bluuebunny/crossref_metadata_2025 huggingface dataset:
The potential role of locally induced matrix metalloproteinase 3 in respiratory mucosal immunity (39.17)
Authors: Guo Xueshui , Knudsen David E. B. , Wang Xiuqing
Published: January 2023

Recent studies suggest that matrix metalloproteinase 3 (MMP3) is involved in intestinal immunity against bacterial infections as well as in immune cell migrations in delayed hypersensitivity. The goal of this study is to assess the potential role of MMP3 in the development of mucosal immunity at respiratory tract using CpG oligodeoxynucleotide (ODN) as a model system. Groups of BALB/c mice were administrated intranasally with either CpG ODN or GpC ODN or PBS. MMP3 was up-regulated at both 1 and 3 days post immunization in bronchoalveolar lavages (BAL) and respiratory mucosal tissues of CpG ODN immunized mice as determined by ELISA, Western blot and immunohistochemistry (IHC) staining. Furthermore, an increase of both CD11c+ antigen presenting cells and CD4+ T cells was observed in lungs of CpG vaccinated mice compared with GpC ODN or PBS controls. To confirm that MMP3 expression is associated with increased immune cell migration in lungs, mice were treated with MMP3-specific inhibitor II 6hr after CpG immunization. Lung tissues were collected 3 days later and subjected to IHC analysis to determine the expression of MMP3 and the number of CD11c antigen presenting cells and CD4+ T cells. Results showed that MMP3 expression was significantly reduced after treatment with inhibitor II, suggesting that MMP3 inhibitor effectively reduced the expression of MMP3. Accordingly, a reduced number of CD11c+ antigen presenting cells and CD4+ T helper cells was observed in inhibitor-treated mice, indicating a possible role of MMP3 in immune cell migration. Ongoing experiments are focused on validating these observations and investigating the mechanistic basis of this MMP3-associated mucosal immunity. It is anticipated that the results may provide new insights on the molecular basis of mucosal immunity at respiratory tract. (Supported by NSF/EPSCoR EPS-0091948)
Distinct mucosal microenvironments differentially regulate memory T cell differentiation state and maintenance (39.20)
Authors: Fraser Kathryn , Casey Kerry A , Vezys Vaiva , Masopust David
Published: January 2023

T cell location and differentiation state are critical factors involved in protection against viral infection. We found that the phenotype of LCMV-specific CD8 and CD4 T cells varied by location, even amongst different mucosal tissues. Virus-specific CD8 T cells upregulated αE integrin and downregulated Ly6C in the small intestine but this did not occur in the spleen, lung, large intestine, or vaginal mucosa. αE integrin was required for maintenance of CD8 T cells in the small intestine IEL (see Casey et al. abstract). TGF β was responsible for inducing this tissue-specific phenotype. Interestingly, virus-specific CD4 T cells did not modify αE integrin and Ly6c in response to TGF β. Virus-specific CD4 T cells exhibited a decrease in αE integrin expression compared to CD8 T cells and this correlated with decreased maintenance of memory antigen-specific CD4 T cells within this compartment. Taken together, these data may explain why the abundant CD4 T cell effector response was poorly maintained specifically within the small intestine epithelium and highlights a mechanism by which tissue microenvironment regulates maintenance and compartmentalization of T cells in mucosal tissues.
CD69 and CD103 cooperatively regulate CD8 T cell responses in the lungs after viral infection (39.25)
Authors: Lee Young-Tae , Suarez-Ramirez Jenny E , Redman Jason M , Aguila Carolina C , Hadley Gregg A , Cauley Linda S
Published: January 2023

T cell mediated immunity to influenza infection disappears within a few months, even though large numbers of virus-specific memory CD8 T cells remain in the circulation for at least two years. It has been suggested that antigen persistence may play an instrumental role in protective cellular immunity. This idea is supported by the presence of activated virus-specific CD8 T cells in the respiratory tract during the months following infection, when processed influenza antigens are presented to virus-specific CD8 T cells in the draining lymph nodes. A majority of the endogenous virus-specific CD8 T cells that were at the mucosal surface of the lungs one month after infection expressed both CD69 and CD103. In contrast, bystander memory CD8 T cells that entered the lungs during parabiosis did not acquire this phenotype. We have crossed CD69KO and CD103KO mice with F5 TcR transgenic mice which are specific for the influenza NP peptide. Transfer studies show that CD103-deficiency results in reduced numbers of CD69+ NP-specific CD8 T cells in the lungs. In contrast, CD69-deficiency results in slightly delayed T cell activation followed by a late accumulation of CD103-negative NP-specific CD8 T cells in the lung parenchyma. This work was supported by NIH grants AI065895 & AI071213 (LSC), RO1 AI-36532 (GH).
TRAIL-expressing CD8+ regulatory T cells participate in the maintenance of ocular immune privilege (39.40)
Authors: Brincks Erik L , Ferguson Thomas A , Griffith Thomas S
Published: January 2023

Immunological tolerance to antigens encountered in the eye, the prototypic immune privileged site, inhibits potentially damaging immune responses that would otherwise be initiated to these antigens at other anatomical locations. The induction of tolerance depends on the FasL-mediated apoptotic death of inflammatory cells that enter the eye in response to antigenic challenge. Moreover, the tolerance is not due to the failure to prime, but is mediated by the generation of CD8+ regulatory T cells (Treg) that can transfer tolerance to a naïve recipient. The present study investigated the mechanism of action of the induced CD8+ Treg. Unlike wildtype (WT) mice, TRAIL-/- or DR5-/- mice did not develop tolerance to antigen injected into the eye, even though the responding lymphocytes underwent apoptosis in the eyes of all mice. We also found that splenocytes from tolerant WT mice produced significantly less IFN, compared to TRAIL-/- mice, after restimulation. Interestingly, CD8+ T cells from TRAIL-/- mice were unable to transfer tolerance to naïve recipient WT mice, but CD8+ T cells from WT or DR5-/- mice could transfer tolerance. Subsequent investigation found that tolerant animals contained CD8+ Treg cells capable of killing antigen-pulsed splenocytes via TRAIL. Together, our data indicate that CD8+ Treg enforce tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.
Estrogenic microenvironment influences immunity (39.41)
Authors: Papenfuss Tracey L , Taylor Cassandra L
Published: January 2023

Estrogens affect the immune system and immune-mediated/autoimmune diseases. We have found that in vivo estriol (E3) alters dendritic cell (DC) phenotype and function that impact CD4+ T cells. This study evaluates the in vitro effects of E3 (and 17-beta estradiol; E2) on immune function. Spleen (SPL) and lymph node (LN) cells from C57BL/6 mice were CFSE stained and cultured for 3-5 days with media, 100 uM E3 or 100 uM E2 and stimulated with media, anti-CD3 or conA. Decreased proliferation was seen in the E3 (and E2) treated cells as demonstrated by increased mean fluorescence intensity (MFI), fewer daughter cell populations and decreased cell yield in both anti-CD3 and conA stimulated groups. Decreased cell number was not due to cell death as determined by trypan blue and 7-AAD staining. E3 and E2 cultures produced less IL-10 and IL-12p40 by ELISA and phenotypic evaluation of CD11c+ and CD11b+ cells by flow after 48 hours of in vitro E3 stimulation demonstrated only slight effects of E3 on CD80, CD86, PD-L1 and PD-L2. Interestingly, E2 treated cells showed increased CD80 and/or CD86 expression in CD11c+ and CD11b+ cells in the LN and SPL. These studies demonstrate that estrogens can have anti-proliferative effects on SPL and LN populations and influence cytokine and costimulatory molecule expression on antigen presenting cells. Further studies are necessary to compare the in vitro and in vivo effects of estrogens such as E3.
Oral tolerance induction is defective during the neonatal period in the mouse (39.42)
Authors: KIM MEE KUM , LEE JOON YOUB , PURTON JARED F , MARTIN CHRISTOPHER , KIM DAVID M , SURH CHARLES D
Published: January 2023

Congenital T cell (T)-deficient mice, such as RAG-1- or SCID mice, succumb to inflammatory bowel disease (IBD) upon adoptive transfer of syngenic naive T cells as a consequence of chronic inflammation in the intestine from unfettered response to commensal bacterial antigens. Although the prevailing view for the onset of IBD is believed to be the absence of regulatory T cells (Tregs), the finding that donor T cells undergo considerable chronic activation even in the presence of Tregs suggests an abnormality in the presentation of enteric antigens also appears to be involved. To study if such a defect is also evident during the lymphopenic neonatal period, we compared the induction of oral tolerance between B6 neonates and adults to host commensal or exogenously provided nominal Ags by adoptively transferring polylconal or Ag-specific donor T cells. Strikingly, donor T cells in neonatal hosts proliferated much faster and more readily acquired effector function than donor T cells in adults. These finding suggests that oral tolerance in normal mice is acquired later with the development of T immunity.
ERK1/2 and NFκB are key signaling pathways in the regulation of CXCL8 expression and secretion by intestinal epithelial cells in response to P2Y6 receptor activation (39.43)
Authors: Grbic Djordje M , Gendron Fernand-Pierre
Published: January 2023

Extracellular nucleotides are recognized as inflammatory-active molecules. We have investigated the role of extracellular uridine 5'-triphosphate (UDP) and its associated P2Y6 receptor in the secretion of CXCL8 by intestinal epithelial cells. We have also characterized signaling pathways linking UDP to the expression of CXCL8. The effect of the P2Y6 activation by UDP on chemokine expression and release by epithelial cells was determined using combination of western blots, luciferase assays, Q-PCR and ELISA. We have monitored the activation of different signaling molecules by western blots, luciferase assays and EMSA, along with different pharmacological inhibitors. Stimulation of Caco-2 cells with UDP lead to PKCδ and PKCζ, as well as ERK 1/2 and NFκB-p65 phosphorylation. Mutations of the NFκB binding site in the minimal promoter of the human CXCL8 completely abolished the luciferase activity stimulated with UDP. EMSA assays showed the importance of NFκB binding site for the initiation of transcription of CXCL8 by P2Y6. In this study, we showed the most detailed signaling pathway known so far for P2Y6 receptor its implication in UDP stimulate CXCL8 expression and release by intestinal epithelial cells.
The effect of c-Kit inhibition on mast cells in skin, colon, and lung (39.46)
Authors: Trueblood Esther S , Branstetter Daniel , Itano Andrea , Ng Gordon Y.
Published: January 2023

Stem Cell Factor (SCF) signaling via its receptor tyrosine kinase, c-Kit, is vital for mast cell development, function and survival. The effect of inhibition of c-Kit function using subcutaneous administration of a specific blocking monoclonal antibody weekly for up to 28 days was examined in African Green monkeys (Cercopithecus aethiops sabaeus). Treatment-related effects on mast cell populations were evaluated in tissue samples collected from wounded skin and colon biopsies on days 0, 7, 14, 21, and 28 (colon only) and skin, lung, and gastrointestinal tract collected at necropsy on day 28. Mast cells were visualized by Astra blue staining and immunohistochemistry for CD117, tryptase, and/or chymase. Specific c-Kit inhibition resulted in depletion of mast cells in unchallenged skin, colon, and lung after 28 days. Additionally, c-Kit antibody treatment inhibited the injury-induced expansion of dermal mast cells adjacent to wounded skin. Mast cells have been implicated as important mediators in a wide range of inflammatory diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, and fibrosis. This is the first report of use of an antibody to deplete tissue mast cells in relevant organs in non-human primates.
TGF-βRII expressing Peyer’s Patch CD4+ T cells are essential for the induction of mucosal unresponsiveness (39.49)
Authors: Gilbert Rebekah Sims , Fujihashi Kohtaro
Published: January 2023

It has been shown that TGF-β signaling plays key roles in the induction of oral tolerance (OT). We assess the ability of CD4-dominant negative TGF-βRII (TbRII) mice to develop mucosal unresponsiveness to orally administered antigen to understand the role of TGF-β signaling in the early events of OT.

Mice were fed a large dose (30 mg) of OVA and were orally immunized three times at weekly intervals with OVA (1mg) plus native cholera toxin (nCT) one week after tolerization. One week after last immunization plasma and fecal extract samples were collected and mononuclear cells were isolated from spleen, Peyer's Patches (PPs), and mesenteric lymph nodes for analysis by ELISA, ELISPOT and flow cytometry. DTH responses were determined by ear swelling after challenge with OVA. In order to examine the roles of TGF-β signaling in early events of OT induction, CD4+ T cells from C57BL/6 mice were examined for the expression of TGF-βR II at 24-hour intervals for 3 days and on days 5 and 7 after feeding 30mg OVA.

OVA-specific IgG and S-IgA titers in plasma and fecal extracts, respectively, as well as DTH response of OVA-fed TbRII mice were essentially the same as TbRII mice fed PBS. These responses were significantly higher than those of C57BL/6 wildtype mice fed OVA. Expression of TGF-βRII by CD4+ T cells of spleen and MLN of wildtype mice was low or variable, respectively. In contrast, expression of TGF-βRII by PP CD4+ T cells was significantly increased over base-line beginning at 24 hours and continued to remain high one week after tolerization.

These results indicate a critical role for TGF-β signaling in OT induction. Further, our findings may indicate important roles for the PPs in the initiation of mucosal unresponsiveness

This work is supported by NIH grants DE 0176707, DE 012242 and AG 025873.
The thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease (39.53)
Authors: Olivares-Villagomez Danyvid , Mendez-Fernandez Yanice V , Parekh Vrajesh V , Lalani Saif , Vincent Tiffaney L , Cheroutre Hilde , Van Kaer Luc
Published: January 2023

Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8alpha-alpha homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically deficient in TL expression. Our findings show that TL expression plays a critical role in maintaining IEL effector functions. Moreover, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.
Pulmonary Dendritic Cells Trans-present IL-15 to Directly Promote the Survival of Effector Influenza Virus-Specific CD8 T Cells in the Lungs (39.6)
Authors: McGill Jodi , Legge Kevin L.
Published: January 2023

We have previously shown that aDC depletion at 48 hours post influenza virus infection results in increased mortality and decreased virus-specific CD8 T cell responses. Reconstitution with pulmonary DC rescues the CD8 T cell response in a manner that requires direct cell-cell contact, MHC I and viral antigen. In aDC depleted mice, influenza-specific CD8 T cells undergo increased apoptosis in the lungs and express decreased levels of the anti-apoptotic factor Bcl-2. Reconstitution with pulmonary DC results in decreased levels of T cell apoptosis, suggesting that pulmonary DC provide a survival signal to effector CD8 T cells in the lungs during infection. Here, we demonstrate that pulmonary DC express IL-15 and IL-15Rα, and blocking these molecules on their surface ablates the rescue of pulmonary CD8 T cell immunity. Together, our results suggest that virus-specific CD8 T cells require interactions with viral antigen-bearing, IL-15 trans-presenting DC in the lungs to up-regulate anti-apoptotic signals such as Bcl-2, promoting T cell survival and accumulation. Our results suggest a "two-hit" model of CD8 T cell activation during influenza infection where T cells require a primary interaction with DC in the lymph node to initiate programmed activation; and a novel, secondary interaction with viral antigen-bearing, IL-15 trans-presenting DC in the lungs to survive, accumulate and mediate viral clearance and recovery.
A pivotal role of stromal NFkappaB-inducing kinase in sphingosine 1-phosphate-medaited peritoneal B cell trafficking for the intestinal IgA production (39.9)
Authors: Kunisawa Jun , Gohda Masashi , Kurashima Yosuke , Ishikawa Izumi , Higuchi Morio , Kiyono Hiroshi
Published: January 2023

Our previous study showed that sphingosine 1-phosphate (S1P) regulates peritoneal B cell trafficking for the subsequent intestinal IgA production. In this study, we demonstrate that stromal NFkappaB-inducing kinase (NIK) is specifically involved in this pathway. Although peritoneal B cells from NIK-mutated alymphoplasia (aly) mice expressed substantial levels of S1P receptor and showed normal migration toward S1P, aly peritoneal B cells showed decreased sensitivity to FTY720, an S1P modulator. NIK-mutated stromal cells showed aberrant expression of VCAM-1, ICAM-1, and CXCL13, leading to the impaired ability to support S1P-mediated peritoneal B cell emigration. Therefore, aly peritoneal B cells exhibited normal S1P-mediated peritoneal B cell trafficking from peritoneum to intestine for IgA production when they were adoptively transferred into SCID mice. Further, transfer of wild-type stromal cells into the peritoneum restored S1P-mediated trafficking of aly peritoneal B cells. These findings suggest that NIK in stromal cells has a specific role in the regulation of S1P-mediated peritoneal B cell-derived intestinal IgA production.

This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Ministry of Health and Welfare of Japan.
CCL1: a novel therapeutic target for the modulation of Treg function: Implications for immmunotherapy of cancer (40.10)
Authors: Hoelzinger Dominique B , Dominguez Ana Lucia , Smith Shannon E , Zorro Manrique Soraya , Lustgarten Joseph
Published: January 2023

Effective anti-tumor immunotherapy must overcome the immunosuppressive networks created by the tumor. Previous studies from our group indicate that intratumoral injection of CpG-ODN reduces Treg levels within the tumor by 80%. This decrease in Tregs is mainly mediated by the activation of antigen presenting cells secreting IL-6. To explore the effect of IL6 on Tregs, an expression microarray analysis of genes involved in TGFÎ2 dependent Treg conversion, which are also sensitive to IL6, was done. It yielded gene candidates which can be exploited to modulate the function of Tregs. In vitro analysis of two such candidates: chemokine ligand 1 (CCL1) and integrinÎ±E (ITGÎ±E), shows that blocking these proteins reduces de novo conversion of Tregs (CCL1 and ITGÎ±E) and inhibits the suppressive function of Tregs (CCL1 only). In vivo assays using the neu tolerant breast cancer mouse model demonstrates that injection of Î±CCL1 or Î±ITGÎ±E blocking antibodies significantly reduced the level of Tregs within the tumor and the tumor draining lymph nodes. Furthermore, the combination of intratumoral injections of CpG-ODN and Î±CCL1 induce complete tumor rejection in neu mice. These results demonstrate that blockage of CCL1 signaling modulated the function of Tregs and subsequently enhances immunotherapeutic approaches.
Sex-related differences in regulatory T cell (Treg) function and resistance to B16 melanoma in the absence of B7-H1 co-signaling (40.18)
Authors: Lin Pei Yi , Livi Carolina , Thibodeaux Suzanne R , Wierman Margaret E , Kious Mark , Jeansonne Duane P , Shin Tahiro , Brumlik Michael J , Zhang Bin , Daniel Benjamin J , Curiel Tyler J
Published: January 2023

We tested Treg function in B7-H1-/- (KO) mice. 6-10 wk male (M) or female (F) KO and M or F wildtype (WT) had equal numbers of CD4+CD25hiFoxP3+ T cells (Tregs) with similar CD25 and Foxp3 MFI. CTLA-4 trended lower in F vs M KO. M and F WT and M KO Tregs suppressed T cell proliferation equally in vitro but F KO Tregs were ~30% less suppressive vs the other 3 groups. Transfer of M KO Tregs fully protected RAG1-/- mice from CD4+RBhi T cell colitis but F KO Tregs did not, with up to 30% weight loss demonstrating poor in vivo Treg function. In M or F WT or KO with subcutaneous B16 melanoma, tumor was different at day 15 in F (159 mm3) vs M (458 mm3, p = 0.04) and F KO vs F WT (593 mm3, p = 0.01). We detected OVA-specific immunity in 0/3 M KO bearing OVA+ B16 vs 3/3 in F KO based on in vivo OT-I cell proliferation. Tregs in pre-pubertal 3-wk F KO had similar phenotype and function vs post-pubertal WT and M KO Tregs. Culture with TGF-β +IL-2 converted 6-wk CD4+CD25- F KO T cells into CD4+CD25hiFoxP3+ T cells with suppression similar to WT, but lower CD25. Thus, F KO T cells can become functional Tregs ex vivo. This previously unknown sex/T cell co-signaling interaction could explain sex-related differences in cancer, autoimmune disease, infection susceptibility and response to hormone antagonists. We hypothesize Treg dysfunction to improve immunity and tumor resistance in KO and estrogen as a B7-H1 co-factor in Treg development. CTLA-4 defects remain undefined.

Supported by CA105207, FD003118, and Voelcker and Greehey Trusts
Interferon-alpha improves immunologic and clinical response to regulatory T cell (Treg) depletion in ovarian cancer (41.15)
Authors: Wall Shawna , Thibodeaux Suzanne R , Lin Pei Yi , Kious Mark , Jeansonne Duane , Brumlik Michael J , Livi Carolina , Daniel Benjamin J , Curiel Tyler J
Published: January 2023

Treg depletion improves anti-tumor immunity but is unlikely to be curative alone. Denileukin diftitox (DT) depletes Tregs in BL6 mice and prolongs survival 11% at 5 µg/mouse weekly starting 2 weeks after challenge with ID8 ovarian cancer. Weekly human interferon (hIFN)-α 20,000 U/day on 4 consecutive days of 7 plus weekly DT survival improved 26% vs DT alone. Neither DT nor DT+hIFN-α improved survival in RAG1-/- mice with ID8, showing a requirement for adaptive immunity. hIFN-α alone reduced in vitro Treg suppression ~15% vs PBS, but did not clearly enhance DT-mediated Treg depletion, or increase T cell activation by CD69 expression. A patient in our clinical trial of DT for ovarian cancer failed, with CA-125 increasing 4.5-fold after 4 monthly cycles. Addition of weekly pegylated IFN-α2a to failed monthly DT improved blood Treg depletion 50% over DT alone and reduced CA-125 by 9-fold, demonstrating immunologic and clinical efficacy. To provide additional mechanistic insight we studied dendritic cell (DC) activation in hIFN-α treatment. In mice with ID8 tumor, hIFN-α significantly increased CD86 expression on myeloid and plasmacytoid DC. IFN-α may thus have an expanded role in treatment of epithelial carcinomas when used with Treg depletion, through enhanced Treg depletion or DC activation among other mechanisms.

Supported by FD003118, the Fanny Rippel Foundation, the Voelcker Trust and the Greehey Fund
Diversification of TCR repertoire in melanoma patients treated with chemotherapy before peptides-vaccination (41.19)
Authors: Nistico Paola , Palermo Belinda , Del Bello Duilia , Gualtieri Novella , Ferraresi Virginia , Belardelli Filippo , Proietti Enrico , Natali Pier Giorgio , Sottini Alessandra , Ghidini Claudia , Serana Federico , Imberti Luisa
Published: January 2023

Combining chemotherapy and immunotherapy is an attractive antitumor strategy. Recently we have reported that the administration of dacarbazine (DTIC) one day before peptide-vaccination in disease-free melanoma patients induced an increase of peptide-specific effector-memory CD8+ lymphocytes. To investigate the functional differences between anti-Melan-A CD8+ T cell response elicited by vaccination alone (Arm 1) or by chemotherapy plus vaccination (Arm 2), we generated T cell clones from 5 patients of both Arms, before and after the treatments. A significant enhancement of tumor lytic activity of Melan-A+ clones generated by patients of Arm 2 after the treatment was observed, whereas no significant differences of tumor recognition efficiency were evident among the two arms before treatment. Ninety-six sequences were obtained and 47 clonotypes with different CDR3 regions were observed. A progressive enhancement of TCR repertoire diversity was found only in patients treated with DTIC before vaccination, whereas patients treated with vaccine alone showed a reduction of TCR repertoire diversity accompanied by a reduction of tumor lytic activity in one patient. These results suggest that a diverse Melan-A-specific T cell repertoire occurs in melanoma patients treated with DTIC plus vaccination, which may offer a new clinically advantageous anti-tumor strategy.
Peripheral NK Cell Activation and Expansion by Artificial Antigen Presenting Cells, 2D11 (41.25)
Authors: Zhang Hua , Levine Bruce , June Carl , Mackall Crystal
Published: January 2023

Natural killer (NK) cells play an important role in early immune defenses against both nonself cells and autologous cells infected with viruses, bacteria, or parasites or in malignant transformation. NK cells mediate direct cytotoxic attack without prior sensitization on their targets through activating receptors, NKG2D, NCR, TRAIL, and inhibitory receptors ("absence of self"), and/or produce cytokines and chemokines that contribute to cytotoxicity and immunoregulation. A number of studies have reported that activation/expansion enhances NK cytotoxicity, but optimal methods for activating and expanding NK cells have not been well defined. Here we demonstrate that artificial APCs (2D11), previously reported and used to expand antigen-specific T cells, also efficiently expand NK cells via NKG2D and 4-1BB signaling mediated by MICA/B and 4-1BBL expression respectively on 2D11 aAPCs. In the presence of exogenous IL15, which is trans-presented by IL-15Rα on 2D11, robust expansion of highly lytic activated NK cells occurs. Compared to resting NK cells, IL15/2D11-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and exerted enhanced cytotoxicity against K562, Ewing's tumor cell lines, including autologous tumors, ALL, as well as NK-resistant Daudi cells. In previous studies, expression of KIR ligands on tumor cells has been put forth as a mechanism of tumor immune escape from NK cell mediated killing. Our preliminary results demonstrate that expression of KIR ligands on tumor targets no longer inhibits killing by NK cells activated with 2D11. Ongoing studies are underway to test the hypothesis that NK cells activated by IL15/2D11 will prove more effective therapeutics due to breaking up of KIR-mediated inhibition of tumor cell killings.
Immunomodulatory activity of Resveratrol and its potential for combination with anti-tumor immunotherapy (41.26)
Authors: Soto Brenda Liz , Hank Jackie A , Polans Arthur S , Sondel Paul M
Published: January 2023

Resveratrol (RV) is a natural compound which is currently being investigated for many possible health promoting activities. It has anti-tumor activity in vitro on cancer cells and in vivo in some animal models of cancer. RV at a dose of 50mg/kg (i.g.) showed antitumor activity when treating nude mice bearing human neuroblastoma (Nb) xenografts. Prior studies on the effect of RV on the immune system found RV to be immunosuppressive in vitro and ex vivo in a dose-dependent manner, but not immunosuppressive in vivo. We are currently examining the ability of RV to be combined with immunotherapy to treat cancer. Using proliferative and cytotoxicity assays we are defining a dose in vitro and a regimen in vivo that does not suppress the immune system and is additive or synergistic in mediating anti-tumor effects when combined with immunotherapy. In our in vitro studies, 50uM or higher of RV inhibited the ability of PHA to stimulate proliferation of human PBMCs and inhibited IL2-stimulated murine splenocytes from dividing as measured by the ability of the cells to pass the dye CFSE to the daughter cells. These cells were found to be arrested in the G1 phase of their cycle, at those same concentrations. We are currently examining the effects of various concentrations of RV on target and effector cells in ADCC and NK killing assays. Also, studies on the antitumor and immunomodulatory effect of RV in vivo in Nb tumor-bearing animals are underway.
Synergy of radiation and immune therapy in tumor eradication (41.27)
Authors: Ruocco Maria Grazia , Kawashima Noriko , Huang Julie , Formenti Silvia C , Dustin Michael L , Demaria Sandra
Published: January 2023

We have investigated the mechanism of synergy between CTLA-4 blockade and local radiation therapy (RT) in treatment of breast cancer using the 4T1 mouse model. We have utilized CXCR6 GFP+ mice and CFP+ 4T1 tumor cells and intravital 2-photon laser scanning microscopy to follow the interaction of T cells with tumor cells. In mice treated with anti-CTLA-4 antibody 9H10 T cell speed was increased preventing stable interactions with tumor cells or stromal elements, critical for efficient tumor killing. RT also increased T cell velocity, but not to the same extent as 9H10 treatment. When RT was combined with 9H10 we observed increased T cell arrest in contact with tumor cells followed by tumor elimination. It has been shown that CTLA-4 ligation can increase T cell motility and this can overcome a TCR mediated stop signal. We confirmed that 9H10 treatment increased T cell motility on ICAM-1 coated surfaces and 9H10 was dominant over stop signals provided by soluble anti-CD3 antibodies. However, when a 9H10 go signal was subordinated to a stronger stop signal provided by solid phase anti-CD3 T cells did stop. These data suggest that anti-CTLA-4 therapy may impair killing of weak immunogenic tumors by suppressing IS formation. However, this effect can be overcome by RT-mediated enhancement of tumor antigen presentation. We have found that RT increases MHC class I and ICAM-1 expression on 4T1 cells, and are currently testing this in vivo.
WT1 peptide mix used for in vitro generation of WT1 CTL (41.29)
Authors: Stamer Mindy M , Herse Zachary , Dunham Kimberly , Bao Lei , Lucas Kenneth G
Published: January 2023

We developed a method to in vitro prime and expand WT1-specific CD8+ T cells using a WT1 peptide mix which could facilitate the expansion of WT1 specific T cells from donors of different HLA backgrounds. Monocyte-derived dendritic cells or PBMC isolated from 4 normal donors were pulsed with WT1 overlapping peptides prior to T cell stimulation. CD8 selected T cells were primed using WT1 peptide pulsed dendritic cells and then re-stimulated 11 days later using WT1 peptide pulsed autologous PBMC. IL-7 and IL-2 were added to the T cell cultures 4 days after the first stimulation to promote CTL differentiation. 7 days after the second CD8 stimulation, WT1 specificity was measured by tetramer staining. All donors exhibited tetramer positive T cells (donor 1=3.11%, donor 2=6.32%, donor 3=2.17%, donor 4=1.08%). WT1-specific cytotoxicity measured by 51Cr-release assays was also assayed 7 days post the second stimulation; however, WT1-specific cytotoxicity was not observed. WT1-specific CD8 T cells from 3 of the 4 donors were cloned and enriched by limited dilution. Clones were removed from OKT3 and WT1-specific cytotoxicity was measured in 51Cr-release assays. At least 1 clone from each of the 3 donors exhibited WT1 specific cytotoxicty (donor 1=54%, donor 2=20%, donor 3=26%). In conclusion, CD8 selected PBMC stimulated by antigen presenting cells pulsed with a WT1 peptide mix stained positive for HLA-A2 WT1-tetramers and exhibited WT1-specific cytotoxicity after cloning and subsequent expansion.
Phase 1 study of infusion of HER-2/neu specific T cells in patients with advanced stage HER-2/neu overexpressing cancers who have received a HER-2/neu vaccine (41.3)
Authors: Dang Yushe , Salazar Lupe G , Coveler Andrew , Waisman James , Higgins Doreen , Childs Jennifer , Bates Nicole , Slota Meredith , Disis Mary L
Published: January 2023

Adoptive T cell therapy has great potential for the treatment of patients with advanced-stage cancers. We have previously reported that HER-2/neu (HER2) specific T cell expansion from peripheral blood mononuclear cells (PBMN) can be greatly facilitated by in vivo antigen specific MHC class II epitope-priming. To test whether vaccine primed T cells could be administered to patients, we assessed the safety and feasibility of infusion of ex vivo expanded HER2 specific T cells in patients who have advanced HER2 overexpressing cancers. Three escalating doses of T cells were given at 10 day intervals with cyclophosphamide used for host lymphodepletion prior to the first dose of T cells. To date, 5 of 10 subjects have been enrolled. The maximal T cell doses infused were 1x109- 41x109 cells (median 10x109) with and average 35% of CD4+ and 60% of CD8+ T cells. IFN-gamma ELISPOT was used to monitor antigen specific T cells in both the infused product and to track the cells in vivo. HER2 specific T cells could be generated at high levels in vivo after infusion. Moreover, the magnitude of HER2 specific CD4+ and CD8+ T cells post-infusion was maintained in responding patients, with precursor frequencies 1:253 and 1:286 in total PBMN after 130 and 450 days respectively. Epitope spreading, which was induced with immunization, was also maintained and in some cases augmented after adoptive T cell therapy. TCR Vβ analysis performed showed the outgrowth of several HER2 specific clones in the CD4+ population post infusion. Our preliminary data suggests that adoptive transfer of autologous HER2 specific polyclonal T cells is safe and results in persistent high levels of tumor specific T cell immunity in vivo.
Generation of WT1-specific CTL from CMV stimulated CD8 cells (41.32)
Authors: Stamer Mindy M , Herse Zachary , Dunham Kimberly , Bao Lei , Lucas Kenneth G
Published: January 2023

WT1 responsive CTL were generated from CMVpp65 stimulated CD8 cells isolated from three CMV-seropositive HLA-A2 normal donors. CD8 selected T cells were stimulated with dendritic cells pulsed with CMVpp65 overlapping peptides. 10 days after the primary stimulation, CD8 T cells were restimulated with CMV pp65 peptide pulsed PBMC. The CMV-stimulated CTL exhibited CMV-specific but not WT1-specific cytotoxicity after the second stimulation as measured by 51CR release assay (Donor 1=54% and 7%, Donor 2=77% and 14%). However, all CMV-stimulated CTL stained with PE-RMFPNAPYL, a WT1A2 tetramer (Donor 1=2.51%, Donor 2=3.85%, Donor 3=0.49%). EBV-CTL generated from an HLA-A2 donor, and WT1 CTL generated from a non-HLA-A2 donor did not stain with the WT1A2 tetramer (0.02% and 0.01% respectively). Therefore, the WT1A2 tetramer was cross-reactive with pp65-CTL and exhibited HLA-A2 specificity. To enrich the CMV-CTL WT1A2 tetramer staining population, we PE-selected donor 1 PE-WT1 tetramer binding pp65 stimulated CTL. The WT1-selected cells were cloned by limiting dilution. 7 of 8 WT1-selected clones stained with the WT1A2 tetramer ranging from 0.89%-3.56%. WT1-selected clones showed three types of specific cytotoxicity: 1) against pp65-only (3 clones=22-34%), 2) against WT1-only (2 clones=43-44%), or 3) against both WT1 and CMV (1 clone=55% and 24% respectively). 2 of the 8 clones did not exhibit either WT1 or CMV specific cytotoxicity. Only 1 of the 2 clones that exhibit WT1 specific cytotoxicity exhibited WT1-specifc IFN-γ production, as measured by ELISPOT. All clones that displayed CMV-specific cytotoxicity also exhibited CMV-specific IFN-γ production. To our knowledge this is the first description of WT1 cross-reactive, CMV-specific CTL.
Intratumoral (IT) injection of hu14.18-IL2 immunocytokine (IC) enhances antitumor activity and augments NK infiltration into tumors: analyses of cellular internalization of hu14.18-IL2 by human NK cells (41.44)
Authors: Buhtoiarov Ilia N. , Neal Zane C. , Gan Jacek , Buhtoiarova Tatiana N. , Hank Jacquelyn A. , Yamane Brett H. , Rakhmilevich Alexander L. , Patankar Manish S. , Gubbels Jennifer A. A. , Gillies Stephen D , Sondel Paul M.
Published: January 2023

Hu14.18-IL2 (EMD 273063) IC, consisting of GD2-specific mAb and IL2, is in trials for treatment of melanoma and neuroblastoma. Preclinical studies showed augmented antitumor activity when IC is injected IT vs. IV. Human IL2R+CD16+NK cells mediate ex vivo ADCC in the presence of IC that binds them to GD2+ tumor cells. We studied the kinetics of IC presence on the surface of human M21 melanoma and CD25+CD16-NK cell lines, NKL and RL12, after IC binding to the cells via GD2 and IL2R, respectively. For NK cells, ~50% of IC was internalized by 6 h, and ~90% by 24 h of cell culture. Excess free IL2 in the culture did not slow internalization kinetics for these NK cells. IL2-deprivation for 24 h prior to IC arming of NK cells decreased IL2Rα expression and overall IC binding but did not affect internalization kinetics of the bound IC. For NK cells that were prelabeled with IC, the decrease of surface IC levels over 24 h correlated with reduction of ADCC in vitro. Unlike NK cells, M21 cells retained ~70% of IC on the surface following 24 h of culture.

When CFSE-labeled NKL cells were injected IV into M21-bearing SCID mice, they migrated better into the tumor when IC was delivered IT than when they were pre-armed with IC before implantation. These preliminary studies suggest that once IC gets to tumor sites, it may stay on the tumor surface for a prolonged time, and that IT injections of IC enhance the recruitment of NK cells to the tumor site.
The effects of HBV core protein on TRAIL-induced apoptosis and its molecular mechanisms (41.45)
Authors: Sun Wensheng , Du Juan , Liang Xiaohong , chen Youhai H
Published: January 2023

Hepatitis B virus (HBV) infection afflicts more than 300 million people worldwide .TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in hepatocyte death during HBV infection. The goal of our study is to determine for the first time whether HBV core protein (HBc) is involved in modulating the TRAIL apoptotic pathway. Using cck-8 and TUNEL assay we firstly reported the HBV core protein (HBc) is a potent inhibitor of TRAIL-induced apoptosis. The further study showed that HBc decreased the expression of DR5, which is one of the most important TRAIL receptors. Additionally , the dual-luciferase assay indicated HBc significantly repressed the promoter activity of the human DR5 gene.Our further results examed by the microarray analysis indicated that HBc also repressed P53 gene. Whether the HBc-p53 pathway plays a significant role in regulating DR5 expression needs to be further studied. These results indicate that HBc may prevent hepatocytes from TRAIL-induced apoptosis by blocking DR5 expression, which in turn contributes to the development of chronic hepatitis and HCC.

This work was supported by grants from the National Natural Science Foundation of China ( No.30700973, No.30772031, No.30128023 and No.30700357). Corresponding author E-mail : WSW@sdu.edu.cn. Wensheng Sun , Juan Du and Xiaohong Liang contributed equally to this work.
The role of CD11c+ hepatic dendritic cells in immue tolerance to hepatocellular carcinoma (41.48)
Authors: Zheng Hong , Avella Diego M , Schell Todd D , Jiang Yixing , Staveley-O'Carroll Kevin F
Published: January 2023

Immune tolerance is a major mechanism involved in the development of hepatocellular carcinoma (HCC). We have previously showed, in a mouse model of spontaneous HCC, that T cell response can be induced in mice with small HCC, but not in mice with large HCC, indicating that immune tolerance occurs when tumors grow to a certain size. The mechanisms responsible for this phenomenon were not clear. In this study, we compared freshly isolated liver dendritic cell (DC) subsets in mice with no tumor, small HCC, and large HCC. We found that total numbers of CD11c+ hepatic DC were comparable in these 3 groups. However, CD11cint B220+ subset of 'plasmacytoid' DC (pDC) significantly decreased in both small and large HCC-bearing mice. Importantly, CD11chi B220- subset of mature DC significantly increased in mice with small HCC. In contrast, CD11chi B220- subset of mature DC remains same in mice with large HCC, the CD11cint B220- subset of immature DC increased instead in these mice. The surface phenotype of each subset of DCs were examined and demonstrated higher level of CD86 and CD40 expression by CD11chi subset of mature DC comparing with CD11cint subsets. These findings indicate that modulating subset distribution of liver DC, such as inhibiting the expansion of CD11chi B220- subset of mature DC, may play a role in immune tolerance to HCC.
Homeostatic proliferation transiently rescues tolerant CD8+ T cells (41.57)
Authors: Schietinger Andrea , Fowler Carla , Teague Ryan , Tan Xiaoxia , Blattman Joseph , Greenberg Philip
Published: January 2023

Peripheral T cell tolerance to self antigens is required to prevent the destruction of normal tissue. Since most tumor antigens are overexpressed self antigens one critical challenge is to develop strategies that break T cell tolerance to such antigens without causing autoimmunity. We previously developed a murine T cell tolerance model in which CD8+ T cells express a transgenic TCR specific for a viral protein that is expressed as a transgene selectively in the liver. Peripheral CD8+ T cells in these mice are tolerant and do not proliferate in response to the antigen. However, strategies that "force" tolerant T cells to proliferate, for example by stimulation with IL-15, abrogated tolerance.

To understand the mechanism by which repetitive cell division rescues tolerant T cells, we studied the effect of homeostatic proliferation (HP) on tolerant T cells. Transgenic tolerant T cells were adoptively transferred into lymphopenic mice in the presence or absence of a tolerizing environment. We found that: (i) expression of self-antigen dramatically augmented HP of tolerant T cells, (ii) HP rescued tolerant T cells even in a tolerogenic environment, and (iii) rescued tolerant T cells persisted without causing autoimmunity. Intriguingly, the observed HP-dependent rescue was transient; tolerant T cells became functional during the "acute phase" of HP but subsequently reacquired their tolerant phenotype even in the absence of a tolerizing environment.

In summary, our studies suggest that HP may lead to a temporary reprieve from epigenetic repression of functional activity that is subsequently reimposed after the proliferative drive terminates.
Evaluation of dendritic cell based cancer vaccines with a huPBL-NOD/SCID/IL2r-gamma-null xenotransplant model (41.7)
Authors: Harui Airi , Kiertscher Sylvia M. , Basak Saroj K , Roth Michael D
Published: January 2023

Dendritic cells (DC) are antigen presenting cells that have shown great potential as cancer vaccines when tested in conventional animal models. However, relating the induction of antitumor responses in mice to the efficacy in humans has been difficult. Here-in we describe the development of a xenotransplant model to detect the capacity for a DC-based vaccine to generate human anti-tumor T cell responses in vivo. Immunodeficient NOD/SCID/IL2r-gamma-null mice allow efficient engraftment and expansion of human peripheral blood leukocytes (huPBL). Over 3 weeks, the lymphoid organs of recipient mice are reconstituted with more than 70% of the spleen cells expressing human CD45. To evaluate anti-tumor responses, mice were engrafted with huPBL, implanted with human tumor cells, and then vaccinated with autologous DC expressing tumor antigens. Spleen cells showed a normal CD4:CD8 ratio, while CD8+ cells predominated in the tumor infiltrating lymphcytes (TIL). There was a significant decrease in naïve CD45RA T cells and an increase regulatory T at the tumor site. Immunized mice showed a decrease in tumor size, an increase in tumor-specific T cells, but not complete tumor resolution. This model may allow the character of TIL and the induction of human immune responses against cancer to be more realistically evaluated in vivo. This study is funded by NIH/NIDA R01-DA03018 and by the NIH/NCI UCLA SPORE in Prostate Cancer.
Ten color flow cytometry reveals CD126 as a marker for mature SP4 and SP8 thymocytes (42.10)
Authors: Tan Chibing , Taylor Ashlee A , Van De Wiele C. Justin , Teague T. Kent
Published: January 2023

We have developed a 10 color flow cytometric staining protocol for the simultaneous detection and characterization of 21 thymocyte subpopulations. These subpopulations include 17 previously described subsets and 4 new subsets. Importantly, this methodology uses only commercially available monoclonal antibodies and therefore could be easily employed by any investigator with access to a standard 4 laser flow cytometer. Using this novel staining scheme we have analyzed mouse thymocytes at discreet stages of development for expression of multiple cytokine receptors. As previously described, we found IL-7Ralpha to be present on some early subsets, upregulated briefly after beta-selection, downregulated on ISP and DP cells, and then re-expressed after positive selection. IL-4Ralpha was also expressed within the early DN1 and DN2 subsets. However, its expression was downregulated prior to beta-selection (in the DN3a subset) and was re-expressed in the DP cells prior to positive selection. IL-6Ralpha (CD126) showed a dramatically different pattern of expression. It was low or negative on all thymocyte subsets with the exception of the CD24low SP4 and SP8 subsets. The CD24low cells represent the more mature SP populations and therefore CD126 expression appears to be a good marker for the detection of mature thymocytes.
Quantitative Phosphoproteomic Analysis of Cellular Signaling (42.21)
Authors: Nguyen Vinh Hai , Salomon Arthur R.
Published: January 2023

Although many details of T cell signaling have been described previously, a systematic, quantitative wide-scale analysis of global tyrosine phosphorylation events that are triggered by activation of the T cell receptor is lacking. Here we introduce and evaluate a new quantitative approach for phosphoproteomic analysis of signaling pathway structure. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or receptor stimulation time course experiments, often with restricted number of time points. Our approach combines genetic analysis of isogenic signaling pathway mutants with quantitative phosphoproteomic method that examines disruption of downstream phosphorylation events through a time course of receptor activation using recently developed visual pathway analysis tools. This new approach is evaluated in the context of the T cell signaling pathway and a T cell clone lacking the upstream Zap-70 tyrosine kinase and its reconstituted counterpart. In our approach, label free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Metabolic labeling of proteins using SILAC method allows for the calculation of ratios used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. Quantitative phosphoproteomic signatures indicative of downstream inhibition, downstream activation, and mutant compensation are revealed and validated with proteomic pathway visual analysis tools using established T cell signaling pathway structure as a scaffold.