The effectiveness of chimeric Ag receptor (CAR)–transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR–T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR–T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR–T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

A major obstacle for chimeric antigen receptor (CAR) T cell therapy in solid tumors is the lack of truly tumor-specific target antigens, which translates to the targeting of tumor-associated antigens (TAAs) overexpressed on tumors but shared with normal organs, raising safety concerns. In addition, expression of TAAs in solid tumors is particularly heterogeneous. In this regard, it is critical to deeply understand the sensitivity of CAR T cells, especially against low-density targets and the possible therapeutic window of antigen density targeted by CAR T cells. In this review, we discuss the recent findings of mechanisms of antigen recognition through CAR, including immunological synapse formation, and the impact of target antigen density for induction of distinct T cell functions. We also discuss rational strategies to adjust and expand the therapeutic window for effective and safe targeting of solid tumors by CAR T cell platforms.

We found low-prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating somatic mutations in MTOR cause FCD type IIb.

Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA.

No abstract

T cells genetically modified with a CD19 chimeric antigen receptor (CD19CAR) are remarkably effective against B-cell malignancies in clinical trials. However, major concerns remain regarding toxicities, such as hypogammaglobulinemia, due to Bcell aplasia or severe cytokine release syndrome after overactivation of CAR T cells. To resolve these adverse events, we aimed to develop an inducible CAR system by using a tetracycline regulation system that would be activated only in the presence of doxycycline (Dox). In this study, the second-generation CD19CAR was fused into the third-generation Tet-On vector (Tet-CD19CAR) and was retrovirally transduced into primary CD8 þ T cells. Tet-CD19CAR T cells were successfully generated and had minimal background CD19CAR expression without Dox. Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19 þ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells. The Dox(þ) Tet-CD19CAR T cells also had significant antitumor activity in a xenograft model. However, without Dox, Tet-CD19CAR T cells lost CAR expression and CAR T-cell functions in vitro and in vivo, clearly segregating the "On" and "Off" status of Tet-CD19CAR cells by Dox administration. In addition to suicide-gene technology, controlling the expression and the functions of CAR with an inducible vector is a potential solution for CAR T-cell therapy-related toxicities, and may improve the safety profile of CAR T-cell therapy. This strategy might also open the way to treat other malignancies in combination with other CAR or TCR gene-modified T cells.

FDA approval of chimeric antigen receptor T cells (CART cells) is the culmination of several decades of technology development and interrogation of the properties of these gene therapies. CART cells exist as personalized "living drugs" and have demonstrated astounding anti-tumor efficacy in patients with leukemia and lymphoma. However, the future promise of CART efficacy for solid tumors, the greatest unmet burden, is met with a number of challenges that must be surmounted for effective immune responses. In this review, we discuss the next-generation developments of CARs to target solid tumors, including fine-tuned and combinational-targeting receptors. We consider the structural intricacies of the CAR molecules that influence optimal signaling and CART survival, and review pre-clinical cell-intrinsic and cell-extrinsic combinational therapy approaches.

We retrospectively reviewed 104 biopsy specimens of previously untreated skin acute graft-versus-host disease (GVHD) within 100 days after allogeneic stem cell transplantation, and analyzed the relationship between types of infiltrating cells and clinical outcomes. Counting the total number of CD8 ؉ T cells, CD163 ؉ macrophages, and CD1a ؉ dendritic cells in 4 fields under original magnification IntroductionMacrophages are phagocytic cells with various abilities, such as phagocytosis, antigen-presenting, and secretion of cytokines. 1,2 Recently, it was revealed in human sequential biopsy data that recipient macrophages contributed to acute graft-versus-host disease (GVHD) by antigen-presenting and secreting cytokines, causing the activation and proliferation of CD8 ϩ T cells. 3 We focused on macrophage involvement in acute GVHD, especially on the relationship between the macrophage infiltration of skin lesions and refractory GVHD. MethodsBetween January 1997 and October 2007 at the Japanese Red Cross Nagoya First Hospital, we used skin biopsy specimens within 100 days after allogeneic stem cell transplantation (allo-SCT) of skin lesions clinically considered acute GVHD without any GVHD treatment from 104 patients who underwent allo-SCTs. We analyzed the relationship between types of infiltrating cells and clinical outcomes by counting the total number of CD8 ϩ T cells, CD163 ϩ macrophages, and CD1a ϩ dendritic cells in 4 fields of a skin biopsy specimen under original magnification ϫ200. Immunohistochemical analysis using paraffin sections was performed using monoclonal antibodies against CD8, CD163, and CD1a (Novocastra). CD163 is a member of the scavenger receptor cystein-rich superfamily and is an exclusive marker for macrophages, playing a major role in the scavenging components of damaged cells. [4][5][6][7] The endpoints of this study were the outcomes of acute GVHD and overall survival (OS). Acute GVHD was diagnosed and graded according to the consensus criteria. 8 We defined refractory GVHD as that exhibited by patients who had persistent lesions after primary steroid treatments. To establish parameters, we analyzed the numbers of infiltrating , disease risk (low vs high), human leukocyte antigen (HLA) disparity (match vs mismatch), donor source (related vs unrelated), graft source (bone marrow vs peripheral blood), age at allo-SCT (Յ 50 years vs Ͼ 50 years), conditioning regimen (conventional regimens vs reduced intensity regimens), and skin GVHD stage at biopsy (stages 1-2 vs stages 3-4). A significance level of P Ͻ .05 was used for all analyses, which were based on all data available as of August 31, 2008. Protocols were approved by the Japanese Red Cross Nagoya First Hospital's Institutional Review Board, and all patients provided informed consent in accordance with the Declaration of Helsinki. Table 1 summarizes the characteristics of patients and information gathered about GVHD. We divided patients into 4 groups according to the amount of infiltrating cells (FM and FT, 60.6%; MT and FM, 18.2%; MT...