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Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science ...

Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin

German Cancer Research Center

German Cancer Consortium, partner site Berlin

T cell surveillance is often effective against virus-associated tumors because of their high immunogenicity. It is not clear why surveillance occasionally fails, particularly against hepatitis B virus- or hepatitis C virus-associated hepatocellular carcinoma (HCC). We established a transgenic murine model of virus-induced HCC by hepatocyte-specific adenovirus-induced activation of the oncogenic SV40 large T antigen (TAg). Adenovirus infection induced cytotoxic T lymphocytes (CTLs) targeted against the virus and TAg, leading to clearance of the infected cells. Despite the presence of functional, antigen-specific T cells, a few virus-infected cells escaped immune clearance and progressed to HCC. These cells expressed TAg at levels similar to HCC isolated from neonatal TAg-tolerant mice, suggesting that CTL clearance does not select for cells with low immunogenicity. Virus-infected mice revealed significantly greater T cell infiltration in early-stage HCC compared with that in late-stage HCC, demonstrating progressive local immune suppression through inefficient T cell infiltration. Programmed cell death protein-1 (PD-1) and its ligand PD-L1 were expressed in all TAg-specific CD8+ T cells and HCC, respectively, which contributed to local tumor-antigen-specific tolerance. Thus, we have developed a model of virus-induced HCC that may allow for a better understanding of human HCC.

Virus-induced hepatocellular carcinomas cause antigen-specific local tolerance.

Proteasome-catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by T cell receptor (TCR)-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm, TCRs were generated against putative KRAS- and RAC2-derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope-specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRAS- and RAC2-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2-derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes.

In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo.

Blood endothelial cells display remarkable plasticity depending on the demands of a malignant microenvironment. While studies in solid tumors focus on their role in metabolic adaptations, formation of high endothelial venules (HEVs) in lymph nodes extends their role to the organization of immune cell interactions. As a response to lymphoma growth, blood vessel density increases; however, the fate of HEVs remains elusive. Here, we report that lymphoma causes severe HEV regression in mouse models that phenocopies aggressive human B cell lymphomas. HEV dedifferentiation occurrs as a consequence of a disrupted lymph-carrying conduit system. Mechanosensitive fibroblastic reticular cells then deregulate CCL21 migration paths, followed by deterioration of dendritic cell proximity to HEVs. Loss of this crosstalk deprives HEVs of lymphotoxin-β-receptor (LTβR) signaling, which is indispensable for their differentiation and lymphocyte transmigration. Collectively, this study reveals a remodeling cascade of the lymph node microenvironment that is detrimental for immune cell trafficking in lymphoma.

Lymphocyte access to lymphoma is impaired by high endothelial venule regression.

Gliomas are intrinsic brain tumors with a high degree of constitutive and acquired resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Glioma subtypes are recognized by characteristic mutations. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy.

T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas.

Insight into processes that determine CD8+ T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often use high-avidity CD8+ T cells in adoptive-transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon noninflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8+ T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen-mismatched leukocytes. CD8+ memory commitment was analyzed in Ebag9-deficient mice that exhibited enhanced tumor cell lysis. This property endowed Ebag9-/- mice with extended control of Tcl-1 oncogene-induced chronic lymphocytic leukemia progression. In Ebag9-/- mice, an expanded memory population was obtained for anti-HY and anti-SV-40 T antigen-specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8+ T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9+/+ and Ebag9-/- T cells. In Ebag9-/- cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation and anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8+ memory lineage.

EBAG9 controls CD8+ T cell memory formation responding to tumor challenge in mice.

Diffuse midline glioma is the leading cause of solid cancer-related deaths in children with very limited treatment options. A majority of the tumors carry a point mutation in the histone 3 variant (H3.3) creating a potential HLA-A*02:01 binding epitope (H3.3K27M). Here, we isolated an H3.3K27M-specific T cell receptor (TCR) from transgenic mice expressing a diverse human TCR repertoire. Despite a high functional avidity of H3.3K27M-specific T cells, we were not able to achieve recognition of cells naturally expressing the H3.3K27M mutation, even when overexpressed as a transgene. Similar results were obtained with T cells expressing the published TCR 1H5 against the same epitope. CRISPR/Cas9 editing was used to exclude interference by endogenous TCRs in donor T cells. Overall, our data provide strong evidence that the H3.3K27M mutation is not a suitable target for cancer immunotherapy, most likely due to insufficient epitope processing and/or amount to be recognized by HLA-A*02:01 restricted CD8 T cells.

H3.3K27M mutation is not a suitable target for immunotherapy in HLA-A2 patients with diffuse midline glioma.

Recurrent neoepitopes are cancer-specific antigens common among groups of patients and therefore ideal targets for adoptive T cell therapy. The neoepitope FGEYIPTV carries the Rac1P29S amino acid change caused by a c.85C>T missense mutation, which is the third most common hotspot mutation in melanoma. Here, we isolated and characterized TCRs to target this HLA-A*02:01-binding neoepitope by adoptive T cell therapy. Peptide immunization elicited immune responses in transgenic mice expressing a diverse human TCR repertoire restricted to HLA-A*02:01, which enabled isolation of high-affinity TCRs. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cells and we observed regression of Rac1P29S expressing tumors  after adoptive T cell therapy (ATT). Here we found that a TCR raised against a heterologous mutation with higher peptide-MHC affinity (Rac2P29L) more efficiently targeted the common melanoma mutation Rac1P29S. Overall, our study provides evidence for the therapeutic potential of Rac1P29S-specific TCR-transduced T cells and reveal a novel strategy by generating more efficient TCRs by heterologous peptides.

Targeting the recurrent Rac1P29S neoepitope in melanoma with heterologous high-affinity T cell receptors.

Response to: Correspondence on 'H3.3K27M mutation is not a suitable target for immunotherapy in HLA-A2+ patients with diffuse midline glioma' by Chheda .

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the danger posed by human coronaviruses. Rapid emergence of immunoevasive variants and waning antiviral immunity decrease the effect of the currently available vaccines, which aim at induction of neutralizing antibodies. In contrast, T cells are marginally affected by antigen evolution although they represent the major mediators of virus control and vaccine protection against virus-induced disease.

Article	Year
Virus-induced hepatocellular carcinomas cause antigen-specific local tolerance. The Journal of clinical investigation\| PubMed ID: 23454765	2013
In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo. eLife\| PubMed ID: 33875134	2021
Lymphocyte access to lymphoma is impaired by high endothelial venule regression. Cell reports\| PubMed ID: 34706240	2021
T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 34782365	2022
EBAG9 controls CD8+ T cell memory formation responding to tumor challenge in mice. JCI insight\| PubMed ID: 35482418	2022
H3.3K27M mutation is not a suitable target for immunotherapy in HLA-A2 patients with diffuse midline glioma. Journal for immunotherapy of cancer\| PubMed ID: 36302563	2022
Targeting the recurrent Rac1P29S neoepitope in melanoma with heterologous high-affinity T cell receptors. Frontiers in immunology\| PubMed ID: 36875127	2023
Response to: Correspondence on 'H3.3K27M mutation is not a suitable target for immunotherapy in HLA-A2+ patients with diffuse midline glioma' by Chheda . Journal for immunotherapy of cancer\| PubMed ID: 36918223	2023
Early protective effect of a ("pan") coronavirus vaccine (PanCoVac) in Roborovski dwarf hamsters after single-low dose intranasal administration. Frontiers in immunology\| PubMed ID: 37520530	2023
A phase I study of MAGE-A1-targeted T1367 T-cell receptor-based cell therapy in patients with advanced multiple myeloma. Haematologica\| PubMed ID: 39279443	2024

Gerald Willimsky

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