TCR Retrogenic Mice as a Model To Map Self-Tolerance Mechanisms to the Cancer Mucosa Antigen GUCY2C

Abraham, Tara S.; Flíckinger, John C.; Snook, Adam E.

doi:10.4049/jimmunol.1801206

Cited by 6 publications

(4 citation statements)

References 57 publications

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“…These may include deletion, anergy, FoxP3+ regulatory (Treg) induction, or others, creating additional opportunities to enhance Ad5-GUCY2C-PADRE efficacy (Treg depletion, for example). In that context, a TCR “retrogenic” mouse model was recently developed to explore mechanisms underlying GUCY2C-specific CD4 + T-cell tolerance and solutions to overcome tolerance [39], potentially providing alternatives to incorporation of PADRE to elicit GUCY2C-specific immunity.…”

Section: Discussionmentioning

confidence: 99%

Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients

Snook¹,

Baybutt²,

Xiang³

et al. 2019

j. immunotherapy cancer

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Background The colorectal cancer antigen GUCY2C exhibits unique split tolerance, evoking antigen-specific CD8 + , but not CD4 + , T-cell responses that deliver anti-tumor immunity without autoimmunity in mice. Here, the cancer vaccine Ad5-GUCY2C-PADRE was evaluated in a first-in-man phase I clinical study of patients with early-stage colorectal cancer to assess its safety and immunological efficacy. Methods Ten patients with surgically-resected stage I or stage II (pN0) colon cancer received a single intramuscular injection of 10 11 viral particles (vp) of Ad5-GUCY2C-PADRE. Safety assessment and immunomonitoring were carried out for 6 months following immunization. This trial employed continual monitoring of both efficacy and toxicity of subjects as joint primary outcomes. Results All patients receiving Ad5-GUCY2C-PADRE completed the study and none developed adverse events greater than grade 1. Antibody responses to GUCY2C were detected in 10% of patients, while 40% exhibited GUCY2C-specific T-cell responses. GUCY2C-specific responses were exclusively CD8 + cytotoxic T cells, mimicking pre-clinical studies in mice in which GUCY2C-specific CD4 + T cells are eliminated by self-tolerance, while CD8 + T cells escape tolerance and mediate antitumor immunity. Moreover, pre-existing neutralizing antibodies (NAbs) to the Ad5 vector were associated with poor vaccine-induced responses, suggesting that Ad5 NAbs oppose GUCY2C immune responses to the vaccine in patients and supported by mouse studies. Conclusions Split tolerance to GUCY2C in cancer patients can be exploited to safely generate antigen-specific cytotoxic CD8 + , but not autoimmune CD4 + , T cells by Ad5-GUCY2C-PADRE in the absence of pre-existing NAbs to the viral vector. Trial registration This trial (NCT01972737) was registered at ClinicalTrials.gov on October 30th, 2013. https://clinicaltrials.gov/ct2/show/NCT01972737 Electronic supplementary material The online version of this article (10.1186/s40425-019-0576-2) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients

Snook¹,

Baybutt²,

Xiang³

et al. 2019

j. immunotherapy cancer

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“…After splenocytes were isolated from immunized mice on the following day, plates were washed with PBS. Splenocytes were then plated in triplicate in a 0.1% DMSO solution of CTL-TEST medium (Cellular Technology Limited) with 10 ug/mL of peptide and incubated at 37°C for 24 h. For TCR avidity studies, splenocytes were pulsed with decreasing concentrations of GUCY2C 254-262 peptide (10 ug/mL to 3 pg/mL) (23)(24)(25). The next day, splenocytes were removed, and development reagents were added to detect IFNg-producing spot-forming cells (SFCs).…”

Section: Ifng Elispot Assaymentioning

confidence: 99%

T-Cell Responses to Immunodominant Listeria Epitopes Limit Vaccine-Directed Responses to the Colorectal Cancer Antigen, Guanylyl Cyclase C

et al. 2022

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The Gram-positive bacterium Listeria monocytogenes (Lm) is an emerging platform for cancer immunotherapy. To date, over 30 clinical trials have been initiated testing Lm cancer vaccines across a wide variety of cancers, including lung, cervical, colorectal, and pancreatic. Here, we assessed the immunogenicity of an Lm vaccine against the colorectal tumor antigen GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination did not prime naïve GUCY2C-specific CD8+ T-cell responses towards the dominant H-2Kd-restricted epitope, GUCY2C254-262. However, Lm-GUCY2C produced robust CD8+ T-cell responses towards Lm-derived peptides suggesting that GUCY2C254-262 peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C254-262 immunity completely suppressed GUCY2C254-262 responses. Comparison of immunogenic Lm-derived peptides to GUCY2C254-262 revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2Kd compared to GUCY2C254-262 peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2Kd binding, producing GUCY2CF255Y, significantly improved stability with H-2Kd and rescued GUCY2C254-262 immunogenicity in the context of Lm vaccination. Collectively, these studies suggest that Lm antigens may compete with and suppress the immunogenicity of target vaccine antigens and that use of altered peptide ligands with enhanced peptide-MHC stability may be necessary to elicit robust immune responses. These studies suggest that optimizing target antigen competitiveness with Lm antigens or alternative immunization regimen strategies, such as prime-boost, may be required to maximize the clinical utility of Lm-based vaccines.

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“…As the retrovirus used in this assay is replication‐incompetent, the transduction procedure can be performed in a standard BSL‐2 tissue culture hood. So far, this method has been successfully applied to express more than 70 TCRs and has been used in over 20 publications in the past three years by multiple laboratories (Abraham et al., ; Bettini et al., ; Drobek et al., ; Giles, Neves, Marshak‐Rothstein, & Shlomchik, ; Gras et al., ; Guan et al., ; Guo et al., ; Jones, Alli, Li, & Geiger, ; Lee et al., ; Miao et al., ; Milasta et al., ; Ng, Leno‐Duran, Samanta, Almo, & Strominger, ; Packard et al., ; Snook et al., ; Sprouse et al., , ; Tan et al., ; Van Braeckel‐Budimir et al., ; Wolf, Emerson, Pingel, Buller, & DiPaolo, ; Yang et al., ). More recently, the method was adapted to express human TCRs in HLA‐humanized mice, which significantly expands the utility of the method in translational applications (Sprouse et al., ).…”

Section: Commentarymentioning

confidence: 99%

“…Importantly, retroviral gene delivery has made investigation of single TCRs in vivo more accessible to a higher number of laboratories and is compatible with an unlimited number of genetic backgrounds. Our laboratory and others have successfully applied the TCR retrogenic approach to study T cell responses to infectious, autoimmune, and model antigens (Abraham, Flickinger, Waldman, & Snook, 2019;Lee, Sprouse, Banerjee, Bettini, & Bettini, 2017;Sprouse et al, 2018;Yang et al, 2018). Over the years, the efficiency of generating TCR retrogenic mice has improved.…”

Section: Introductionmentioning

confidence: 99%

Generation of T Cell Receptor Retrogenic Mice

Kong

Bettini

2019

CP in Immunology

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The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus‐mediated stem cell–based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector‐encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

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TCR Retrogenic Mice as a Model To Map Self-Tolerance Mechanisms to the Cancer Mucosa Antigen GUCY2C

Cited by 6 publications

References 57 publications

Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients

Split tolerance permits safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients

T-Cell Responses to Immunodominant Listeria Epitopes Limit Vaccine-Directed Responses to the Colorectal Cancer Antigen, Guanylyl Cyclase C

Generation of T Cell Receptor Retrogenic Mice

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