Major limitations of currently investigated␣␤T cells redirected against cancer by transfer of tumor-specific ␣␤TCR arise from their low affinity, MHC restriction, and risk to mediate self-reactivity after pairing with endogenous ␣ or ␤TCR chains. Therefore, the ability of a defined ␥9␦2TCR to redirect ␣␤T cells selectively against tumor cells was tested and its molecular interaction with a variety of targets investigated. Functional analysis revealed that a ␥9␦2TCR efficiently reprograms both CD4 ؉ and CD8 ؉ IntroductionThe major challenge in the field of adoptive immunotherapy is the generation of tumor-reactive ␣␤T cells which can be applied to a broad variety of cancer patients. To facilitate the rapid generation of tumor-reactive ␣␤T cells, it has been proposed that ␣␤T cells can be reprogrammed with genes encoding for a tumor-specific ␣␤TCR or a chimeric receptor. 1 Several such receptors are already being used to redirect ␣␤T cells in phase 1 clinical trials. 1,2 However, reprogramming ␣␤T cells with defined ␣␤TCRs is substantially hampered by their restriction to HLA types, thus limiting the number of patients who can be treated with one ␣␤TCR. In addition, pairing of introduced with endogenous ␣␤TCR chains can induce life-threatening autoreactivity. 3,4 One attractive alternative to mediate a selective antitumor reactivity with a high-affinity TCR might arise from the ability of ␥␦T cells to mediate antitumor reactivity while ignoring a healthy environment. [5][6][7] Isolated ␥9␦2T cells efficiently kill tumor cells of hematologic malignancies and from solid tumors. 7 However, the function and proliferation capacity of ␥␦T cells is frequently heavily impaired in cancer patients 8 making autologous ␥␦T cells less attractive for immune interventions. On the other hand, as end-stage cancer patients can easily elicit ␣␤T-cell immune responses against, for example, viral Ags, 9,10 ␣␤T cells might serve as carriers for broadly tumor-reactive ␥␦TCRs.The recognition of mevalonate metabolites (phosphoantigens) 11 which are overexpressed in a broad range of tumor cells has been suggested as an important mechanism by which multiple ␥9␦2TCR can sense malignant transformation as the recognition involves TCR domains which are conserved in most ␥9␦2TCRs. [12][13][14] In addition, ␥9␦2TCR G115 has been also suggested to bind to a complex of Apolipoprotein AI (ApoAI) and F1-ATPase, 15 a complex mitochondrial enzyme found on the surface of many malignant cells. 16 This knowledge might allow a rational design of ␥␦T cell-based immunotherapies. Therefore, we investigated whether a defined ␥9␦2TCR can be efficiently expressed in ␣␤T cells, mediate tumor-specific proliferation of ␣␤T cells, and redirect both effector CD8 ϩ and helper CD4 ϩ ␣␤T-cell subsets against a broad panel of tumor cell lines while ignoring normal cells in vitro and in vivo. MethodsCell lines, Abs, the retroviral transduction and expansion of ␣␤T cells, functional T-cell assays 11,[17][18][19][20] as well as the animal model used are described in supp...

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Immunotherapy with innate immune cells has recently evoked broad interest as a novel treatment option for cancer patients. ␥9␦2T cells in particular are emerging as an innate cell population with high frequency and strong antitumor reactivity, which makes them and their receptors promising candidates for immune interventions. However, clinical trials have so far reported only limited tumor control by adoptively transferred ␥9␦2T cells. As a potential explanation for this lack of efficacy, we found unexpectedly high variability in tumor recognition within the physiologic human ␥9␦2T-cell repertoire, which is substantially regulated by the CDR3 domains of individual ␥9␦2TCRs. In the present study, we demonstrate that the reported molecular requirements of CDR3 domains to interact with target cells shape the physiologic ␥9␦2T-cell repertoire and, most likely, limit the protective and thera- IntroductionImmunotherapy with innate immune cells has become widely used because this approach obviates the need to match a cellular product to a defined HLA haplotype, allowing adoptive immunotherapies to be used in virtually any cancer patient without extensive in vitro selection or manipulation of the cellular product. 1-4 ␥9␦2T cells are promising as an innate cell population for this purpose because they are usually observed at high frequencies in the human peripheral blood and provide a strong antitumor reactivity against various solid and hematologic cancers. 5 However, within ␥9␦2T-cell populations, individual clones display great diversity in the repertoire because of the activating or inhibitory receptors expressed. 6 Selecting innate cell products for certain cell types, such as those with a low level of inhibitory receptors, therefore seems plausible, especially considering the limited efficacy of adoptively transferred innate immune cells in clinical trials. 7,8 An alternative proposal is to engineer cells to express defined activating innate receptors that mediate strong antitumor reactivity, such as a defined ␥9␦2TCR, 9 which could pave the way for readily available and more effective cellular products. However, the molecular details of how a ␥9␦2TCR interacts with its target are not fully understood, making it challenging to select defined ␥9␦2T cells or to engineer T cells with defined ␥9␦2TCRs.In "classic" immunoreceptors such as ␣␤TCRs or Igs, the complementary determining regions (CDRs) determine affinity and specificity for a specific (peptide) epitope. V(D)J recombination allows the creation of a highly variable CDR repertoire ensuring recognition of an immense collection of antigens. ␥9␦2T cells also possess a rearranged TCR that mediates recognition. The phosphoantigen isopentenyl pyrophosphate (IPP) has been suggested to be a key player in ␥9␦2TCR-mediated activation, 5,10,11 but no direct interaction between a ␥9␦2TCR and IPP or any other phosphoantigen has ever been demonstrated. It was previously suggested that positively charged residues within the ␥9␦2TCR are crucial for the response to negatively...

Human papillomavirus (HPV) type 16 infection is strongly associated with the development of cervical carcinoma (CxCa) in women. The HPV16-derived oncoproteins E6 and E7, responsible for both onset and maintenance of malignant transformation, are expressed constitutively in CxCa cells and represent tumor-associated Ags. As a result, E6 and E7 constitute potential targets for adoptive CTL-mediated immunotherapy of CxCa. However, the availability to date of well-characterized HPV16-specific, CxCa-reactive human CTLs is extremely limited. The current study describes the in vitro generation and isolation of HPV16 E7-specific, CxCa-reactive human CTL clones from low-frequency healthy donor-derived CD8β-positive precursors. For this purpose, an in vitro CTL induction protocol was used involving mature monocyte-derived dendritic cells as stimulator cells loaded with an HLA-A2.1-restricted, E711–20-derived high-affinity altered peptide ligand. A double tetramer-guided isolation procedure and subsequent limiting-dilution cloning resulted in Ag-specific CTL clones. Stringent CTL characterization clearly indicated Ag-specific, HLA-A2.1-restricted reactivity against different HPV16-transformed CxCa cell lines. To allow expansion of E711–20-specific CTL clones to numbers required for prolonged in vitro as well as in vivo application, their life span was significantly extended by ectopic expression of human telomerase reverse transcriptase. Collectively, our results show that optimized CTL induction and stringent CTL selection procedures, followed by human telomerase reverse transcriptase-mediated life span extension will allow continued availability of low-frequency HPV16-specific, CxCa-reactive human CTL clones. This may enhance the prospects of HPV16-specific adoptive CTL immunotherapy in CxCa patients.

T-cells play a critical role in tumor immunity. Indeed, the presence of tumor-infiltrating lymphocytes is a predictor of favorable patient prognosis for many indications and is a requirement for responsiveness to immune checkpoint blockade therapy targeting programmed cell death 1. For tumors lacking immune infiltrate, or for which antigen processing and/or presentation has been downregulated, a promising immunotherapeutic approach is chimeric antigen receptor (CAR) T-cell therapy. CARs are hybrid receptors that link the tumor antigen specificity and affinity of an antibody-derived single-chain variable fragment with signaling endodomains associated with T-cell activation. CAR therapy targeting CD19 has yielded extraordinary clinical responses against some hematological tumors. Solid tumors, however, remain an important challenge to CAR T-cells due to issues of homing, tumor vasculature and stromal barriers, and a range of obstacles in the tumor bed. Protumoral immune infiltrate including T regulatory cells and myeloid-derived suppressor cells have been well characterized for their ability to upregulate inhibitory receptors and molecules that hinder effector T-cells. A critical role for metabolic barriers in the tumor microenvironment (TME) is emerging. High glucose consumption and competition for key amino acids by tumor cells can leave T-cells with insufficient energy and biosynthetic precursors to support activities such as cytokine secretion and lead to a phenotypic state of anergy or exhaustion. CAR T-cell expansion protocols that promote a less differentiated phenotype, combined with optimal receptor design and coengineering strategies, along with immunomodulatory therapies that also promote endogenous immunity, offer great promise in surmounting immunometabolic barriers in the TME and curing solid tumors.