bluuebunny/crossref_metadata_2025 huggingface dataset:
CD137 promotes proliferation and survival of human B cell (34.14)
Authors: Zhang Xiaoyu , Voskens Caroline J. , Sallin Michelle , Strome Scott E
Published: January 2023

CD137 (4-1BB) mediated co-stimulation plays an important role in directing the fate of antigen stimulated T cells and NK cells, yet its role on B cell function is currently unknown. The goal of our study was first to evaluate which external signals regulate CD137 expression on human B cells and second to define the biological effect of CD137-mediated co-stimulation on human B cells. Flow cytometric analysis was used to evaluate cell surface expression of CD137. Cell proliferation was assessed by 3H-TdR incorporation and CFSE dilution assay. Cell apoptosis was determined by Annexin V and 7-AAD staining. Here we report that CD137 is expressed on human B cells stimulated with anti-Ig. CD137 expression is enhanced by CD40 ligation and IFN-γ, and is inhibited by IL-4 and IL-10. The expression of CD137 on activated human B cells is functionally relevant, as engagement with its ligand at the time of activation stimulates B cell proliferation. Furthermore, CD137 stimulation enhances B cell survival which is associated with expression of the anti-apoptotic proteins Bcl-xl and Mcl-1. Our study demonstrates a direct role for CD137-mediated co-stimulation in defining the fate of antigen-stimulated human B cells.
Interactions between LMP1 and CD40 signals (34.15)
Authors: Stunz Laura , Bishop Gail
Published: January 2023

The EBV encoded protein LMP1 signals to B lymphocytes in a manner similar in many respects to the normal cellular protein CD40. Under circumstances in which LMP1, not expressed in normal latency, is re-expressed in vivo, LMP1 and CD40 are expressed in the same cells. Thus, we have used both cell line and transgenic mouse models to investigate the interaction of LMP1 and CD40 signals. CD40 and LMP1 signals cooperate in stimulation of JNK , IkBa and p38 MAPK phosphorylation in B cell lines, as well as in primary mouse B cells and BMDCs, though TRAFs 2 and 3 are degraded in a manner similar to CD40 signaling alone. Though additivity is seen in early signaling through these two pathways, in vivo effects of LMP1 + CD40 signaling do not always show cooperation. Expression of a hybrid molecule with the extracellular domain of mouse CD40 and the intracellular signaling domain of LMP1 in B cells, macrophages and dendritic cells on a CD40 negative background leads to lymphadenopathy, splenomegaly, spontaneous germinal centers, and autoantibody formation. However, when CD40 is also expressed some of these in vivo effects are ameliorated (splenomegaly, some cytokines), or delayed (autoantibody production). One interesting effect of mCD40-LMP1 signaling in vivo is elevated serum IL17, which we hypothesized is secondary to elevated IL6 production in the mCD40-LMP1tg mice. However, mice expressing both mCD40-LMP1 and mCD40 transgenes also have elevated IL6, but serum IL17 levels are not elevated, suggesting that other factors or pathways are involved.
Regulation of HSH2 Adaptor Protein Expression in B cells is Mediated by Posttranscriptional Mechanisms (34.18)
Authors: Lewis Karon C , King Rodney , Justement Louis
Published: January 2023

Previous studies have shown that the adaptor protein hematopoietic src homology 2 (HSH2) is expressed at low levels in resting B cells. HSH2 expression is up-regulated in response to various stimuli that promote splenic B cell survival and differentiation including α-CD40 mAb, LPS, CpG, and Blys. HSH2 expression was found to be dependent upon NF-¿B stimulation in that inhibitors of NF-¿B blocked up-regulation of HSH2 expression. HSH2 expression did not increase upon stimulation of B cells via the BCR unless cells were previously stimulated through CD40. Preliminary data reveal that HSH2 protein expression is decoupled from transcript abundance. Real-time PCR analyses show that upon α-CD40 stimulation of splenic B cells, HSH2 transcript levels remain relatively constant, whereas protein expression increases. This suggests that there are one or more posttranscriptional mechanisms in place that control HSH2 expression at the protein level. One possibility is that the untranslated regions of HSH2 mRNA contain regulatory regions responsible for translational control, which is currently under investigation. A second finding is that HSH2 turns over rapidly at the protein level in response to conditions in which the 26S proteosome is blocked with pharmacological antagonists. Thus, it is apparent that HSH2 is subject to proteolytic degradation and that this is under tight regulation. In summary, HSH2 expression is controlled by posttranscriptional mechanisms that maintain low basal expression in resting B cells, but promote increased expression in cells exposed to agonists that promote survival and differentiation.
Superoxide production influences cell cycle entry downstream of BCR ligation (34.20)
Authors: Richards Sabrina , Clark Edward A.
Published: January 2023

Purpose: To investigate the contribution of BCR-induced superoxides to BCR-dependent signaling outcomes. Methods: gp91phoxiKO mice were utilized. Purified B cells from WT or gp91phoxiKO mice were loaded with CFSE, and stimulated via the B cell receptor (BCR). Proliferation was determined by CFSE dilution and G1 entry by Pyronin-Y staining. Western blotting of BCR-stimulated B cells was performed to measure cell cycle proteins. WT and gp91phoxiKO mice were immunized with TI-2 and TD antigens and serum Ab titers measured using ELISA. Results: More gp91phoxiKO B cells entered G1 and knockout B cells proliferated more after BCR stimulation. Reduction in levels of p27Kip1 occurred more quickly in BCR-stimulated gp91phoxiKO B cells. gp91phoxiKO mice displayed enhanced Ag-specific Ab production after TI-2 immunization. Conclusions: Superoxide derivatives negatively regulate signaling pathways leading to BCR-dependent proliferation. The perturbation in signaling in ROS-deficient B cells lies downstream of calcium signaling but upstream of p27Kip1.

These studies supported by a Cancer Research Institute Training Grant and NIH grants GM37905 and AI44257.
Preformed CD40 ligand is expressed in effector but not regulatory CD4+ T cells, and is sufficient to induce antigen-specific B cell activation (34.4)
Authors: Koguchi Yoshinobu , Thauland Timothy J. , Gardell Jennifer L. , Buenafe Abigail C. , Slifka Mark K. , Parker David C.
Published: January 2023

Previously, we showed that preformed CD40 ligand (pCD40L) exists in effector CD4+ T cells and can be sent to the cell surface within minutes following antigen recognition. We tested which subsets of Th cells possess pCD40L. In vitro primed and polarized Th1 and Th17 but not Th2 and induced regulatory T (iTreg) cells possess pCD40L. Consistently, Th1 and follicular helper T cells from LCMV infected mice and Th1 and Th17 cells from mice with EAE have pCD40L. However, Th2 cells induced in vivo by papain plus OVA immunization do possess pCD40L. In contrast, natural Treg cells as well as iTreg cells from an in vivo oral tolerance model do not have pCD40L. To examine the function of pCD40L, we cultured cyclosporine A-treated Th1 cells, which maintain pCD40L expression but not de novo production of cytokines and CD40L, with a mixture of peptide-pulsed and -unpulsed B cells from WT and CD40-deficient mice. This assay revealed an antigen-specific, CD40-dependent, activation of B cells by pCD40L. Interestingly, anti-CD40L mAb can only partially neutralize this CD40-dependent activation, suggesting that the adhesion ring of the immune synapse may block access of the antibody to delivery of pCD40L to antigen-presenting B cells. pCD40L may be used by effector Th cells to deliver an activation signal to antigen-presenting APCs through the immune synapse during brief interactions that are not long enough for de-novo CD40L to be synthesized.
Direct interaction with dendritic cells through CD28-CD80/CD86 supports plasma cell survival (34.9)
Authors: Nair Jayakumar R , Carlson Louise M , Rozanski Cheryl , Boise Lawrence H , Chanan-Khan Asher , Lee Kelvin P
Published: January 2023

The long term survival of plasma cells in their bone marrow niches, necessary to maintain persistent antibody titres, depends on specific survival signals that are produced by their interaction with bone marrow stromal cells (BMSC), interactions that remain largely uncharacterized. Both normal and malignant plasma cells (like myeloma cells) express the T-cell co-stimulatory molecule CD28 and are also known to interact with dendritic cells (DC) in bone marrow biopsies. We also know that ligating CD80/CD86 on DCs with soluble CD28/CTLA4 ligands can up-regulate IL-6, an essential plasma cell survival factor. In our studies, co-culturing CD28+ plasma cells (myeloma cell lines or primary plasma cell isolates) with immature DCs significantly up-regulated DC production of IL-6 (400 - 500 pg/ml as opposed to 40 - 70 pg/ml), which was blocked with anti-CD28 antibodies. Cell-free plasma cell-DC co-culture supernatants supported plasma cell proliferation and survival. Furthermore, plasma cell CD28 binding to CD80/CD86 on DCs induces DC expression and activity of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), which inhibits T cell activation by depleting the essential amino acid tryptophan from the microenvironment. Our study suggests a pro-survival role for DC in regulating plasma cell survival by modulating their bone marrow micro-environment
Early Intestinal Epithelial Response to Toxoplasma gondii Infection (39.29)
Authors: Leifer Cynthia A , Ju Chia-hsin , Chockalingam Annapoorani
Published: January 2023

Intestinal epithelial cells are critical for nutrient uptake, but also provide a physical and chemical barrier to infection. When that barrier is breached, epithelial cells can respond and regulate local and systemic immune responses. Toxoplasma gondii (T. gondii) is an orally acquired apicomplexan protozoan intracellular parasite that first encounters intestinal epithelium and then disseminates systemically. Little is known about the responses elicited early during infection in the intestine since studies to date have focused primarily on systemic immune responses through intraperitoneal infection. Furthermore, we lack information on the response of human intestinal cells to live T. gondii infection. Here we examined the early response of human intestinal epithelial cells following infection with T. gondii. Using an in vitro system, we show that human small intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-κB nuclear translocation, and secretion of interleukin (IL)-8. Both ERK1/2 activation and IL-8 secretion responses were PI3 kinase-independent yet MyD88- and TLR2-dependent. Although parasite genotype correlates with pathogenicity in mice, all genotypes of T. gondii elicited a similar response through TLR2 and MyD88. Oocysts of T. gondii stimulated cells via TLR2, similar to tachyzoites. Furthermore, we were able to identify many T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells recognize and respond to T. gondii during infection, the outcome of which likely modulates intestinal and systemic immunity.
Intramuscular delivery of a plasmid-encoded molecular adjuvant elicits immune responses at mucosal sites in a macaque model (39.51)
Authors: Schoenly Kimberly Ann , Kutzler Michele A , Cisper Neil J , Yan Jian , Khan Amir S , Draghia-Akli Ruxandra , Lewis Mark G , Weiner David B
Published: January 2023

As the primary site of HIV-1 replication is the mucosa, a desirable feature of a HIV-1 vaccine would be the generation of immune responses at mucosal sites. We created an optimized CCL27 DNA construct as well as optimized SIV gag, pol, and env plasmids for immunizing rhesus macaques. Macaques (n=5/group) were immunized with SIV plasmids +/- CCL27. Both immunization groups had significant IFN-g responses against the encoded SIV antigens (~12,000 SFU/106 PBMCs). CD8+ T cell proliferation as measured by CFSE staining was similar between the groups at ~22%. While systemic immune responses were robust, co-delivery of CCL27 did not adjuvant peripheral responses similar to what was observed in mice. Interestingly, macaques that received CCL27 had greater antigen specific IgA at mucosal sites including bronchial lavage and fecal samples. In addition, CD4+ T cells secreting effector cytokines were enhanced in the CCL27 group at mucosal, but not peripheral sites when compared to the antigenic group alone. The CCL27 group also had more polyfunctional CD8+ T cells in the bronchial lavage than the DNA group post-challenge. These data show that a novel chemokine adjuvant may be capable of targeting immune responses to mucosal sites. This strategy may also be applicable to other diseases than HIV-1 where mucosal immune responses are desirable.
Chemokines and recruitment of regulatory T cells to the tumor (40.14)
Authors: Fan Xiaozhou , Allison James P.
Published: January 2023

Foxp3+ regulatory T cells (Treg) are crucial to maintain peripheral tolerance by restraining the reactivity of other immune cells, but this suppressive function is in contrary a great hurdle for successful tumor immunity. Studies in humans have correlated high frequency of Treg to poor prognosis. In a transplantable mouse melanoma model B16/BL6, the tumor is also highly infiltrated with Treg. The aim of this study is to identify the chemokines that specifically recruit Treg to the tumor site and to develop immune intervention strategies to manipulate this traffic. Two C-C motif chemokine receptors, CCR4 and CCR8, have been reported to be expressed preferentially in human Treg. We found that both the two receptors are present on the majority of tumor-infiltrating Treg, with significantly higher frequency than the CD4+ Foxp3- effector T cells (Teff) and the CD8+ compartments. Furthermore, the CCR4+ or CCR8+ Teff are not actively producing IFN-γ, indicating most of the tumor-reactive effector T cells are negative for these receptors. On the other hand, the ligands for CCR4, CCL17 and CCL22, and the one for CCR8, CCL1, are upregulated in the tumors and/or the vaccine sites of the GM-CSF-secreting cellular vaccine (GVAX) as reflected by real-time PCR. These data together suggest that the chemokine-receptor network involving CCR4 and CCR8 could be a potential target for immunotherapy against cancer.
CD73 on tumor cells impairs anti-tumor T cell immunity: a novel mechanism of tumor-induced immune suppression (40.17)
Authors: Zhang Bin , Jin Dachuan , Fan Jie , Liu Aijie , Daniel Benjamin , Shin Tahiro , Curiel Tyler
Published: January 2023

CD73, originally defined as a lymphocyte differentiation antigen, is thought to function as a costimulatory molecule on T lymphocytes and an adhesion molecule that is important for lymphocyte binding to endothelium. We show here that CD73 is widely expressed on many tumor cell lines and upregulated in cancerous tissues. Because CD73 ecto-5'-nucleotidase catalyzes AMP breakdown to immunosuppressive adenosine, we propose that CD73-generated adenosine may prevent tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that genetic ablation of CD73 on ID8 ovarian tumor cells by siRNA reduced its enzymatic activity and increased survival and decreased tumor burden in immunocompetent but not immunodificient Rag1-/- mice. CD73 knockdown improved tumor antigen-specific T cell proliferation and CTL in vivo killing function and decreased apoptosis of tumor activated microenvironmental T cells. Notably, there was no therapeutic benefit of adoptive T-cell immunotherapy in mice bearing tumors without CD73 knockdown. By contrast, combining tumor CD73 knockdown and T cell transfer cured all mice. Moreover, blockade of A2A adenosine receptor with its antagonist SCH58261 also augmented the efficacy of T cell therapy. These findings identify a potential mechanism for CD73-mediated tumor immune evasion and point to a novel cancer immunotherapy strategy by targeting tumor CD73.
CD8+ T cell precursor frequency determines the quality of the anti-tumor immune response (40.33)
Authors: Rizzuto Gabrielle A , Merghoub Taha , Hirschhorn-Cymerman Daniel , Liu Cailian , Leskohin Alexander M , Zhong Hong , Panageas Katherine S , Perales Miguel-Angel , Altan-Bonnet Gregoire , Wolchok Jedd D , Houghton Alan N
Published: January 2023

Poor clinical response to cancer vaccines may in part be due to a paucity of self-antigen specific T cells surviving negative selection. Here, we demonstrate the frequency of endogenous, mouse CD8+ T cells recognizing the melanoma/melanocyte antigen gp100 to be <0.0001% (~1 in 1 million). We developed a model to study the responsiveness of different frequencies of CD8+ T cells (pmel-1) against this self antigen. We show that endogenous CD8+ T cell frequency is too low for creating a robust anti-tumor immune response upon vaccination, but supplementing this repertoire with low numbers of naïve pmel-1 cells increases vaccine-elicited tumor immunity. By combining adoptive transfer of only 1000 pmel-1 cells, lymphopenia, and vaccination with mutated self-antigen, we were capable of inducing regression of melanoma tumors in mice.

Curiously, treatment was impaired as the number of tumor-specific cells transferred was increased. At high doses, competition between clones significantly hindered responses. We show that tumor-specific cells primed at low precursor frequencies where competition is not operative, undergo more rounds of proliferation, acquire poly-functionality, and eradicate tumors more effectively. This work demonstrates that precursor frequency can be exploited to augment clinical vaccine strategies. Transfer of optimized numbers of naïve tumor-specific T cells, followed by in vivo activation is a new approach that should be applied to immunotherapy.
IL-21 induced Bcl3 expression blocks type 1 effector differentiation of CD8+ T cells and enhances their tumor efficacy (40.35)
Authors: Shrikant Protul A
Published: January 2023

IL-21 induced Bcl3 expression blocks type 1 effector differentiation of CD8+ T cells and enhances their tumor efficacyCheryl Eppolito, Qingsheng Li and Protul A Shrikant11Department of Immunology, Roswell Park Cancer Institute, 322 Cancer Cell Center, Elm and Carlton Streets, Buffalo, NY. 14263

The γc cytokine; IL-21, induces Jak1/Jak3 mediated STAT1, STAT3 and STAT4 phosphorylation as well as T-bet expression. Surprisingly, IL-21 treated antigen stimulated CD8+ (OT-1) T cells fail to produce IFNγ, although they demonstrate robust cytolytic activity. In addition, IL-21 treatment reduces KGLR1, CXCR3 and CD44 expression while increasing L-selectin, IL-7Ra and CD71 expression, suggesting its ability to dampen effector maturation. The IL-21 induced Jak3 activity is essential for PI3K induced Bcl3 expression. The IKβ member Bcl3 is known to associate with NFkB p50 homodimers and stabilize them for activation and/or repression of specific gene programs that favor anti-inflammatory responses in myeloid cells. The IL-21 induced Bcl3 regulates IFNγ production in OT-1 cells by interfering with STAT1 and STAT4 induced sustained T-bet expression and the loss of Bcl3 renders IL-21 treated OT-1 cells to demonstrate greater effector phenotype. However, the cytolytic maturation induced by IL-21 is only marginally affected by Bcl3 expression. Moreover, IL-21 induced Bcl3 promotes survival of antigen stimulated OT-1 cells and in the absence of Bcl3 the IL-21 conditioned OT-1 cells are less persistent and are ineffective at controlling tumor growth after adoptive transfer. Hence, our results demonstrate a novel pathway by which IL-21 induces "partial" effector maturation, which has greater tumor efficacy. This establishes a new paradigm for the generation of modest effector CD8 T cells that persist for effective ACT mediated tumor therapy.

Supported by NIH-NCI 5R01CA104645 to PAS
Murine mammary carcinoma cells and CD11c+ dendritic cells elicit distinct responses to lipopolysaccharide and exhibit differential expression of genes required for TLR4 signaling (40.4)
Authors: Kurt Robert A , Palha De Sousa Chiquita , Blum Christopher , Sgroe Erica
Published: January 2023

Particular attention has been given to Toll-like receptors (TLR) on dendritic cells (DC) because of their ability to bridge innate and adaptive defenses. Since TLR are also expressed by epithelial cells, and because the majority of cancers are carcinomas, and thus of epithelial origin, we were interested in comparing responsiveness of a carcinoma and DC to a TLR agonist. For this purpose we used the mammary carcinoma 4T1 and CD11c+ DC. Both 4T1 and DC expressed genes encoding multiple TLR and secreted the proinflammatory chemokines CCL2 and CXCL1 in response to the TLR4 agonist lipopolysaccharide (LPS). However, DC, but not 4T1 secreted IL-1beta, TNF-alpha, and upregulated CD80 and CD86 expression following LPS treatment. Gene arrays were used to delineate potential reasons for the differential responsiveness of the cells to LPS. The arrays showed that genes encoding TLR4, CD14, myeloid differentiation primary response gene 88 (Myd88), and Toll-like receptor adaptor molecule 2 (TICAM-2) were expressed at greater levels by the DC; results which were verified by quantitative RT-PCR. These data demonstrate that 4T1 and CD11c+ DC are distinctly responsive to LPS, and that this may be a consequence of differential expression of genes encoding proteins important for TLR signaling.
Ex vivo activity of early cytokine conditioned effector CD8+ T cells inversely correlates with their anti-tumor activity in vivo (41.1)
Authors: Diaz-Montero C. Marcela , Salem Mohamed L. , Naga Osama , Montero Alberto J. , Cole David J.
Published: January 2023

Enhanced effector activity such as cytokine secretion, cell proliferation , cytolytic activity, etc. in response to in vitro stimulation has been widely used as a means to predict in vivo activity after immune mediated interventions, such as adoptive transfer or systemic vaccination. Contrary to this, here we show that the enhanced in vivo survival and anti-tumor activity of cytokine conditioned early activated CD8+ T cells inversely correlates with their effector functions in vitro. When compared with late effector PmelIL-12, early effector showed decreased production of IFNγ and TNFα, and decreased expression of the degranulation marker CD107a in response to in vitro reactivation. However, after transfer into lymphodepleted hosts, early effector PmelIL-12 survived for at least 20 days and were able to effectively control the progression of B16 tumors in a superior manner than late effector PmelIL-12. Thus, effector activity in response to ex vivo antigenic restimulation does not necessarily predict in vivo activity. Other parameters such as surface expression of functional markers (CCR7, CD62L) and /or survival (CD127) could serve as better predictors of in vivo activity. Developing and validating accurate predictors of in vivo lymphocyte function would greatly facilitate our ability to rationally design novel immune based therapies for clinical trials that have significant anti-tumor activity.
Targeting ALDH1A1 for Immunotherapy of Cancer Stem Cells (41.20)
Authors: visus carmen , Wang XinHui , Paola Miyazato , Wang YangYang , Szczepanski Miroslaw , Ferrone Soldano , Deleo Albert
Published: January 2023

Background: ALDH bright cells identify cancer stem cells in breast cancer and other tumors as well. In the past, we were able to generate ALDH1A188-96 peptide specific T cells. Now we hypothesize that ALDH1A188-96 peptide-specific T cells can target cancer stem cells which are otherwise untreatable using available therapies.

Methods: ALDH bright cells were identified by FACS analysis and sorted from head and neck and breast cancer cell lines. Those cells were injected in NOD/SCID mice in order to study their tumorigenicity. The reactivity of the ALDH peptide-specific T cells was tested in IFN-g ELISPOT assays.

Results: ALDH bright cells were isolated from head and neck and breast cancer cell lines and found highly tumorigenic in NOD/SCID mice. ALDH1A188-96 peptide-specific CD8+ T cells recognized ALDH bright cells in ELISPOT assays but did not recognized ALDH negative cells or the bulk tumor cell lines unless those cells were pulsed with the exogenous ALDH1A188-96 peptide.

Conclusion: We are able to target ALDH bright cells for immunotherapy utilizing ALDH1A1 specific T cells and this may result in control of tumor growth and metastasis.
Increased lymphopenia results in higher percentages of FoxP3+ T cells in patients reconstituted by PBMC adoptive transfer and vaccinated (41.36)
Authors: Thompson James  A , Assmann Ilka , Haley Daniel , Moudgil Tarsem L. , Jensen Shawn M. , Poehlein Christian H. , Hu Hong-Ming , Walker Edwin B. , Sacks Natalie , Hege Kristen , Curti Brendan , Urba Walter J. , Fox Bernard A.
Published: January 2023

In clinical trials therapeutic tumor vaccines have failed to provide evidence of efficacy. Based on preclinical tumor models showing that vaccination during homeostasis-driven proliferation improved therapeutic efficacy, we performed a translational phase I/II clinical trial in men with metastatic hormone-refractory adenocarcinoma of the prostate. This strategy has the theoretical advantage that it eliminates regulatory T cells (Treg). Patients were randomized to cohort A, B or C. Cohort A patients (n = 5) received prostate GVAX TM (two prostate cancer cell lines that secrete GM-CSF, Cell Genesys Inc.) vaccination only. Cohort B (n = 5) received cyclophosphamide followed by reinfusion of autologous PBMC and vaccine (GVAX). Cohort C patients (n=5) received same treatment as Cohort B with the addition of fludarabine. We hypothesized that the most lymphopenic patients would have the fewest Treg. While all cohorts showed an increase in the total number and frequency of peripheral Treg, the most lymphopenic patients (cohort C) had the greatest fold increase in peripheral Treg at weeks 2-6 (2.3,1.2,0.7 / p<0.05). Current studies are examining whether adoptive transfer of CD25 depleted PBMC will reduce Treg recovery and improve clinical response.

Supported by CA119123, Robert W. Franz, Chiles foundation, Prostate Cancer Foundation and Providence Medical Foundation
Vaccination with Mimotopes Prevents Tumor Growth by Enhancing the Activation of T cells that Respond to Natural Tumor Antigens (41.40)
Authors: Slansky Jill E. , Jordan Kimberly R. , Kappler John W.
Published: January 2023

Vaccination with mimotopes, or peptide mimics of self-tumor antigens, is a potential strategy for antigen-specific immunotherapy of cancer. We tested mimotopes with substitutions in residues involved in T cell interactions, rather than in conventional MHC-anchor residues, for protection against the transplantable tumor, CT26. Although vaccination with all of the mimotopes elicited tumor-specific T cells more effectively than the wild type tumor antigen, AH1, only some mimotopes prevented tumor growth. Vaccination with the protective mimotopes generated considerably more cross-reactive T cells that produced the cytokine IFNgamma after stimulation with lower concentrations of the AH1 peptide. These T cells expressed TCR molecules with a more restricted Vbeta repertoire ex vivo, but were similar in CDR3 sequence to the T cells generated by vaccination with the AH1 peptide. Importantly, the T cells responding to the non-effective mimotopes did not inhibit the antitumor responses of the protective mimotopes. These results suggest that protective mimotopes generate antitumor immunity, in part, by enhancing the activation and differentiation of a subset of T cells that naturally respond to the tumor, not a new repertoire of T cells only elicited by the mimotope.
Induction of anti-tumor immunity by a protein transfer mediated cancer vaccine (41.49)
Authors: Bozeman Erica Necole , Cimino Ashley M. , Tien Linda , Shashidnamurthy Rangaiah , Selvaraj Periasamy
Published: January 2023

The induction of a robust anti-tumor immune response is essential to the development of an effective cancer vaccine. Our lab has extensively studied a protein transfer approach that would allow for the rapid and stable incorporation of a myriad of immunostimulatory proteins, such as costimulatory molecules and cytokines, to the surface of tumor cell membranes via a glycosylphosphatidylinositol (GPI) -anchor. Using the 4T07 murine breast cancer model, all BALB/C mice immunized subcutaneously with tumor membranes modified to express B7.1, IL-2 and IL-12 alone or in combination were protected against direct and secondary challenges with live tumor cells. Additionally, these modified tumor membranes induced regression of a wild-type tumor growing at a distant site. These results suggest that the addition of immunostimulatory proteins directly to the tumor cells using a GPI-anchor can be proven effective in inducing an anti-tumor immune response that provides protective as well as concomitant immunity in a tumor bearing host.
Gap-junction communication between autologous endothelial and tumor cells induce cross-recognition and elimination by specific CTL (41.58)
Authors: Chouaib Salem , Benlalam Houssem
Published: January 2023

Cellular interactions in the tumor stroma play a major role in cancer progression but can also induce tumor rejection. To explore the role of endothelial cells in these interactions, we used an in vitro 3D collagen matrix model containing a cytotoxic T lymphocyte CTL clone (M4.48), autologous tumor cells (M4T) and an endothelial cell (M4E) line that are all derived from the same tumor. We demonstrate here that specific killing of the endothelial cells by the CTL clone required the autologous tumor cells and involved antigen cross-presentation. The formation of gap-junctions between endothelial and tumor cells is required for antigenic peptide transfer to endothelial cells that are then recognized and eliminated by CTL. Our results indicate that gap-junctions facilitate an effective CTL-mediated destruction of endothelial cells from the tumor microenvironment which may contribute to the control of tumor progression.
T-bet controls CD4+CD25+Foxp-3+ T regulatory cells and Th17 cells in lung adenocarcinoma (41.59)
Authors: Finotto Susetta , Boross Ildiko , Koslowski Michael , Karwot Roman , Maxeiner Joachin , Türeci Olez , Lehr Hans Anton , Finotto Susetta
Published: January 2023

The range of identified effector CD4+ T cell lineages has been expanded to enclose the Th17 cells whose function in lung tumor has not been investigated yet.

In this study, we report increased expression of the two newly identified transcription factors of the Th17 lineage, RORA and RORC2, in the lung of patients affected by lung adenocarcinoma.

Targeting IL-17A in a murine model of lung adenocarcinoma, resulted in local expansion of CD4+ effector T cells producing IFNγ and reduction of CD4+CD25+FOXP3+ regulatory T cells. In support of a protective role of IFNγ in this model, T-bet (-/-) mice exhibited increased tumor load, decreased survival and increased residual CD4+CD25+FOXP3+ T regulatory cells after anti-IL-17A antibodies treatment as compared to wild type littermates.

Thus, blockade of lung Th17 responses requires T-bet to reduce T-regulatory cells for limiting lung cancer.
Textpresso Site Specific Recombinases: a text mining tool to aid in identifying Cre expressing mice and conditional alleles (42.23)
Authors: Condie Brian G , Urbanski William M
Published: January 2023

The use of site-specific recombinases such as Cre and Flp in mouse genetics has risen dramatically. The number of mouse strains designed to express Cre recombinase activity in specific tissues and the number of mouse strains carrying conditional "floxed" alleles has increased explosively. As a result, the scientific literature describing these valuable research resources has burgeoned to more than 6000 peer reviewed publications. The large size and rapid growth of this literature makes it very easy to miss key papers that contain critical information relevant to your research. To assist investigators in identifying appropriate Cre recombinase expressing mice, conditional floxed alleles and other research resources based on site specific recombinase technology we have generated a new online resource based on the widely used Textpresso text mining software (http://www.textpresso.org). Our text mining tool, Textpresso Site Specific Recombinases (Textpresso SSR) allows researchers to search a database containing the full text of over 6000 peer-reviewed publications. This database contains most of the publications that describe or use Cre expressing mice as well as papers describing mouse strains carrying conditional mutant alleles. The database can be searched using keywords or combinations of keywords and categories. For each publication that matches the search terms Textpresso SSR displays the abstract as well as the individual sentences that match the query. Textpresso SSR also provides a unique tool for the in depth text mining of publications that describe the results of tissue specific or temporal specific mouse gene knockouts. Textpresso SSR can be accessed at http://ssrc.genetics.uga.edu.

This work was supported by the University of Georgia.
Delivery to Macrophages and Efficacy of Orally Administered RNAi Compounds (42.27)
Authors: Kamens Joanne , Khvorova Anastasia , Pavco Pamela , Rodgers Amanda , Ford Glenna , Bulock Karen , Smith-Anzures Bernice , Lapierre Jennifer , Lam Jessica , Libertine Lyn , Flannery-Rossi Kehlen , Metterville Jake , Byrne Michael , Czech Michael , Ostroff Gary
Published: January 2023

Data will be presented on a method for RNAi compound delivery utilizing RNA encapsulated in beta-glucan shells. When orally administered to mice, these encapsulated nucleic acids traverse the intestinal epithelia by an M cell transcytosis mechanism and are then endocytosed by macrophages. Macrophages containing RNAi compounds migrate to sites of inflammation, and the targeted RNA is specifically inhibited by RNA interference. This formulation presents some unique advantages for therapeutic RNAi including oral mode of administration, low dose (ug/kg/day) and specific delivery to immune cells possibly affording safety advantages for some targets. This novel mode of RNA administration can be used to down-regulate target genes involved in multiple inflammatory disease indications.
Consistency and sensitivity of immune response measurements among multiple laboratories using a standardized ELISpot assay kit (42.28)
Authors: Patke Deepa S , Dryga Sergey A , Marking Christine , Jeffrey Lisa , Manak Mark
Published: January 2023

ELISpot assays measure antigen specific cell mediated immunity, and are useful indicators of vaccine and therapeutic efficacy. Methods that yield consistent and reproducible results are required to compare data in longitudinal studies or among laboratories. Here, we describe the performance of a validated ELISpot assay using PBMC controls and standardized antigenic stimulants among laboratories participating in an External Quality Assurance (EQA) Program. Responses from four PBMC controls (high, medium, low and negative responders) were compared in an IFN-γ ELISpot assay using SeraCare (SC) kits and individual laboratory specific assay protocol and kits in an ELISpot EQA. SC kit performance was also compared to competitor kits in previous rounds of an EQA, each with 13 participants. SC kits showed high sensitivity in detection of low responders (Spot forming cells [SFC] range 10.9 - 49.2/well) compared with in house kit results (SFC range 0.4 - 67.2). Competitor kits yielded larger numbers of Too Numerous To Count results with high responders and greater variability in comparison to SC kits. SC kits yielded a broad linear dose response curve (10-1000 SFC/well) as a result of smaller spot size and improved resolution. SC kits and standardized assay protocols used by multiple laboratories allowed improved sensitivity of detection of low level antigenic responses, higher consistency and a broad linear range.
Reduced inflammation and altered cytokine expression in neonates compared to adults during paramyxoviral infection (43.13)
Authors: Bhattacharya Somashubhra , Janowski Ann , Shornick Laurie P
Published: January 2023

Neonates are especially susceptible to respiratory viral infection. However, there is little known about the neonatal immune response to respiratory virus. We compared the adult and neonatal responses to Sendai Virus (SeV). 6-8 week old C57BL/6 adults and 2-day-old C57BL/6 pups were infected with 500 pfu SeV/g body weight. Adults exhibited transient weight loss, which peaked at day 7 post-infection. Neonatal mice were in a rapid growth period and did not lose body weight during the course of infection. However, neonatal mice infected with SeV showed a slowing of growth after day 8 compared to neonates inoculated with UV-inactivated virus. Viral titer, measured by plaque forming assay, showed similar viral loads and rates of viral clearance in the adults and neonates. Adults exhibited significant neutrophilic inflammation in the lung that peaked at day 7 post-infection. Surprisingly, neonatal lungs had significantly less inflammation in the lung despite comparable levels of virus. ELISA of lung homogenates showed higher levels of IL-1b in the adults. In contrast, neonates had higher levels of TGF-b consistent with reduced inflammation in the neonatal lungs. These results suggest that neonates may be protected from lung inflammation, despite having significant viral load during primary paramyxoviral infection. Further studies will address the ability of the neonates to mount a secondary challenge to SeV. This research was funded by Saint Louis University.
Protection or immunopathology upon heterologous virus infection: A decision of memory cells (43.15)
Authors: Wlodarczyk Myriam F , Kraft Anke , Chen Hong , Selin Liisa K
Published: January 2023

Respiratory viral infection can cause subclinical to severe lung disease. As hosts are continuously exposed to environmental pathogens, a history of past infection might alter the immune response to a subsequent infection. This concept termed heterologous immunity has been largely overlooked. Prior immunity to influenza A (FLU) was shown to have a profound impact on acute infection with LCMV increasing virus titers and immunopathology, a phenomenon that we have called deranged immunity. We observed that LCMV infected FLU-immune mice displayed an alteration of the kinetics of their immune response with increased and more activated CD8 T cells. This is consistent with the activation of cross-reactive memory T cells. We showed that the FLU-induced memory CD8 and/or CD4 T cells played a role in enhancing lung immunopathology, and virus titers. We also identified a cross-reactive CD8 T cell response between the two viruses that maybe playing a role in modulating disease. Consistent with this result, the Vbeta TCR repertoire of some FLU epitope-specific memory CD8 T cells was altered upon LCMV infection. Enhanced IFNg produced by these crossreactive memory cells seemed to play a role in mediating the enhanced immunopathology. These finding have important implications for understanding variability in infectious disease susceptibility and in designing more efficient vaccines against respiratory infectious diseases.
NK1.1+ cells are required for neutrophil recruitment to sites of early viral infection (43.4)
Authors: Hosiawa-Meagher Karoline A. , Clingan Jon , Matloubian Mehrdad
Published: January 2023

LCMV replication is controlled by type I and II interferon (IFN) responses that drive antigen specific T cell expansion and function. It is unclear whether innate immune cells contribute to the initiation of T cell responses. We focused on two innate cell populations, natural killer (NK) cells and CD11b+ cells (Ly6G+ Ly6C+ neutrophils and Ly6G- Ly6C+ monocytes), hypothesizing that sensing of LCMV infection in the spleen drives recruitment of innate cell populations to sites of early infection. C57BL/6-WT or IFN-αR1, IFN-γR, MyD88 or MAVS deficient mice were infected with 2x106 PFU LCMV-Armstrong. Mice were sacrificed at 14, 24, 72 hrs post infection (pi) at which time neutrophil and NK cell recruitment to LCMV infected cells in the spleen was determined by immunofluorescence staining. In WT mice, neutrophils and NK cells were recruited to sites of infection as early as 14 hrs, but dissipated by 72 hrs pi. Depletion of NK1.1+ cells prior to infection abrogated neutrophil recruitment at 24 hrs pi. Furthermore, recruitment of neutrophils to sites of infection was fully dependent on MyD88 and partially dependent on MAVS, but was independent of type I or II IFN receptors. To conclude, our data demonstrate that neutrophil recruitment to sites of early LCMV infection is completely dependent on NK cells and MyD88 signaling, partially dependent on MAVS signaling, and independent of IFN responses.

Supported by Arthritis Foundation
Adaptive Immune Features of Natural Killer Cells (44.1)
Authors: Sun Joseph C , Beilke Joshua N , Lanier Lewis L
Published: January 2023

In an adaptive immune response, naïve T cells proliferate during infection and generate long-lived memory cells that undergo secondary expansion following re-encounter with the same pathogen. Although Natural Killer cells traditionally have been classified as cells of the innate immune system, they share many similarities with cytotoxic T lymphocytes. In a mouse model of cytomegalovirus (MCMV) infection, we demonstrate that, like T cells, NK cells bearing the virus-specific Ly49H receptor proliferate 100-fold in the spleen and 1000-fold in the liver following infection. Following a contraction phase, Ly49H+ NK cells reside in lymphoid and non-lymphoid organs for several months. These self-renewing "memory" NK cells rapidly degranulate and produce cytokines upon reactivation. Adoptive transfer of these NK cells into naïve animals followed by viral challenge results in a robust secondary expansion and protective immunity. These findings reveal novel properties of NK cells previously attributed only to cells of the adaptive immune system.
Attenuation of lymphocytic choriomeningitis virus relaxes interferon-dependence of adaptive immune responses and of virus control (44.12)
Authors: Bergthaler Andreas , Flatz Lukas , Steinborn Ralf , Lutz Hans , Suter Mark , Pinschewer Daniel D
Published: January 2023

Interferons (IFNs) are essential for CD8+ T cell responses against lymphocytic choriomeningitis virus (LCMV) and for humoral responses against vesicular stomatitis virus (VSV), and they have a profound impact on control of these infections. Recently, we have developed LCM viruses carrying the VSV envelope gene (rLCMV/VSVG) as live-attenuated arenavirus vaccines. Here, we assessed how innate and adaptive immunity contribute to control of rLCMV/VSVG.

Even in mice lacking IFN type I and II receptors, rLCMV/VSVG was efficiently controlled and induced neutralizing antibodies at titers similar or even higher than in wildtype mice. Similarly, significantly higher frequencies of specific CD8+ T cells were detected which remained protective for more than 400 days. Mice lacking both IFN type I receptor and RAG1 (AR) failed to control the virus and became viremic while RAG1 single-deficient mice exhibited circulating viral RNA but not overt viremia. Type II IFN was found to have an effect on rLCMV/VSVG control only on an AR background, while NK cells did not seem to contribute.

Thus, viral attenuation for vaccine use was found to considerably relax the IFN-dependence of virus control and of antiviral adaptive immune responses. This provides a better understanding of the attenuated behavior of these vaccine candidates and highlights the varying impact of IFN in LCMV infection.
MHC H-2k prompts Ly49G2+ NK cell responsiveness to murine cytomegalovirus and acquisition of virus-specific effector T cells (44.16)
Authors: Stadnisky Michael David , Xie Xuefang , Bullock Timothy N. , Brown Michael G.
Published: January 2023

An MHC H-2k locus is critical in NK cell-mediated resistance to murine CMV (MCMV) in MA/My mice. Recently we found that congenic mouse strains C57L.M-H2k(R7) and C57L.M-H2k(R2) which express different MHC class I D alleles, also exhibit remarkable variation in MCMV resistance. Interestingly, MCMV resistance (Cmvr) in R7 mice corresponds with selective expansion and activation of inhibitory Ly49G2 receptor bearing NK cells after virus infection. Here we examined a potential role for Ly49G2+ NK cells in MCMV resistance, maintenance of splenic dendritic cell subsets and establishment of virus-specific CD8+ T cells. We show that Ly49G2+ NK cells acquire functional competence most efficiently in Dk-expressing R7 animals. In addition, we have observed a loss of splenic CD8α+ and CD11b+ dendritic cells shortly after MCMV infection in susceptible (Cmvs) R2 mice, whereas R7 animals maintain these subsets. We further report that efficient MHC H-2k NK-mediated virus immunity led to a robust and significant expansion of MCMV-specific CD8 T cells by day 6 in R7 mice in contrast with R2 or C57L (Cmvs) animals, which failed to mount efficient CD8 T cell immune responses. We conclude that MHC H-2k enhances virus specific T cell immunity in R7 congenic mice, in addition to establishing functionally competent Ly49G2+ NK cells, which can rapidly respond to MCMV infection. Supported by NIH R01 AI050072.
Opposing Effects Of IL-17/Th17 Response In Modifying Host Resistance Against Respiratory Chlamydia Infection Via Divergent Immune Mechanisms (44.22)
Authors: Wang Jun , Zhou Xiaohui , Chen Rose , Ramirez Elizabeth Acosta , Beer Jennifer , Moore Jessica M , Brown Tyler , Kolls Jay K , Stadnyk Andrew
Published: January 2023

The role of IL-17/Th17 response in host resistance against Chlamydia trachomatis (Ct) infection is investigated in C57BL/6 and C3H/HeN mice. We showed that C3H/HeN mice developed a skewed Th17/Th1 immune profile which led to a massive accumulation of neutrophils in the lung, impaired bacterial clearance, severe body weight loss and high mortality rate. In comparison, a relatively moderate IL-17/Th17 response was developed in C57BL/6 mice. By using recombinant adenovirus vector-mediated IL-17 transgene delivery approach, we have found that IL-17/Th17 response has both protective and detrimental roles in host resistance against Ct. On one hand, IL-17 protects host from Ct infection via stimulating pulmonary DC function for establishing a protective immunity. On the other hand, IL-17 stimulates neutrophils which counteract the protective effect of IL-17 via promoting intracellular growth of Ct. Of importance, the final outcome of two opposing effects manifests in a time-dependent and dose-dependent manner. Over expressing IL-17 at later stages of Ct infection in the airway could convert C57BL/6 mice into Ct-susceptible phenotype, indicating that an exacerbated Th17 response is responsible for an enhanced Ct susceptibility. This is the first report that a multi-facet role of IL-17 is documented in modifying host immune responses via divergent immune mechanisms in a single disease model.