The sorting receptor Sortilin exhibits a dual function in exocytic trafficking of interferon-γ and granzyme A in T cells.

Immunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules.

Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy.

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8(+) cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFß receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

CD96 expression determines the inflammatory potential of IL-9-producing Th9 cells.

Recent findings demonstrated proinflammatory functions of interleukin (IL)-9-producing T helper type (Th) 9 cells in the pathogenesis of intestinal bowel diseases (IBDs). However, also antiinflammatory properties have been ascribed to Th9 cells, pointing to a functional heterogeneity. To dissect the specific expression pattern and, especially, diversity of murine antigen-specific Th9 cells, we applied single cell transcription profiling. Th9 cells displayed reduced expression of typical activation markers, such as Cd40 ligand and Cd96, whereas expression of Cd25 and Cd83 was increased compared with other Th subsets. Importantly, we identified two subsets of Th9 cells differing above all in their CD96 expression. The heterogeneous CD96 expression was specific for Th9 cells and not observed for other Th subtypes, such as Th1 cells. Lower CD96 expression was also observed in human IL-9+ compared with IFN-γ+ T cells. Although Il9 was highly transcribed by all Th9 cells, IL-9 mRNA and protein expression was increased in CD96low cells. Transfer of CD96low Th9 cells into recombination activating gene 1-deficient (Rag1-/- ) mice caused severe weight loss, intestinal and colonic inflammation, and destruction of allogeneic skin grafts and thus showed high inflammatory potential. This was associated with their expansion and tissue accumulation. Contrastingly, CD96high Th9 cells did not cause colitis and showed reduced expansion and migratory potential. Blockade of CD96 completely restored the expansion and inflammatory properties of CD96high Th9 cells. Collectively, our data suggest an inhibitory role for the cosignaling receptor CD96 in Th9 cells, raising new opportunities in the treatment of IL-9-associated inflammations such as IBD.

Cooperation of genes in HPV16 E6/E7-dependent cervicovaginal carcinogenesis trackable by endoscopy and independent of exogenous estrogens or carcinogens.

Human papillomavirus (HPV) infection is necessary but insufficient for progression of epithelial cells from dysplasia to carcinoma-in situ (CIS) to invasive cancer. The combination of mutant cellular and viral oncogenes that regulate progression of cervical cancer (CC) remains unclear. Using combinations of HPV16 E6/E7 (E+), mutant Kras (mKras) (K+) and/or loss of Pten (P-/-), we generated autochthonous models of CC without exogenous estrogen, carcinogen or promoters. Furthermore, intravaginal instillation of adenoCre virus enabled focal activation of the oncogenes/inactivation of the tumor suppressor gene. In P+/+ mice, E6/E7 alone (P+/+E+K-) failed to cause premalignant changes, while mKras alone (P+/+E-K+) caused persistent mucosal abnormalities in about one-third of mice, but no cancers. To develop cancer, P+/+ mice needed both E6/E7 and mKras expression. Longitudinal endoscopies of P+/+E+K+ mice predicted carcinoma development by detection of mucosal lesions, found on an average of 23 weeks prior to death, unlike longitudinal quantitative PCRs of vaginal lavage samples from the same mice. Endoscopy revealed that individual mice differed widely in the time required for mucosal lesions to appear after adenoCre and in the time required for these lesions to progress to cancer. These cancers developed in the transition zone that extends, unlike in women, from the murine cervix to the distal vagina. The P-/-E+K+ genotype led to precipitous cancer development within a few weeks and E6/E7-independent cancer development occurred in the P-/-E-K+ genotype. In the P-/-E+K- genotype, mice only developed CIS. Thus, distinct combinations of viral and cellular oncogenes are involved in distinct steps in cervical carcinogenesis.

Oncogene-specific T cells fail to eradicate lymphoma-initiating B cells in mice.

To date, little is known about the interaction between (pre-)malignant B cells and T cells. We generated transgenic mice that allow B cell-specific induction of the oncogene SV40 large T-antigen (TAg) to analyze the role of oncogene-specific T cells during sporadic B-cell lymphoma development. Constitutive TAg expression in CD19-Cre × LoxP-Tag mice resulted in TAg-tolerant CD8+ T cells and development of B-cell lymphomas. In contrast, CD19-CreERT2 × LoxP-Tag mice retained TAg-competent CD8+ T cells at time of oncogene induction and TAg expression in few B cells of adult mice resulted in exceptionally rare lymphoma formation late in life. Increased lymphoma incidence in the absence of TAg-specific T cells suggested T cell-mediated inhibition of lymphoma progression. However, TAg-initiated B cells were not eliminated by T cells and detected long term. Our results demonstrate a failure of the immune system to eradicate lymphoma-initiating B cells, retaining the risk of lymphoma development.

Clinical translation and regulatory aspects of CAR/TCR-based adoptive cell therapies-the German Cancer Consortium approach.

Adoptive transfer of T cells genetically modified by TCRs or CARs represents a highly attractive novel therapeutic strategy to treat malignant diseases. Various approaches for the development of such gene therapy medicinal products (GTMPs) have been initiated by scientists in recent years. To date, however, the number of clinical trials commenced in Germany and Europe is still low. Several hurdles may contribute to the delay in clinical translation of these therapeutic innovations including the significant complexity of manufacture and non-clinical testing of these novel medicinal products, the limited knowledge about the intricate regulatory requirements of the academic developers as well as limitations of funds for clinical testing. A suitable good manufacturing practice (GMP) environment is a key prerequisite and platform for the development, validation, and manufacture of such cell-based therapies, but may also represent a bottleneck for clinical translation. The German Cancer Consortium (DKTK) and the Paul-Ehrlich-Institut (PEI) have initiated joint efforts of researchers and regulators to facilitate and advance early phase, academia-driven clinical trials. Starting with a workshop held in 2016, stakeholders from academia and regulatory authorities in Germany have entered into continuing discussions on a diversity of scientific, manufacturing, and regulatory aspects, as well as the benefits and risks of clinical application of CAR/TCR-based cell therapies. This review summarizes the current state of discussions of this cooperative approach providing a basis for further policy-making and suitable modification of processes.

Fas expression by tumor stroma is required for cancer eradication.

The contribution of molecules such as perforin, IFN-γ (IFNγ), and particularly Fas ligand (FasL) by transferred CD8(+) effector T (T(E)) cells to rejection of large, established tumors is incompletely understood. Efficient attack against large tumors carrying a surrogate tumor antigen (mimicking a "passenger" mutation) by T(E) cells requires action of IFNγ on tumor stroma cells to avoid selection of antigen-loss variants. Because "cancer-driving" antigens (CDAs) are rarely counterselected, IFNγ may be expected to be dispensable in elimination of cancers by targeting a CDA. Here, initial regression of large, established tumors required neither IFNγ, FasL, nor perforin by transferred CD8(+) T(E) cells targeting Simian Virus (SV) 40 large T as CDA. However, cytotoxic T(E) cells lacking IFNγ or FasL could not prevent relapse despite retention of the rejection antigen by the cancer cells. Complete tumor rejection required IFNγ-regulated Fas by the tumor stroma. Therefore, T(E) cells lacking IFNγ or FasL cannot prevent progression of antigenic cancer because the tumor stroma escapes destruction if its Fas expression is down-regulated.

Differently immunogenic cancers in mice induce immature myeloid cells that suppress CTL in vitro but not in vivo following transfer.

Tumors frequently induce immature myeloid cells (iMC), which suppress specific and unrelated cytotoxic T lymphocyte (CTL) responses and are termed myeloid-derived suppressor cells (MDSC). Mainly analyzed by in vitro assays in tumor transplantation models, little is known about their function in autochthonous tumor models in vivo. We analyzed iMC in 3 SV40 large T (Tag)-driven conditional autochthonous cancer models with different immune status: (1) Early Tag-specific CTL competence and rare stochastic Tag activation leading to sporadic cancer, which induces an aberrant immune response and CTL tolerance; (2) Cre/LoxP recombinase-mediated hepatocellular carcinoma (HCC) development in neonatal Tag-tolerant mice; and (3) Tag-activation through Cre recombinase-encoding viruses in the liver and HCC development with systemic anti-Tag CTL immunity. In the first but not two latter models, tumors induced CTL hyporesponsiveness to tumor-unrelated antigens. Regardless of the model, tumors produced interleukin-6 and vascular endothelial growth factor but not granulocyte macrophage­colony-stimulating factor (GM-CSF) and induced iMC (CD11b(+)Gr-1(int)) that suppressed CTL responses in vitro. None of the iMC from the different tumor models suppressed CTL responses in adoptive cell transfer experiments unless GM-CSF was provided in vivo. Together, iMC expand independent of the type of antitumor response and are not immunosuppressive in a cell-autonomous fashion.

High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination.

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

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

Recurrent neoepitopes are cancer-specific antigens common among groups of patients and therefore ideal targets for adoptive T cell therapy. The neoepitope FSGEYIPTV 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 in vivo 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.

Early protective effect of a ("pan") coronavirus vaccine (PanCoVac) in Roborovski dwarf hamsters after single-low dose intranasal administration.

Introduction: 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. Materials and methods: We generated a multi-epitope vaccine (PanCoVac) that encodes the conserved T cell epitopes from all structural proteins of coronaviruses. PanCoVac contains elements that facilitate efficient processing and presentation of PanCoVac-encoded T cell epitopes and can be uploaded to any available vaccine platform. For proof of principle, we cloned PanCoVac into a non-integrating lentivirus vector (NILV-PanCoVac). We chose Roborovski dwarf hamsters for a first step in evaluating PanCoVac in vivo. Unlike mice, they are naturally susceptible to SARS-CoV-2 infection. Moreover, Roborovski dwarf hamsters develop COVID-19-like disease after infection with SARS-CoV-2 enabling us to look at pathology and clinical symptoms. Results: Using HLA-A*0201-restricted reporter T cells and U251 cells expressing a tagged version of PanCoVac, we confirmed in vitro that PanCoVac is processed and presented by HLA-A*0201. As mucosal immunity in the respiratory tract is crucial for protection against respiratory viruses such as SARS-CoV-2, we tested the protective effect of single-low dose of NILV-PanCoVac administered via the intranasal (i.n.) route in the Roborovski dwarf hamster model of COVID-19. After infection with ancestral SARS-CoV-2, animals immunized with a single-low dose of NILV-PanCoVac i.n. did not show symptoms and had significantly decreased viral loads in the lung tissue. This protective effect was observed in the early phase (2 days post infection) after challenge and was not dependent on neutralizing antibodies. Conclusion: PanCoVac, a multi-epitope vaccine covering conserved T cell epitopes from all structural proteins of coronaviruses, might protect from severe disease caused by SARS-CoV-2 variants and future pathogenic coronaviruses. The use of (HLA-) humanized animal models will allow for further efficacy studies of PanCoVac-based vaccines in vivo.

In vivo imaging of an inducible oncogenic tumor antigen visualizes tumor progression and predicts CTL tolerance.

Visualizing oncogene/tumor Ag expression by noninvasive imaging is of great interest for understanding processes of tumor development and therapy. We established transgenic (Tg) mice conditionally expressing a fusion protein of the SV40 large T Ag and luciferase (TagLuc) that allows monitoring of oncogene/tumor Ag expression by bioluminescent imaging upon Cre recombinase-mediated activation. Independent of Cre-mediated recombination, the TagLuc gene was expressed at low levels in different tissues, probably due to the leakiness of the stop cassette. The level of spontaneous TagLuc expression, detected by bioluminescent imaging, varied between the different Tg lines, depended on the nature of the Tg expression cassette, and correlated with Tag-specific CTL tolerance. Following liver-specific Cre-loxP site-mediated excision of the stop cassette that separated the promoter from the TagLuc fusion gene, hepatocellular carcinoma development was visualized. The ubiquitous low level TagLuc expression caused the failure of transferred effector T cells to reject Tag-expressing tumors rather than causing graft-versus-host disease. This model may be useful to study different levels of tolerance, monitor tumor development at an early stage, and rapidly visualize the efficacy of therapeutic intervention versus potential side effects of low-level Ag expression in normal tissues.

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

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 (H3.3K27M26-35). 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.

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

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.

Isolation of Neoantigen-Specific Human T Cell Receptors from Different Human and Murine Repertoires.

(1) Background: Mutation-specific T cell receptor (TCR)-based adoptive T cell therapy represents a truly tumor-specific immunotherapeutic strategy. However, isolating neoepitope-specific TCRs remains a challenge. (2) Methods: We investigated, side by side, different TCR repertoires-patients' peripheral lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs), PBLs of healthy donors, and a humanized mouse model-to isolate neoepitope-specific TCRs against eight neoepitope candidates from a colon cancer and an ovarian cancer patient. Neoepitope candidates were used to stimulate T cells from different repertoires in vitro to generate neoepitope-specific T cells and isolate the specific TCRs. (3) Results: We isolated six TCRs from healthy donors, directed against four neoepitope candidates and one TCR from the murine T cell repertoire. Endogenous processing of one neoepitope, for which we isolated one TCR from both human and mouse-derived repertoires, could be shown. No neoepitope-specific TCR could be generated from the patients' own repertoire. (4) Conclusion: Our data indicate that successful isolation of neoepitope-specific TCRs depends on various factors such as the heathy donor's TCR repertoire or the presence of a tumor microenvironment allowing neoepitope-specific immune responses of the host. We show the advantage and feasibility of using healthy donor repertoires and humanized mouse TCR repertoires to generate mutation-specific TCRs with different specificities, especially in a setting when the availability of patient material is limited.

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

PURPOSE: 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. EXPERIMENTAL DESIGN: Using peptide-based ELISpot assays, we screened for potential recurrent glioma neoepitopes in MHC-humanized mice. Following vaccination, droplet-based single-cell T-cell receptor (TCR) sequencing from established T-cell lines was applied for neoepitope-specific TCR discovery. Efficacy of intraventricular TCR-transgenic T-cell therapy was assessed in a newly developed glioma model in MHC-humanized mice induced by CRISPR-based delivery of tumor suppressor-targeting guide RNAs. RESULTS: We identify recurrent capicua transcriptional repressor (CIC) inactivating hotspot mutations at position 215 CICR215W/Q as immunogenic MHC class II (MHCII)-restricted neoepitopes. Vaccination of MHC-humanized mice resulted in the generation of robust MHCII-restricted mutation-specific T-cell responses against CICR215W/Q. Adoptive intraventricular transfer of CICR215W-specific TCR-transgenic T cells exert antitumor responses against CICR215W-expressing syngeneic gliomas. CONCLUSIONS: The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of human leukocyte antigen (HLA)-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T-cell adoptive therapy.

Commensal microflora and interferon-gamma promote steady-state interleukin-7 production in vivo.

IL-7 is a major regulator of lymphocyte homeostasis; however, little is known about the mechanisms that regulate IL-7 production. To study Il7 gene regulation in vivo, we generated a novel IL-7-reporter mouse, which allows the non-invasive quantification of Il7 gene activity in live mice and, additionally, the simultaneous activation/inactivation of target genes in IL-7-producing cells. With these IL-7-reporter mice, we identify thymus, skin and intestine as major sources of IL-7 in vivo. Importantly, we show that IFN-gamma and the commensal microflora promote steady-state IL-7 production in the intestine. Furthermore, we demonstrate that the blockade of IFN-gamma signaling in intestinal epithelial cells strongly reduces their IFN-gamma-driven IL-7 production. In summary, our data suggest a feedback loop in which commensal bacteria drive IFN-gamma production by lymphocytes, which in turn promotes epithelial cell IL-7 production and the survival of IL-7-dependent lymphocytes.

Sporadic immunogenic tumours avoid destruction by inducing T-cell tolerance.

The recognition and elimination of tumours by T cells, a process termed cancer immunosurveillance, is effective against certain virus-associated cancers. Spontaneous tumours often induce a specific immune response and are therefore also immunogenic. However, it is not clear whether they can be controlled by T cells. The immunosurveillance hypothesis postulates that tumours, if they eventually grow, escaped T-cell recognition by losing immunogenicity. Here we show, by generating a mouse model of sporadic cancer based on rare spontaneous activation of a dormant oncogene, that immunogenic tumours do not escape their recognition but induce tolerance. In this model, tumours derive from single cells and express a tumour-specific transplantation rejection antigen. Whereas vaccinated mice remain tumour-free throughout their lifetime, naive mice always develop a progressively growing tumour. We also show that despite specific recognition by T cells, the tumours do not lose their intrinsic immunogenicity and are rejected after transplantation in T-cell-competent recipients. Furthermore, in the primary host tumour-induced tolerance is associated with the expansion of non-functional T cells. Together, our data argue against immunosurveillance of spontaneous cancer.

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

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 KRASG12V- and RAC2P29L-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 KRASG12V- and RAC2P29L-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2P29L-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.

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

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-ß-receptor (LTßR) 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.