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To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rβγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led—in the absence of lymphodepletion or exogenous cytokine support—to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.

SummaryInterleukin-2 receptor (IL-2R)-mediated intracellular signaling is a key regulator of T-cell fate decisions. While the potent signals generated by IL-2 engagement execute effector differentiation, elevated metabolic activities and rapid cellular expansion, IL-15 binding induces a stemness/memory phenotype and a quiescent metabolic state. Here, we demonstrate that weak but sustained signaling generated by a non-IL-2Rα-binding variant of IL-2 (IL-2v) drive proliferation/metabolic and stemness transcriptional programs, thereby reprogramming CD8+T cells into a hybrid ‘metabolically active stem-like state’. We further show that IL-2v-induced T cells are capable of superior engraftment, persistence, and tumor control when utilized in adoptive cell therapy. Taken together, our study highlights the ability to fine-tune cytokine engagement of cognate receptors in order to generate therapeutically relevant T-cell states and further reveals the metabolic plasticity of the T-cell memory program.

Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4+ and CD8+ T cells. LDRT elicited predominantly CD4+ cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4+ cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell–infiltrated tumors.Significance:Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4+ effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors.This article is highlighted in the In This Issue feature, p. 1

Supplementary Figure from Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy

Supplementary Data from Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy

Supplementary Table from Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy

Adoptive cell therapy (ACT) usingex vivoexpanded tumor-infiltrating T lymphocytes (TILs) can mediate responses in metastatic melanoma, but long-term efficacy remains limited to a fraction of patients. Here we interrogated tumor-microenvironment (TME) cellular states and interactions of longitudinal samples from 13 metastatic melanoma patients treated with TIL-ACT in our clinical study (NCT03475134). We performed single-cell RNA-seq and spatial proteomic analyses in pre- and post-ACT tumor tissues and showed that responders exhibited higher tumor cell-intrinsic immunogenicity. Also, endogenous CD8+TILs and myeloid cells of responders were characterized by increased cytotoxicity, exhaustion and costimulation and type-I IFN signaling, respectively. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders have rich baseline intratumoral and stromal tumor-reactive T-cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study reveals CD8+T-cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.One-Sentence SummaryResponse to adoptive TIL therapy in melanoma is determined by CD8+TIL-myeloid cell networks

Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. We discovered that NFAT5, an NFAT family member lacking an AP-1 docking site, is highly expressed in exhausted T cells from murine and human tumors and is a central player in tumor-induced exhaustion. While NFAT5 overexpression reduced tumor control, NFAT5 deletion improved tumor control by promoting the accumulation of tumor-specific CD8+ T cells that expressed less TOX and PD-1 and produced more cytokines particularly among precursor exhausted cells. Conversely, NFAT5 had no effect on chronic infection-induced T cell exhaustion. Mechanistically we found that TCR triggering induced NFAT5 expression and that hyperosmolarity stimulated transcriptional activity of NFAT5. We propose that NFAT5 takes over NFAT1/2 to promote exhaustion specifically in tumor-infiltrating CD8+ T cells.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer we show that tumor-specific CD8(+) TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1(+)CD8(+) TIL can be however polyfunctional. PD-1(+) TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8(+) TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APCs. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.