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Supplementary Materials and Methods, References

Flow chart of transcriptome profiling after antigen-specific stimulation of redirected T cells with different co-stimulations

List of differentially regulated cell cycle genes with Log2FC values.

supplementary figure legends

Purpose: Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed.Experimental Design: Fibroblast activation protein (FAP)–specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized in vitro and antitumor efficacy was tested in vivo in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM.Results: All the three CARs demonstrated FAP-specific functionality in vitro. Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. In vivo, only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality.Conclusions: Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior in vitro functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted. Clin Cancer Res; 24(16); 3981–93. ©2018 AACR.

Epstein–Barr virus (EBV) was the first human tumor virus being discovered and remains to date the only human pathogen that can transform cells in vitro. 55 years of EBV research have now brought us to the brink of an EBV vaccine. For this purpose, recombinant viral vectors and their heterologous prime-boost vaccinations, EBV-derived virus-like particles and viral envelope glycoprotein formulations are explored and are discussed in this review. Even so, cell-mediated immune control by cytotoxic lymphocytes protects healthy virus carriers from EBV-associated malignancies, antibodies might be able to prevent symptomatic primary infection, the most likely EBV-associated pathology against which EBV vaccines will be initially tested. Thus, the variety of EBV vaccines reflects the sophisticated life cycle of this human tumor virus and only vaccination in humans will finally be able to reveal the efficacy of these candidates. Nevertheless, the recently renewed efforts to develop an EBV vaccine and the long history of safe adoptive T cell transfer to treat EBV-associated malignancies suggest that this oncogenic γ-herpesvirus can be targeted by immunotherapies. Such vaccination should ideally implement the very same immune control that protects healthy EBV carriers.

The Epstein-Barr virus (EBV) is one of the predominant tumor viruses in humans, but so far no therapeutic or prophylactic vaccination against this transforming pathogen is available. We demonstrated that heterologous prime-boost vaccination with the nuclear antigen 1 of EBV (EBNA1), either targeted to the DEC205 receptor on DCs or expressed from a recombinant modified vaccinia virus Ankara (MVA) vector, improved priming of antigen-specific CD4(+) T cell help. This help supported the expansion and maintenance of EBNA1-specific CD8(+) T cells that are most efficiently primed by recombinant adenoviruses that encode EBNA1. These combined CD4(+) and CD8(+) T cell responses protected against EBNA1-expressing T and B cell lymphomas, including lymphoproliferations that emerged spontaneously after EBNA1 expression. In particular, the heterologous EBNA1-expressing adenovirus, boosted by EBNA1-encoding MVA vaccination, demonstrated protection as a prophylactic and therapeutic treatment for the respective lymphoma challenges. Our study shows that such heterologous prime-boost vaccinations against EBV-associated malignancies as well as symptomatic primary EBV infection should be further explored for clinical development.

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Immune responses to Epstein-Barr virus (EBV) infection synergize with the main genetic risk factor HLA-DRB1*15:01 (HLA-DR15) to increase the likelihood to develop the autoimmune disease multiple sclerosis (MS) at least sevenfold. In order to gain insights into this synergy, we investigated HLA-DR15 positive human immune compartments after reconstitution in immune-compromised mice (humanized mice) with and without EBV infection. We detected elevated activation of both CD4+ and CD8+ T cells in HLA-DR15 donor-reconstituted humanized mice at steady state, even when compared to immune compartments carrying HLA-DRB1*04:01 (HLA-DR4), which is associated with other autoimmune diseases. Increased CD8+ T cell expansion and activation was also observed in HLA-DR15 donor-reconstituted humanized mice after EBV infection. Despite this higher immune activation, EBV viral loads were less well controlled in the context of HLA-DR15. Indeed, HLA-DR15-restricted CD4+ T cell clones recognized EBV-transformed B cell lines less efficiently and demonstrated cross-reactivity toward allogeneic target cells and one MS autoantigen. These findings suggest that EBV as one of the main environmental risk factors and HLA-DR15 as the main genetic risk factor for MS synergize by priming hyperreactive T-cell compartments, which then control the viral infection less efficiently and contain cross-reactive CD4+ T cell clones.

A proportion of EBV+ B cells are infectible by CXCR4-tropic HIV-1 in vitro and in vivo, and these HIV-infected B cells can transmit infection to previously virus-naïve humanized mice., HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell–mediated immune control.

Volume 10, no. 1, e01941-18, 2019, https://doi.org/10.1128/mBio.01941-18. After careful review of Fig. 3, it was noted that for the …

Epstein-Barr virus (EBV) infects the majority of the human population and usually persists asymptomatically within its host. Nevertheless, EBV is the causative agent for infectious mononucleosis (IM) and for lymphoproliferative disorders, including Burkitt and Hodgkin lymphomas. The immune system of the infected host is thought to prevent tumor formation in healthy virus carriers. EBV was one of the first viruses described to express miRNAs, and many host and viral targets were identified for these in vitro. However, their role during EBV infection in vivo remained unclear. This work is the first to describe that EBV miRNAs mainly increase viremia and virus-associated lymphomas through dampening antigen recognition by adaptive immune responses in mice with reconstituted immune responses. Currently, there is no prophylactic or therapeutic treatment to restrict IM or EBV-associated malignancies; thus, targeting EBV miRNAs could promote immune responses and limit EBV-associated pathologies., The human persistent and oncogenic Epstein-Barr virus (EBV) was one of the first viruses that were described to express viral microRNAs (miRNAs). These have been proposed to modulate many host and viral functions, but their predominant role in vivo has remained unclear. We compared recombinant EBVs expressing or lacking miRNAs during in vivo infection of mice with reconstituted human immune system components and found that miRNA-deficient EBV replicates to lower viral titers with decreased frequencies of proliferating EBV-infected B cells. In response, activated cytotoxic EBV-specific T cells expand to lower frequencies than during infection with miRNA-expressing EBV. However, when we depleted CD8+ T cells the miRNA-deficient virus reached similar viral loads as wild-type EBV, increasing by more than 200-fold in the spleens of infected animals. Furthermore, CD8+ T cell depletion resulted in lymphoma formation in the majority of animals after miRNA-deficient EBV infection, while no tumors emerged when CD8+ T cells were present. Thus, miRNAs mainly serve the purpose of immune evasion from T cells in vivo and could become a therapeutic target to render EBV-associated malignancies more immunogenic.

Purpose: Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed. Experimental Design: Fibroblast activation protein (FAP)–specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized in vitro and antitumor efficacy was tested in vivo in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM. Results: All the three CARs demonstrated FAP-specific functionality in vitro. Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. In vivo, only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality. Conclusions: Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior in vitro functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted. Clin Cancer Res; 24(16); 3981–93. ©2018 AACR.