Aviad Pato

Imprinting of Lymphocytes with Melanoma Antigens Acquired by Trogocytosis Facilitates Identification of Tumor-Reactive T Cells

Trogocytosis is a contact-dependent intercellular transfer of membrane fragments and associated molecules from APCs to effector lymphocytes. We previously demonstrated that trogocytosis also occurs between tumor target and cognate melanoma Ag-specific cytotoxic T cells (CTL). In this study, we show that, following trogocytosis, immune effector cells acquire molecular components of the tumor, including surface Ags, which are detectable by specific mAbs. We demonstrate that CD8+ and CD4+ T cells from melanoma patients’ PBMC and tumor-infiltrating lymphocytes (TIL) capture melanoma Ags, enabling identification of trogocytosing lymphocytes by staining with Ag-specific Abs. This finding circumvents the necessity of tumor prelabeling, which in the past was mandatory to detect membrane-capturing T cells. Through the detection of melanoma Ags on TIL, we sorted trogocytosing T cells and verified their preferential reactivity and cytotoxicity. Furthermore, tumor Ag–imprinted T cells were detected at low frequency in fresh TIL cultures shortly after extraction from the tumor. Thus, T cell imprinting by tumor Ags may allow the enrichment of melanoma Ag-specific T cells for research and potentially even for the adoptive immunotherapy of patients with cancer.

Human T cell crosstalk is induced by tumor membrane transfer.

Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC). Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs) and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8+ T cells to act as APCs (CD8+T-APCs). We demonstrate that, following trogocytosis, CD8+T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1) Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2) Fratricide (killing) of CD8+T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8+ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response.

Membrane-attached cytokines expressed by mRNA electroporation act as potent T-cell adjuvants

Proinflammatory cytokines are widely explored in different adoptive cell therapy protocols for enhancing survival and function of the transferred T cells, but their systemic administration is often associated with severe toxicity which limits their clinical use. To confine cytokine availability to the therapeutic T cells, we expressed 3 key cytokines, IL-2, IL-12, and IL-15, as integral T-cell membrane proteins. To prevent permanent activation of growth signaling pathways, we delivered these genes to T cells through mRNA electroporation. The engineered cytokines could be detected on the surface of mRNA-transfected cells and binding to their cell-surface receptors mainly occurred in cis. The 3 human cytokines supported the ex vivo growth of activated human CD8 and CD4 T cells for at least 6 days posttransfection, comparably to high-dose soluble IL-2. Similarly, membrane IL-2, membrane IL-12, and, to a lesser extent, membrane IL-15, were comparable with their soluble counterparts in supporting proliferation of splenic mouse CD8 T cells. Following electroporation of human CD8 T cells and antimelanoma tumor-infiltrating lymphocytes, membrane cytokines synergized with constitutively active toll-like receptor 4 in inducing interferon-&ggr; secretion. Efficient cooperation with TLR4 was also evident in the upregulation of the activation molecules CD25, CD69, CD137 (4-1BB), and CD134 (OX40). Taken together, membrane cytokines expressed through mRNA transfection emerge as effective tools for enhancing T-cell proliferation and function and may have potential use in adoptive T-cell therapy.

Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells

Adoptive T cell therapy of cancer employs a large number of ex‐vivo‐propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell‐extrinsic and intrinsic mechanisms often diminish the in‐vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll‐like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand‐binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin‐1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti‐tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR‐4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR‐4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)‐γ, triggered the surface expression of CD25, CD69 and 4‐1BB and up‐regulated a panel of cytokines and chemokines. In tumour‐infiltrating lymphocytes prepared from melanoma patients, caTLR‐4 induced robust IFN‐γ secretion in all samples tested. Furthermore, caTLR‐4 enhanced the anti‐melanoma cytolytic activity of tumour‐infiltrating lymphocytes and augmented the secretion of IFN‐γ, tumour necrosis factor (TNF)‐α and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) for at least 4 days post‐transfection. Our results demonstrate that caTLR‐4 is capable of exerting multiple T cell‐enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy.

Spontaneous Activation of Antigen-presenting Cells by Genes Encoding Truncated Homo-Oligomerizing Derivatives of CD40.

The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-&bgr;2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high–molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1&bgr;, TNF&agr;, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow–derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte–derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-&bgr;2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.

Potent Activation of Human T Cells by mRNA Encoding Constitutively Active CD40

New strategies for augmenting the actual performance of therapeutic T cells in vivo are needed for improving clinical outcome of adoptive cell therapy. Cumulative findings suggest that CD40 plays an intrinsic role in T cell costimulation. Recently, we demonstrated the ability of truncated, auto-oligomerizing CD40 derivatives to induce strong activation of APCs in a ligand-independent manner. We reasoned that constitutively active CD40 (caCD40) can similarly exert enhancing effects on human antitumor T cells. To test this assumption, we transfected human T cells with in vitro–transcribed caCD40 mRNA. In polyclonal T cells, caCD40 triggered IFN-γ secretion and upregulated CD25 and 4-1BB. In antimelanoma tumor-infiltrating lymphocytes (TILs), caCD40 induced massive production of IFN-γ, exerting a pronounced synergistic effect when coexpressed with constitutively active TLR4 devoid of its extracellular ligand binding. In unselected “young” TILs, caCD40 reproducibly increased surface expression of CD25, OX40, 4-1BB, CD127, and CD28. Three days post-mRNA electroporation of CD8 TILs, caCD40 elevated IFN-γ and TNF-α production and cytolytic activity in the presence of autologous but not HLA-I–mismatched melanoma. Enhanced killing of autologous melanoma by young TILs was observed 4 d posttransfection. These findings suggest that caCD40 can function as a potent T cell adjuvant and provide essential guidelines for similar manipulation of other key members of the TNFR family.

Abstract A095: New genes for enhancing T cell function in adoptive cell therapy of cancer

A growing number of clinical trials point to the exquisite ability of adoptively transferred T cells to eradicate vast masses of tumor cells, including aggressive metastatic tumors. In one therapeutic approach T cells are derived from tumor biopsies (tumor-infiltrating lymphocytes, or TILs) and express endogenous TCRs. In others T cells are expanded from the peripheral blood lymphocyte pool and redirected to recognize tumor antigens by virtue of exogenous TCR or chimeric antigen receptor (CAR) genes. Although reports on enduring clinical responses are encouraging, a significant portion of patients who do not respond, or respond only partially and transiently, underscore the critical challenge of improving T cell function and survival at the tumor site. In attempt to improve the clinical efficacy of adoptive cell therapy we created three classes of genetic adjuvants designed to operate autonomously in gene-modified T cells. One class encodes constitutively-active (ca) toll-like receptors devoid of their ligand-binding domain, the other produces membrane-anchored immunostimulatory cytokines and the third encodes a novel homo-oligomerizing configuration of tumor necrosis factor receptors, inducing constitutive ligand-free activation. For gene delivery into human TILs or peripheral blood T cells we have been using electroporation of in-vitro-transcribed mRNA. Here we present data obtained in preliminary ex-vivo experiments designed to assess the function of caTLR4, membrane IL-2, IL-12 (single chain) and IL-15 and trimeric caCD40 and their level of cooperation. The mere expression of caTLR4 mRNA in polyclonal human CD8 and CD4 T cells induced the production of IFN-γ, triggered the surface expression of CD25, CD69 and 4-1BB and upregulated a panel of cytokines and chemokines. In all samples of anti-melanoma TILs tested, caTLR4 induced a burst of IFN-γ secretion, which usually ceased within 24 hours post-transfection. Furthermore, caTLR4 enhanced the cytolytic activity of TILs and augmented the secretion of IFN-γ, TNF-α and GM-CSF in the presence of autologous, but not mismatched, melanoma for at least 4 days post-transfection. The membrane cytokines were found to act mainly in-cis and each could support the proliferation of human CD8 and CD4 T cells for at least 6 days post-transfection, at a magnitude comparable to a saturating dose of soluble IL-2. Following mRNA electroporation of anti-melanoma TILs, membrane cytokines cooperated with caTLR4 in inducing IFN-γ secretion and upregulating the activation and costimulatory molecules CD25, CD69, 4-1BB and OX40. Different combinations of caTLR4, membrane cytokines and caCD40 mRNA induced spontaneous cytokine secretion in mRNA-transfected TILs, typically acting in a synergistic manner. Even when the spontaneous, initial effect waned, a considerably higher fraction of mRNA-transfected, compared to control-transfected TILs could still respond robustly to autologous, but not allogeneic, melanoma. In the presence of the three classes of adjuvants, transfected short-term cultured ‘young’ TILs revealed exceptional upregulation of 4-1BB, OX40, CD25 and CD28 and an increase in IFN-γ production and no, or only minimal change in the inhibitory receptors PD-1 and CTLA-4. Here, too, the different adjuvants displayed marked synergy, with caCD40 often exerting a prominent effect even when delivered as a single gene. Enhanced killing of autologous melanoma cells by electroporated young TILs was observed up to ten days post-electroporation. Our findings suggest that this new panel of genetic adjuvants can substantially improve the functional properties of antitumor T cells for extended periods, offering a new tool in all approaches for cancer immunotherapy employing adoptive T cell transfer. Citation Format: Gideon Gross, Aviad Pato, Hadas Weinstein-Marom, Noam Levin, Alon Margalit, Orit Itzhaki, Michal Besser, Tamar Peretz, Arthur Machlenkin, Michal Lotem. New genes for enhancing T cell function in adoptive cell therapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A095.