Andrea Schietinger

Priming of naive T cells inside tumors leads to eradication of established tumors

The tumor barrier comprised of nonantigenic stromal cells may contribute to the failure of tumor rejection. The tumor-necrosis factor superfamily member LIGHT (also known as TNFSF-14) is a ligand of stromal cell–expressed lymphotoxin-β receptor and T cell–expressed herpes viral entry mediator (HVEM). Here we show that forced expression of LIGHT in the tumor environment induces a massive infiltration of naive T lymphocytes that correlates with an upregulation of both chemokine production and expression of adhesion molecules. Activation of these infiltrating T cells, possibly through HVEM, leads to the rejection of established, highly progressive tumors at local and distal sites. Our study indicates that targeting the tumor barrier may be an effective strategy for cancer immunotherapy.

Induced sensitization of tumor stroma leads to eradication of established cancer by T cells

Targeting cancer cells, as well as the nonmalignant stromal cells cross-presenting the tumor antigen (Ag), can lead to the complete destruction of well-established solid tumors by adoptively transferred Ag-specific cytotoxic T lymphocytes (CTLs). If, however, cancer cells express only low levels of the Ag, then stromal cells are not destroyed, and the tumor escapes as Ag loss variants. We show that treating well-established tumors expressing low levels of Ag with local irradiation or a chemotherapeutic drug causes sufficient release of Ag to sensitize stromal cells for destruction by CTLs. This was shown directly using high affinity T cell receptor tetramers for visualizing the transient appearance of tumor-specific peptide–MHC complexes on stromal cells. Maximum loading of tumor stroma with cancer Ag occurred 2 d after treatment and coincided with the optimal time for T cell transfer. Under these conditions, tumor rejection was complete. These findings may set the stage for developing rational clinical protocols for combining irradiation or chemotherapy with CTL therapy.

Tolerance and Exhaustion: Defining Mechanisms of T cell Dysfunction

CD8 T cell activation and differentiation are tightly controlled, and dependent on the context in which naïve T cells encounter antigen, can either result in functional memory or T cell dysfunction, including exhaustion, tolerance, anergy, or senescence. With the identification of phenotypic and functional traits shared in different settings of T cell dysfunction, distinctions between such dysfunctional states have become blurred. Here, we discuss distinct states of CD8 T cell dysfunction, with an emphasis on: (i) T cell tolerance to self-antigens (self-tolerance); (ii) T cell exhaustion during chronic infections; and (iii) tumor-induced T cell dysfunction. We highlight recent findings on cellular and molecular characteristics defining these states, cell-intrinsic regulatory mechanisms that induce and maintain them, and strategies that can lead to their reversal.

Rescued Tolerant CD8 T Cells Are Preprogrammed to Reestablish the Tolerant State

Too Much Tolerance? In the immune system, loss of tolerance to self can have devastating consequences, such as the development of autoimmune diseases. In some cases, however, we may wish to be able to break tolerance, for example, to activate immune cells to fight tumors. Schietinger et al. (p. 723, published online 19 January; see the Perspective by Lee and Jameson) used a combination of genetic mouse models and adoptive immune cell transfers to better understand the mechanisms regulating tolerance in T lymphocytes. In contrast to the prevailing paradigm, the maintenance of T lymphocyte tolerance did not require the continuous presence of antigen. Tolerance was able to be broken when previously tolerized cells were placed in an environment depleted of immune cells. However, when lymphocyte numbers were restored, cells were once again tolerized, even in the absence of antigen. These data, together with gene expression profiling, suggest that tolerance is associated with a specific gene expression program that, although possible to override temporarily, is reimposed by epigenetic mechanisms. Maintenance of T cell tolerance is likely regulated by epigenetic mechanisms. Tolerant self-antigen–specific CD8 T cells fail to proliferate in response to antigen, thereby preventing autoimmune disease. By using an in vivo mouse model, we show that tolerant T cells proliferate and become functional under lymphopenic conditions, even in a tolerogenic environment. However, T cell rescue is only transient, with tolerance reimposed upon lymphorepletion even in the absence of tolerogen (self-antigen), challenging the prevailing paradigm that continuous antigen exposure is critical to maintain tolerance. Genome-wide messenger RNA and microRNA profiling revealed that tolerant T cells have a tolerance-specific gene profile that can be temporarily overridden under lymphopenic conditions but is inevitably reimposed, which suggests epigenetic regulation. These insights into the regulatory mechanisms that maintain or break self-tolerance may lead to new strategies for the treatment of cancer and autoimmunity.

Bystander killing of cancer requires the cooperation of CD4+ and CD8+ T cells during the effector phase

Killing of nonmalignant stroma requires cooperation between CD4+ and CD8+ T cells during the effector phase in the tumor microenvironment.

Equilibrium between Host and Cancer Caused by Effector T Cells Killing Tumor Stroma

The growth of solid tumors depends on tumor stroma. A single adoptive transfer of CD8(+) CTLs that recognize tumor antigen-loaded stromal cells, but not the cancer cells because of MHC restriction, caused long-term inhibition of tumor growth. T cells persisted and continuously destroyed CD11b(+) myeloid-derived, F4/80(+) or Gr1(+) stromal cells during homeostasis between host and cancer. Using high-affinity T-cell receptor tetramers, we found that both subpopulations of stromal cells captured tumor antigen from surrounding cancer cells. Epitopes on the captured antigen made these cells targets for antigen-specific T cells. These myeloid stromal cells are immunosuppressive, proangiogenic, and phagocytic. Elimination of these myeloid cells allowed T cells to remain active, prevented neovascularization, and prevented tumor resorption so that tumor size remained stationary. These findings show the effectiveness of adoptive CTL therapy directed against tumor stroma and open a new avenue for cancer treatments.

Chromatin states define tumour-specific T cell dysfunction and reprogramming

Tumour-specific CD8 T cells in solid tumours are dysfunctional, allowing tumours to progress. The epigenetic regulation of T cell dysfunction and therapeutic reprogrammability (for example, to immune checkpoint blockade) is not well understood. Here we show that T cells in mouse tumours differentiate through two discrete chromatin states: a plastic dysfunctional state from which T cells can be rescued, and a fixed dysfunctional state in which the cells are resistant to reprogramming. We identified surface markers associated with each chromatin state that distinguished reprogrammable from non-reprogrammable PD1hi dysfunctional T cells within heterogeneous T cell populations from tumours in mice; these surface markers were also expressed on human PD1hi tumour-infiltrating CD8 T cells. Our study has important implications for cancer immunotherapy as we define key transcription factors and epigenetic programs underlying T cell dysfunction and surface markers that predict therapeutic reprogrammability.

Antibody recognition of a unique tumor-specific glycopeptide antigen

Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the “Tn antigen.” Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen.

Densely Granulated Murine NK Cells Eradicate Large Solid Tumors

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

A sensitivity scale for targeting T cells with chimeric antigen receptors (CARs) and bispecific T-cell Engagers (BiTEs).

Although T cells can mediate potent antitumor responses, immune tolerance mechanisms often result in the deletion or inactivation of T cells that express T-cell receptors (TCRs) against potentially effective target epitopes. Various approaches have been devised to circumvent this problem. In one approach, the gene encoding an antibody against a cancer-associated antigen is linked, in the form of a single-chain variable fragment (scFv), to genes that encode transmembrane and signaling domains. This chimeric antigen receptor (CAR) is then introduced into T cells for adoptive T-cell therapy. In another approach, the anti-cancer scFv is fused to a scFv that binds to the CD3ε subunit of the TCR/CD3 complex. This fusion protein serves as a soluble, injectable product that has recently been termed bispecific T-cell engager (BiTE). Both strategies have now been tested in clinical trials with promising results, but the comparative efficacies are not known. Here, we performed a direct comparison of the in vitro sensitivity of each strategy, using the same anti-cancer scFv fragments, directed against a tumor-specific glycopeptide epitope on the sialomucin-like transmembrane glycoprotein OTS8, which results form a cancer-specific mutation of Cosmc. While both approaches showed specific responses to the epitope as revealed by T cell-mediated cytokine release and target cell lysis, CAR-targeted T cells were more sensitive than BiTE-targeted T cells to low numbers of antigens per cell. The sensitivity scale described here provides a guide to the potential use of these two different approaches.