bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-depleting anti-CD4 antibody alleviates ovalbumin-induced airway inflammation and T-helper type 2 responses in mice (140.13)
Authors: Wu Cheng-Jang , Lin Chun-Yen , Kuo Ming-Ling
Published: January 2023

Bronchial asthma is an emerged chronic inflammatory disorder of the airways. It is becoming urgent to identify target molecules for the preventive and therapeutic purposes. Non-depleting anti-CD4 monoclonal antibody, YTS177, has been used to induce immunotolerance in allograft transplantation through antigen-specific Treg cells. This study aims for determining whether YTS177 is able to inhibit the ovalbumin (OVA)-induced allergic inflammation in an asthmatic murine model. The injection of YTS177 before and after OVA-sensitization was able to suppress airway hyperresponsiveness, pulmonary eosinophilia, OVA-specific immunoglobulin (Ig) E synthesis, and the production of T helper (Th) 2 cytokines from OVA-activated splenocytes. Furthermore, YTS177 had the therapeutic capacity to relieve the attack of asthmatic symptoms when it was administrated after the mice were sensitized and challenged with OVA. However, the depletion of CD25+ cells did not reverse YTS177-induced hyporesponsive status. The state might be resulted from the angery of CD4+ T cells with the increased level of anergy factor-Cbl-b in activated CD4+ cells cultured with YTS177. Moreover, the high level of cell cycle inhibitor p27Kip1 and increased number of G1 phase arrested-T cells were detected. Therefore, p27Kip1 might be involved in the preventive and therapeutic efficacy of YTS177 for allergic diseases.
Identification of TSLP-dependent mediators in a calcipotriol (Dovonex®) driven mouse model of skin inflammation (140.16)
Authors: Jessup Heidi K , Comeau Michael R
Published: January 2023

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is sufficient to induce asthma or atopic dermatitis (AD)-like phenotypes when selectively overexpressed in transgenic mice. Although T and B cells have been reported to be dispensable for the TSLP-induced inflammation in these models, little is known about the downstream pathways or mediators involved in the inflammatory response driven by TSLP. Topical treatment with vitamin D3 (1α,25-(OH)2D3) or MC903, the low-calcemic analog of vitamin D3, has been shown to induce TSLP expression in epidermal keratinocytes. This induction resulted in an AD-like phenotype similar to that seen in TSLP transgenic mice. 0.005% calcipotriol (Dovonex®) is a synthetic, low-calcemic vitamin D3 analog which is FDA-approved for the treatment of psoriasis. Following topical application of Dovonex® onto mouse ears, TSLP mRNA expression increased over time. Concomitant increases in CCL17/TARC, CCL22/MDC, and IL-6 mRNA were also observed. These increases were dependent on TSLP as an antibody that blocked TSLP signaling abrogated the response. Similarly, a variety of TSLP-dependent protein analytes were increased in the skin following application of Dovonex®. Together these data identify TSLP-dependent mediators that may play a role in the development of skin inflammatory diseases such as atopic dermatitis.
A Novel Anti-Inflammatory Role for Andrographolide in Asthma via Inhibition of The Nuclear Factor-kappaB Pathway (140.17)
Authors: Wong WS Fred , Bao Zhang , Guan Shou Ping , Cheng Chang , Kemeny D Michael , Leung Bernard P
Published: January 2023

Persistent activation of nuclear factor (NF)-κB has been associated with the development of asthma. Andrographolide, the principal active component of a medicinal plant Andrographis paniculata, has been shown to inhibit NF-κB activity. We hypothesized that andrographolide may attenuate allergic asthma via inhibition of the NF-κB signaling pathway. BALB/c mice sensitized and challenged with OVA developed airway inflammation. Andrographolide inhibited OVA-induced increases in total cell count, eosinophil count, and IL-4, IL-5 and IL-13 levels in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, chitinases, Muc5ac and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. In human lung epithelial cells, andrographolide blocked TNF-α-induced phosphorylation of inhibitory κB (IκB) kinase-β (IKKβ), and downstream IκBα degradation, p65 subunit of NF-κB phosphorylation, and p65 nuclear translocation and DNA-binding activity. Our findings implicate a potential therapeutic value of andrographolide in the treatment of asthma and it may act by inhibiting NF-κB pathway at the level of IKKβ activation. (This work was supported by a BioMedical Research Council grant BMRC06/1/21/19/443)
Specific blockade of protein kinase C (PKC)-theta with a small molecule antagonist attenuates lung inflammation in a mouse model of established allergic airway disease (140.21)
Authors: DeClercq Charlene , Demers Delia , Bree Andrea , Fish Susan , Wei Shui-Quing , Brennan Aggie , Kasaian Marion , Chen Joan , Wang Daniel , Boschelli Diane , Chaudhary Divya , Williams Cara
Published: January 2023

PKCθ is primarily expressed in T cells and is recruited to the immunological synapse upon T cell receptor signaling. PKCθ deficient mice display a diminished pulmonary inflammatory response following allergen challenge. We investigated the allergic airway response in mice when PKCθ is specifically blocked with a small molecule inhibitor (compound A). Mice were sensitized with ovalbumin (OVA) in alum on days 0 and 14, then challenged with OVA on days 26, 27, and 28 (primary challenges). Four weeks later mice were aerosolized once with OVA (rechallenge). Airway inflammation was monitored after both primary challenges and rechallenge. Compound A was dosed 30 mg/kg intraperitoneally twice daily in 3 dosing regimens: for 4 days concurrent with and after the primary challenges only; for 3 days starting before the OVA rechallenge only; and both of the above regimens. Administration of compound A significantly inhibited the influx of eosinophils, lymphocytes and neutrophils into the airways. Ex vivo antigen induced Th2 cytokine production by splenocytes was also reduced in cells obtained from mice treated with Compound A in either the primary challenge phase, or both primary and rechallenge phases, but not the rechallenge phase alone. We conclude that blocking T cell activation with a PKCθ inhibitor may attenuate airway inflammation in established allergic disease and may be beneficial in treating chronic allergic asthma.
AIMP1 as a negative regulator of airway inflammation via suppression of Th2 responses (140.3)
Authors: Hong Hye Jin , Kim Myun Soo , Kim Eugene , Kim Tae Sung
Published: January 2023

AIMP1 (aminoacyl-tRNA synthetase-interacting multifunctional protein1)/p43 is found to related to Lupus-like autoimmune disease. Previously we demonstrated that AIMP1/p43 activated dendritic cells and macrophages, leading to the induction of Th1 responses. In this study, we investigated a role of AIMP1/p43 in the pathogenesis of lung airway inflammation by characterizing a phenotype of AIMP1 knockout mouse. Asthma-like pathological changes were observed in the lung and the sensitivity to methacholine was also severely increased in AIMP1 knockout mouse. Histologic studies showed that AIMP1 knockout mice had even thicker alveolar walls than the wild type mice. The expression level of gob-5, a gene related to mucus production, was highly enhanced in AIMP1 knockout mice. Additionally GATA-3, a transcription factor inducing Th2 development, was dramatically increased in the lung homogenates of AIMP1 knockout mice. Furthermore, in the AIMP1 knockout mice, the total IgE level was increased, along with the levels of IgG1 and IgG2a. The levels of IL-4 production were significantly higher in lymphocytes of AIMP1 knockout mice after OVA restimulation, compared with those in the wild type mice. Taken together, these results indicate that the Th2 responses were strongly increased in AIMP1 knockout mice and suggest that AIMP1 functions as a negative regulator of the Th2-response in asthma-like airway inflammation.
Divergent Roles for Airway Epithelial MMP7 and Retinoic Acid in Experimental Asthma (140.5)
Authors: Goswami Sangeeta , Angkasekwinai Pornpimon , Shan Ming , Greenlee Kendra J , Barranco Wade T , Polikepahad Sumanth , Seryshev Alexander , Sur Sanjiv , Woodruff Prescott , Dong Chen , Corry David B , Kheradmand Farrah
Published: January 2023

The innate immune response of airway epithelial cells to aeroallergen likely initiates the development of T cell responses that are central to allergic inflammation. Although proteolytically active allergens induce the expression of interleukin (IL)-25/IL-17E, we show that epithelial matrix metalloproteinase 7 (MMP7) is expressed in human asthma, and is required for maximal activity of this cytokine in promoting T helper type 2 cell differentiation. Allergen-challenged MMP7-/- mice showed reduced airway hyperreactivity, allergic inflammatory cytokine, and increased expression of retinal dehydrogenase (RALDH)-1. Inhibition of RALDH-1 restored the asthma phenotype in MMP7-/- mice and inhibited lung T regulatory cell responses while exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating IL-25 while simultaneously inhibiting retinoid-dependent T regulatory cell development.
Disparate immune responses to Mi-HAg occur in vitro and in vivo (141.1)
Authors: Xu Hong , Bozulic Larry D , Zhu Ziqiang , Huang Yiming , Hussain Lala R , Ildstad Suzanne T
Published: January 2023

Historically, insights into the mechanism of alloresponses to minor histocompatibility antigen (Mi-HAg) disparities have focused on T cell activity and used in vitro assays. It was believed that prior in vivo immunization was required for T cell responses to Mi-HAg to occur in vitro. Here, we investigated the generation of adaptive immune responses to Mi-HiAg using an in vivo model. We found that AKR (H-2k) mice rejected skin grafts from MHC-matched, Mi-HAg-disparate B10.BR (H-2k) mice and generated significantly high levels of anti-donor Ab, suggesting that skin graft rejection was associated with the initiation of strong adaptive humoral responses. As B cell activation requires signaling from activated T cells, in vitro and in vivo MLR assays were performed to examine the immune response to Mi-HAg. AKR cells exhibited no reactivity to Mi-HAg disparate stimulators in in vitro MLR assays. In contrast, T cells from AKR mice exhibited strong reactivity in in vivo MLR assays to Mi-HAg in B10.BR mice. The reason for this disparity in response to Mi-HAg in vitro vs. in vivo MLR is not clear. The in vitro culture environment may lack some factors that were present in vivo and that are critical for generation of T cell immune responses to Mi-HAg. T cells were indeed activated in response to Mi-HAg, as CD44highCD62L-/low effector/memory T cells were generated.

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A novel method to assess the level of immunosupression in transplant patients using Amnis ImageStream technology (141.24)
Authors: Pankewycz Oleh , Minderman Hans , Wallace Paul , Feng Lin , Laftavi Mark R.
Published: January 2023

Contemporary immunosuppressive protocols rely on drug dosing guidelines gleaned from large clinical trials, but the effect of this therapy in a transplant recipient remains difficult to predict. To assess global immunosuppression in individual patients, we measured the degree to which NFkB translocation in peripheral T cells was impaired by conventional immunosuppressive therapy. NFkB is a ubiquitously expressed transcription factor normally sequestered in the cytoplasm. Upon activation, NFkB inhibitors are degraded, releasing NFkB to translocate to the nucleus. The abundance of NFkB in the nucleus vs. the cytosol reflects the activation state of a particular cell type. Peripheral blood cells were isolated from 17 subjects, stimulated in culture for 24 hrs with PMA and ionomycin and stained for T cell surface markers and the major intracellular isoform of NFkB, RelA or p65. Using Amnis Imagestream technology, we compared nuclear NFkB in resting and activated CD3, CD4 and CD8 T cell subsets. Compared to normal controls (n=8), transplant recipients on tacrolimus, MPA and prednisone showed reduced translocation of NFkB to the nucleus. Patients with stable allograft function (n=5) had a 36-75% reduction in nuclear NFkB translocation in all 3 T cell subsets. In contrast, patients with acute rejection (n=2) had an increase in ability to translocate NFkB in CD3 and CD8 cells and only a 9% suppression in CD4 cells. Patients with viral infections (n=2) had severely compromised NFkB translocation with 100% suppression in CD3 and CD4 subsets and 92% suppression in CD8 T cells. NFkB inhibition strongly correlates with overall immunosuppression and clinical outcomes. The novel Amnis Imagestream assay may be a useful guide to individualizing immune therapy or directing minimization strategies. (unfunded)
T cells reconstituting the periphery in thymoglobulin treated kidney transplant recipients are preferentially donor-senescent when compared to basiliximab treated patients (141.25)
Authors: Keslar Karen , Cherkassky Leonid , Valujskikh Anna , Shoskes Daniel , Fairchild Robert , Poggio Emilio
Published: January 2023

Induction with anti-thymocyte globulin (ATG) results in better 1 yr renal transplant outcomes than basiliximab in immunologically sensitized transplant patients. This observation contrasts with the potential homeostatic proliferation of surviving donor-reactive T cells in the post-ATG lymphopenic environment. Donor-specific reactivity was measured longitudinally in 8 (n=4 for basiliximab, n=4 for ATG) kidney transplant recipients who completed a 6 mo follow-up. Peripheral blood cells at pretransplant and monthly posttransplant were tested for donor-reactivity by IFN-γ ELISPOT assays. Patients induced with ATG or basiliximab had similar decreases in their absolute lymphocyte counts (and total T cells) at 1 wk post-tx. While these counts returned to near baseline by 3 mo in basiliximab treated patients, counts in ATG-treated patients were 61% of baseline at 6 mo. Mean post-tx donor-specific alloreactive T cell frequencies at 6 mo follow-up were similar to pretransplant levels for basiliximab patients but were <20% of pretransplant levels for ATG-treated patients. Furthermore, peripheral T cells in ATG-treated patients are not preferentially reconstituted with RO+ memory cells as might be expected in an environment promoting homeostatic proliferation. These data suggest that reconstitution of the periphery in ATG-treated patients is not preferentially donor-reactive.
Host-derived plasmacytoid dendritic cells prolong cardiac allograft survival and regulate alloreactive T cell responses via B7 homolog-1 (141.28)
Authors: Matta Benjamin M , Sumpter Tina L , Colvin Bridget L , Thomson Angus W
Published: January 2023

This study evaluated the contribution of the inhibitory molecule B7 homolog-1 (B7-H1) to the regulation of alloimmune responses by pDC. mPDCA-1+ splenic pDC from wild type (WT) or B7-H1 knockout (KO) mice were pulsed with donor antigen (Ag). Cell phenotype was analyzed for Ag-presenting and co-regulatory molecules and T cell allostimulatory capacity was assessed in mixed leukocyte reaction (MLR). WT or KO pDC pulsed with donor Ag were injected into syngeneic mice and CD4+ T cells were purified 7 days later for secondary MLR. T cell proliferation was assessed by 3H-thymidine incorporation and cytokines quantified by ELISA. BALB/c hearts were transplanted into untreated C57BL/6 controls or into mice receiving syngeneic pDC pulsed with donor Ag 7 days before grafting. pDC expressed low levels of CD40, CD80, CD86, MHC I/II, and B7-H1 and showed poor CD4+ T cell stimulatory capacity compared to conventional myeloid DC. pDC from KO mice exhibited greater T cell stimulatory capacity compared to WT controls. T cells from mice given immature, donor Ag-pulsed KO pDC showed greater proliferation and secreted more IFN-γ in secondary MLR compared to WT controls. Recipient-derived, immature WT pDC pulsed with donor Ag prolong allograft survival. These results reveal that B7-H1 on pDC contributes to regulation of alloreactive T cell responses. This work was funded by the transplantation immunology training grant NIAID T32 AI 074490.
Prolonged Exposure of Myeloid Dendritic Cells To Rapamycin enhances LPS-induced IL-12p70 production, but does not promote Th1 responses (141.29)
Authors: Turnquist Heth Roderick , Macedo Camila , Cardinal Jon S. , Geller David A , Metes Diana , Thomson Angus W.
Published: January 2023

Rapamycin (RAPA) is a tolerance- and Treg-sparing immunosuppressant. Prolonged exposure of myeloid dendritic cells (mDC) to RAPA confers resistance to phenotypic and functional maturation following exposure to inflammatory stimuli. Infusion of RAPA-conditioned mDC (RAPA-DC) promotes transplant survival and inhibits GVHD. However, RAPA is also reported to have pro-inflammatory properties, mediated by increased DC IL-12. Our aim was to define how DC cytokine production and CD4+ T helper (Th) cell polarization is modified by prolonged RAPA exposure.

Methods: Bone-marrow derived murine mDC were generated in 7 day (d) cultures, with RAPA added d2-d7. Following purification, RAPA-DC or control mDC (CTR-DC) remained unstimulated or were exposed to LPS. Co-stimulatory molecule expression and intracellular IL-12p40 was determined by flow cytometry. Th polarization was assessed using these DC to stimulate alloreactive CD4+ T cells, followed by analysis of T cell proliferation, apoptosis, and intracellular cytokines. Western blot was used to assess total and phosphorylated signaling molecules.

Results: RAPA-DC displayed reduced costimulatory molecules and allostimulatory capacity before and after exposure to LPS. IL-12p40 and p70 were reduced in unstimulated RAPA-DC compared to CTR-DC. LPS induced a significant increase in IL-12p70 production by RAPA-DC, due to failed inhibition of glycogen synthase kinase-3. However, RAPA-DC stimulated with LPS remained poor allostimulators that induced apoptosis in alloreactive CD4+ T cells and failed to promote Th1 responses.

Conclusions: Prolonged exposure of mDC to RAPA, although promoting LPS-induced IL-12p70 production, inhibits T cell stimulatory capacity and Th1 polarization.
Treg versus TH17 lymphocyte lineages are cross-regulated by LIF versus IL-6 (141.34)
Authors: Metcalfe Su M , Gao Wenda , Thompson Lorraine , Zhou Qiang , Putheti Prabhakar , Fahmy Tarek , Strom Terry
Published: January 2023

In an in vivo/ex vivo murine heart allograft model of allo-tolerance versus allo-rejection, that retains the complexity of the in vivo environment, novel molecular links to Foxp3 and tolerance have been revealed. These include leukaemia inhibitory factor (LIF), a member of the IL-6 cytokine family. However, IL-6 itself was linked to allo-rejection. This raises several questions: (i) does LIF play an active role in immune tolerance? and (ii) does the differential between LIF and IL-6 occur within the T cell itself? Using CD4+Foxp3+ Treg (nTreg) from Foxp3-GFP knockin mice we tested the effect of exogenous LIF, versus exogenous IL-6, on the respective lineage-specific transcription factors, Foxp3 (Treg) and RORγt (TH17). LIF supported Foxp3 transcription but not RORγt: in contrast IL-6 profoundly repressed Foxp3 and induced RORγt. For the LIF-specific receptor subunit, gp190, the response to LIF versus IL-6 was the polar opposite: gp190 transcripts were increased by LIF but strongly inhibited by IL-6, a result that indicated a potential mechanism for cross-regulation, wherein IL-6 would reduce LIF signalling by repressing synthesis of the LIF receptor. Overall, we suggest LIF is a nodal regulator of the Treg lineage, acting to oppose IL-6, the nodal regulator of the TH17 lineage. Our discoveries identify novel drug targets and we provide proof of concept using targeted LIF-nanotherapy to enhance donor-specific Treg cells in vivo. Funding BHF, NIHR, NIH, JDRF.
Manipulating The Liver to Induce Systemic Tolerance to Heart Allografts Through CD4+CD25+ Regulatory T cells and NKT Cells (141.39)
Authors: Li Wei , Carper Katie , Wang Weigang , Meng Zihui , Reyes Jorge D , Perkins James D
Published: January 2023

The liver has been demonstrated in many models to favor the induction of peripheral tolerance. The mechanisms mediating this phenomenon remain undefined. In this study, we delivered the C3H spleen cells (SCs) into the B6 liver through the portal vein (p.v.) in contrast to tail vein (i.v.) injection to characterize the liver leukocytes by flow cytometry and their function by MLR, ELISPOT, and immunohistochemistry assays. Heterotopic heart transplantation was performed from C3H donors to B6 recipients at day 7 post-treatment. The TCRαβ +NK1.1+ cells (NKT) were significantly increased in the liver, and CD4+CD25+Foxp3+ regulatory T cells (Treg) were markedly increased in the both liver and spleen after p.v. but not i.v. injection of C3H SCs. The proliferative activity of T cells from the p.v. treated mice was significantly suppressed and IL-2 was decreased, IL-4 and IL-10 were increased in vitro, suggesting that the role of the Treg may contribute to these responses. Further, significantly increased IL-4, IL-10, and IFN-γ production was detected from liver nonparenchymal cells under αGalCer stimulation, suggesting the role of liver NKT cells. Heart allograft survival was significantly prolonged in the C3H SCs p.v. treated recipients than in the i.v. treated recipients. Therefore, the liver can serve as a site of systemic tolerance induction. Both NKT cells and CD4+CD25+Foxp3+ Treg appear to be involved in the regulation of peripheral immune responses after antigen immunization of the liver.
In Vivo Introduction of an Extrachromosomal Plasmid Expressing Long-term Human Alpha-1-antitrypsin protects Islet Allografts (141.48)
Authors: Moser Hadas , Shahaf Galit , Bellacen Keren , Mizrahi Mark , Lewis Eli C
Published: January 2023

Alpha-1-antitrypsin (AAT) exerts anti-inflammatory and tolerogenic activities during islet allograft transplantation in diabetes mouse models. Although a serine protease inhibitor, evidence suggests that AAT possesses activities that are independent of protease inhibition. The previously reported plasmid, pEF-hAAT, contains the genomic sequence of human AAT (hAAT) and sustains circulating levels after single hydrodynamic tail-vein injection (HDI) in mice. To asses whether expression of hAAT by pEF-hAAT protects islets from acute rejection, pEF-hAAT (100 μg, n = 6) or PBS (n = 4, control) were hydrodynamically introduced to mice. Liver expression and circulating hAAT levels were determined. Supernatant of transfected Hepa1c cells exhibited anti-elastase activity. Sixteen days post HDI mice were injected streptozotocin (225 mg/kg) and grafted with 450 allogenic islets. Circulating hAAT levels (1-350 μg/ml) persisted for over 30 days. Normoglycemia was achieved and islets were accepted in 6 out of 6 recipients that expressed hAAT. All 4 control animals exhibited acute islet allograft rejection by day 12. We conclude that plasmid-derived long-term expression of as low as 1 μg/ml hAAT exhibits islet-allograft protection. Using site-directed mutagenesis of pEF-hAAT we will examine whether activities that are independent of protease inhibition partake in the protection of islet allografts.
COMPENSATION FOR PKC-theta DEFICIENCY IN ALLOIMMUNE RESPONSES (141.6)
Authors: Yin Deng Ping , Ma Lian Li , Gao Xiudan , Williams Phillip , Chari Ravi
Published: January 2023

We have recently shown that PKC?-/- T cell-reconstituted Rag2-/- mice failed to reject fully MHC mismatched (H-2d) cardiac allografts. Sub-optimal blockade of costimulation, insufficient to prolong survival of cardiac allografts in wild-type (WT) mice, induced donor specific tolerance in PKC?-/- mice. The current results showed that cardiac allografts were rejected in PKC?-/- T cell-reconstituted Rag2-/- mice by an adoptive transfer of PKC?-/- B cells. Cardiac allografts were accepted in PKC? and B cell double knockout mice (PKC?-/-/µ MT) and PKC? and CD28 double knockout (PKC?-/-CD28-/-) mice, whereas acute rejection occurred in either CD28 or B cell single knockout mice. Bioluminescence imaging and flow cytometry analyses showed reduced NF-(B activation in the cardiac allografts, decreased frequency of Th1, Th17, and Treg subsets in the spleen cells, and diminished the percentage of IE-reactive V-beta11 subset in the peripheral blood lympocytes in PKC?-/-, and PKC?-/-CD28-/- mice. A single TLR engagement with CpG ODNs prevented tolerance induction in both PKC?-/-/µ MT and PKC?-/-CD28-/- mice, associated with the elevation of NF-(B activation and specific T cell subsets. We conclude that B cells have the ability to compensate for the deficiency of PKC?, which is CD28 dependent. CpG ODNs restore allograft rejection bypassing PKC?, CD28, and B cell signaling pathways.
Ag-specific humoral immune responses: effects of dialyzable spleen extracts from immunized mice (33.12)
Authors: Valderrabano-Ortiz Estela , Perez-Tapia Sonia Mayra , Calderon-Amador Juana , Estrada-Parra Sergio , Flores-Romo Leopoldo , Estrada-Garcia Iris
Published: January 2023

Murine dialyzable spleen extracts (DSE) are similar to Transfer Factors in that they can transfer cell mediated immunity (CMI) from an immunocompetent organism into a naïve one in an antigen-specific manner. Lymphoid organs from rats treated with transfer factor have shown histological changes and increased lymphoblasts and plasmocytes. In this work we investigated if treating mice with DSE obtained from OVA immunized mice (DSEova) could modify the humoral immune response against OVA in naïve mice. Methods. DSEova preparation: splenic cell suspensions from OVA immunized BALB/c mice were disrupted by freeze-thaw cycles, extracts were then dialyzed with a membrane pore of 10 kDa. BALB/c mice were treated with 20μg of DSEova alone. Serum samples were obtained between days 0-36, and IgM and IgG anti-OVA were assayed by ELISA. Mice were sacrified on days 9, 15 and 23 post-immunization, and germinal centers (GC) in lymph nodes were identified by immunohistochemistry using PNA and THY-1. Results. Compared to control saline-treated animals, DSEova treated mice produced IgM and IgG anti-OVA. Compared to saline-treated mice, DSEova-mice showed increased numbers of GC, but of smaller size than OVA-treated mice. Conclusion. In this in-vivo model, compared to control-mice, DSEova would appear to stimulate the Ag-specific antibody responses and the appearance of GC.
CD4+ T cells during priming help naive CD8 +T cell activation (33.19)
Authors: Chen Shu-Ching , Liao Chi-Chia , Lai Yo-Ping
Published: January 2023

Upon encountering their antigens, naïve T cells are activated and driven to clonal expansion and differentiation into armed effector cells. According to current understanding, induction of an optimal CD4+ T-cell response requires both antigen-specific and costimulatory signals. While, stimulating naïve CD8+ T cells with specific antigens and costimulatory signal is not sufficient to induce optimal clonal expansion and effector functions. In this study, an in vitro approach was utilized to dissect the cellular and molecular requirement for CD8+ T-cell priming and differentiation. Naïve CD62LhiCD44lo CD8+ T cells were sorted and stimulated by anti-CD3 plus anti-CD28 antibodies (anti-CD3/CD28). Firstly, mitomycin C-treated splenocytes did not aid CD8+ T-cell activation. Secondly, supernatant from activated CD8+ T cells-depleted splenocytes may contain certain mediator(s) that effectively helps CD8+ T-cell activation. Furthermore, we demonstrated that the certain mediator was provided by activated CD4+ T cells and acted at priming to promote clonal expansion of naïve CD8+ T cells. These new findings advance our understanding of the regulation of CD8+ T cell activation and differentiation.
TIM1 engagement on invariant NKT cells inhibits IFN-γ production in the presence of TCR stimulation in vitro and in vivo (33.21)
Authors: Kim Hye Sung , Chung Doo Hyun
Published: January 2023

T cell immunoglobulin domain and mucin domain (TIM)1, expressed on Th2 cells, regulates immune responses by modulating cytokine production. However, the functional roles of TIM1 have not been examined in NKT cells. Therefore, we investigated the immunologic effects of TIM1 on NKT cells using anti-TIM1 mAbs. Mouse NK1.1+TCR-β+ and α-GalCer/CD1d dimmer+ NKT cells constitutively expressed TIM1 on cell surface. TIM1 engagement on NKT cells using a series of anti-TIM1 mAbs suppressed IFN-γ production in the presence of TCR stimulation in vitro, whereas its effects on Th2 cytokines production of NKT cells were various depending on anti-TIM1 mAb clone. The injection of various anti-TIM1 mAbs into B6 mice also reduced IFN- γ levels in the sera with the coinjection of α-galactosylceramide (α-GalCer), and one of anti-TIM1 mAbs enhanced Th2 cytokines. Moreover, TIM1 engagement increased p27 and GATA-3 expression but reduced T-bet expression in NKT cells in the presence of TCR engagement. The adoptive transfer of NKT cells preincubated with a series of anti-TIM1 mAbs aggravated bleomycin-induced pulmonary fibrosis in NKT cell-deficient mice by suppressing IFN-γ production. Taken together, TIM1 costimulation inhibits IFN- γ production by NKT cells, which may regulate immune responses in vivo, and be a useful therapeutic candidate for various immune diseases.
Agonistic monoclonal antibody against DR5 induces ROS aggregation, sustained JNK activation and Endo G release in Jurkat leukemia T lymphocytes (33.22)
Authors: Zheng Dexian , Chen Caifeng , Chen Feng , Liu Yanxin
Published: January 2023

We previously reported that AD5-10, a novel agonistic mAb to DR5, possessed a strong cytotoxic activity in various tumor cells. In this study, we further demonstrated the ROS generation, sustained activation of JNK, loss of mitochondrial membrane potential, and release of Endo G in Jurkat leukemia T lymphocytes upon AD5-10 stimulation. The dominant negative JNK, but not p38, enhanced NF-κB activation, suppressed caspase-8 recruitment in DISC, reduced the dysfunction of mitochondria, therefore, inhibited the cell death in these cells. The data may throw light on the therapeutic strategy for T lymphocyte leukemia and solid tumors.

We further investigated the sensitivity of lung cancer cells to AD5-10. A549 and SCLC showed a moderate sensitivity and H460 resistant to the mAb, which was not related to DR5 expression, but c-FLIPL expression and cleavage. Inhibition of c-FLIPL by siRNA significantly enhanced AD5-10-induced cell death. The sensitizing effect was associated with decreased expression of Bid and Bcl-XL, change of mitochondrial membrane potential, release of cytochrome c, and caspase activation. Immunohistochemistry assay revealed that c-FLIPL was expressed in 87.9% of the lung carcinoma tissues from the patients, but little in the normal controls, suggesting that inhibition of c-FLIPL might be a potential strategy for lung cancer therapy.
Dynamic αβ T cell receptor quaternary structure is linked to its mechanosensor function (33.27)
Authors: Kim Sun Taek , Takeuchi Koh , Sun Zhen-Yu J. , Touma Maki , Wagner Gerhard , Reinherz Ellis L.
Published: January 2023

The T cell receptor (TCR) mediates antigen recognition and T cell activation via its dimeric αβ, CD3εγ, CD3εδ and CD3ξξ subunits. A structural mechanism integrating both functions remains elusive. Here, anti-CD3 mAbs, NMR cross-saturation plus chemical shift mapping and mutational analyses were used to show that site-specific binding topology and TCR quaternary change are essential for activation. Two agonist mAbs footprint to the membrane distal CD3ε lobe, whereas a non-agonist mAb binds to the cleft between CD3ε and CD3γ. T cell triggering is not linked to mAb affinity or CD3ε binding stoichiometry per TCR but requires an intact TCRβ-CD3εγ module. CD3γ ectodomain mutants or an Fab directed toward the lever-like Cβ FG loop near the agonist mAb site inhibit antigen-dependent activation by preventing quaternary change upon ligand binding. Flexible elongated TCRα (25-26 aa) and TCRβ (19 aa) membrane stalks versus rigid compact CD3ε, CD3γ, and CD3δ (5-10 aa) G-strands and CxxC membrane proximal motifs permit dynamic TCRαβ movement over asymmetric CD3 heterodimers upon p-MHC binding. Extracellular mechanical torque on the TCR resulting from specific p-MHC binding during T cell scanning of APCs can initiate quaternary structure changes within the TCR, triggering downstream signaling. These findings suggest that the TCR is a specialized mechanosensor, converting mechanical energy from immune surveillance into a biochemical signal at no energetic cost.
The co-regulatory molecule TIGIT contributes to suppressor function of regulatory T cells. (33.29)
Authors: Grogan Jane L , Yu Xin
Published: January 2023

TIGIT is a co-regulatory molecule expressed on regulatory, memory and activated naïve T cells. Poliovirus receptor (PVR, CD155) was identified as the high-affinity receptor for TIGIT. TIGIT can induce immunoregulatory dendritic cells via ligation of PVR that can inhibit T cell responses in vitro and in vivo, in part due to IL-10 induction. We further examined the role of TIGIT on human T regulatory cells (Treg). Isolated human TIGIT+ T cells inhibited CD4+CD25-TIGIT- naïve T cell proliferation in vitro in the presence of APC. An anti-TIGIT antagonist antibody inhibited Treg suppression in vitro. Transwell experiments show that TIGIT-mediated suppression of effector cells requires DC-Treg cell-cell interaction. Furthermore, the TIGIT-mediated Treg suppression could be replaced by TIGIT.Fc alone. These data suggest that TIGIT plays a role in T regulatory cell function via interacting with PVR on APC.
CD46-induced regulatory T cells suppress Mycobacterium tuberculosis-specific effector T cell responses (33.5)
Authors: Truscott Steven M , Abate Getahun , Price Jeffrey D , Kemper Claudia , Atkinson John P , Hoft Daniel F
Published: January 2023

Human regulatory T cells can be generated through a number of known pathways in vivo and in vitro. Simultaneous engagement of the TCR and complement regulator CD46 on human CD4+ T cells induces adaptive Treg development. These CD46-induced Treg, also known as 'complement-induced Treg' or 'cTreg,' have been shown to suppress bystander CD4+ T cells activated in vitro with crosslinking antibodies to CD3 and CD28. Here we offer the first description of cTreg suppression of human effector T cells in an APC-dependent system of pathogen infection. We found that soluble molecules secreted by cTreg suppress Mycobacterium tuberculosis-specific CD4+, CD8+, and γδ T cell responses. These immunosuppressive effects were mediated in part by IL-10 and in part by other as yet unidentified soluble factors acting synergistically with IL-10. In addition, our results indicate that different mechanisms may be involved in the suppression of αβ and γδ T cells. We further show that APC functions were not affected by cTreg, indicating direct suppression of pathogen-specific T cells. Modulating the immunosuppressive functions of this Treg subset could lead to improved tuberculosis therapeutics or vaccines.
Autoregulatory B-1 cells (34.13)
Authors: Sindhava Vishal J , Bondada Subbarao
Published: January 2023

Immunological tolerance in the periphery is mediated by clonal inactivation mechanisms as well as by regulatory cells. Recent studies showed that high IL-10 producing B-cell subsets with varying phenotype can regulate different immune responses in numerous mouse models. We found that, in comparison to B-2 cells, peritoneal B-1a cells are hyporesponsive to TLR stimulation in proliferation and antibody secretion, but produce very high amounts of IL-10. We hypothesized that the high IL-10 levels work in an autocrine manner and autoregulate B-1 cells that are prone to produce autoantibodies. Accordingly, neutralization of IL-10 enhanced peritoneal B-1, but not splenic B-2 cell proliferation and differentiation to all TLRs tested. Moreover, IL-10-/- peritoneal B-1 B-cells responded better than wild type B-1 cells to TLR stimulation. Co-stimulation with CD40 and BAFF, but not IL-5, overcame the inhibitory effect of IL-10. The increased IL-10 production was unique to peritoneal B-1 B-cells, since splenic B-1 B cells behaved like splenic B-2 cells, in terms of IL-10 production and proliferation. This autoregulation appears to have physiological significance since IL-10 knock out peritoneal B-1 cells controlled Borrelia hermsii infection better than wild type B-1 cells. Thus the IL-10 mediated autoregulation of B-1 cells may have a role in the control of autoimmunity and infection. (Supported by NIH grants to SB).
Cytokine-secreting follicular T cells shape the antibody repertoire (34.19)
Authors: Reinhardt Richard Lee , Liang Hong-Erh , Locksley Richard M.
Published: January 2023

High-affinity antibodies are critical for host protection against pathogens and toxins, and underlie successful vaccines. Generation of such antibodies requires T cell-dependent help, which mediates germinal center (GC) reactions where mutation and selection of B cells expressing receptors with high-affinity for antigen occurs. Follicular CD4 T (TFH) cells, which comprise only 5-10% of GC cells, have been imaged in stable conjugates with GC B cells, but no functional analysis of B/TFH cell interactions in GC has been performed. Using an IL-4 reporter system, we show that TFH cells comprise essentially all of the cytokine-secreting T cells in lymph nodes and are functionally distinct from T cells secreting the same cytokine in peripheral tissues. TFH cells with different cytokine profiles could be visualized in the same GC and could be isolated as conjugates with B cells undergoing cytokine-specific immunoglobulin isotype class switching, expressing high levels of AID, and evidence of somatic hypermutation. These findings support a model wherein GC B cells compete for cytokines produced by rare antigen-specific GC TFH cells that mediate highly localized effects and regulate the size, affinity and isotype of the B cell antibody response.
Negative regulation of IgA production by the Fc&[alpha]/&[mu]R, an Fc receptor for IgA and IgM (39.13)
Authors: Kurita Naoki , Honda Shin-ichiro , Cho Yukiko , Usui Kenta , Shibuya Akira
Published: January 2023

Fc&[alpha]/&[mu] receptor (Fc&[alpha]/&[mu]R) is a novel Fc receptor for both IgA and IgM. Fc&[alpha]/&[mu]R transcript is expressed in the gut as well as spleen and lymph node. The immunohistological analysis showed that Fc&[alpha]/&[mu]R is highly expressed on follicular dendritic cells (FDCs) in germinal centers and at lesser extent on B cells of Peyer's patches. Since Peyer's patches are known as major inductive sites of IgA-producing cells, we investigated the roles of Fc&[alpha]/&[mu]R in IgA production.

We found that Fc&[alpha]/&[mu]R-deficient mice showed elevated serum, but not fecal, IgA. Moreover, after oral administration of T- independent antigens, Fc&[alpha]/&[mu]R-deficient mice showed elevated antigen-specific serum, but not fecal, IgA production. Fc&[alpha]/&[mu]R-deficient mice showed comparable half life of serum IgA to wild type mice, indicating that the production of serum IgA is increased in Fc&[alpha]/&[mu]R-deficient mice. Indeed, IgA-positive B cells were increased in Peyer's patches, mecenteric lymph nodes and spleen in Fc&[alpha]/&[mu]R-deficient mice. Bone marrow chimeric mice demonstrated that serum IgA was elevated in mice whose non-hematopoietic, but not hematopoietic, cells lack Fc&[alpha]/&[mu]R expresssion. These results suggested that Fc&[alpha]/&[mu]R expressed on FDC in Peyer's patches may regulate IgA production against intragastrically administered antigens. The molecular and cellular basis of these phenomena are under investigation.
Immunomodulatory effects of dietary fibre supplementation on lymphocyte populations, cytokine and immunoglobulin A production(39.16)
Authors: Gannon Mark , Brooks Steve , Kalmokoff Martin , Raju Jayadev , Selinger Brent , Inglis Douglas , Green-Johnson Julia
Published: January 2023

Gastrointestinal microflora has been shown to have a bi-directional relationship with the host immune system. A variety of fermentable carbohydrate polymers largely pass through the small intestine, providing fermentable substrates for gut microflora. Dietary fibre supplementation may provide a strategy for manipulating the intestinal bacterial profile, changing the interaction with the mucosal immune system, thereby modulating the host immune system. Over a six week trial, 30 weanling BioBreeding rats were fed either control diet alone or supplemented with oat bran or wheat bran fibre. We have shown that wheat bran fibre increases mesenteric lymph node (MLN) B lymphocyte numbers, and decreases systemic IL4 production, MLN T lymphocytes, and intestinal IgA, concurrent with increased colonic bacterial population diversity. We also observed no evidence of pro-inflammatory responses resulting from dietary fibre supplementation. This lends support to the hypothesis that dietary fibre may modulate the host immune system through modification of the gut microflora profile.

The research is funded by the Advanced Foods and Materials Network
The role of TL1A-DR3 TNF-family interactions in inflammatory bowel disease (39.3)
Authors: Meylan Françoise , Song Yun-Jeong , Malm Ian , Acharya Krishika , Kahle Erin , Fuss Ivan , Strober Warren , Belkaid Yasmine , Siegel Richard
Published: January 2023

DR3 is a TNF-receptor specifically expressed on lymphocytes which can costimulate lymphocyte activation. Its ligand, TL1A, can be produced by at least 3 different cell subsets: endothelial cells, lymphocytes and DC. TL1A and DR3 have been reported to be upregulated in biopsy samples and in animal models of IBD, providing circumstantial evidence of the involvement of this TNF ligand-receptor pair in IBD. We have generated TL1A transgenic mice that constitutively express TL1A by either T-cells or DC. Both of these transgenic lines develop spontaneous IBD. Constitutive expression of TL1A by T cells leads to a severe pathology found primarily in the ileum and duodenum in all founders, from an early age. Consistent with the role of TL1A as a T cell costimulator, the T cell population had an activated phenotype and increased effector subsets. Surprisingly, despite the inflammation, we observed increased FoxP3+ cells. Interestingly, IL-13 and to a lesser extent IL-17 are the cytokine mRNA that are the most elevated in the gut. In contrast, constitutive expression of TL1A by DC leads to a milder pathology that is more restricted to the ileum, and is detectable in older mice. Ectopic expression of TL1A appears to be able to overcome T-cell self-tolerance and drive IBD with no other overt stimuli. These findings suggest that TL1A may be an interesting target for therapy.
Selection and application of M cell targeting peptide ligand to induce immune responses after mucosal immunization of the ligand-fused antigen (39.31)
Authors: Jang Yong-Suk , Kim Sae-Hae , Seo Ki-Weon , Lee Kyung-Yeol
Published: January 2023

Mucosa is the most successful application area of vaccination through the induction of the systemic and mucosal immune responses. In this study, we have searched for reliable peptides which could be used as mucosal adjuvant to potentiate the orally introduced antigen to induce the immune response. Using the phage display library, we have selected peptides which can bind onto cultured M cell model using Caco-2 and Raji co-culture system which had been confirmed by UEA-1, WGA, and M cell specific antibody by confocal scanning light microscopy. Interestingly, one of the selected peptides showed a high homology with outer membrane protein of Yersinia enterocolitica-like strains which are well-known human pathogen causing various gastrointestinal systemic syndromes and capable of invading and destroying M cells. In order to confirm the role of selected peptide to enhance the immune response induction after oral administration, fusion proteins between a peptide ligand and EGFP or VP1 antigen of foot-and-mouth disease were produced and tested in ex vivo intestinal loop and in vivo mouse systems. (This work was supported by the grant from ARPC.)
Retinoic acid as an adjuvant to imprint mucosal homing properties to vaccine-induced T cells (39.32)
Authors: Tan Xuefang , Blattman Joseph , Greenberg Phil
Published: January 2023

Objective: All-trans retinoic acid (ATRA) induces the expression of gut-homing receptors on T cells during activation in vitro. We investigated the effect of ATRA as an in vivo adjuvant during vaccination on the formation and migration of memory T cells.

Methods: T cells were analyzed in control and ATRA treated mice immunized with recombinant adenovirus expressing the LCMVgp33 epitope as well as following heterologous virus challenge. To directly compare the proliferative capacity and homing of memory cells from ATRA or vehicle-treated mice, these cells were co-transferred into recipients and responses analyzed following MVAgp33 challenge.

Results: ATRA treatment during priming resulted in increased memory T cells in mucosal tissues and somewhat decreased splenic memory CD8 T cells. T cells primed in the context of ATRA exposure mounted stronger responses to systemic challenge with MVAgp33, and were more resistant to challenge with VVgp33 at vaginal surfaces. In co-transfer experiments, memory CD8 T cells from ATRA treated mice exhibited greater proliferation and increased gut homing.

Conclusion: ATRA exposure during priming promotes the generation of memory T cells with greater proliferative potential and enhances mucosal localization. Thus ATRA may be a useful as an adjuvant during vaccination to enhance T cell responses at mucosal sites. This work is supported by the Bill & Melinda Gates Foundation.
Powerful mucosal immune response in macaques (Macaca mulatta) in response to SIV gp96-Ig immunization (39.35)
Authors: Strbo Natasa , Vaccari Monica , Pahwa Savita , Kolber Michael , Fisher Eva , Gonzalez Louis , Felber Barbara , Pavlakis George , Franchini Genoveffa , Podack Eckhard
Published: January 2023

Induction of mucosal immunity is critical for protection from enteric pathogens. We have developed a vaccine design that utilizes the unique property of the endoplasmic reticulum chaperon, heat shock protein (HSP) gp96, to bind antigenic peptides and deliver them to APCs.

The aim of our study was to evaluate safety and systemic and mucosal SIV-immunity with secreted gp96-Ig vaccines in non-human primates.

We have generated a secreted form gp96 by replacing the ER retention sequence KDEL with the IgG1-Fc tag. HEK-293 cells were transfected with gp96-Ig and with the cDNAs encoding the SIV antigens gag, pol, env and retanef (reassorted rev, tat, nef). Irradiated, transfected 293 cells that secrete 1, 5 or 50 micrograms gp96-Ig-SIV-peptide complexes in 24h, were injected intraperitoneally in Mamu-A*01+ macaques at 0, 4 and 24 weeks. The frequency of SIVGag- and SIVTat- tetramer+ cells in the rectal mucosa reached 30-50% in some macaques after the third immunization. Tetramer+ cells expressed appropriate functional (granzymeB) and migration markers (alpha4beta7, CD103). The cells secrete IL-2 and IFN-gamma in response to different peptide (gag, tat, env) stimulation.

We conclude that the cell secreted SIV-gp96-Ig vaccine is safe and can induce strong poly-specific, multifunctional CD8 responses in mucosal compartments that are thought to be critical for protection from SIV/HIV infection.