bluuebunny/crossref_metadata_2025 huggingface dataset:
Ex vivo expansion of B cells from hematopoietic stem cells to supplement cord blood transplants (141.21)
Authors: Beaudette-Zlatanova Britte C , Le Phong T , Zhang Shubin , Stiff Patrick J , Knight Katherine L
Published: January 2023

Successful cord blood (CB) transplantation in adults is hampered by low numbers of hematopoietic stem cells (HSC) and infection due to delayed establishment of immunocompetence. By performing CDR3 spectratype analysis of T and B lymphocytes from CB transplant recipients, we demonstrated that these patients do not develop a diverse repertoire until 6 months or more after transplant. To provide rapidly engrafting antigen-responsive cells to CB recipients and to increase the number of transplanted cells, we developed an in vitro system to expand lymphoid progenitors from CB HSC. CB HSC (CD34+CD45RA+CD38-) are co-cultured for one to two weeks with either HEK 293T cells or a non-tumorigenic human thymic epithelial cell line (TEC) in media supplemented with a cocktail of cytokines and growth factors. In this study, we focused on B-lineage cells (CD79a+) and found, on average, B-lineage cells expanded more than 70 fold in HEK 293T co-cultures and nearly 100 fold in TEC co-cultures. We conclude that B-lineage cells can be expanded effectively in vitro; consequently, we are testing whether these cells are functional after engraftment in NOD/SCID/IL2Rγ-/- mice. We hypothesize that compared to patients receiving HSC alone, patients receiving transplants supplemented with in vitro-generated lymphoid progenitors will become immunocompetent sooner.

Funding by NIH AI068390 (KLK) and AG023809 (PTL)
T cell responses to hub gene knockout in in-vitro propagated Jurkat Cells: Application to Systems Biology (141.50)
Authors: Khanna Ajai , Lwin Wint Wah , Park Ken , Wauson Matthew , Gao Qin , Perkins David
Published: January 2023

Systems biology is unraveling complex gene interconnections. T cell genes are upregulated during rejection. Suppression of hub and node genes in the T cell genome could result in T cell hyporesponsiveness.

Objective: To knockdown hub or node genes via RNA interference in Jurkat cells and study their effect on T cell proliferation.

Methods: A molecular interaction network was built with Cytoscape using microarray data on Jurkat cells to identify upregulated hub genes: ATP5C1, IL6ST, PRKCZ, MYC, and MAPK1 that served as targets for RNAi knockdown. Jurkat cell lines were lipotransfected with siRNAs for the selected hub genes. Cell proliferation was measured by ATP release bioluminescence assay and BRDU labeling.

Results: At 12 hours following incubation with siRNA MAPK1, there was a reduction in ATP production compared to controls. At 24 hours, siRNAs incorporating genes for ATP5C1, IL6ST, PRKCZ, and MYC showed reduction in Jurkat cell proliferation compared to controls. No difference was observed in MAPK1 group and in the group transfected with a combination of siRNAs for all the identified hub genes.

Conclusions: Downregulation of hub genes by siRNA transfection is possible and could open opportunities to target these genes and modify T cell responses. This will help us in integrating systems biology to identify genes that could be potential therapeutic targets.
The role of Mad, Mnt and c-Myc proteins in OX40 stimulated T cells (33.26)
Authors: Vasilevsky Nicole A , Weinberg Andrew
Published: January 2023

We sought to determine the molecular mechanisms by which OX40 mediates T cell function. Gene array analysis of OX40-activated Ag-specific T cells suggested a role for the transcription factor c-Myc and antagonist Mad family proteins. D011.10 T cells were adoptively transferred into BALB/c recipients and immunized with soluble OVA and an agonist anti-OX40 antibody, which enhances Ag-specific T cell proliferation and survival. Western blot analysis of Ag-specific T cells purified ex vivo revealed that Mad4, Mnt and c-Myc proteins were up-regulated after OX40 engagement. Mad4 and Mnt protein levels peaked at days 3-4 and decreased as the cells contracted in size. C-Myc protein levels remained unchanged over time after OX40 stimulation. We hypothesize that the up-regulation of c-Myc in activated T cells drives proliferation and consequently activation-induced cell death, which is counteracted by Mad4 and Mnt proteins after anti-OX40 engagement, allowing T cells to survive the "blast crisis" phase. Indeed, siRNA knockdown of Mad4 and Mnt proteins led to decreased survival of OX40-activated T cells. We also found that OX40 stabilized both Mad4 and Mnt proteins against degradation and we demonstrated the presence of a serine residue in Mad4 that was essential in mediating accelerated protein degradation. These data provide evidence that Mad4 and Mnt play a role to enhance memory T cell survival after OX40 engagement.
Direct and indirect effects of TLR agonists on FasL, TNF-alpha and TRAIL expression in CD4 T cells (33.7)
Authors: Weinlich Ricardo , Sant'Anna Maíra M , Ferra Luciana C , Campopiano Júlia C , Bortoluci Karina R , Amarante-Mendes Gustavo P
Published: January 2023

The lifespan of CD4 T lymphocytes is partially controlled by the engagement of the so-called Death Receptors (DR) on their surface. De-regulation of DR ligands, such as FasL, TNF-alpha and TRAIL, is associated with many pathological processes, including Immunodeficiency and Autoimmunity. We have recently shown that TcR/CD3-mediated FasL upregulation in CD4 T cells is prevented by PGE2 derived from LPS-stimulated antigen-presenting cells (APCs). Here we show that Toll-like receptor (TLR) agonists other than LPS can interfere with the expression of FasL, TNF-alpha and TRAIL. Except TLR7, stimulation of TLRs in APCs resulted in induction of PGE2, thereby indirectly blocked the expression of DR ligands on T cells. On the other hand, direct stimulation of TLR7 on T cells lead to repression of CD3-mediated FasL expression and consequently reduced AICD. Interestingly, this effect was independent of NF-kB. Altogether, these results suggest that pathogen-associated molecular patterns can alter the expression of DR ligands, either by direct stimulation on CD4 T cells or by inducing the release of PGE2 by APCs.

Supported by FAPESP and CNPq.
MAP kinase phosphatase 1 is necessary for T cell activation and function (35.1)
Authors: Zhang Yongliang , Reynolds Joseph , Chung Yeonseok , Dong Chen
Published: January 2023

Mitogen-activated protein kinases (MAPK) are evolutionary conserved immune regulators. MAPK phosphatases that negatively regulate MAPK activities have emerged as important players in both innate and adaptive immune responses. MKP-1, also known as DUSP1, was previously shown to negatively regulate innate immunity by inhibiting pro-inflammatory cytokine production. Here we found that MKP-1 positively regulate T cell activation, function and negatively regulate apoptosis. MKP1-deficient dendritic cells exhibited significantly reduced ability to activate T cells than wild-type cells. In T cells, MKP-1 deficiency led to enhanced activation of ERK and JNK but not p38, and impaired the activation, proliferation and function of T cells in vitro and in vivo. MKP-1 deficient T cells exhibited enhanced activation induced cell death. Consistently, MKP-1-/- mice were found to be resistant to experimental autoimmune encephalomyelitis but were defective in anti-influenza immunity in vivo. Our results thus demonstrate MKP-1 as a critical positive regulator of T cell activation and function and may be targeted in treatment of immune diseases.
Enhanced humoral immune responses against T-independent antigens in Fcα/μR-deficient mice (39.14)
Authors: Honda Shin-ichiro , Kurita Naoki , Shibuya Akira
Published: January 2023

IgM is an antibody class common to all vertebrates that plays a primary role in host defences against infection. Binding of IgM with an antigen initiates the complement cascade, accelerating cellular and humoral immune responses. However, the functional role of the Fc receptor for IgM in such immune responses remains obscure. Here we show that mice deficient in Fcα/μR, an Fc receptor for IgM expressed on B cells and follicular dendritic cells (FDCs), had enhanced germinal center formation and affinity maturation and memory induction of IgG3+ B cells after immunization with T-independent (TI) antigens. Moreover, Fcα/μR-deficient mice showed prolonged antigen retention by marginal zone B (MZB) cells and FDCs. In vitro studies demonstrated that interaction of the IgM immune complex with Fcα/μR partly suppressed TI antigen retention by MZB cells. We further showed that downregulation of complement receptor (CR)1 and CR2 or complement deprivation by in vivo injection with anti-CR1/2 antibody or cobra venom factor attenuated antigen retention by MZB cells and GC formation after immunization with TI antigens in Fcα/μR-/-_mice. Taken together, these results suggest that Fcα/μR negatively regulates TI antigen retention by MZB cells and FDCs, leading to suppression of humoral immune responses against T-independent antigens.
The influence of commensal bacteria on anti-viral immunity (39.26)
Authors: Abt Michael C , Beiting Daniel , Kobuley Dymtro , Yu Yimin , Zaph Colby , Wherry John , Artis David
Published: January 2023

Alterations in the composition of intestinal commensal bacteria in humans are associated with enhanced susceptibility to multiple inflammatory diseases suggesting that signals derived from commensal bacteria may influence the development and/or function of the immune system. Supporting this, germ-free or gnotobiotic mice exhibit reduced numbers of lymphocytes in the intestinal intraepithelial compartment. However, whether alterations in the acquisition or composition of commensal bacteria influence immunity to infection remains poorly defined. To test this, mice housed under conventional or gnotobiotic conditions were infected i.p. with Lymphocytic Choriomeningitis Virus (LCMV). At day 7 post-infection, gnotobiotic mice exhibited a significant reduction in the frequency and number of LCMV-specific CD8+ T cells in multiple tissues including the spleen and intraepithelial compartment. Furthermore, LCMV-specific CD8+ T cells from gnotobiotic mice were less capable of producing IFN-γ following LCMV peptide stimulation. In addition, at day 36 post-infection, when conventionally-housed mice have established a LCMV-specific CD8+ memory T cell population, gnotobiotic mice had a significantly impaired population of LCMV-specific CD8+ memory T cells. Diminished LCMV-specific CD8+ effector and memory T cell responses were not the result of inherent defects in gnotobiotic mice as oral administration of a cocktail of antibiotics to conventionally-housed mice also displayed a defective LCMV-specific CD8+ T cell response following infection. Collectively, these data suggest an integral role of commensal bacteria in influencing virus specific CD8+ T cell effector and memory responses.
Response of human epithelial cells and monocytes to UV radiation in a co-culture model of inflammatory acne (39.47)
Authors: Dapul Gina E , Brown Paula , Yaping E. , Lee Wei-Li , Shalita Alan R. , Nowakowski Maja
Published: January 2023

Earlier studies in our laboratories showed that ultraviolet (UV) light has anti-inflammatory properties, as demonstrated by inhibition of IL-1 and ICAM-1 expression by human keratinocytes (hTERT, human telomerase reverse transcriptase - transformed HaCaT cells). The goal of the study was to construct an improved model system that involved a modified co-culture of more than one cell line, exposure to UVA and UVB, and measurement of nitric oxide production as a marker for inflammation. The ability of SC cells to produce nitric oxide in culture was confirmed by incubation in the presence of phorbol myristic acetate (PMA, Sigma) (40-160 nM) for 48 hrs, which resulted in 1.6 fold increase in accumulation of nitric oxide. Human epithelial cells (A549, ATCC) were seeded in a 24-well tissue culture plate (0.1 x 106 cells/well) 48 hrs before use. Cells were exposed to UVA (from 0 to 40J/m2), UVB (from 0 to 200mJ/cm2), and bacterial lipopolysaccharide (LPS, Sigma) (0 to 500ng/ml) and incubated for 48 hours. Supernatants of the irradiated and control A549 cells were co-cultured with human monocytes (SC, ATCC) which were seeded at 0.2 x 106 cells per well in a 24-well tissue culture plate. The modified co-culture was incubated for 48 hours. The supernatants were collected and nitric oxide content was measured (modified Griess assay). Under these conditions, neither LPS nor UV radiation triggered nitric oxide production (<10 μM). The modified co-culture system is a useful model for studying cell-cell interactions and responses to inflammatory stimuli and inhibitory agents or treatments. Supported in part by New York State Dept. of Health, ECRIP Fellowship to G.E.P.
Comparison of the Expression Profile of JunB, c-Jun, and S100A8 (Calgranulin A) in Psoriasis Vulgaris and Guttate Psoriasis (39.48)
Authors: Lee Kyung Ho , Park Chul Chong , Chun Ji Hoon , Woo So-Youn
Published: January 2023

Background: Psoriasis is a chronic, inflammatory, immune-mediated skin disease. Recently, several psoriasis-linked genetic loci have been reported; PSORS4 contains S100A8 (calgranulin A), and the PSOR6 (19p13) locus harbors JunB (19p13.2). S100A8 is considered to be a marker of inflammation in a variety of diseases. The expression of JunB and c-Jun have been reported to be reduced in psoriatic lesions.

Objective: We attempted to assess the role and correlation of S100A8, JunB, and c-Jun in the pathogenesis of guttate psoriasis and psoriasis vulgaris by studying whether any difference of immunohistochemical expression existed.

Methods: Skin biopsy specimens from patients with psoriasis vulgaris (n=37) and guttate psoriasis (n=17), and a normal skin controls (n=9) were utilized in the study. Formalin-fixed and paraffin-embedded tissue sections were prepared and JunB, c-Jun, and calgranulin A were immunohistochemically stained in order to compare the expression of those three proteins in each group.

Results: Reduced JunB expression was observed in patients with psoriasis vulgaris and guttate psoriasis, as compared to patients in the control group; however, c-Jun expression was reduced only in the psoriasis vulgaris group. The expression of S100A8 increased in the psoriasis groups as compared to the control group. In addition, the expression of S100A8 was different between the psoriasis vulgaris and guttate psoriasis groups; S100A8 was expressed more profoundly in the guttate psoriasis group (p<0.05).

Conclusion: Our results indicate that S100A8 contributes to the pathogenesis of guttate psoriasis, and it may be a good target for therapy for guttate psoriasis provoked by microorganisms.
Natural Killer T (NKT) cells mediate anti-tumor activity via killing of tumor-associated macrophages (40.19)
Authors: Metelitsa Leonid S , Song Liping , Salo Jill , Engell Kelly , Wu Hong-wei , Sposto Richard , Seeger Robert C , Asgharzadeh Shahab
Published: January 2023

Tumor infiltration with CD1d-reactive Vα24-invariant NKT cells associates with favorable outcome in neuroblastoma and other cancers. However, the function of NKTs in tumor immunity remains largely unknown. We demonstrate that CD68+ tumor-associated monocytes/macrophages (TAMs) represent the majority of CD1d-expressing cells in primary neuroblastomas. TAMs in vitro and in vivo stimulated neuroblastoma growth via IL-6 production and were the source of IL-6 in primary tumors and metastatic bone marrows in patients. Gene expression analysis with TaqMan® low-density arrays of 129 primary MYCN non-amplified neuroblastomas revealed that high level expression of TAM genes (CD14, CD16, IL-6, IL-6R, and TGFβ) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing was CD1d-restricted since it was inhibited by anti-CD1d mAb. Co-transfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and delayed tumor growth compared to mice transferred with monocytes alone. Thus, killing of TAMs reveals a novel mechanism of NKT cell anti-tumor activity that relates to the disease outcome and should be considered for NKT-based therapeutic strategies. Supported by NIH grants: RO1 CA116548 (LSM) and PO1 CA81403 (RCS).
SYNERGY BETWEEN MOLECULAR TARGETS OF COSTIMULATION IN PROMOTING T CELL PERSISTENCE (40.31)
Authors: Song Jim , Croft Michael
Published: January 2023

Costimulation regulates multiple cellular processes including proliferation, expansion and survival of T cells, and molecular targets of costimulation might be useful to augment T cell activities. Two defined targets of costimulatory signals in CD4 T cells are the anti-apoptotic bcl-2 family molecule bcl-xL, and survivin, an Inhibitor of Apoptosis family member that might regulate cell division and survival. However, the relative importance and relationship between these molecules in CD8 T cells is not clear. To understand whether they have overlapping or synergistic functions, we used retrovirus-mediated transduction to introduce bcl-xL and survivin separately, or together linked by a 2A picornavirus self-cleaving peptide, into antigen (Ag)-responding CD8 T cells. In vitro, we found that CD8 effector T cells expressing both bcl-xL and survivin strongly expanded at an early stage and had a long-term survival advantage. In vivo, with response to tumor-expressed Ag following adoptive T cell transfer, Ag-reactive CD8 T cells expressing both bcl-xL and survivin displayed a greatly enhanced tumor protective activity compared CD8 T cells expressing either molecule introduced separately. These results indicate that bcl-xL and survivin can critically contribute in a synergistic, non-redundant, manner to augment the accumulation and persistence of CD8 T cells following encounter with Ag. The data provide new insights into why costimulatory signals might need to be sustained over time or provided periodically via ligation of multiple receptors.
Combination of CpG-ODN+anti-4-1BB but not CpG-ODN+anti-OX40 enhances antitumor immune responses in neu mice modulating the inhibitory function of Tregs (40.7)
Authors: Smith Shannon E , Hoelzinger Dominique B , Dominguez Ana Lucia , Lustgarten Joseph
Published: January 2023

Previous studies have established that 4-1BB and OX40, members of the TNFR family, enhance antitumor immune responses. Recent studies from our laboratory indicate that intratumoral injections of CpG-ODN induce antitumor responses in Her-2/neu tolerant mice. However, this treatment alone is not sufficient to completely eradicate tumors. Here we investigate whether the combination of CpG+anti-OX40 or anti-4-1BB enhance the antitumor responses in neu mice. Our results indicate that CpG+anti-4-1BB but not CpG+anti-OX40 eradicate tumor in neu mice. CpG+anti-4-1BB treatment significantly increased the number of CD8 cells within lymphoid organs as compared to control or anti-OX40 treated animals. More importantly, we observed that anti-4-1BB but not anti-OX40 inhibits the suppressive function of Tregs. Microarray analysis of Tregs revealed specific transcriptional differences brought about by treatment with anti-OX40 or anti-4-1BB. 4-1BB modulates differential gene expression in Tregs of over 300 genes that remain unaffected by OX40. Potential gene candidates include interleukin-9 and protein-tyrosine phosphatase receptor-type zeta-1, which are transcriptionally down and up regulated in the presence of anti-4-1BB. These proteins may play a role in Treg function, and could explain the enhanced anti-tumor effect observed in the presence of anti-4-1BB.
The role of TLR2 in the immunostimulatory effect of Polysaccharide krestin (PSK) (41.13)
Authors: Lu Hailing , Yang Yi , Gad Ekram , Disis Mary L.
Published: January 2023

Polysaccharide krestin (PSK) is a mushroom extract that has long been used in Asia as an anti-cancer drug, although the mechanism of action is unclear. Using neu-transgenic

(neu-tg) mice, a model of human HER2 positive breast cancer, we investigated the anti-tumor effect of PSK and its potential mechanisms. We found that oral administration of PSK significantly inhibited the growth of both implanted and spontaneous breast tumors in these mice. In vitro PSK treatment stimulated splenocytes proliferation and secretion of TNFalpha. PSK activated dendritic cells (DC) from neu-tg mice as shown by increased expression of CD86, CD80, and MHCII. The expression of toll-like receptor 2 (TLR2) on DC was also increased. To study the potential activating effect of PSK on TLRs, we used HEK293 cells transfected with different TLRs and incubated them with PSK (0.5 to 1500ug/ml, 16 hour). PSK activated NFkB in HEK cells transfected with TLR2 in a dose-dependent manner, but had no effect on HEK transfected with other TLRs (4, 7, or 8). To further investigate the role of TLR2 in mediating the immunostimulatory effect of PSK, we used splenocytes from TLR2 knockout (k/o) mice and WT mice and treated them with PSK (25 to 400ug/ml, 72 hour). PSK stimulated the secretion of TNFalpha in splenocytes from WT mice dose-dependently but not in splenocytes from k/o mice. These results suggest that TLR2 is critical in mediating the immunostimulatory effect of PSK.
Immune correlates of clinical responses and survival in ovarian cancer patients treated with intraperitoneal interleukin-2 (41.2)
Authors: Vlad Anda M , Budiu Raluca A , Thaller Julia , Edwards Robert P
Published: January 2023

Ovarian cancer is the most lethal gynecologic cancer and new and improved adjuvant therapies are needed. The confinement of disease to the peritoneal cavity has led to the therapeutic use of intraperitoneal (IP) infusion of biologic agents. Interleukin-2 (IL-2) is a T-cell growth factor important in anti-tumor immunity. The aim of our study was to determine the clinical efficacy of IP IL-2 in a phase II trial for platinum-resistant ovarian cancer and to identify immune correlates of response and survival in 31 IL-2 treated patients. Peripheral blood T cells, collected throughout the treatment were phenotyped and cytokines measured by FACS. Sera and IP fluids were assayed by ELISA for IgE and IgG antibodies and by cytometric bead arrays for VEGF and eotaxin. We found significant associations between baseline IFNγ-secreting CD8 T cells and survival (p=0.04) and between number change during treatment of circulating T cells and survival (p=0.05). Peripheral blood eosinophils were markedly increased at the end of treatment (p<0.0001) and associated with increased eotaxin (p=0.03). Although serum VEGF increased with treatment (p=0.025), measurements in the IP fluid suggest a decrease in local levels of VEGF (p=0.053). This study provides evidence for IP IL-2 in platinum-resistant ovarian cancer and identifies several immune correlates of survival. NIH R21 CA74105-02S1, ACS, Chiron Therapeutics, Magee Health Foundation, PA Dept. of Health.
Development of a transgenic mouse with high affinity TCR reactive to human tyrosinase epitope (41.39)
Authors: MEHROTRA SHIKHAR , Li Mingli , Lyons Gretchen E , Spivey Natalie , Palma Telma Martins da , Naga Osama S , Norell Haakan R , Diaz-Montero C. Marcela , Cole David J , Nishimura Michael I
Published: January 2023

Most tumor-associated antigen epitopes recognized on human melanomas by autologous T lymphocytes are derived from nonmutated self-proteins. Given the belief that T cells bearing high affinity T cell receptor (TCR) would be more effective at targeting self antigens expressed by tumors, understanding tolerance with reference to T cells expressing high affinity TCRs against self-tumor antigens is important for optimizing the anti-tumor T cell response. Thus, using a high affinity TCR isolated from a class-I restricted CD4+ T cells from tumor infiltrating lymphocytes of a patient with metastatic melanoma and reactive to human tyrosinase-derived peptide YMDGTMSQV we developed a novel TCR transgenic mice. Our results show that HLA-A2 restricted TCR passed thymic selection in C57BL/6J (H2-Kb) wild type mice and could be tracked in the periphery. Around 25% of the peripheral CD4+ T cells and more than 75% of the peripheral CD8+ T cells from these TCR transgenic mice expressed functional human tyrosinase TCR. Since tyrosinase is a self-antigen and C57BL/6J mice lack HLA-A2/tyrosinase complexes on cells in their thymus -we developed HLA-A2+TCR+ double transgenic mice by breeding our TCR transgenic mouse and HLA-A2 transgenic mouse strains. Surprisingly, more than 90% of TCR transgenic T cells in the HLA-A2+TCR+ double transgenic mice lacked expression of CD4 or CD8 co-receptors and had CD3+CD4-CD8- phenotype. These double transgenic strains also developed spontaneous autoimmune vitiligo. Thus, our novel TCR transgenic mouse model would allow us to undertake various developmental and translational studies.
Dissociated induction of cytotoxicity and DTH by CFA and CpG (41.8)
Authors: Tigno-Aranjuez Justine T , Lehmann Paul V. , Tary-Lehmann Magdalena
Published: January 2023

Vaccinations typically rely on immunization with live virus for eliciting protective CD8+ T cell immunity. There is increasing interest to use subunit vaccination strategies to achieve such responses. Complete Freund's Adjuvant (CFA) and CpG are considered some of the most potent adjuvants for eliciting immunity. Whereas a wealth of information is available on how these adjuvants affect CD4+ T cell responses, their effects on engaging CD8+ T cell immunity are not completely understood. We immunized C57BL/6J mice with the class I restricted peptides Uty or SIINFEKL using these two adjuvants and tested for cytokine secretion, proliferation, in vivo cytotoxicity and delayed-type hypersensitivity (DTH). Our data show that CFA induced CD8+ T cells to proliferate, mediate DTH, and to secrete IFN-gamma, IL-2 and IL-17. Despite these markers of CD8+ T cell activation, CFA failed to induce an early cytotoxic CD8+ T cell response. In contrast, CpG promoted a vigorous cytolytic response without activating substantial cytokine production, proliferation or DTH. These data have implications for CD8+ T cell sub-unit vaccine design in which cytotoxicity versus DTH plays a key role in host defense. Funding for this work was supported by National Institutes of Health Grant AI47756 and by Cellular Technology Ltd. account OSA483072 to M.T.L.
Ex Vivo-Generation of Gut-Homing Regulatory T-Cells: Characterization and Therapeutic Evaluation in Mice With Established Colitis (42.2)
Authors: Grisham Matthew B. , Karlsson Fridrik , Ostanin Dmitry V. , Jackson Sherry , Gray Laura
Published: January 2023

The objective of this study was to develop an ex vivo method to generate large numbers of Foxp3-expressing Tregs that can be used to evaluate their therapeutic efficacy in a T-cell-dependent model of chronic colitis. We found that polyclonal activation of CD4+CD25- Foxp3- T-cells in the presence of small amounts of IL-2, TGFβ and all trans retinoic acid (RA) converts >90% of these T-cells to Foxp3-expressing Tregs (termed iTregs) as well as induces a 4-5-fold increase in proliferation following a 4 day incubation period in vitro. Furthermore, we found that the presence of RA enhances the surface expression of the gut-homing adhesion molecules α4β7 and CCR9. Finally, we observed that these iTregs are significantly more potent at suppressing T-cell activation in vitro and reversing established colitis in vivo. This protocol routinely generates 30-40 million iTregs from one mouse spleen assuming that the spleen yields approximately 10 million CD4+Foxp3- T-cells and that >90% of these T-cells convert to Foxp3-expressing iTregs as well as undergo a 4-fold proliferation during the 4 day incubation period. We conclude that this protocol generates sufficient numbers of iTregs that can be used in vitro and in vivo to assess the therapeutic efficacy and immunological mechanisms responsible for suppression of inflammation in different models of autoimmune disease (Supported by PO143785).
The neutrophil serine protease inhibitor SerpinB1 protects against inflammatory lung injury and morbidity in influenza virus infection (43.10)
Authors: Gong Dapeng , Benarafa Charaf , Hartshorn Kevan L , Remold-O'Donnell Eileen
Published: January 2023

SerpinB1 is an efficient inhibitor of neutrophil serine proteases. SerpinB1-/- mice fail to clear bacterial lung infection with increased inflammation and neutrophil death. Here, we investigated the role of serpinB1 in influenza virus infection, where infiltrating neutrophils and monocytes facilitate virus clearance but can also cause tissue injury. Influenza virus (H3N2 A/Phil/82) infection caused greater and more protracted body weight loss in serpinB1-/- vs. WT mice (20% vs. 15%; nadir on day 4 vs. day 3). Increased morbidity was not associated with defective virus clearance. Cytokines (IFNγ, TNFα, IL-17, IFNα, G-CSF) and chemokines (MIP-1α, KC, MIP-2) were increased in serpinB1-/- mice vs. WT on days 2-7 post-infection but not on day 1. In WT mice, histology indicated large infiltration of neutrophils peaking on day 1 and maximal airway injury on day 2 that resolved on day 3 coincident with the influx of monocytes/macrophages. In serpinB1-/- mice, neutrophils also peaked on day 1; epithelial injury was severe and sustained with accumulation of dead cells on day 2 and 3. Immunophenotyping of lung digests on day 2 and 3 showed delayed recruitment of monocytes, macrophages and DC in serpinB1-/- mice, but increase of activated CD4 (day 2-3) and CD8 (day 3) T cells. Our findings demonstrate that serpinB1 protects against morbidity and inflammatory lung injury associated with influenza infection. Funded by NIH AI072552.
Subcapsular sinus macrophages prevent lethal vesicular stomatitis virus (VSV) infection (43.11)
Authors: Iannacone Matteo , Moseman Elliott Ashley , Tonti Elena , van Rooijen Nico , von Andrian Ulrich H
Published: January 2023

When a pathogen escapes innate host responses at the site of entry, it has the potential to spread systemically by accessing tissue-draining lymph vessels that channel interstitial fluid to the venous circulation. Before reaching the blood, the tissue-derived lymph must first pass through draining lymph nodes (LNs). LN act as filter stations preventing pathogens from reaching the systemic circulation and they are the staging grounds for adaptive immune responses, providing a specialized environment for the presentation of antigens to B and T cells. It is unclear how viruses are cleared from afferent lymph and how antiviral immune responses are generated. We have previously identified a population of CD11b+CD169+ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of LNs that capture viral particles within minutes after subcutaneous (sc) injection. Using VSV, an acutely cytopathic RNA virus closely related to rabies virus, we show here that these macrophages are critical to prevent viral access to nerve termini located in the SCS. Indeed, mice depleted of SCS macrophages develop an ascending CNS paralysis that leads to death in 50% of the animals 6-10 days after sc infection. Macrophage-depleted mice die despite mounting an increased neutralizing antibody (nAb) response. The notion that nAb are dispensable for protection against a sc infection (the natural route of transmission of this virus) is further demonstrated by the fact that antibody-deficient mice, which die upon intravenous infection, survive a sc VSV infection as long as SCS macrophages are present. While nAb are unnecessary for survival, B cells are required, and their protective role presumably relies on maintenance of LN architecture and normal macrophage phenotype.
The role of IL-17 in the protective immune response to Mycobacterium tuberculosis (44.18)
Authors: Windish Hillarie Plessner , Zheng John , Bertholet Sylvie , Baldwin Susan L , Ireton Gregory C , Vedvick Tom , Reed Steven G , Coler Rhea N
Published: January 2023

Mycobacterium tuberculosis (Mtb) infection kills more than 1.5 million people each year. While BCG prevents disseminated disease in children, a vaccine that confers long lasting protection from tuberculosis remains elusive. Previous work has demonstrated that expression of IL-17 in response to Mtb antigens supported resistance during subsequent infection. This suggests that an effective tuberculosis vaccine should induce IL-17. We hypothesized that a vaccine, comprised of ID83 and a TLR4 agonist/adjuvant protects against tuberculosis, in part by inducing IL-17. We characterized expression of IL-17 during the response to vaccination of C57Bl/6 mice. We found that IL-17 was produced by γδ T cells, Th17 cells, and also CD8 T cells in response to immunization. IL-17 expression was far more robust in the memory response to Mtb antigens, either during re-stimulation with vaccine antigens or during Mtb infection, than in the primary response. In continuing studies, we investigate the role of IL-17 in response to Mtb antigens and its role in protection that results from ID83 vaccination.

This work was supported by NIH Contract N01-AI-25479, and NIH grants AI-44373, AI-067251 and AI-078054.
MyD88 is required for viral-induced B cell activation and intestinal IgA production (44.20)
Authors: Blutt Sarah Elizabeth , Akira Shizuo , Dustin Lynn , Conner Margaret E
Published: January 2023

The role of TLR signaling in the induction of specific antibody responses is controversial and defined primarily using model antigens. Our study objective was to determine whether TLR signaling is required to generate pathogen-specific antibody responses. To assess whether TLR signaling is required for a viral-specific intestinal B cell responses, we examined whether B cell responses were aberrant after rotavirus infection of mice lacking expression of specific adapter proteins used in TLR signaling (MyD88, TIRAP, TRIF). Rotavirus infection of MyD88-/- mice did not result in a significant induction of either B cell activation or intestinal rotavirus-specific IgA. In contrast, mice lacking TIRAP and TRIF expression had wild type levels of both B cell activation and intestinal IgA production. B cell activation was normal in mice with a defect in BCR signaling (xid) or mice expressing an OVA-specific BCR, indicating that rotavirus-induced B cell activation does not require a rotavirus-specific BCR. These findings indicate that BCR-independent signaling through MyD88 plays a critical role in pathogen induced B cell activation and the generation of pathogen-specific intestinal IgA.

This work was supported by NIH AI10604, AI24998, and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
TREATMENT WITH A SPHINGOSINE ANALOG DIMINSHES IMMUNOPATHOLOGY FOLLOWING INFLUENZA VIRUS INFECTION (45.2)
Authors: Walsh Kevin Barry , Marsolais David , McGavern Dorian , Hatta Yasuko , Kawaoka Yoshihiro , Rosen Hugh , Oldstone Michael B.A.
Published: January 2023

Highly pathogenic strains of influenza virus can result in high mortality rates due to severe tissue damage caused by direct injury induced by the virus or by the immune response against the virus (immunopathology). Immune-mediated pulmonary tissue damage is a result of cytokine/chemokine release and the antiviral activity by cells of the innate and adaptive immune responses. Here we report that intratracheal treatment with a sphingosine analog, AAL-R, results in the inhibition of dendritic cell (DC) activation and antigen presentation to T cells in the mediastinal lymph nodes and the lung. Diminished DC function restricts virus-specific T cell proliferation causing a subsequent reduction in the accumulation of T cells in the lungs. The immunosuppressive effect involves signaling through S1P receptors on DC, but is independent of S1P1, S1P2 and S1P3. Inhibition of the accumulation of virus-specific T cells in the lungs occurs without impinging the production of anti-viral neutralizing antibodies. AAL-R treatment hinders cytokine/chemokine production which leads to a reduction in polymorphonuclear leukocyte and macrophage infiltration resulting in reduced lung injury. Importantly, local AAL-R administration into the lung was effective in controlling CD8+ T cell accumulation in the lungs when given four days following influenza virus infection. Administration of sphingosine analogs during influenza virus infection offers a potentially powerful therapeutic for alleviating pulmonary immunopathology.
Age-associated defects in Tcell metabolism (46.10)
Authors: Faulkner Matthew F , Durdik Jeannine M
Published: January 2023

Aging at the cellular and molecular level causes a decline in immune function. We are characterizing the responses of naïve T-cells to activation. Activated T-cells of aged mice initiate proliferation as well as their young counterparts, but have increased apoptotic rates. However, the mechanisms behind this post- activation death are not completely understood. We will show alterations in metabolic activities in T-cells of aged mice through differences in the expression of glucose transporter (Glut 1), glucose uptake and lactate production. Additionally, we will correlate these findings with autophagocytic vesicle formation by analyzing microtubule-associated protein -1 light chain 3 (LC3) as well as comparing relative amounts of actively respiring mitochondria using Mitotracker Red CM-H2XRos, a membrane potential dependant dye, and total mitochondrial mass by staining with the mitochondrial-selective fluorescence label Mitotracker Green. Levels of reactive oxygen species will be then quantified by staining with ROS specific dye DCF-DA and correlated to the ratio of mitochondrial mass to membrane potential.
Tracing memory T cell fate using Ox40-cre and novel reporter alleles (46.17)
Authors: Klinger Mark , Chmura Stephen A , Killeen Nigel
Published: January 2023

OX40 is a member of the Tumor Necrosis Factor Receptor Family that is expressed on activated and regulatory T cells. We have used an Ox40-cre allele with new Cre-dependent reporter alleles to discriminate memory T cells that differ in how much OX40 they expressed during immune responses to viruses. The reporters reveal flux in CD4+ memory T cells resulting in a gradual time-dependent enrichment for cells that previously expressed high amounts of OX40. Such flux was much less evident in CD8+ memory T cells. Differences in amounts of prior OX40 expression were nonetheless found to correlate with phenotypic differences in both CD4+ and CD8+ memory T cells. The results make clear the potential for using the Ox40-cre and reporter alleles for tracing the fate of activated T cells and relating it to differences in prior OX40 expression.
ICOS/B7h Bidirectional Signal to Induce Secretion of IL-1β and IL-17 (46.3)
Authors: Chiocchetti Annalisa , Mesturini Riccardo , Nicola Stefania , Dianzani Umberto
Published: January 2023

B7h is constitutively expressed by antigen presenting cells (APC) and binds ICOS expressed by activated T cells. ICOS triggering costimulates T helper (TH) cell activation and modulates their cytokine secretion, whereas the effect of B7h triggering in APC is unknown. This work investigated B7h reverse signalling in dendritic cells (DC). We induced DC maturation with LPS in the presence or absence of a soluble ICOS-Ig fusion protein; then, both cytokine secretion and allostimolatory activity were evaluated. B7h stimulation substantially modulated LPS-induced DC maturation by influencing cytokine secretion. The most striking effect was 80-fold increase of IL-1b secretion, but other changes were 3-fold increase of IL-10 and 2-fold decrease of TNF-a. In IL-1b secretion, B7h triggering acted as a "second signal" by activating caspase-1, required to cleave pro-IL-1b whose synthesis was induced by LPS. B7h triggering on LPS-activated DC also modulated their capacity to activate allogeneic T cells in mixed lymphocyte cultures (MLC) by decreasing the proliferative response and changing cytokines secretion, including 20-fold decrease of IFN-g and 5-fold increase of IL-17. This effect was partly ascribable to the cytokine milieu secreted by DC and particularly to the high IL-1b levels, known to be involved in differetiation of human Th17 cells. However, ICOS triggering too played a role in IL-17 secretion since blocking of the B7h/ICOS interaction inhibited IL-17 secretion in MLC with DC. Moreover, in naive Th cells activated with anti-CD3 antibodies, ICOS stimulation induced IL-17 secretion in the presence of exogenous IL-1b. In this work we showed that B7h-mediated reverse signaling induced IL-1b by DCs which in turn favors IL-17 secretion by Th cells. On the other side, we demonstrated that ICOS costimulation directly favors Th17 differentiation.
Live imaging of peripheral T cell-DC interactions in the islets: An emerging axis of autoimmune infiltration (48.15)
Authors: Friedman Rachel S. , Melli Kristin , Sorensen Caitlin M. , Tang Qizhi , Krummel Matthew F.
Published: January 2023

Defining processes of islet infiltration and destruction in Type 1 diabetes (T1D) is important for its prevention and preventing destruction of transplanted islets in diabetic individuals. We hypothesize that T1D onset and persistence is maintained by T cell-dendritic cell (DC) interactions in both the draining pancreatic lymph node (PLN) and the islets. In the steady-state, we found that intra-islet DCs are a uniform semi-mature population that constitutively take up β cell antigens in preparation for antigen presentation. Islet-antigen specific CD8 T cells activated in the PLN trafficked to the islets where they induced maturation of islet DCs. Leukocytic infiltration of the islets and islet DC maturation were dependent on IFN-γ production by T cells. Blockade of antigen processing following T cell activation in the PLN also blocked islet DC maturation but not islet infiltration. This suggests that the DCs require productive antigen-presenting interactions with T cells in the islets for maturation to occur. Through 2-photon imaging of the islets, we visualized sustained interactions of islet-antigen specific CD8 T cells with islet DCs and characterized features of cytotoxicity and dwell-time in this setting. Taken together, these data suggest that directed and synaptic delivery of IFN-γ to intra-islet DCs induces their maturation.

This work was funded by the Larry L. Hillblom Foundation and the Juvenile Diabetes Research Foundation.
Natural killer cells suppress autoimmune myocarditis in mice (48.23)
Authors: Ligons Davinna L. , Talor Monica V. , Rose Noel R. , Cihakova Daniela
Published: January 2023

Autoimmune myocarditis is induced by the adoptive immune response; however, there is evidence that the innate immune system plays a major role in modulating disease. Natural killer (NK) cells can function in early innate responses, as well affecting the later adaptive immune response. Depletion of NK cells has been shown to exacerbate the development of virus-induced myocarditis. We hypothesized that NK cells also modulate the virus-free model of myosin-induced experimental autoimmune myocarditis (EAM). To study the action of NK cells, we immunized BALB/c mice on days 0 and 7 with myocarditogenic peptide MyHC (614-629) in complete Freund adjuvant. The mice were treated with PBS, rabbit IgG or anti-asialo GM1 antibody every day for 6 days prior to the first immunization and every 2 days following day 0 until day 21. We found that depleting NK cells significantly increased the severity of disease, and serum antibodies specific to the myocarditogenic peptide. Splenic CD4+ T cells were significantly more activated in NK depleted mice compared to controls. Cytokine analysis of heart homogenates revealed a significant increase in eotaxin and IL-5 in the hearts of mice that were depleted of NK cells. Our data suggest that NK cells suppress the adoptive immune response and modulate eosinophil migration and T cell activation during EAM in mice.
Lupus immunotherapy using CD4 targeted nanoparticles (48.29)
Authors: Look Michael W. , Stern Eric , Wang Qin , DiPlacido Leah , Craft Joseph E. , Fahmy Tarek M.
Published: January 2023

The pathogenesis of systemic lupus erythematosus is mediated by collaborations between CD4 T cells and B cells with autoantibody production. We propose that the use of biodegradable, nanoparticulate materials that are targeted against CD4 T cells and carry immunosuppressive drugs and proteins can be used to treat lupus. Biodegradable nanoparticles exhibit excellent therapeutic features such as biocompatibility, targeting potential, and controlled release rates of its encapsulated cargo. Here, we report results demonstrating the therapeutic efficacy of using nanoparticles to suppress lupus in NZB/W F1 mice, a lupus prone animal model. NZB/W F1 mice were administered weekly treatments of nanoparticle therapy, and disease progression and survival were monitored. Results show that nanoparticle therapy can delay disease progression and improve survival, using an immunosuppressive drug dosage in nanoparticles that is at least 16-fold less than the amount of immunosuppressive drug administered in buffer. These results suggest that nanoparticle immunotherapy can be used to effectively treat lupus at a lower dosage amount and frequency of dosage than conventional therapeutic regimens.

This research is funded by the Wallace H. Coulter Foundation. ML is funded by a National Defense Science and Engineering Graduate (NDSEG) fellowship from the U.S. Department of Defense.
B cells play a critical role for renal injury in an experimental model of systemic sclerosis (48.3)
Authors: Chen Cindy Y , Delaney Tracy A , Jane Connor , Kiener Peter , Coyle Anthony J , Herbst Ronald
Published: January 2023

Systemic sclerosis (SSc) is a complex autoimmune disease involving autoreactive B and T cells and excessive extracellular matrix deposition in target organs such as kidneys. We have developed a novel human collagen V (hCollV) induced autoimmune model which shares some common pathological and immunological features with Ssc. Mice immunized with hCollV have increased proteinuria, renal fibrosis, serum autoantibodies (anti- RNP, -SSA, -collagen I, II) (p<0.01), and renal IgG depositions by day 42. Levels of proinflammatory cytokines and chemokines associated with tissue fibrosis, such as IL-6 and MCP-1, were elevated in the sera of immunized mice when compared to controls, as were gene expression levels in the kidneys by qPCR analysis. Depletion of B cells by afucosylated anti-hCD19 antibody resulted in reduced proteinuria, tissue fibrosis, and IgG depositions in the kidneys of immunized mice, decreased cytokines and chemokines levels, and greatly declined serum autoantibodies. Therefore, our data indicated that the B cells play crucial roles in causing renal damage in the hCollV-induced systemic sclerosis model, suggesting a potential therapeutic target to benefit Ssc patients.
Mapping Susceptibility Loci for Immune Mediated Nephritis in 129/svJ Mice (49.19)
Authors: Xie Shangkui , Li Li(Lisa) , Xie Chun , Li Li , Han Jie , Wang Tao , Zhou Joseph , Mohan Chandra
Published: January 2023

Laboratory strains of mice differ in their susceptibility to immune-mediated nephritis. Whereas NZW, DBA1 and 129/svJ strains of mice are highly susceptible to anti-GBM induced nephritis, most other laboratory strains, including C57BL/6 and BALB/c have significantly less disease. In order to uncover susceptibility genes for immune-mediated nephritis, a panel of 156 (129 x BALB/c)F2 mice were bred, challenged with anti-GBM antibodies and phenotyped for nephritis. About 40% of these F2 progeny had evidence of severe nephritis. Genotyping and linkage analysis revealed 2 strongly recessive 129/svJ loci for glomerulonephritis, one on chromosome 2 (linked to rs6295520, P=0.0008) and the second on chromosome 14 (linked to rs3701623, P=0.0004). Proteinuria was strongly linked to 3 recessive 129/svJ loci, including rs3677657 on chr. 7 (P=0.0026), rs3711424 on chr. 13 (P=0.0001), and the chromosome 14 locus for GN. Among these, the loci on Chr 7 and 14 were also strongly linked to glomerular crescent formation, operating additively.

To summarize, recessive loci on chromosomes 2, 7, 13 and 14 account for the enhanced susceptibility to nephritis in 129/svJ mice. Congenic dissection studies and candidate gene testing are in progress.