bluuebunny/crossref_metadata_2025 huggingface dataset:
Neonatal Tolerization with Bacterial Antigens Stimulates an Expansion of CD11b+ Leukocytes with Inhibitory Activity in IL-10 Gene-Deficient Mice (39.38)
Authors: Sydora Beate C , Foshaug Rae R , Fedorak Richard N
Published: January 2023

Tolerance to antigens of the endogenous flora can be induced by intra-peritoneal injection of 100 microliter of a sterile lysate solution (2mg/ml protein) prepared from fecal material of conventionally-raised mice with enteric micro flora into neonates. In IL-10 gene-deficient mice this treatment results in delayed onset of intestinal inflammation and reduced levels of pro-inflammatory cytokine release with a concomitant increase in CD11b+ leukocytes in spleen. To investigate whether these CD11b+ leukocytes play an active role in the reduction of bacteria-induced immune responses we isolated CD11b+ leukocytes from spleens of 20 week old mice that were tolerized to bacterial antigens as neonates and added these cells in limited dilutions to spleen cell cultures stimulated with bacterial antigens.

We found that in bacterial antigen-tolerized, Il-10 gene-deficient mice the amount of CD11b+ leukocytes in spleen doubled compared to the amount found in untreated mice and mice injected with germ-free lysates. Sorted CD11b+ spleen cells from tolerized mice were able to inhibit antigen-stimulated spleen cell proliferation and release of pro-inflammatory cytokines in a concentration-dependent manner suggesting that CD11b+ cells play a regulatory role in the response to bacterial antigens in this IBD model. This work is supported by the Crohn's and Colitis Foundation of Canada.
Surfactant protein A differentially modulates the functional outcome of T cell activation (39.45)
Authors: Mukherjee Sambuddho , Giamberardino Charles , Evans Kathy S , Goto Hisatsugu , Pastva Amy , Lo Bernice , Wright Jo Rae
Published: January 2023

The pulmonary surfactant lipoprotein complex lowers the surface tension at the alveolar/airway interface of the lung and participates in pulmonary host defense. Previous studies have shown that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that surfactant proteins may modulate the activation threshold of T cells depending upon the strength of the activation signal.

The strength and duration of lymphocyte activation are crucial in determining the fate of responding lymphocytes. Activation of specific signaling pathways, including NFkB, NFAT, STAT and MAPK pathways, determines the quality, magnitude and duration of immune responses, imparts a tunable quality, and essentially constitutes the 'strength of signal' (SoS). This study highlights a differential role of SP-A across multiple modes of T cell activation. Modulation of signal strength imparted by different activating agents ex vivo and in vivo in different mouse models, as well as in vitro with human cells show a strong correlation between strength of signal and functional effects of SP-A interactions. While proliferation is enhanced in the presence of SP-A at low signal strengths, the inhibition reported in the literature is observed at higher SoS. The enhanced proliferation is observed within a kinetic envelope, and is largely absent in SP-A-/- mice, while the inhibition is primarily caused by lack of cell cycle progression, with no effect on cell death or early cytokine production. This is intrinsic to the global T cell population, and is not mediated by proportions of Treg or effector memory cells. Changes in capacitative [Ca++]i flux are likely to be a key component of these phenotypic effects.

[Support: NIH HL-68072]
A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts (39.5)
Authors: Dullaers Melissa , Li Dapeng , Xue Yaming , Ni Ling , Gayet Ingrid , Banchereau Jacques , Oh Sangkon
Published: January 2023

Mucosal IgA secreted by local plasma cells (PCs) is a critical component of mucosal immunity. Although IgA class switching can occur at mucosal sites in a T cell-independent manner, high-affinity PCs are optimally generated in germinal centers (GCs) in a T cell-dependent fashion. However, the mechanism of how CD4+ helper T cells induce mucosal-homing IgA-PCs remains unclear.

We show here that TGFb1 and IL-21, produced by follicular helper T cells (TFH), synergize to generate abundant IgA-plasmablasts (PBs) that express CCR10. In the presence of IL-21, TGFb1 promotes naive B cell proliferation and differentiation. In addition it overrides IL-21-induced IgG class switching in favor of IgA. Furthermore, in combination with IL-21, TGFb1 downregulates CXCR5, while upregulating CCR10 on PBs, enabling their exit from GCs and migration towards local mucosa. This is supported by the presence of CCR10+IgA+ cells in tonsil GCs.

In conclusion, TFH, through IL-21 and TGFb1 contribute to the generation of human mucosal-homing IgA+ PCs. Thus, mucosal vaccines should aim to induce robust TFH responses.
α-GalCer is an effective mucosal adjuvant for repeated nasal or oral delivery of HIV peptide antigens (39.54)
Authors: Courtney Amy N , Thapa Prakash , Nehete Pramod N , Li Chun , Zhou Dapeng , Sastry K. Jagannadha
Published: January 2023

Mucosal vaccine delivery is important for strong immune responses at biologically relevant sites, however weakly immunogenic antigens delivered by the mucosal route may induce tolerance. Therefore, appropriate adjuvants should be included for effective mucosal immunization. Mice were immunized by the oral or nasal route with alpha-Galactosylceramide (α-GalCer) as an adjuvant for co-administering with a CTL-inducing HIV envelope peptide (R15K). We observed antigen-specific cellular immune responses in multiple systemic and mucosal tissues. Contrary to the known potential of repeated parenteral dosing with α-GalCer to induce NKT cell anergy that could compromise adoptive immunity, we observed that multiple doses delivered by mucosal routes were more efficient in priming broader antigen-specific immune responses. We also obtained results showing that poly-lactic acid based nanoparticles conjugated with α-GalCer can repeatedly stimulate NKT cells both in vitro and in vivo without inducing anergy. Mechanistic studies showed that nanoparticle-formulated α-GalCer is efficiently presented by mouse CD11c+ dendritic cells and CD11b+ macrophages, but very poorly by B220+ B cells. These results demonstrate the effectiveness of α-GalCer as an adjuvant for repeated administration of vaccines for protection against mucosally transmitted pathogens.
Vitamin D is required for the development of functional CD8{alpha}{alpha} intestinal intraepithelial lymphocytes (39.7)
Authors: Bruce Danny W , Cantorna Margherita T
Published: January 2023

Inflammatory bowel disease (IBD) is an autoimmune disease that affects the gastrointestinal tract. Vitamin D an immunoregulatory steroid hormone has been shown to have suppressive affects on IBD and vitamin D deficiency has been shown to have a direct correlation with the severity of inflammation in experimental models of the disease. Several regulatory cells including CD8αα + TCRαβ + T cells that are found in the epithelial layer of the gut, have been shown to suppress experimental models of IBD. Here we show that mice that lack the vitamin D receptor (VDR) or are vitamin D deficient have significantly reduced CD8αα + TCRαβ + T cells. When vitamin D is provided to deficient mice this population of T cells recovers. Precursors of CD8αα + TCRαβ + T cells in the thymus of VDR deficient mice appear to be normal. However, gut homing of the CD8αα + TCRαβ + T cells precursors is reduced. Those CD8αα + TCRαβ + T cells that are in the gut of mice that lack the VDR produce fewer cytokines and appear to be dysfunctional. Vitamin D and signaling through the VDR is required for the development of functional CD8αα + TCRαβ + T cells.
CLEC2A Is a Novel Skin-Specific, Stimulatory Ligand of Human NK Cells (39.8)
Authors: Steinle Alexander , Stejfova Veronika , Kuttruff Sabrina , Schittek Birgit , Spreu Jessica
Published: January 2023

The Natural Killer Gene Complex (NKC) encodes for numerous C-type lectin-like receptors (CTLR) variably expressed on lymphocytes and myeloid cells. Well-known representatives of NKC-encoded CTLR are NKG2D, CD69, and Ly49 molecules. However, the NKC also contains a considerable number of genes which are barely characterized. Recently, we described the CLEC2A gene encoding for a new member of the human CLEC2 family of NKC-encoded CTLR. CLEC2A, in contrast to other human CLEC2 family members such as CD69/CLEC2C and AICL/CLEC2B, is virtually not expressed by peripheral leukocytes, but its expression appears restricted to skin. We now report that CLEC2A encodes for a non-disulfide-linked, homodimeric surface CTLR which is expressed on freshly isolated skin cells. Addressing a potential immune-associated function, we find that CLEC2A stimulates cytotoxicity and cytokine release of human NK cells. However, we failed to ascertain CLEC2A engagement by known activating NK receptors. Instead, we identified a hitherto undescribed NKC-encoded CTLR (NKp65) that specifically interacts with CLEC2A and activates NK cytotoxicity. In summary, we define CLEC2A as a skin-specific and NK cell-stimulating CTLR engaging NKp65, a novel activating CTLR which may allow for dedicated immunosurveillance of human skin.

This work was supported by funds (SFB 685/A1) of the German Research Foundation (DFG).
Identification of in vivo 6-thioguanine-resistant T cells reactive to melanoma-associated antigens (40.15)
Authors: Zuleger Cindy L , Macklin Michael D , Pei Qinglin , Newton Michael A , Albertini Mark R
Published: January 2023

T cells from melanoma patients (MEL) can mediate in vitro anti-tumor and regulatory functions. We hypothesize that in vivo hypoxanthine-guanine phosphoribosyltransferase-deficient T cells (MT) from MEL are enriched for T cells undergoing repetitive in vivo cell division in response to melanoma. MT were selected with 6-thioguanine (TG) from MEL PBMC as clonal isolates or as mass cultures, and from tumor-bearing tissues as mass cultures. Wild-type T cells (WT) were obtained analogously, without TG selection. TCR beta chain (TCRB) cDNA sequence analysis identified significantly enriched T cell clonal amplifications in MT compared to WT: 3 of 6 PBMC clonal isolate assays (p ≤ 0.05); 6 of 6 PBMC mass cultures (p < 0.001); 5 of 5 tumor mass cultures (p < 0.005). Identical TCRB sequences were found within and between MEL, in PBMC and tumor, demonstrating trafficking of T cell clones and suggesting shared immunogenic melanoma antigens. Using peptide-MHC pentamers, a significant enrichment of melanoma-specific T cells in MT compared to WT was demonstrated in 3 of 5 tumor mass cultures (p < 0.001). Preliminary data from MT in a tumor suggest that some MT possess specific reactivity to melanoma peptide-pulsed targets in vitro. We conclude that MT from tumor sites are enriched for T cells responding in vivo to melanoma and provide candidate probes into the immunobiology of melanoma.

Supported by a VA Merit Grant and gifts to the UWCCC.
Using T Cell Responses In The Lymph Node To Predict Patient Outcome In Colorectal Cancer (40.16)
Authors: Kemp Roslyn A , Yoon Han-Seung , Anjomshoaa Ahmad , Reeve Anthony E
Published: January 2023

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide - over half of those diagnosed will die from the disease. An important role for the immune response in controlling CRC has been demonstrated in numerous animal models and in human patients. The extent of T cell infiltration in the primary tumour is a better predictor of survival in CRC patients than traditional staging. Further, recent data by our lab and others have shown a correlation between immune response gene expression and favourable patient outcome. Despite the presence of an immune response, tumours still develop and grow. We hypothesised that the T cell response in the lymph nodes, i.e. the site of immune response initiation, may be a better predictor of patient outcome than that of the primary tumour. To this end, we analysed T cell subsets in the lymph nodes of Dukes' Stage B colorectal cancer (non metastatic) in a cohort of patients from New Zealand who had clinical follow-up. We found that, as is true for CD8+ T cells infiltrating the primary tumour, the number of CD8+ T cells on the lymph nodes correlated with a favourable patient outcome. However, the number of total CD4+ T cells in the lymph node was not correlated with patient outcome. The subtypes of CD4+ and CD8+ T cells, e.g. regulatory, effector, memory, are yet to be investigated. However, our data shows that the anti-tumour immune response appears to be initiated effectively in the lymph node and implies that the tumour environment may act directly to down-modulate an effective immune response.
Assessment of telomerase inactivation in T cell responses and antitumor immunity (40.28)
Authors: Gong Jianlin , Koido Shigeo , Liu Chunlei
Published: January 2023

Telomerase knockout (mTerc-/-) mice expressing polyoma middle T oncogene (PyMT) in 4 generations were immunized with fusions of dendritic cells and MUC1-positive mammary tumor cells (FC/MMT). Both CD4 and CD8 T cells from immunized MMT mice divided vigorously as measured by CFSE labeling. Comparable cell division was observed in T cells from immunized G0 mTerc+/-MMT, G1 and G2 mTerc-/-MMT mice. In contrast, cell division decreased in T cells from G3 mTerc-/-MMT mice. CTL against mammary tumor cells were induced in LNC and Splenocytes from immunized but not non-immunized early generations of mTerc-/-MMT mice. However, CTL in the late generations of immunized mTerc-/-MMT mice decreased. Finally, the efficacy of immunotherapy in the prevention and treatment of spontanenous mammary carcinomas was assessed. In the prevention study, the time required for the mammary tumors in MMT, G0 mTerc+/-MMT, G1, G2 and G3 mTerc-/-MMT mice immunized with FC/MMT were 124.7±3.8, 118.6±10.8, 139.9±25.3, 138±12.1 and 146.8±21.2 days, respectively. The time required for the mammary tumor development in MMT, G0 mTerc+/-MMT, G1, G2 and G3 mTerc-/-MMT mice immunized with FC/MMT in the treatment study were 112.4±10.4, 113.6±8.4, 136.3±10, 144.3±12.7 and 158.7±15.7 days, respectively. In contrast, the latency time for tumor development in MMT, G0 mTerc+/-MMT, G1, G2 and G3 mTerc-/-MMT mice without immunization were 73.07±28.03, 96.07±28.79, 115.6±8.88, 117.14±32.1 and 127.11±35.84 days, respectively. The difference of time required for tumor development between mice with or without vaccination in both prevention and treatment studies is statistically significant.
Ligation of the OX40 co-stimulatory receptor reverses self-Ag and tumor-induced CD8 T cell anergy in vivo (40.30)
Authors: Redmond William , Gough Michael , Weinberg Andrew
Published: January 2023

Tumor-specific CD8 T cell peripheral tolerance occurs through a variety of mechanisms including clonal deletion, suppression, and the induction of anergy and is an important barrier that limits the generation of anti-tumor immunity. Several groups have demonstrated that prostate cancer can render tumor-specific CD8 T cells anergic, suggesting that strategies to reverse tumor-induced anergy may augment anti-tumor immunity. Recent work has demonstrated that signaling through the OX40 (CD134) co-stimulatory receptor, a member of the TNFR super-family, can augment both CD4 and CD8 T cell expansion, differentiation, and the generation of memory cells. However, whether OX40 ligation can reverse CD8 T cell anergy, and more specifically, tumor-induced CD8 T cell anergy, remains unclear. In the current study, we demonstrate that OX40 ligation can reverse CD8 T cell anergy to a prostate-specific self-antigen in non-tumor bearing hosts. Furthermore, OX40 engagement reversed tumor-specific CD8 T cell anergy and restored the proliferative capacity of tumor-reactive CD8 T cells in vivo, which attenuated the extent of tumor growth and enhanced the survival of tumor-bearing hosts. This is the first demonstration that OX40 ligation can rescue the function of anergic self or tumor-reactive CD8 T cells in vivo and suggests that OX40-mediated therapy may provide a novel means of boosting anti-tumor immunity by restoring the responsiveness of previously anergic tumor-specific CD8 T cells.

Supported by an American Cancer Society - Sam E. and Kathleen Henry post-doctoral fellowship, a Prostate Cancer Foundation Young Investigator Award (W.L.R.), and grants from the NIH (A.D.W.).
NKT activation together with blockade of PD-L1 pathway lead to enhanced tumor responses (40.32)
Authors: Latchman Yvette E , Durgan Kevin , Warner Paul
Published: January 2023

Invariant NKT cells (iNKT cells) are a unique population of lymphocytes that have a biased TCR gene expression, are restriction by CD1d molecules, and rapidly produce a spectrum of cytokines following activation. Various studies have shown that positive costimulatory pathways can influence the activation of iNKT cells. However, little is understood about the regulation of NKT cells by negative costimulatory pathways. Here we show that PD-L1 and PD-1. but not PD-L2, are expressed on iNKT cells in spleen, liver and thymus. In vivo activation with α-GalCer lead to increased cytokine production and expansion of iNKT cells in the absence of the PD-L1 pathway. To study whether PD-L1 deficiency on NKT cells would affect antigen specific T cell responses we utilized CD8+ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 T cells after adoptive transfer into wild type recipients. However, this expansion was significantly augmented when OT-1 T cells were adoptively transferred into PD-L1-/- recipients. In a B16 melanoma metastasis model, there was an increased clearance of lung nodules when PD-L1-/- recipients were given dendritic cells loaded with α-GalCer and TRP-2 peptide. These data demonstrate that blocking PDL1:PD-1 interactions between NKT cells and cells of the adaptive immune system are important in mounting an effective anti-tumor immune response.
Interaction of CD103 with E-cadherin triggers CCR5 recruitment at the immunological synapse resulting in intratumoral retention of tumor-specific CTL (40.37)
Authors: MAMI-CHOUAIB Fathia , Le Floc'h Audrey , Jalil Abdelali , Combadière Christophe , Franciszkiewicz Katarzyna
Published: January 2023

We previously characterized two tumor-specific T-cell clones from PBL and tumor-infiltrating lymphocytes (TIL) of a lung cancer patient with identical TCR and similar lytic potential, but with different anti-tumor reactivity. We demonstrated that the interaction of alphaE(CD103)beta7 integrin, expressed on the TIL clone but not on the PBL counterpart, with the epithelial marker E-cadherin on human lung tumor cells, plays a crucial role in TCR-mediated cytotoxicity. In the present study we show that both TIL and PBL clones are able to migrate towards autologous tumor cells and that chemokine receptor CCR5 is involved in this process. Interestingly, adoptive transfer of the PBL clone in the cognate tumor engrafted in SCID-NOD mice and subsequent TCR engagement and TGF-β1 secretion triggers CD103 expression on T-cell surface and thus potentiation of anti-tumor cytotoxic activity. Furthermore, confocal microscopy analyses indicated that the interaction of αEβ7 integrin with E-cadherin, but not LFA-1 with ICAM-1, promotes CCR5 recruitment at the immunological synapse formed between TIL and tumor cells, leading to inhibition of T-cell sensitivity to CCL5 chemotactic gradient. Our results provide evidence for a role of tumor microenvironment in regulating anti-tumor CTL function and suggest a role of CD103 in intratumoral retention of tumor-specific T cells by a mechanism involving CCR5.
Cbl-b-deficiency in tumor-reactive CD8+ T cells improves efficacy and bypasses the requirement for IL-2 administration during adoptive therapy of progressive leukemia (40.38)
Authors: Stromnes Ingunn M. , Blattman Joseph N. , Tan Xiaoxia , Greenberg Philip D.
Published: January 2023

OBJECTIVE: We have examined if decreasing Cbl-b expression in tumor-reactive CD8 T cells used for adoptive immunotherapy of progressive leukemia can enhance efficacy and bypass the requirement for IL-2 administration.

METHODS: Mice expressing a transgenic receptor, TCRgag, which renders CD8 T cells reactive with FBL leukemia, were crossed with Cbl-b-deficient and CD28-deficient mice. TCRgag Cbl-b+/+ and Cbl-b-/- cells were tested for function and expanded in vitro prior to use in adoptive therapy.

RESULTS: Cbl-b-/- TCRgag cells exhibited a lower threshold for T cell activation, greater IL-2 production and enhanced proliferation, expansion and survival. The enhanced response of Cbl-b-/- TCRgag cells compared to Cbl-b+/+ cells in vitro revealed both IL-2-dependent and IL-2-independent effects. In vivo, adoptively transferred Cbl-b+/+ TCRgag CD8 T cells can only prevent lethal disease in mice bearing disseminated leukemia if low dose IL-2 is subsequently administered to promote proliferation and maintain cell survival. By contrast, Cbl-b-/- TCRgag cells cured mice independent of a requirement for IL-2 administration.

CONCLUSIONS: These results suggest that targeting Cbl-b in antigen-specific T cells may provide a strategy to enhance efficacy of CD8 T cell adoptive immunotherapy in humans with cancer.
Deletion of adenosine A2A receptors on CD8 T cells enhances their accumulation in tumors and tumor-draining lymph nodes, as well as their ability to control tumor outgrowth (40.6)
Authors: Vincent Isaah S , Linden Joel M , Engelhard Victor H
Published: January 2023

Previous work has demonstrated that ablation of adenosine A2A receptor (A2AR) expression in tumor-bearing mice enhances tumor control. However, it is not yet clear what cells and functions are influenced by A2AR signaling. Here we show that the ability of adoptively transferred OT-I CD8 T cells to control ovalbumin expressing B16 melanomas in A2aR+/+ recipients is enhanced if the A2AR gene is ablated in the OT-I cells. This demonstrates directly that the ability of CD8 T cells to control tumors is influenced by adenosine. To further understand the functions of CD8 T cells influenced by A2AR deletion, we examined the activation, proliferation, and survival of A2AR+/+ and A2AR-/- OT-I cells in tumors and tumor-draining lymph nodes (TDLN) after co-transfer. While activation and proliferation of both populations in TDLN was comparable, the accumulation of A2AR-/- cells in both TDLN and tumor was enhanced relative to A2AR+/+ cells. Preliminary data suggest that this is due to enhanced survival of A2AR-/- CD8 T cells in TDLN. This enhanced accumulation does not occur in the lymph nodes of mice immunized with bone marrow derived dendritic cells or Vaccinia virus, suggesting that it reflects alterations in the level of adenosine specifically in TDLN. We theorize that enhanced accumulation of A2AR-/- CD8 T cells is at least partially responsible for their increased ability to control tumors.

Support: NIH R01 CA78400, F31 CA130117
Pharmacodynamics, Pharmacokinetics, and Tolerability of a B-Cell Specific Antibody-Targeted Chemotherapeutic Agent, Anti-CD79b-MCC-DM1, in Cynomolgus Monkeys (41.12)
Authors: Williams Marna , Li Dongwei , Fuji Reina , Fuh Franklin , Prabhu Saileta , Zheng Bing , Elkins Kristi , Yu Shang-Fan , Chuh Josefa , Tan Christine , Hongo Jo-anne , Raab Helga , Young Judy , Ross Sarajane , Kozak Katherine , Eaton Dan , Spencer Susan , Poon Kirsten A. , Fielder Paul , Tibbitts Jay , Berry Karen , Manning William , Ramakrishnan Vanitha , Ebens Allen , Polson Andrew G.
Published: January 2023

Antibody-based therapeutic approaches have revolutionized the treatment of non-Hodgkin's lymphomas (NHL) as well as other hematological malignancies. However, a large variability in clinical response has been observed and patients with refractory or relapsed lymphomas have limited therapeutic options.

Antibody drug conjugates (ADC) may prove valuable in this respect. The B cell surface antigen, CD79b, is an attractive target for this approach as it is rapidly internalized upon antibody engagement and has an appropriate expression pattern, being expressed only on normal and malignant B-cells. Anti-CD79b-MCC-DM1 is an ADC consisting of a chimeric monoclonal anti-CD79b antibody, 10D10, conjugated via a thioether linker to a cytotoxic drug, DM1 (a potent tubulin polymerization inhibitor).

To investigate the pharmacodynamics (PD), pharmacokinetics (PK), and tolerability of 10D10-MCC-DM1 in nonhuman primates, we administered 10D10-MCC-DM1 or unconjugated antibody 10D10 to cynomolgus monkeys. 10D10-MCC-DM1 and 10D10 resulted in sustained depletion of B-cells and B cell subsets in blood, with more substantial depletion in the group given 10D10-MCC-DM1. These findings correlated well with the depletion of B-cells observed in lymphoid tissues (bone marrow, lymph nodes, and spleen). Both 10D10-MCC-DM1 and 10D10 were well tolerated. Taken together, these preclinical data suggest that targeting CD79b with ADCs may provide a safe and effective therapy for B-cell malignancies in humans.
Priming anti-tumor responses with MHC I single chain trimer vaccines (41.16)
Authors: Ordaz Maria L , Li Gang , Andreansky Samita , Lybarger Lonnie
Published: January 2023

The inefficiency of the MHC I presentation pathway and the low affinity of some tumor peptide/MHC complexes can lead to a reduced density of tumor-associated peptide/MHC complexes on tumor cells, which can partly explain the failure to prime CTL (cytotoxic T lymphocyte) responses against tumors. Here, we are evaluating a set of MHC I Single Chain Trimers (SCTs) used in conjunction with dendritic cell (DC) or DNA vaccination for their ability to prime a CTL response specific for tumor antigens. A SCT consists of an MHC I molecule covalently linked to a peptide and our goal is to test whether tumor-associated antigen presentation by MHC I SCTs will result in better CTL priming and immune protection than more conventional peptide-specific vaccination protocols. The melanoma antigenic peptide TRP2 (Tyrosinase related protein 2, aa180-188) was chosen in this study for presentation on Kb SCTs; OVA-SCT (Kb-SIINFEKL) was chosen as a control. In vivo experiments suggest that mice vaccinated with TRP2 SCT-transduced DCs present better tumor protection, compared to controls. SCT vaccines, combined with Treg depletion are in progress to evaluate specific priming and tumor protection.

Our preliminary data suggest that SCTs represent a promising technology capable of priming strong and specific CTL responses, with the potential of eliciting systemic tumor protection.

Supported by the Arizona Biomedical Research Commission.
Chimeric Mesothelin Virus-like Particle Vaccine Induces Strong Anti-tumor Immunity against Pancreatic Cancer by Activating Plasmacytoid Dendritic Cells and Reducing CD4+foxp3+ ICOS- Regulatory T cells (41.28)
Authors: Zhang Sheng , Cubas Rafael , Li Min , Chen Changyi , Yao Qizhi
Published: January 2023

Abstract

Our previous study has shown that mesothelin (MSLN) is highly expressed in pancreatic cancer and can be used as an immunotherapeutic target. Here, we further deciphered the effective anti-tumor immune responses elicited by vaccination of virus-like particle (VLP) that highly express murine MSLN (mMSLN) on their surface. In an orthotopic pancreatic cancer mouse model, we found that mMSLN-VLP immunization showed strong anti-pancreatic cancer effects leading to significantly reduced tumor volume and longer survival compared with the VLP control. Elevated MSLN-specific IFN-γ secreting CD8+ T cells, MSLN-specific tetramer positive CD8+ T cells, and MSLN-specific cytotoxic T cells were found in mMSLN-VLP immunized groups. More importantly, progressively decreased percentage of CD4+foxp3+ regulatory T cells (Tregs) cells was found in all VLP immunized groups in both spleen and tumor sites with multiple VLP immunizations. Interestingly, this reduced CD4+foxp3+ Treg population was composed of mostly high ICOS-expressing cells. Furthermore, VLPs activated plasmacytoid dendritic cells (pDCs) and increased IL-6 production, which led to the survival of CD4+foxp3+ICOS+ Tregs. Blockage of IL-6 partially rescued the reduction of ICOS-foxp3+ Treg in splenocytes. Thus, the efficacy of a MSLN-VLP tumor vaccine may lie not only in the induction of a tumor specific CD8+ T cell response, but also in eliciting strong immune responses by activating pDCs and the reduction of CD4+foxp3+ICOS- Treg sub-populations to control MSLN over-expressing tumor progression.
Tumor-derived Autophagosomes is a Potent Vaccine in Cancer Immunotherapy (41.33)
Authors: Li Yuhuan , Pang Puiyi , Urba Walter , Fox Bernard A , Hu Hong-Ming
Published: January 2023

Active immunotherapy (AIT) using vaccines has been exploited to eradicate primary tumors and metastases; however, overall clinical response rate of AIT is less than 4%. This disappointing result underscores the need for novel vaccines. We have developed an autophagosome-enriched vaccine (DRibble), and evaluated its efficiency in cross-presentation of tumor-associated antigens (TAA) and its anti-tumor efficacy. We found that autophagosomes can accumulate and be released from a number of tumor cell lines after prolonged inhibition of proteasome-mediated protein degradation. By transmission electron microscopy, we confirmed the presence of numerous double-membrane autophagosomes in DRibble preparations from tumor cells. The DRibbles sequestered long-lived and short-lived proteins, defective ribosomal products, as well as heat shock proteins HSP90 and HSP94, as evidenced by western blotting. Using CFSE dilution of labeled antigen-specific TCR transgenic CD8+ T cells as a read out, we showed that DRibbles were highly efficient in cross-presenting TAA in vitro and in vivo. Vaccination with DCs loaded with gp100-DRibbles resulted in a doubling of median survival time of mice bearing 6-day s.c. B16F10 tumors (59 days for vaccinated mice vs 26 days for untreated mice). Remarkably, 83% of mice bearing 6-day s.c. 3LL tumors were cured when they received DRibble vaccines, whereas all untreated mice succumbed to disease. Based on these strong preclinical results, our Institute has initiated a clinical trial for non-small cell lung cancer using autologous tumor-derived DRibbles. This work was supported by NIH grants CA 107243 (HMH), CA 123864 (WJU), ACS grant LIB-106810 (HMH), the Providence Medical Foundation, and the Kuui Foundation.
Adoptive T Cell Therapy Targeting WT1 in Leukemia and MDS patients (41.34)
Authors: Ragnarsson Gunnar , Nguyen Hieu , Chaney Colette , Ho Bill , Greenberg Phil
Published: January 2023

Objectives: A clinical trial is being performed to primarily determine the safety in leukemia patients of targeting WT1, a transcription factor over-expressed in leukemia cells that promotes the malignant phenotype, by adoptive transfer of WT1-specific CD8+ clones (CTL). The secondary objectives are to assess in vivo persistence of transferred CTL, localization of transferred cells to bone marrow (BM), and potential antileukemic activity.

Methods: HLA-A0201+ patients with relapsed Acute Leukemia, Chronic Myeloid Leukemia or Myelodysplastic Syndrome after allogeneic hematopoietic cell transplant receive 5 infusions, with the last two followed by IL2, of CTL (3.3x10 8-1.0x1010 cells/m2) derived from their matched donor.

Results: Four patients have now completed and a fifth patient started therapy. No significant treatment related toxicities were observed in patients who completed therapy. WT1 was elevated in PBMC in 1 patient prior to therapy (a marker of minimal residual disease), and became undetectable after therapy. A second patient exhibited reduction in leukemic blasts after CTL infusion (11.8% to 1.8% of PBMC). The infused CTL persisted for up to 2 weeks in PBMC and were detected in the BM after CTL infusions.

Conclusion: The preliminary results suggest infusion of WT1-specific CD8+ clones is safe; and that CTL can localize to the BM and may have anti-leukemic activity.

Support: NCI CA18029 & LLS 4442-09
Histone deacetylase inhibitor, LBH589, synergizes with an immunotherapy treatment in an in vivo murine brain tumor model (41.35)
Authors: Lisiero Dominique Naomi , Soto Horacio , Fang Shanna , Ribas Antoni , Liau Linda , Prins Robert
Published: January 2023

Cancer immunotherapy treatments may show low response rates due to an anti-apoptotic tumor environment. We hypothesized a treatment of immunotherapy and histone deacetylase inhibitor, LBH589, could induce tumor cell expression of pro-apoptotic molecules and sensitize these cells to immune mediated cell death.

Treatment with adoptive transfer immunotherapy and LBH589 resulted in synergistically reduced s.c. tumor burden, and prolonged survival in an intracranial tumor model. In vivo studies in non-tumor bearing mice treated with this combination therapy showed a 2-fold increase in the number of adoptively transferred cells in comparison to an immunotherapy alone treatment group. Furthermore, combination treatment also showed a 2-fold increase in ex vivo interferon-γ secretion with and without stimulation of hgp100 peptide in comparison to an immunotherapy alone group. However, overall levels of histone 3 lysine acetylation in these adoptively transferred CD8+ T-cells do not change between treatment groups.

Our results suggest LBH589 enhances immunotherapy treatment by decreasing tumor burden and increasing survival in a murine melanoma brain tumor model. Furthermore, treatment with LBH589 increases CD8+ T-cell secretion of interferon-γ in the absence and presence of peptide, and this does not appear to be caused by a change in overall histone 3 lysine acetylation levels.
Early Hematopoietic Zinc Finger (EHZF/ZNF521) prevents tumor cell recognition by Natural Killer cells. (41.43)
Authors: carbone ennio , La Rocca Rosanna , Bond Heather M. , Giovanni Morrone , fulciniti mariateresa , mazzei valerio , Tadepally Laksmikanth , mesuraca maria , ferrone soldano , gulletta elio , catalano lucio , rotoli bruno , grieco michele
Published: January 2023

EHZF/ZNF521 is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is downregulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed upregulation of HLA class I cell surface expression. This phenotypic change was associated with increased level of HLA class I heavy chain, in absence of detectable changes in the expression of other antigen processing machinery (APM) components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B-lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I antigen-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I antigen upregulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells.
Mutant gp70 as a source of a novel CT26 tumour antigen, GSW11 (41.46)
Authors: Bailey Ian G , Reeves Emma , Elliott Tim , James Edd
Published: January 2023

There is accumulating evidence that CD4+ CD25+ regulatory T cells (Treg) play an important role in anti-tumour immunity by preventing effective T cell responses to tumour antigens. Tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. The depletion of Tregs prior to challenge with the murine colorectal tumour, CT26, stimulates a robust, protective T cell response which is also protective to challenge with other tumours of different histological origins, such as B cell lymphomas and a renal cell carcinoma. This cross protection has not been seen with other tumour cell lines. We have identified a CT26-derived cross-protective antigen, GSW11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. This protein has previously been shown to encode CT26-specific CD8 and CD4 antigens, implicating it as a 'hot-spot' for CT26 tumour antigens. Interestingly, we have identified a truncated version of gp70 which may be responsible for generation of GSW11. Expression studies have revealed increased gp70 expression in CT26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (GSW11) generated.
Metastatic hepatoma cell model and its mechanism of immunotolerance (41.47)
Authors: Cao Lili , Sun Wensheng , Han Lihui , Zhang Zhiyong
Published: January 2023

Metastatic hepatoma cells are the most dangerous parts in hepatocarcinoma patients.We want to study the mechanism of metastatic hepatoma cells to escaping from host immune attacks. Firstly, we constructed the metastatic hepatoma cell model by using plates pre-coated with Poly-HEMA and found that they were aggregated, proliferation-resistant, apoptosis-resistant, cell cycle arrested and more invasive. These metastatic hepatoma cells could settled down and proliferated again under suitable conditions. Subsequently,we investigated the response of metastatic hepatoma cells to the typical immune surveillant molecule TNF-related apoptosis-inducing ligand (TRAIL). The data indicated that metastatic hepatoma cells not only resist to TRAIL-induced apoptosis, but also express lower membrane binding TRAIL and excrete more soluble TRAIL (sTRAIL) to exert a stealth "tumor counterattack" effect to attack immune effector cells. Deeply study found that the membrane binding death receptors DR4 and DR5 were down-regulated on metastatic BEL7402 and SMMC7721 cells and that their active level of caspase-8,-9,-3 was highly restrained. So we concluded that metastatic hepatoma cells could escape from host immune system by the death receptor pathway.

This work was supported by grants from the National Natural Science Foundation of China (No.30873025, No.30700357 and No.30772031), Corresponding author E-mail:WSW@sdu.edu.cn.
Use of DLE as treatment in murine endotoxic shock (41.54)
Authors: Robledo Avila Frank Hanz , Vazquez Leyba Said Kayum , Valderrabano Ortiz Estela , De la Fuente Granada Marisol , Perez Tapia Sonia Mayra , Estrada Garcia Iris , Estrada Parra Sergio
Published: January 2023

The LPS is a structural component of the external membrane of the Gram negative bacteria and is the most powerful of the inflammation inductors. High doses of LPS have as consequence a high production of inflammatory cytokines, which causes the endotoxic shock. The Dializable Leukocyte Extracts (DLE) is the dialyzable fraction with lower molecular weight to12 KDa which come from leukocytes lysates. In this work a model of endotoxic shock LPS was induced established in C57BL6 male 6 weeks of age, the dose for endotoxic shock induction was 130mg/kg mouse weight. The doses of 0.001ug and 0.0001ug mDLE showed protection against endotoxic shock death in 10% of the mice and the 0.01ug mDLE dose protected against of endotoxic shock death from 10 to 30% of the mice. Levels of TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, IL-10 and TGF-beta were measured in serum of mice endotoxic shock induced and treated with mDLE, the groups in which LPS was administrated and then treated with 0.01ug, 0.001ug or 0.0001ug of mDLE showed smaller levels of inflammatory cytokines and more higher levels of antiinflammatory cytokines than the LPS treated control group. The splenic index was measured in the endotoxic shock induced and mDLE treated groups, the group LPS and the 0.001ug of mDLE treated showed the higher splenic index in comparison with the LPS control group. So the DLE can be use as a potential therapeutic agent as adjuvant in the treatment of sepsis.
Redirection of human T lymphocytes towards HER2 by T cell receptor gene transfer for adoptive T cell transfer (41.6)
Authors: Conrad Heinke , Meyerhuber Peter , Kast Barbara , Mueller Julia , Peschel Christian , Uckert Wolfgang , Bernhard Helga
Published: January 2023

The clinical goal of our studies is the adoptive transfer of primary T cells transduced with a HER2-specific T cell receptor (TCR) for patients with HER2-overexpressing breast cancer.

HLA-A2-, CD8 T cells were stimulated with allogeneic HLA-A2+ dendritic cells (DC) pulsed with the peptide HER2369-377. HER2369-377-reactive T cells were screened, cloned and further tested in functional assays.

The TCR from the HER2-reactive CTL clone KU1 was cloned into a retrovirus. Primary T lymphocytes were transduced with this construct and functionally compared with the parental CTL clone KU1.

The TCR-transduced primary T cells recognized the peptide HER2369-377 exogenously loaded onto T2 cells or endogenously expressed by tumor cells and transfectants. Similar to the parental CTL clone KU1, the transgenic T cells were not only able to recognize HER2369-377, but also the corresponding peptides from HER3 and HER4. Following TCR optimization (codon optimization, murinization), the TCR-transduced T cells displayed an enhanced tumor cell recognition.

In conclusion, the fine specificity of a HER2-reactive TCR is conserved following transduction into primary T lymphocytes. These results facilitate the design of HER2-directed immunotherapies based on TCR-transduced T cells. The observed cross-recognition especially with HER3 may be beneficial as HER2 and HER3 overexpressing tumors are particularly aggressive.

DFG
Tumor-associated antigen MUC1 contains self epitopes subject to self-tolerance and tumor-associated “foreign” epitopes that elicit effective immunity: important distinction for cancer vaccines (41.60)
Authors: Ryan Sean O. , Turner Michael S. , Revers Leigh , Gariepy Jean , Finn Olivera J.
Published: January 2023

Human adenocarcinomas express tumor-associated antigen (TAA) MUC1 that presents to the immune system peptide epitopes as well as glycopeptide epitopes carrying tumor specific carbohydrates. In MUC1-Tg mice responses to MUC1-peptides are suppressed while vaccination with tumor-associated glycopeptides results in effective anti-MUC1 immunity. Transgene negative mice respond equally to both antigens. Using our newly generated TCR transgenic mice with peptide and glycopeptide specific TCRs, we show for the first time that peptide specific CD4 T cells transferred to MUC1-Tg mice are suppressed through mechanisms of peripheral tolerance that are not induced against MUC1-glycopeptide specific CD4 T cells. This tolerance is due to MUC1-peptide epitope presentation in the periphery of healthy MUC1-Tg mice, previously thought to occur primarily in tumor bearing mice. MUC1 glycopeptide epitopes are tumor specific and thus treated as foreign in MUC1-Tg mice, resulting in effective activation of CD4 T cells. Furthermore, glycopeptide-specific T cells provide help to enhance peptide-specific T cell responses. We conclude that TAA contain epitopes that are subject to self tolerance and also tumor specific (foreign) epitopes not affected by self tolerance. It is important to maintain this distinction in attempts to develop effective and safe cancer vaccines. (Supported by NIH T32CA82084, RO1CA56103, CBCRA/CCS)
Real time, single-cell characterization of basophilic exocytosis through carbon-fiber microelectrode amperometry (CFMA) (42.1)
Authors: Ersin Özlem H , Ge Shencheng , Haynes Christy L
Published: January 2023

Histamine released by previously sensitized basophils plays a crucial role in inflammation and immediate hypersensitivity reactions. Whereas ensemble-based immunohistochemical assays characterize the average release profile of cell populations, differences among individual basophils or basophil sub-populations remain largely unexplored based on technological limitations. Previous work in our lab has used microelectrochemical techniques to measure real time quantal release from single cells isolated from separate platelet and mast cell populations. Herein we report the use of carbon-fiber microelectrodes to measure the concentration and time course of single-cell histamine secretion by primary culture basophils in response to varying physiological and electrical stimuli. Future work will utilize CFMA and the patch clamp technique to probe single-cell degranulation in a polydimethylsiloxane (PDMS) microfluidic chip model of graduated complexity.
Bioassay development of cytokines produced by Th17 cells (42.16)
Authors: Soto Hortensia , Leettola Catherine , Huang Jianyong , LeFevre Nora , Ji Shaoquan
Published: January 2023

The description of a new subpopulation of IL-17 producing T helper cells opened a new avenue in the understanding of immune mechanisms and immune pathologies. This recently described subpopulation has been called Th17 cells, and has a different cytokine profile from the previously described subpopulations Th1 and Th2. Th1 produce IFNγ and Th2 produce IL-4, IL-5, IL-6, and IL-13. Murine Th17 cells produce a large series of cytokines: IL-17A, IL-17F, IL-21, IL-22, IL-26, IL-6 and tumor necrosis factor (TNF)α. In humans, IL-17A, IL-17F, IL-21, IL-22 have been identified as effector cytokines that are preferentially produced by Th17 cells. In this study, we describe bioassays for cytokines produced by Th17 cells. IL-17A activity was measured by its ability to induce IL-6 in human dermal fibroblast (HDF). HDF were incubated with serial dilutions of IL-17A for 48 h, and the IL-6 was measure by ELISA. hIL-17A has ED50 of 2-4 ng/ml. IL-22 bioactivity was measure using human Colo-205 cells. IL-22 is known to induce IL-10 production in endothelial cells. Colo-205 cells were treated with IL-22 for 48 h, and IL-10 was measured by ELISA. hIL-22 shows an ED50 of 0.062-0.177 ng/ml. Mouse IL-21 was measured in a proliferation assay using CTLL-2. The ED50 for mouse IL-21 is 40 ng/ml. We have successfully expressed these cytokines and shown that they have potent biological activity. BioLegend.
A flow cytometric application for screening of new anti-inflammatory compounds (42.18)
Authors: Sepulveda Homero , Rabenstein Jacob , Apgar John , Princen Fred
Published: January 2023

Septic shock is a major public health problem. Organ failure during sepsis has been attributed to an extensive inflammatory response. Consequently, inflammatory reactions are important targets for drug development.

Inflammatory cytokine production is the result of the activation of signaling pathways. The Toll-Like receptor (TLR) family can recognize every known human pathogen. They trigger the host defense by activating signaling pathway such as NF-kB, MAPK, AKT and JAK/STAT. The stimulation of these pathways by pathogens will lead to an activation of transcription factors and the release of cytokines that can positively or negatively influence innate and adaptive immunity. Therefore, there is great interest in developing cell signaling inhibitors as new classes of anti-inflammatory and immuno-regulatory compounds.

We have used BD Cytometric Bead Array and BD Phosflow technologies to propose a flexible application that will facilitate the screening of compound libraries to find new anti-inflammatory drug candidates. To test this approach, we used well-characterized MAPK and PI3K/AKT inhibitors in an in vitro sepsis model. Our results demonstrated that we can simultaneously monitor the effect of a given compound on cell signaling molecules and on the inflammatory cytokines released. Moreover, we were able to specifically identify the cell population involved in the process.

In conclusion, this screening approach allows researchers to quantitatively assess and correlate the release of inflammatory cytokines with the signaling state of the cell.