)
Authors: Nkyimbeng-Takwi EusebiusHenry , Shanks Kathleeen , Smith Elizabeth , Lipsky Michael , DeTolla Louis , Keegan Achsah , Chapoval Svetlana
Published: January 2023

Semaphorin 4A belongs to a family of secreted and membrane-bound glycoproteins which were originally found to be expressed in the nervous system and function as axon guidance molecules. Recent studies identified Sema4A to be expressed and function in the immune system. An abundant Sema4A expression was found on APC but not on resting T cells. The expression was upregulated with corresponding cell activation. Sema4A on APC has been reported to enhance activation and differentiation of T cells in vitro and generation of Ag-specific T cells in vivo. We hypothesized that Sema4A plays a critical regulatory role in allergic airway response. To test our hypothesis, we employed Sema4A-/- and WT mice in the well-defined mouse model of OVA-induced experimental asthma. We observed a significant increase in eosinophilic airway infiltration accompanied by bronchial epithelial cell hyperplasia in allergen-treated Sema4A-/- mice as compared to similarly treated WT mice. This enhanced inflammatory response was associated with a selective increase in BAL IL-13 content. Surprisingly, an augmented AHR was detected in both PBS- and OVA-treated Sema4A-/- mice versus similarly treated WT mice. Allergic response was downregulated by re-introduction of recombinant Sema4A protein. However, the in vitro recall T cell response to OVA was not affected by Sema4A deficiency. These data provide a new insight into Sema4A biology and defines it as a critical regulator of Th2-driven lung pathophysiology.
Alterations in pulmonary immunity in response to vaccinia virus (103.8)
Authors: Walline Crystal , Benson Heather , Sehra Sarita , Mwanthi Muithi , Fisher Amanda , Clapp D. Wade , Brutkiewicz Randy , Kaplan Mark , Wilkes David , Blum Janice
Published: January 2023

Vaccinia virus (VV) infection in mice is an accepted model to study the pathogenesis of variola virus, the causative agent of human smallpox. Although the lung is a key site of poxvirus infection, there is little known about the local immune response to intrapulmonary VV infection. Additionally, because vaccination with VV is contraindicated in patients with atopic allergic skin conditions it is very important to determine the role of atopic lung disease on local vaccinia immunity. We have found that established allergic lung disease results in significantly increased numbers of eosinophils, myeloid dendritic cells (mDCs) and alveolar macrophages. Interestingly, an intrapulmonary VV infection in mice with allergic lung disease does not further increase the number of mDCs or alveolar macrophages, but does trigger a significant increase in plasmacytoid DCs (pDCs) and a significant decrease in eosinophils. Moreover, pulmonary eosinophils, mDCs and macrophages from mice with allergic lung disease were less susceptible to an in vitro VV infection than the respective cells from naïve mice. Whereas pulmonary pDCs and B cells from mice with allergic lung disease were more susceptible to an in vitro VV infection. Alterations in antigen presentation were also observed using splenocytes from animals with induced hypersensitivity. Taken together, these studies strongly suggest that the allergic lung microenvironment may influence anti-viral immunity to an intrapulmonary VV infection.
Optical marking of migrating lymphocytes by two-photon photoconversion for single cell tracking and fate mapping (104.22)
Authors: Phan Tri , Hampton Henry , Tomura Michio , Kanagawa Osami , Mackay Charles , Brink Robert , Chtanova Tatyana
Published: January 2023

We present a method for localised two-photon photoconversion of fluorescent cells within distinct microanatomical compartments in intact lymph nodes. This method allows efficient photoconversion of whole cell populations as well as of single cells while preserving cell viability and motility. Moreover, the combination of high-speed image acquisition and real-time display permits us to target and lock-on to individual cells for photoconversion as they migrate within the lymph node. Optical cell marking makes it possible to differentiate otherwise homogenous responding cells based on their microanatomical location. It also enables discontinuous in vivo tracking of cell fates to distal sites after extended time intervals.