bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of MDSC induced T cell tolerance in cancer (88.26)
Authors: Nagaraj Srinivas , Schrum Adam , Cho Hyun-II , Celis Esteban , Gabrilovich Dmitry I
Published: January 2023

Tumor antigen-specific T-cell tolerance is a critical element of tumor escape. It is known that MDSC can induce antigen-specific CD8+ T cell tolerance in an experimental system with adoptive transfer of transgenic T cells. In this study we addressed the critical question whether a similar phenomenon can be observed in a "natural" non-transgenic experimental model. Balb/C mice were subcutaneously inoculated with two different tumor cell lines, a derivative A2L2 cell line, which expresses high levels of HER2/neu and the parental cell line 66.3. HER2/neu specific CD8+T cells were generated by immunization of mice with peptide. MDSC were able to tolerize antigen-specific T cells in vivo if they were collected from A2L2 but not from 66.3 tumor-bearing mice indicating the tumor-specific nature of T cell tolerance induced by MDSC. Next we investigated the type of signaling caused by MDSC in CD8+T cells that could lead to tolerance. MDSC dramatically reduced the ability of CD8+ T cells to respond to the specific peptide. However, study of various signaling molecules using different experimental systems and tests found that MDSC did not induce negative signaling in T cells. This conclusion was confirmed in adoptive transfer experiments with CD8+T cells expressing dual TCR: LCMV glycoprotein peptide gp33-41 and OVA-derived H-2Kb-restricted peptide ova257-264. We found that MDSC loaded with peptide specific for one TCR induced tolerance only against that specific epitope but CD8+ T cells retained the ability to respond to the peptide specific for the other TCR expressed on the same cell.
Deficiency of adipocyte fatty acid-binding protein, A-FABP, suppresses tumor metastasis (88.9)
Authors: Li Bing , Stout Robert D. , Suttles Jill
Published: January 2023

An association between inflammation and cancer is well-established and it has been shown that tumor infiltrating macrophages (TIM) play a supportive role in tumor development. Recent studies have found that expression of A-FABP by macrophages promotes their inflammatory function. Thus, in the present study we evaluated the influence of A-FABP expression on tumor growth and metastasis. Upon transplantation of the of the murine tumor cell lines LL/2, MC38 and E0771, we found significantly lower levels of tumor metastasis to the lung in A-FABP-/- mice as compared to wild-type mice. In the LL/2 model, decreased metastasis in A-FABP-/- mice was not associated with decreased growth of the primary tumor. Concentrating on the role of macrophages, we found that although wild-type and A-FABP-/- mice showed equal numbers of TIM, A-FABP-/- TIM were poor producers of inflammatory cytokines, including IL-6, TNFa and IL-23 as compared to wild-type TIM. Activation of the MAPKs p38, ERK1/2 and JNK, as well as activation of IKK and NFkB in response to interaction with tumors was diminished in A-FABP-/- macrophages. In addition, A-FABP-/- macrophages produced significantly lower levels of MMP, an established mediator of tumor metastasis, both in vitro and in vivo as compared to wild-type macrophages. These results suggest a critical role for A-FABP in macrophage activities that promote tumor metastasis.
Anti-CD3 antibodies induce tolerance to FVIII in Hemophilia A mice after FVIII plasmid-mediated gene therapy (89.19)
Authors: Peng Baowei , Ye Peiqing , Rawlings David J , Ochs Hans D , Miao Carol H
Published: January 2023

We report here the successful use of anti-CD3 antibodies to modulate the immune responses after gene therapy. Five consecutive medium doses of anti-CD3 treatment concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic-level of FVIII gene expression in treated Hemophilia A mice. Moreover, repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells in treated mice and increased the frequency of CD4+CD25+FoxP3+ regulatory T cells (Tregs). Nevertheless, prior depletion of CD25+ T cells did not abrogated tolerance induction and adoptive transfer of CD4+CD25+ T cells of tolerized mice in early time points to syngeneic plasmid injected mice did not transfer the dominant tolerance. Furthermore, anti-CD3 treated mice could mount normal immune responses against both T-dependent and T-independent neo-antigens. Our data indicate that anti-CD3 immunomodulation can be a safe and effective strategy to prevent transgene-specific immune responses following gene therapy.
Antigen dose over-rides DC phenotype in the control of CD4+ regulatory T cell induction through reduced Akt/mTOR signaling (89.30)
Authors: Turner Michael S , Finn Olivera J , Morel Penelope A
Published: January 2023

There is no consensus regarding which features of dendritic cells (DC) are optimal for inducing Foxp3+ regulatory T cells (Treg). Immature DC (iDC) have been shown to induce Tregs in response to foreign and allo-antigens. However, in the autoimmunity setting, mature (mDC) induce tolerance to self antigens by inducing Treg expansion and Th2 differentiation.

We compared the induction of islet autoantigen-specific Treg by mDC, and iDC populations that have distinct phenotypes and cytokine profiles. CD4+ T cells from BDC2.5 TCR Tg mice were cultured in the absence of exogenous TGFβ or IL-2 with iDC or mDC pulsed with high- or low-affinity antigenic peptides.

DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Higher doses of antigenic peptide induced strong Akt/mTOR signaling and favored the expansion of Foxp3- Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 (OVA) and VFT (MUC1) TCR-Tg T cells and could be recapitulated with anti-CD3/CD28 stimulation in the absence of any DC.

These studies indicate that the strength of Akt/mTOR signaling is a critical T cell-intrinsic determinant for Treg vs Th induction. Furthermore, appropriately adjusting the dose of antigenic peptide can override the effect that DC phenotype and cytokine production may play.
The link between aberrant regulatory DC subset with breaking of tolerance in CD48 deficient mice (89.31)
Authors: Tamir Ayala , Durgan Kevin , Latchman Yvette
Published: January 2023

Dendritic cells (DCs) are antigen presenting cells with a dual role of priming T cells response and induction of peripheral tolerance. While CD11chigh DCs normally stimulate immunity (sDCs), CD11clowCD45rbhigh DCs were recently characterized as a regulatory subtype (rDC), which maintains low levels of costimulatory molecules after maturation. We found rDC levels were at least 2 times lower in bone marrow and spleens of CD48-/- C57BL/6 mice compared to their WT counterparts. Previously, we observed that aging CD48-/- mice developed spontaneous lupus-like autoimmune disease, indicated in elevated levels of autonuclear antibodies. We have shown that treatment of bone-marrow derived DCs with the neuropeptide, vasoactive intestinal peptide (VIP) and IL-10 strongly expanded the rDCs subtype, which produced high and low levels of IL-10 and IL-12, respectively. VIP-treated DCs of WT mice produced significantly higher amounts of IL-10, compared to CD48-/- DCs. We have also demonstrated that adoptive transfer of sDCs and rDCs subtypes together with OT-II-derived CD4+ T cells into WT and CD48-/- mice induced inhibition of OT-II-CD4+ T cell expansion only when WT-derived rDCs were transferred, as compared to expansion induced by their sDCs counterparts. These data suggest that dysfunctional and lower levels of rDCs of CD48-/- mice may be responsible for the development of spontaneous SLE in these mice when they are older.
T-bet controls regulatory T cell function during type-1 inflammatory responses (89.33)
Authors: Koch Meghan A , Perdue Nikole R , Tucker-Heard Glady's , Killebrew Justin R , Urdahl Kevin B , Campbell Daniel J
Published: January 2023

Regulatory T cells (TR) expressing the transcription factor Foxp3 play a critical role in preventing autoimmunity and restraining immune responses during infection. To accomplish these tasks, several subsets of TR work in concert to maintain normal immune homeostasis in secondary lymphoid tissues and at peripheral sites. However, the molecular basis underlying the phenotypic and functional differences of TR subsets remain obscure. Here, we show that during type 1 inflammatory responses, Foxp3+ TR upregulate the TH1-specifying transcription factor T-bet. These Foxp3+T-bet+ cells retain regulatory function, and do not produce the pro-inflammatory TH1 cytokine IFN-γ. Within TR, T-bet directs expression of the TH1-associated chemokine receptor CXCR3, and T-bet+ TR accumulate at sites of microbial replication in mice during persistent infection with Mycobacterium tuberculosis. T-bet-deficient TR show diminished proliferation and are outcompeted by wild type TR during strong TH1 responses in vivo. Furthermore, T-bet expression by TR is required for their proper homeostasis and function during TH1-driven autoimmunity. Thus, our data define a unique population of CD4+ T cells in which the activities of Foxp3 and T-bet are overlaid, resulting in functional TR capable of expanding during type-1 inflammation and suppressing TH1 responses in vivo.

(NIH -DK072295, AI067750, AI069889)
Role of the PKCλ/ι in Th2 establishment and airway hyperresponsiveness (90.11)
Authors: Yang Jun-Qi , Diaz-Meco Maria T. , Moscat Jorge
Published: January 2023

The differentiation of T cells along different lineages is central to the control of immunity. In order to investigate the role of PKCλ/ι, a member of atypical protein kinase C, in T cell function, we used a conditional gene knockout system by loxP-Cre recombination to selectively delete PKCλ/ι in activated T cells. With this system, we found that PKCλ/ι was necessary for Th2 cytokine production. Upon anti-CD3 plus anti-CD28 stimulation, IL-4 and IL-13 production from CD4+ T cells was significantly reduced in conditional KO mice as compared to their WT littermates. However, PKCλ/ι was not involved in Th1 cytokine secretion. T cell proliferation was impaired in conditional PKCλ/ι KO mice. Furthermore, deficiency of PKCλ/ι inhibited OVA-induced allergic airway inflammation in vivo. Our data demonstrated that the activation of the transcription factors NFAT and NF-¿B was impaired in PKCλ/ι-deficient activated T cells. In addition, we presented genetic knockout evidence in ex vivo experiments with primary T cells that PKCλ/ι was critical for the control of cell polarity during T cell activation. Therefore, PKCλ/ι emerges as a critical regulator of Th2 cell activation.
PD-1 localization in the immunological synapse is different in exhausted as compared to effector LCMV specific CD8 T cells (90.12)
Authors: Duraiswamy Jaikumar , Pryshchep Sergey , Ha Sang-Jun , Goronzy Jorg J , Ahmed Rafi
Published: January 2023

Understanding the molecular basis of ineffective T cell response during chronic viral infections may provide new therapeutic targets. Our studies in lymphocytic choriomeningitis virus (LCMV) infection provided the first indication that programmed death-1 (PD-1) pathway regulate T cell dysfunction during chronic infection. These findings were extended to HIV, SIV, HBV and HCV infections and suggest that in vivo blockade of PD-1 pathway may provide a therapeutic approach for human chronic infections. However, further studies are needed to determine how best to modulate PD-1 signaling to activate anti-viral T cells while minimizing immunopathology. Understanding these signaling effects would be greatly advanced by direct imaging of PD-1 in relation to signaling structures in the immunological synapse. Using LCMV model, we investigated PD-1 localization at the T:DC synapse by direct imaging of PD-1 in relation to T cell receptor by confocal microscopy. We compared P14 (LCMV gp33-specific) effector and exhausted cells from mice infected with LCMV Armstrong and clone 13 strains, respectively. Here we show that PD-1:TCR co-localization at the T:DC synapse is different between exhausted and effector CD8 T cells. Our study provides important informations on molecular mechanisms behind T cell exhaustion and may help to identify novel targets to modify PD-1 signaling for the treatment of human chronic infections.
Cycloastragenol extends T cell proliferation by increasing telomerase activity (90.30)
Authors: Valenzuela Hector F. , Fuller Thomas , Edwards Jim , Finger Danielle , Molgora Brenda
Published: January 2023

All normal human somatic cells have a finite number of times that they can divide and when this limit is reached cells are described as cellular senescent. Telomere shortening has been attributed as a main mechanism for cellular aging and for the lack of cellular functions associated with aging. Unlike what is seen in most somatic cells, in activated T cells telomerase activity is upregulated, resulting in increased telomere length. However, this brief period of telomerase induction only delays cellular senescence. Naturally, there is a great deal of interest in finding inducers of telomerase that may help delay the onset of cellular aging. There are various nutraceuticals that claim to both increase the health of individuals and delay the onset of cellular aging. We tested the nutraceuticals resveratrol and cycloastragenol for their ability to enhance T cell functions in vitro. In this study we evaluated the effect of these compounds on cellular proliferative capacity, levels of telomerase activity, surface markers and cytokine secretion of human CD4 and CD8 T cells. Our results show that cycloastragenol moderately increase telomerase activity and proliferative capacity of both CD4 and CD8 T cells. These preliminary results suggest that nutraceuticals inhibit the onset of CD4 and CD8 cellular senescence.
Exosomes secreted from human neural stem cells suppress T cell activation (90.33)
Authors: Kim Su Young , Cho Hyoung-Soo , Yang Seung-Ha , Shin Jin-Young , Kim Jung-Sik , Park Chung-Gyu
Published: January 2023

Exosomes are 50-100 nm diameter small membrane vesicles secreted from a variety of cells. Although exosomes express a common set of proteins, their protein composition is quite unique and varied significantly. Therefore, their function seems to be dynamic and determined by cell type specific proteins. Human neural stem cells (hNSCs) have been reported to have a therapeutic potential for neuro-regeneration in neurodegenerative disorders. However, the underlying mechanisms of neuroprotective effects of hNSCs are not still clear. In this study, we demonstrated that culture supernatant of hNSC, HB1.F3, can suppress the activation and proliferation of human T cells by apoptosis and cell cycle arrest. More interestingly, we found that exosomes secreted by hNSCs and included in culture supernatant play a critical role in the suppression of T cells by inducing G0/G1 arrest. Therefore, we here suggest that hNSCs has an immunomodulatory effect on T cells without cell-to-cell contact and a possible mechanism is mediated by secreted exosomes.
SHIP-deficient dendritic cells suppress T cell proliferation via a nitric oxide independent mechanism (91.9)
Authors: Antignano Frann , Hamilton Melisa , Cohen Carla , Ho Victor , Krystal Gerald
Published: January 2023

Dendritic cells (DCs) are potent stimulators of the immune system but can also be powerful inducers of tolerance. Previous studies have shown that myeloid DC precursors are capable of suppressing T cell proliferation through a contact and nitric oxide (NO) -dependent mechanism. In addition, GM + IL-4, but not Flt3L, -derived DCs have been reported to suppress T cell proliferation by inducing T cell apoptosis. The SH2-containing inositol 5'-phosphatase (SHIP) is a potent suppressor of the PI3K pathway with known functions in regulating myeloid cell development and survival. However, little is known of SHIP's role in the suppressive ability of DC subsets. In this study we co-cultured polyclonally activated T cells with bone marrow derived DCs or DCs isolated from the spleens of WT and SHIP-/- mice to determine SHIP's role in DC-induced T cell suppression. We found that both WT and SHIP-deficient DCs derived with GM-CSF ± IL-4 profoundly suppress T cell proliferation while Flt3L-derived and splenic DCs do not. The mechanism of suppression proved to be contact dependent for WT and SHIP-deficient GM-CSF DCs. However, unlike WT, SHIP-deficient DCs do not produce detectable levels of NO. These results suggest a novel mechanism of myeloid DC-induced T cell suppression at work in SHIP-deficient DCs.

This research is funded by an NCIC grant held by GK.
IFNbeta inhibits DC migration in vitro and in vivo (92.2)
Authors: Yen Jui-Hung , Ganea Doina
Published: January 2023

Interferon β (IFN β) is a widely approved therapeutic option for the treatment of multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE). However, the molecular mechanisms underlying the effects of IFNβ in MS/EAE are still not fully understood. In EAE, the major players are encephalitogenic CD4+ T cells and perivascular myeloid dendritic cells (DC). Therefore, migration of both peripheral T cells and DC into the CNS is essential for development of EAE/MS. Migration of DC from the inflammatory site to draining lymph nodes for antigen presentation and activation of naïve T cells, and to the CNS during chronic neuroinflammation requires CCR7 and MMP-9 expression. In this study, we report for the first time that IFNβ inhibits CCR7 expression and MMP-9 production in DC matured with a cytokine cocktail (TNFα+IL-1+IL-6+PGE2). In addition, DC matured in the presence of IFNβ exhibit a lower migratory capacity compared to mature DC. STAT-1 signaling is essential for IFNβ-mediated inhibition of CCR7 and MMP-9 expression, and subsequent DC migration. Our in vivo data also show that mice treated with IFNβ have a lower number of DC migrating to the draining lymph nodes following exposure to FITC as compared to controls. In conclusion, our results provide an addition mechanism for the beneficial effect of IFNβ in MS/EAE through the inhibition of mature DC migration. This work is support by NIH 2RO1AI052306
Eosinophil-derived IL-16 induction of FIZZ1 expression in alveolar epithelial cells is mediated by CD9 (93.11)
Authors: Liu Tianju , Huaux Francois , Choi Yoon Young , Ullenbruch Matthew , Phan Sem H
Published: January 2023

Recruited eosinophils in pulmonary fibrosis can play a role in tissue remodeling but its role is not fully understood. A novel secreted protein, found in inflammatory zone 1 (FIZZ1) is highly induced in bleomycin-induced lung injury. It is primarily localized to lung epithelial cells and mononuclear phagocytes. It has potential fibrogenic properties due to its ability to induce myofibroblast differentiation and enhance fibroblast resistance to apoptosis. In this study, the ability of eosinophils to regulate epithelial FIZZ1 expression was analyzed. Eosinophil and alveolar epithelial type II cell (AEC) co-cultures induced AEC FIZZ1 expression, which was significantly suppressed only by antibodies to IL-16. Cytokine array analysis confirmed that eosinophils from fibrotic lung expressed IL-16. Moreover IL-16 stimulated AEC FIZZ1 expression in a dose dependent manner, and AECs were found to express CD9, a putative IL-16 receptor. Suppression of CD9 expression by CD9 siRNA significantly inhibited AEC FIZZ1 expression by 67%. However antibodies to CD9 stimulated FIZZ1 expression indicating perhaps that antibody binding could activate CD9 signaling. These findings suggest that eosinophil-derived IL16 is capable of regulating FIZZ1 expression in AECs via binding to CD9. This may represent another pathway by which eosinophils could promote tissue fibrosis.

Supported by NIH grants HL28737, HL31963, HL 52285 and HL77297
Development of a human in vitro blood-nerve barrier model (94.20)
Authors: Ubogu Eroboghene E , Yosef Nejla , Xia Robin H
Published: January 2023

Phenotypic and functional differences exist between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells from the same tissue. Little is known about cellular interactions at the blood-nerve barrier (BNB). An in vitro BNB (IVBNB) model was developed using primary human endoneurial endothelial cells (pHEndECs) freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion and density centrifugation. pHEndECs are spindle shaped, contact inhibited, differentiate to form capillary-like tubes, are Ulex Europaeus Agglutinin 1 and von Willebrand Factor positive and endocytose acetylated low density lipoprotein. They also express alkaline phosphatase, γ-glutamyl transpeptidase, glucose transporter-1, p-glycoprotein and low levels of cellular adhesion molecules. Culturing pHEndECs on collagen coated transwell inserts was used to develop the IVBNB. High transendothelial electrical resistances (~160Ω.cm2; maximal 12 days after seeding) and low solute permeability to fluoresceinated high molecular weight (70 kDa) dextran (~0.7%) were seen. Electron microscopy demonstrated intercellular tight junctions and 50-100 nm-diameter pinocytic vesicles. These features are consistent with the BNB. This model provides an avenue to study mechanisms of leukocyte entry into peripheral nerves.
Simultaneous detection of mouse FoxP3, IL-17 and surface markers by multi-color flow cytometry (96.18)
Authors: Li Li , Wu Xiaowei , Wei Aili , Rohrer Jurg
Published: January 2023

Naïve CD4+ T helper cells can be induced to differentiate towards at least four types of committed helper T cells, namely T helper 1 (Th1), Th2, Th17 and regulatory T cells (Treg) according to the local cytokine milieu. The committed cells are characterized by the expression of specific transcription factors and by the cytokines released. Th17 cells are very efficient inducers of tissue inflammation and crucial initiators of organ specific autoimmunity. FoxP3+ Treg cells are necessary and sufficient to prevent autoimmunity. There is a reciprocal relationship between pathogenic Th17 cells and Foxp3+ Treg cells. Therefore the need to simultaneously detect FoxP3 and IL-17 is becoming increasingly important. Since mouse FoxP3 is sensitive to fixation buffer, it is important to use the appropriated buffers and protocols for its detection. In this report, we described the development of a fix/perm buffer system and protocol for simultaneous detection of mouse FoxP3 and IL-17 with cell surface markers CD4 and CD25. Using the optimized buffer and storage conditions, staining can be done on freshly fixed cells or stored fixed cells. In addition, effects of temperature during the fix/perm steps and the sensitivity of certain clones to the fixation process will be discussed.
Acquistion of Effector and Memory Responses is Reciprocally Regulated by IL-12 and IFN-α/β in Human CD8+ T Cells (96.8)
Authors: Ramos Hilario J , Davis Ann M , Cole Alexander G , Farrar J. David
Published: January 2023

The innate cytokines IL-12 and IFN-α/β act in concert to facilitate clearance of intracellular pathogens. However their precise role in promoting human T cell responses is still unclear. In this study, we demonstrate that these cytokines regulate distinct aspects of effector and memory human CD8+ T cell differentiation. We found that IL-12 programs the development of TEM cells which secrete high levels of IFN-γand TNF-α and attain strong cytolytic activity. Programming of TEM cells by IL-12 correlated to the induction of the IL-12 receptor β 2 (IL-2Rβ2) and was associated with cells which had undergone rapid division. Alternatively, IFN- α/β enhanced a population of TCM cells which expressed high levels of the lymph node homing receptor CCR7. This population did not display immediate effector activity however, upon secondary activation, were capable of rapid proliferation and acquisition of both memory and effector phenotypes. TCM development was regulated by IFN-αand proceeded by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN- α/βreceptor-2 (IFNAR2). We further examined the effects of the strength of TCR signal on TEM and TCM development. In the presence of both IL-12 and IFN- α/β, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both TCM and TEM. Together, our results support a novel model in which IL-12 and IFN- α/βact in a non-redundant manner to regulate the co-linear generation of both effector and memory cells.

This study is supported by a grant from the National Institutes of Health/National Institute of Allergy and Infectious Disease (AI56222) and by a predoctoral fellowship from the National Instititue of Health/Nation Institute of Allergy and Infectious Disease (AI68622).
IL-4 regulates CD8+ T cell homeostasis in BALB/c but not C57BL/6 mice (97.8)
Authors: Morris Suzanne C. , Ruwe Stephanie , Scott Jane , Finkelman Fred D.
Published: January 2023

Because IL-4 potently stimulates murine CD8+ T cell proliferation during an in vivo Th2 response, we evaluated whether IL-4 also contributes to CD8+ T cell homeostasis in naïve mice. Adult, wild-type BALB/c mice have ~2x the number of total and ~5x the number of memory phenotype (CD44highLy6Chigh) splenic CD8+ T cells as IL-4-, IL-4Rα- and Stat6-deficient BALB/c mice. Rare, IL-4-dependent, memory phenotype CD8+ T cells are found in BALB/c thymus by week 2 of life. The effects of IL-4-deficiency on CD8+ T cells can be reproduced by treating pregnant mice and then their offspring with anti-IL-4 mAb for the first 12 days of life, but not by treating adult mice with anti-IL-4 mAb for 2 weeks. In contrast to BALB/c mice, wild-type, IL-4-deficient and IL-4Rα-deficient C57BL/6 mice have similar numbers of memory phenotype CD8+ splenocytes, which are fewer than those found in wild-type BALB/c mice. BALB/c and C57BL/6 adult mice have similar basal IL-4 production and their memory phenotype CD8+ T cells are similar in size and surface markers, except that the C57BL/6 cells express more IL-15Rα and IL-2/15Rβ. At least 3 genes contribute to the BALB/c vs. C57BL/6 difference. Taken together, these observations suggest that IL-4 acts early in BALB/c T cell development to promote memory phenotype CD8+ T cell differentiation and that greater thymic IL-4 production or responsiveness may contribute to the BALB/c Th2 cytokine predisposition.
Inhibition of anti-apoptotic signals in prostate cancer cells by CCL25 blockade enhances the cytotoxic effects of etoposide (98.1)
Authors: Sharma Praveen K , Singh Rajesh , Eaton John W , Lillard, Jr. James W , Novakovic Kristian R , Grizzle William E , Singh Shailesh
Published: January 2023

Despite of current advancement in treatment and management of prostate cancer (PCa); it is still remaining the second leading cause of cancer-related deaths among men in the US. Current chemotherapeutic regimens provided palliative benefit but relatively modest survival advantage for the patients with hormone refractory PCa. This results in an initial response, but always followed by relapse due to chemoresistance and relatively low rate of apoptosis in PCa cells. Therefore, better efficacy of chemotherapeutics can be achieved by combining additional agents, which can block the mechanisms responsible for this low apoptosis rate. We have previously shown that CCR9 is expressed by the PCa cells and support the mechanisms involved in PCa metastasis. In this study, we have demonstrated that CCR9 and its natural ligand; CCL25 interaction up regulates anti-apoptotic proteins and promotes cell survival. Inhibition of these interactions using antibody blocked enhanced the etoposide-induced apoptosis of PCa cells in vitro and reduces tumor burden in vivo. Therefore, targeting this axis with chemotherapeutic agents may enhance the therapeutic outcomes of PCa. [Supported by funds from DOD PCRP Award W81XWH-06-1-0521]
Anti-inflammatory properties of docosahexaenoic acid (DHA) in bone marrow-derived dendritic cells (98.11)
Authors: Kong Weimin , Yen Jui-Hung , Ganea Doina
Published: January 2023

Omega 3 fatty acids have anti-inflammatory effects in chronic inflammatory and autoimmune diseases. Previous studies showed that dendritic cells (DC) differentiated in the presence of DHA express reduced costimulatory molecules and secrete lower levels of cytokines. We report that 24h exposure to DHA of immature DC prevents LPS-induced upregulation of MHCII, CD40, CD80 and CD86. Immature DC are characterized by CCR5 expression and migration to proinflammatory chemokines, whereas mature DC express CCR7 and migrate to lymph nodes in response to CCL19/21. DHA exposure kept CCR5 expression at high level, and prevented CCR7 expression induced by LPS. DHA-treated DC migrated poorly towards CCL19. Proinflammatory cytokines including IL-12p70, IL-23, IL-6 and TNFα produced by mature DC were inhibited by DHA. We found that DHA inhibited IL-12p70 and IL-23 production induced by ligands for TLR4 and 2, and also by ligands for the intracellular TLR3 and 9. In agreement with previous reports showing that omega 3 fatty acids bind to PPARγ, we found that DHA induced nuclear PPARγ translocation, and that a PPARγ antagonist partially reversed the inhibitory effect of DHA on IL-12p70. In conclusion, our studies indicate that exposure to DHA maintains already differentiated DC in an immature state and prevents expression of proinflammatory cytokines and chemokines.

Support NIH 2RO1AI052306.
Interleukin-18 Downregulates Collagen Production in Human Dermal Fibroblasts via ERK pathway (98.14)
Authors: Kim Hee Jung , Song Seok Bean , Lee Jun Young , Cho Baik Kee , Cho Dae Ho , Park Hyun Jeong
Published: January 2023

Excessive accumulation of collagen contributes to the fibrotic process. Several experimental studies have shown that interferon (IFN)-γ is effective in preventing fibrogenesis. Interleukin-18, formerly called IFN-γ -inducing factor, is a key mediator of inflammation and host defense responses. Here, we investigated the regulatory effect of IL-18 on expression of type I and collagen genes in dermal fibroblasts. Exposure of human dermal fibroblasts (HDF) to IL-18 resulted in a reduction of collagen gene expression and protein production was in a dose-dependent manner using real-time RT-PCR and Sircol assay. Next, we determined the signaling pathways involved by small interfering RNA(siRNA), reporter assay, and Western blotting. Silencing of the Ets-1 reversed inhibitory effects of IL-18. We demonstrated that IL-18 induced phosphorylation of extracellular signal-regulated kinase (ERK) within 10 minutes, and inhibitor of ERK blocked the inhibitory effects of IL-18. These results suggest that L-18 downregulates collagen production in HDF directly via Ets-1-dependent pathway and ERK pathway.
Relationship between Amyloid β (Aβ) Protein and Inflammatory Markers in Down Syndrome (DS) (98.19)
Authors: Mehta Pankaj D. , Patrick Bruce A. , Mehta Sangita P. , Barshatzky Marc , Dalton Arthur J.
Published: January 2023

Brain autopsy data from persons with DS showed that the neuropathology of Alzheimer disease (AD) is always present in DS 40 years of age and older. The core protein of the neuritic plaque is Aβ protein. Studies have suppoted the role of immune abnormalities and inflammation in the pathogenesis of DS. We hypothesized that the levels of TNFα and Cystatin C would be higher in DS than controls, and would correlate with Aβ levels. The study included 40 persons with DS (44±8 years old) and age-matched controls. Plasma levels of Aβ40 and Aβ42 , TNFα and Cystatin C were quantitated by using an ELISA. Aβ40 and Aβ42, TNFα and Cystatin C levels were higher in DS than controls (p<.001). Although there was significant relation between Aβ40 and Aβ42 levels (r=.6; p<.01)in DS, there was no relation between Aβ40 and Aβ42 levels with TNFα or Cystatin C. The higher TNFαand Cystatin C levels indicate a chronic state of immune activation in DS. Because there was no relation between Aβ and TNFα or Cyststin C the data suggests that increases in TNFα and Cystatin C reflect more infections in DS than AD neuropathology.
Analysis of proinflammatory cytokines as biocompatibility markers of emdogain (98.21)
Authors: SOLIS SOTO JUAN MANUEL , NAKAGOSHI CEPEDA SERGIO EDUARDO , GARZA ENRIQUEZ MARIANELA
Published: January 2023

Emdogain are enamel matrix proteins (amelogenins) that are taken from developing teeth in pigs. It is indicated for the treatment of intra-bony defects due to moderate or severe periodontitis, as well as the surgical treatment of gum recessions. However, its physiological effects are not completely characterized. This work analyzes the effects of emdogain on the production of proinflammatory cytokines and mineralization factors. Monocytes, isolated from peripheral blood, were incubated with different concentrations of emdogain for 2 days. Immunocytochemical methods were used to detect IL-1beta, IL-6, IL-12, TNF-alpha, and IFN-gamma. Fibroblasts, isolated from gingival tissue, were incubated with different concentrations of emdogain for 7 days. Immunocytochemical methods were used to detect osteopontin, osteonectin, osteoprotegerin, and ameloblastin. Morphometrical analysis showed significant increased production of osteopontin (p<0.01), osteoprotegerin (p<0.05) and osteonectin (p<0.05). Ameloblastin production was similar to controls. Of the proinflammatory cytokines only IL-12 (p<0.001) and IL-6 (p<0.05) were produced in considerable amounts. Production of IL-1beta, IFN-gamma and TNF-alpha was similar to controls. Emdogain has a good biocompatibility, because it only increases the production of pleiotropic cytokines. Besides, it stimulates the production of factors of mineralization like osteopontin, osteoprotegerin and osteonectin.
Collagen antibody-induced arthritis (CAIA) in mice: Triggering of arthritis by HMGB1, a late stage lethal mediator of LPS (99.22)
Authors: Waritani Takaki , Cutler Dawn , Terato Kuniaki
Published: January 2023

High mobility group box 1 protein (HMGB1), identified as a late stage lethal mediator of endotoxin, is currently considered a pro-inflammatory cytokine involved in severe inflammatory reactions such as rheumatoid arthritis. Pathological roles of HMGB1 in collagen antibody-induced arthritis (CAIA) model in mice were studied. DBA/1 mice were injected with sub-arthritogenic dose of anti-type II collagen monoclonal antibody (mAb) cocktail on day 0, and then LPS (50 μg) or HMGB1 (500 μg) by IP injection on day 3. Mice receiving HMGB1 developed arthritis (score: 6-8 out of 16 on day 7-10), although the severity of arthritis is milder than mice triggered by LPS (score: 14-16). Importantly, an anti-HMGB1 mAb capable of preventing death of mice by absorption of lethal dose of LPS prevented the development of arthritis by HMGB1, but failed to prevent the arthritis triggered by LPS, indicating that LPS stimulates a variety of inflammatory pathways. On the other hand, other clones of anti-HMGB1 mAb failed to prevent the arthritis induced by HMGB1, indicating the importance of epitope specificity for neutralizing the biological activity of HMGB1. This new arthritis model induced by the combination of mAb cocktail and HMGB1 provides a new tool to study mechanisms of inflammatory reactions linked to toll-like receptors (TLR) 2/4 and/or receptor for advanced glycation end-products (RAGE) in autoimmune mediated arthritis.
IL-21 is increased in CD4+ T cells and serum from patients with systemic lupus erythematosus (99.36)
Authors: Nguyen Vinh , Siaton Bernadette , Rus Violeta
Published: January 2023

Studies in animal models of lupus have reported increased production of IL-21 in MRL/lpr or BXSB mice and attenuation of autoimmune features in mice treated with IL-21 blocking agents. Little is known about IL-21 production and function in patients with systemic lupus erythematosus (SLE). We determined IL-21 levels in serum samples from 28 patients with SLE, 8 disease and 16 healthy controls by ELISA. Mean level of IL-21 was significantly higher in SLE patients (440±372 pg/ml) compared to normal controls (114 ± 101 pg/ml; p=0.05) and inversely correlated with C3 levels. Furthermore, patients with active disease (SLEDAI >4) had higher levels of serum IL-21 (1332.7±1084pg/ml) than patients with inactive disease (217.6±184 pg/ml). IL-21 and IL-21R expression was determined on ex vivo and in vitro stimulated CD4+ T cells by RT-PCR and immunostaining. IL-21 mRNA expression was 7 fold higher in ex vivo CD4+ T cells while intracellular levels were higher in stimulated memory CD4+ T cells from lupus patients compared to controls. IL-21R expression was upregulated on in vitro stimulated CD4+ T cells in both patients and controls. CD4+ T cells from lupus patients had equal susceptibility to proliferative effects induced by IL-21 when compared with controls. Similarly, lupus B cells were equally responsive to IL-21 when compared to controls with respect to proliferation, plasma cell differentiation, IgG and IL-10 production. In conclusion, elevated levels of IL-21 in lupus patients may promote auto-reactive CD4+ T cells proliferation as well as B cell proliferation and differentiation, thus contributing to the generation of T cell dependent autoreactive B cell responses.
Burkitt lymphoma-associated molecules impair HLA class II activation of CD4+ T cells (100.20)
Authors: God Jason , Cameron Christine , Amria Shereen , Haque Azizul
Published: January 2023

Burkitt Lymphoma (BL) is a very fast growing form of non-Hodgkin's lymphoma. The common characteristic of all BL is the translocation and constitutive activation of the c-myc oncogene. Our present study suggests that BL and Epstein-Barr Virus-transformed B-lymphoblastoid cell lines (B-LCL) constitutively express HLA class II proteins and bind peptide ligands. However, unlike B-LCL, BL cells were not capable of activating CD4+ T cells. While BL and B-LCL possessed similar levels of HLA class II, invariant chain (Ii) and HLA-DM, BL cells expressed lower levels of co-stimulatory molecules (CD80/CD86). Biochemical and functional assays showed that addition of external co-stimulatory signals to BL cells failed to functionally deliver HLA class II antigens to CD4+ T cells. Although both BL and B-LCL were capable of binding peptides at neutral and acidic pH, BL cells were only able to activate T cells at low pH (5.5), suggesting that BL-associated inhibitory molecules were eluted at the acidic pH. BL conditioned medium, as well as acid eluent from BL cells and primary B-cell tumors, inhibited functional class II presentation by both B-LCL and mature dendritic cells, while those eluent from B-LCL and primary B-cells did not. No characteristic inhibitory cytokines were identified in the acid eluent from BL or primary B-cell tumors. Taken together, these results suggest that BL-associated molecules may inhibit HLA class II activation of CD4+ T cells.
Selective depletion of CD4+CD25+Foxp3+ regulatory T cells by low-dose cyclophosphamide is explained by reduced intracellular ATP levels (100.23)
Authors: Huang Bo
Published: January 2023

Adenosine-5'-triphosphate (ATP), the energy molecule and signal element, significantly affects nearly all major biological processes of T cells. Recently, CD4+CD25+Foxp3+ regulatory T (Treg) cells have been demonstrated to play important roles in mediating cancer development and viral infections. Although Cyclophosphamide (CY) has shown promise as a drug to selectively target Treg cells with low-dose in vivo, the underlying molecular mechanism of this selective effect remains unclear. In this report, we provide evidence that the low intracellular ATP concentration accounts for the selective depletion of Treg cells by low-dose CY. Compared to conventional T cells, much lower ATP levels were found in Treg cells. This was due to low expression of one microRNA, miR-142-3p, leading to high adenylyl cyclase 9 to convert more ATP to cAMP, and CD39 on the surface of Treg cells to hydrolyze ATP. As a result, the low level of ATP attenuates the synthesis of glutathione, leading to the decrease of CY detoxification, thus increasing the sensitivity of Treg cells to low-dose CY. Thus, we here identify a molecular pathway through which low-dose cyclophosphamide selectively ablates Treg cells. Our findings also imply that low levels of ATP are probably related to Treg cell function.
Tumor microenvironments direct the recruitment and expansion of human Th17 cells (100.28)
Authors: Peng Guangyong , Su Xinming , Ye Jian , Hsueh Eddy , Zhang Yanping , Hoft Daniel
Published: January 2023

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases, their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients, but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study, we further showed elevated Th17 cell populations in the TILs obtained from melanoma, breast and colon cancers, suggesting that development of tumor-infiltrating CD4+ Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition, we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell-to-cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and NOD2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs, demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments, and provide new insights relevant for the development of novel cancer immunotherapeutic approaches.
Cancer-induced Immunostimulatory CD11b+Gr-1+ cells (100.30)
Authors: Shin Tahiro , Guo Miao , Curiel Tyler , Tomihara Kei
Published: January 2023

Both innate and adaptive immune systems are considered equally important for cancer prevention, immunosurveillance, and control of cancer progression. It is known that while both systems initially eliminate emerging tumor cells efficiently, tumors eventually escape immune attack by a variety of mechanisms including differentiation and recruitment of immunosuppressive CD11b+Gr-1+ myeloid suppressor cells into the tumor microenvironment. However, we show here that CD11b+Gr-1+ cells found in ascites of epithelial ovarian cancer-bearing mice at advanced stages of disease are immunostimulatory rather than being immunosuppressive. These cells consist of a homogenous population of cells that morphologically resemble neutrophils. Moreover, like dendritic cells, immunostimulatory CD11b+Gr-1+ cells can strongly cross-prime, augmenting the proliferation of functional CTLs via signaling through the expression of costimulatory molecule CD80. Adoptive transfer of these immunostimulatory CD11b+Gr-1+ cells from ascites of ovarian cancer-bearing mice results in the significant regression of subcutaneous tumors even without being pulsed with exogenous tumor antigen prior to adoptive transfer. We now show for the first time that adaptive immune responses against cancer can be augmented by these cancer-induced granulocyte-like immunostimulatory myeloid (CD11b+Gr-1+) cells, thereby mediating very effective anti-tumor immunity.
Impact of bortezomib-induced proteasome inhibition in vivo on anti-tumor T cell responses (101.1)
Authors: Shanker Anil , De Kluyver Rachel , Sayers Thomas
Published: January 2023

The perturbation of cellular homeostasis offers a new approach for cancer therapy. Our recent reports showed that proteasome inhibitor bortezomib (Velcade/PS-341) not only sensitizes tumors to death receptor-mediated apoptosis but also increases their susceptibility to NK cell lysis. However, the effects of bortezomib on T cell anti-tumor responses are not known. Thus, in mice established with Renca-HA tumor expressing influenza virus hemagglutinin (HA) as a surrogate tumor antigen, we evaluated the anti-tumor functions of adoptively transferred monoclonal HA-specific CD4 and CD8 T cells following bortezomib administration. Interestingly, no inhibition of T cell proliferation or cytolytic activity in vivo was observed following bortezomib treatments. Nor were there any defects in dendritic cell activation or antigen presentation in bortezomib-treated mice. However, a decrease in total T cell yield and a blockade of cellular homeostatic proliferation was noted in these mice. Thus, bortezomib-inhibitory effects on T cell responses appeared quantitative rather than qualitative. Moreover, combined treatment of mice established with Renca-HA tumor with adoptive T cell transfer and bortezomib resulted in superior anti-tumor responses than either therapy alone. These data demonstrate that, with an appropriate scheduling, it may be possible to combine bortezomib-induced sensitization of tumors to apoptosis with tumor-specific T cell adoptive transfers to improve cancer immunotherapy.
Small molecule inhibitor screen of MHCII processing pathways using influenza virus A/PR/8/34 (130.11)
Authors: Miller Michael , Eisenlohr Laurence
Published: January 2023

During a viral infection, antigen presenting cells (APCs) process foreign intracellular material into short peptides (epitopes) and present them on the cell surface for recognition by CD4+ and CD8+ T cells. We have previously identified three immunodominant epitopes derived from the surface proteins of the influenza virus strain A/PR/8/34. S1 and S3 are derived from endocytosed hemagglutinin protein whereas NA79 is derived from endogenously-produced neuraminidase. Although these three epitopes are restricted to the same MHCII molecule I-Ed, their processing pathways are reported to be dependent on different cellular components such as H2-DM, TAP, and endosomal proteases. To discover additional components of these distinct antigen presentation pathways, we screened a small molecule library for compounds that would selectively inhibit presentation of S1, S3, and NA79. We screened 8,000 compounds from the NCI/DTP Open Chemical Repository and identified 239 primary leads for specific inhibition of epitope presentation ranging from 50% - 100 %. To further validate the primary leads, we performed dose responses to identify 27 potent inhibitors of our known epitopes. Future work will involve purification methods to identify cellular targets that directly interact with our small molecule hits.