Establishment and Characterization of an Anti-idiotypic CD4+CD8−T Cell Line to Murine Anti-α(1 → 3) Dextran Antibody

BackgroundThe Gram-positive bacterium Listeria monocytogenes (Lm) is a promising vector for cancer immunotherapy due to its ability to directly infect antigen-presenting cells, induce potent CD8+ T-cell immunity, and remodel immunosuppressive tumor microenvironments.1 Recent clinical trials have demonstrated safety and immunogenicity of Lm-based cancer vaccines in lung, cervical, pancreatic, and other cancers. In colorectal cancer, the transmembrane receptor guanylyl cyclase C (GUCY2C) is an emerging target for immunotherapy.2 Here, we examined the immunogenicity of a recombinant strain of Listeria monocytogenes secreting GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination induced robust Lm-specific CD8+ T-cell immunity but failed to prime GUCY2C-specific CD8+ T-cell responses. These studies explore the hypothesis that immunodominant Lm antigens suppress primary immunity to subdominant GUCY2C epitopes in Lm-GUCY2CMethodsLm-GUCY2C expresses the extracellular domain of mouse GUCY2C23-429 downstream of an ActA promoter integrated into the genome of the live, attenuated delta actA delta inlB Lm strain. Altered peptide ligands were designed based on NetMHCpan 4.0 peptide-MHC binding algorithms and similarly cloned into Lm. Peptide-MHC class I complex stability was quantified by FACS-based surface peptide-MHC dissociation on the TAP-deficient cell line, RMA-S H-2Kd.3In vivo efficacy studies employed IFNγ-ELISpot quantification of T-cell responses and tumor challenge studies with the CT26 colorectal cancer cell line. Adenovirus expressing GUCY2C was used as a positive control.2 4ResultsLm-GUCY2C vaccination of BALB/c mice generated Lm-specific CD8+ T-cell responses but an absence of GUCY2C-specific immunity. Peptide-MHC stability studies revealed poor stability of the dominant GUCY2C254-262 epitope complexed with H-2Kd compared to H-2Kd-restricted Lm epitopes derived from the LLO and p60 Lm antigens. Mutation of the GUCY2C254-262 peptide at critical anchoring residues for binding H-2Kd revealed that the altered peptide ligand with an F255Y mutation significantly improved the stability of the GUCY2C254-262-H-2Kd complex. Similarly, vaccination of mice with recombinant Lm-GUCY2C expressing the altered peptide ligand (Lm-GUCY2CF255Y) restored GUCY2C immunogenicity and antitumor immunity.ConclusionsImmunodominant Lm antigens may interfere with immune responses directed to the vaccine target antigen GUCY2C by competing with GUCY2C epitope for MHC class I binding and presentation. Moreover, use of a substituted GUCY2C -peptide ligand with enhanced peptide-MHC class I stability restored GUCY2C-specific immunity in the context of Lm-GUCY2C, an approach that can be translated to patients. Importantly, these studies also suggest that ongoing Lm-based vaccine development programs targeting a variety of antigens in other cancer types may be similarly limited by the immunodominance of Lm epitopes.AcknowledgementsThe authors thank Dr. Peter Lauer for providing the pPL2 integration vector used in cloning Lm-GUCY2C and Dr. Sean Murphy for providing the RMA-S H-2Kd cell line.Ethics ApprovalStudies were approved by the Thomas Jefferson University IACUC (Protocol # 01956).ReferencesFlickinger JC, Rodeck U, Snook AE. Listeria monocytogenes as a Vector for Cancer Immunotherapy: Current Understanding and Progress. Vaccines (Basel) 2018;6. doi:10.3390/vaccines6030048.Snook AE, Baybutt TR, Xiang B, Abraham TS, Flickinger JC, Hyslop T, et al. Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients. J Immunother Cancer 2019;7:104. doi:10.1186/s40425-019-0576-2.Müllbacher A, Lobigs M, Kos FJ, Langman R. Alloreactive cytotoxic T-cell function, peptide nonspecific. Scand J Immunol 1999;49:563–9.Flickinger J. JC, Singh J, Carlson R, Leong E, Baybutt T, Barton J, et al. Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity. J Immunother Cancer 2020.

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677 Reverse abscopal effect: intertumoral heterogeneity suppresses systemic CD8 T cell-mediated antitumor immunity and confers PD-1 inhibitor resistance in synchronous melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.677 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A705-A705

Author(s):

Shuyang Qin ◽

Booyeon Han ◽

Alexander Chacon ◽

Alexa Melucci ◽

Alyssa Williams ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Cd8 T Cells ◽

Antitumor Immunity ◽

Immune Escape ◽

Cd8 T Cell ◽

Abscopal Effect ◽

Immunogenic Tumor ◽

Ifn Γ

BackgroundDespite recent advancements in systemic therapy, only a minority of metastatic patients develop meaningful clinical responses to immune checkpoint inhibitors. Inherent genetic instability of melanoma generates genomically and microenvironmentally distinct metastases. These different tumor microenvironments (TMEs) contain numerous T cell suppression mechanisms, such as upregulation of the PD-1/PD-L1 exhaustion pathway. However, as synchronous metastases share one host immune system, intertumoral heterogeneity may result in increasing cross-talk between metastases that impairs systemic antitumor immunity and promotes PD-1 immunotherapy resistance.MethodsYUMM 1.7 (less immunogenic) and YUMMER 1.7 (more immunogenic cell line derived from YUMM following UVB irradiation) melanoma cell lines were simultaneously injected into opposite flanks of the same mice as a model of synchronous melanoma. We assessed tumor growth in wildtype, interferon-gamma (IFN-γ) knockout, and CD8-depleted mice as well as in response to PD-1 inhibitor. We characterized the TME with flow cytometry and performed TCR sequencing on tumor-infiltrating CD8 T cells.ResultsDistinct TMEs were observed for YUMM and YUMMER tumors simultaneously grown in the same mouse. The presence of the less immunogenic YUMM tumor allows the more immunogenic YUMMER tumors to escape IFN-γ and CD8 T cell-mediated rejection, despite abundant tumor-infiltrating, clonally expanded CD8 T cells. Identical immunodominant CD8 T cell clones were found in both YUMM and YUMMER tumors within the same mouse. Synchronous YUMMER-infiltrating CD8 T cells exhibit suppressed phenotypes, including increased persistence of surface PD-1 and decreased surface CD107a expressions. Simultaneously, these synchronous YUMMER tumors additionally upregulate macrophage surface PD-L1 expression, which potentially contributes to tumor immune escape. Lastly, synchronous YUMMER tumors become resistant to PD-1 inhibition, in direct contrast to control YUMMER tumors.ConclusionsIn a host with multiple melanoma lesions, immunogenicity of all tumors contribute to the systemic antitumor immune response. We show that two synchronous tumors with synonymous mutations (<40%), as is the case with metastatic patients, lead to skewed CD8 T cell expansion of the same clones in both tumors. The presence of a less immunogenic tumor prevents CD8 and IFN-γ mediated rejection of the more immunogenic tumor. Furthermore, CD8 T cells in the more immunogenic tumor exhibit decreased effector function and increased resistance to PD-1 blockade, as tumor-infiltrating macrophages concurrently become more immunosuppressive. These results are highly suggestive of a “reverse abscopal effect,” by which immunologically “cold” tumors generate systemic immunosuppression that facilitate PD-1 immunotherapy resistance and immune escape of all other tumors in synchronous metastatic melanoma patients.AcknowledgementsWe would like to thank Dr. Marcus Bosenberg from the Department of Dermatology at Yale University for kindly gifting us with the YUMMER 1.7 murine melanoma cell line.Ethics ApprovalAnimal experiments were approved by the University Committee on Animal Resources and performed in accordance with University of Rochester approved guidelines.

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Characterization of Molecular and Biophysical Cryoinjury Mechanisms In A Human T Cell Line At Slow and Rapid Rates of Cooling

Cryobiology ◽

10.1016/j.cryobiol.2021.11.066 ◽

2021 ◽

Vol 103 ◽

pp. 175

Author(s):

Sandra Tamarin ◽

Aimee L. Eggler ◽

Jens O.M. Karlsson

Keyword(s):

T Cell ◽

Cell Line ◽

Human T Cell ◽

Human T Cell Line

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Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e14565 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e14565-e14565

Author(s):

Amit Adhikari ◽

Juliete Macauley ◽

Yoshimi Johnson ◽

Mike Connolly ◽

Tim Coleman ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Mouse Model ◽

T Cell Activation ◽

Cell Activation ◽

Cd8 T Cell ◽

T Cell Response

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

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