Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy.

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity.

Tumor reactive γδ T cells contribute to a complete response to PD-1 blockade in a Merkel cell carcinoma patient.

Immunotherapies targeting PD-1/PD-L1 are now widely used in the clinic to treat a variety of malignancies. While most of the research on T cell exhaustion and PD-1 blockade has been focused on conventional αß T cells, the contribution of innate-like T cells such as γδ T cells to anti-PD-1/PD-L1 mediated therapy is limited. Here we show that tumor reactive γδ T cells respond to PD-1 blockade in a Merkel cell carcinoma (MCC) patient experiencing a complete response to therapy. We find clonally expanded γδ T cells in the blood and tumor after pembrolizumab treatment, and this Vγ2Vδ1 clonotype recognizes Merkel cancer cells in a TCR-dependent manner. Notably, the intra-tumoral γδ T cells in the MCC patient are characterized by higher expression of PD-1 and TIGIT, relative to conventional CD4 and CD8 T cells. Our results demonstrate that innate-like T cells could also contribute to an anti-tumor response after PD-1 blockade.

Molecular, metabolic, and functional CD4 T cell paralysis in the lymph node impedes tumor control.

CD4 T cells are central effectors of anti-cancer immunity and immunotherapy, yet the regulation of CD4 tumor-specific T (TTS) cells is unclear. We demonstrate that CD4 TTS cells are quickly primed and begin to divide following tumor initiation. However, unlike CD8 TTS cells or exhaustion programming, CD4 TTS cell proliferation is rapidly frozen in place by a functional interplay of regulatory T cells and CTLA4. Together these mechanisms paralyze CD4 TTS cell differentiation, redirecting metabolic circuits, and reducing their accumulation in the tumor. The paralyzed state is actively maintained throughout cancer progression and CD4 TTS cells rapidly resume proliferation and functional differentiation when the suppressive constraints are alleviated. Overcoming their paralysis established long-term tumor control, demonstrating the importance of rapidly crippling CD4 TTS cells for tumor progression and their potential restoration as therapeutic targets.

Molecular, metabolic and functional CD4 T cell paralysis impedes tumor control.

CD4 T cells are important effectors of anti-tumor immunity, yet the regulation of CD4 tumor-specific T (T TS ) cells during cancer development is still unclear. We demonstrate that CD4 T TS cells are initially primed in the tumor draining lymph node and begin to divide following tumor initiation. Distinct from CD8 T TS cells and previously defined exhaustion programs, CD4 T TS cell proliferation is rapidly frozen in place and differentiation stunted by a functional interplay of T regulatory cells and both intrinsic and extrinsic CTLA4 signaling. Together these mechanisms paralyze CD4 T TS cell differentiation, redirecting metabolic and cytokine production circuits, and reducing CD4 T TS cell accumulation in the tumor. Paralysis is actively maintained throughout cancer progression and CD4 T TS cells rapidly resume proliferation and functional differentiation when both suppressive reactions are alleviated. Strikingly, Treg depletion alone reciprocally induced CD4 T TS cells to themselves become tumor-specific Tregs, whereas CTLA4 blockade alone failed to promote T helper differentiation. Overcoming their paralysis established long-term tumor control, demonstrating a novel immune evasion mechanism that specifically cripples CD4 T TS cells to favor tumor progression.

Identification of antigenic epitopes recognized by tumor infiltrating lymphocytes in high grade serous ovarian cancer by multi-omics profiling of the auto-antigen repertoire.

Immunotherapeutic strategies aimed at enhancing tumor cell killing by tumor-specific T cells hold great potential for reducing tumor burden and prolonging survival of cancer patients. Although many potential tumor antigens have been described, identifying relevant targets when designing anti-cancer vaccines or targeted cell therapies remains a challenge. To identify novel, potentially immunogenic candidate tumor antigens, we performed integrated tumor transcriptomic, seromic, and proteomic analyses of high grade serous ovarian cancer (HGSC) patient tumor samples. We identified tumor neo-antigens and over-expressed antigens using whole exome and RNA sequencing and examined these in relation to patient-matched auto-antibody repertoires. Focusing on MHC class I epitopes recognized by CD8+ T cells, HLA-binding epitopes were identified or predicted from the highly expressed, mutated, or auto-antibody target antigen, or MHC-associated peptides (MAPs). Recognition of candidate antigenic peptides was assessed within the tumor-infiltrating T lymphocyte (TIL) population expanded from each patient. Known tumor-associated antigens (TAA) and cancer/testis antigens (CTA) were commonly found in the auto-antibody and MAP repertoires and CD8+ TILs recognizing epitopes from these antigens were detected, although neither expression level nor the presence of auto-antibodies correlated with TIL recognition. Auto-antibodies against tumor-mutated antigens were found in most patients, however, no TIL recognition of the highest predicted affinity neo-epitopes was detected. Using high expression level, auto-antibody recognition, and epitope prediction algorithms, we identified epitopes in 5 novel antigens (MOB1A, SOCS3, TUBB, PRKAR1A, CCDC6) recognized by HGSC patient TILs. Furthermore, selection of epitopes from the MAP repertoire identified 5 additional targets commonly recognized by multiple patient TILs. We find that the repertoire of TIL specificities includes recognition of highly expressed and immunogenic self-antigens that are processed and presented by tumors. These results indicate an ongoing autoimmune response against a range of self-antigens targeted by HGSC TILs.

Rock and sediment dataset of petit-spots in the northwestern Pacific.

Rock and sediment samples were collected from petit-spots in the northwestern Pacific. The sampling was conducted using deep-submergence vehicle (DSV) Shinkai 6500 and its mother ship, research vessel (RV) Yokosuka during YK20-14S and YK21-07S cruises. The collected rock samples are basalt and peperite. Some of the basalts include small mantle xenoliths (∼3 cm in diameter). The dataset of rock and sediment samples from the petit-spots located on >130 Ma northwestern Pacific plate are presented herein. The peperites are a reaction product between petit-spot magma and wet sediment, and the mantle xenoliths are fragmented mantle materials transported by the petit-spot magmas. Therefore, the petit-spot samples are of significant importance to elucidate modification process of the surface condition by petit-spot magma and to characterize the deep lithospheric mantle. The dataset presented herein provides in a sense a unique insight into the whole Pacific plate just before its subduction beneath the Japan arc.

Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

[Development of universal CAR-T cells].

The efficacy of adoptive immunotherapy using CD19-targeting chimeric antigen receptor (CAR)-engineered T cells against B-cell malignancies has already been established in the clinic. However, high economic costs and heterogeneous quality of CAR-T cells derived from individual patients hinder further expansion of their applicability to various cancer types, including solid tumors. Mass CAR-T cell production from healthy donors is a promising approach to overcome these problems, given that allogeneic immunity elicited against donor CAR-T cells by the recipient's immune system is controlled. CAR-T cells genetically ablated with T-cell receptor and human leukocyte antigen molecules, referred to as universal CAR-T cells, may enable the use of allogeneic T cells for off-the-shelf adoptive cancer immunotherapy. However, several concerns, such as poor persistence of infused CAR-T cells and chromosomal abnormalities due to genome editing, remain to be addressed. Thus, recent clinical trials on universal CAR-T cells are summarized and future perspectives to overcome current challenges are discussed in this review.

Pre-encoded responsiveness to type I interferon in the peripheral immune system defines outcome of PD1 blockade therapy.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and 'cold' or 'hot' tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.

Characterization of Proteasome-Generated Spliced Peptides Detected by Mass Spectrometry.

CD8+ T cells recognize peptides displayed by HLA class I molecules and monitor intracellular peptide pools. It is known that the proteasome splices two short peptide fragments. Recent studies using mass spectrometry (MS) and bioinformatics analysis have suggested that proteasome-generated spliced peptides (PSPs) may account for a substantial proportion of HLA class I ligands. However, the authenticity of the PSPs identified using bioinformatics approaches remain ambiguous. In this study, we employed MS-based de novo sequencing to directly capture cryptic HLA ligands that were not templated in the genome. We identified two PSPs originating from the same protein in a human colorectal cancer line with microsatellite instability. Healthy donor-derived CD8+ T cells readily responded to the two PSPs, showing their natural HLA presentation and antigenicity. Experiments using minigene constructs demonstrated proteasome-dependent processing of two PSPs generated by standard and reverse cis splicing, respectively. Our results suggest a broader diversity of HLA class I Ag repertoires generated by proteasomal splicing, supporting the advantage of MS-based approaches for the comprehensive identification of PSPs.

Heterogeneity of Circulating Tumor Cell-Associated Genomic Gains in Breast Cancer and Its Association with the Host Immune Response.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy-number gains. Through FISH, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21% to 69%, median 55.5%, n = 19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70% to 100%, median 93%, samples n = 41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a posttreatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. In addition, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and posttreatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment. SIGNIFICANCE: As breast cancers progress, they become more heterogeneous for multiple regions amplified in circulating tumor cells, and intratumoral spatial heterogeneity is associated with the immune landscape.

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

Changing the landscape of tumor immunology: novel tools to examine T cell specificity.

Immunotherapy has established itself as a stalwart arm in patient care and with precision medicine forms the new paradigm in cancer treatment. T cells are an important group of immune cells capable of potent cancer immune surveillance and immunity. The advent of bioinformatics, particularly more recent advances incorporating algorithms employing machine learning, provide a seemingly limitless ability for T cell analysis and hypothesis generation. Such endeavors have become indispensable to research efforts accelerating and evolving to such an extent that there exists an appreciable gap between knowledge and proof of function and application. Exciting new technologies such as DNA barcoding, cytometry by time-of-flight (CyTOF), and peptide-exchangeable pHLA multimers inclusive of rare and difficult HLA alleles offer high-throughput cell-by-cell analytical capabilities. These outstanding recent contributions to T cell research will help close this gap and potentially bring practical benefit to patients.

Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.

Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma.

HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden.

The immune system is the body's way of defending itself, offering protection against diseases such as cancer. But to remove the cancer cells, the immune system must be able to identify them as different from the rest of the body. All cells break down proteins into shorter fragments, known as peptides, that are displayed on the cell surface by a protein called human leukocyte antigen, HLA for short. Cancer cells display distinctive peptides on their surface as they generate different proteins to those of healthy cells. Immune cells called T cells use these abnormal peptides to identify the cancer so that it can be destroyed. Sometimes T cells can lack the right equipment to detect abnormal peptides, allowing cancer cells to hide from the immune system. However, T cells can be trained through a treatment called immunotherapy, which provides T cells with new tools so that they can spot the peptides displayed by HLA on the previously 'hidden' cancer cells. There are many different forms of HLA, each of which can display different peptides. Current research in immunotherapy commonly targets only a subset of HLA forms, and not all cancer patients have these types. This means that immunotherapy research is only likely to be of most benefit to a limited number of patients. Immunotherapy could be made effective for more people if new cancer peptides that are displayed by the other 'under-represented' forms of HLA were identified. Murata, Nakatsugawa et al. have now used T cells that were taken from tumors in eight patients with melanoma, which is a type of skin cancer. A library of fluorescent HLA-peptides was generated ­ using a new, simplified methodology ­ with 25 forms of HLA that displayed over 800 peptides. T cells were then mixed with the library to identify which HLA-peptides they can target. As a result, Murata, Nakatsugawa et al. found the cancer targets of around 12% of the tumor-infiltrating T cells tested, including those from under-represented forms of HLA. Consequently, these findings could be used to develop new immunotherapies that can treat more patients.

Melting of recycled ancient crust responsible for the Gutenberg discontinuity.

A discontinuity in the seismic velocity associated with the lithosphere-asthenosphere interface, known as the Gutenberg discontinuity, is enigmatic in its origin. While partial mantle melts are frequently suggested to explain this discontinuity, it is not well known which factors critically regulate the melt production. Here, we report geochemical evidence showing that the melt fractions in the lithosphere-asthenosphere boundary were enhanced not only by accumulation of compacted carbonated melts related to recycled ancient marine sediments, but also by partial melting of a pyroxene-rich mantle domain related to the recycled oceanic eclogite/pyroxenites. This conclusion is derived from the first set of Mg isotope data for a suite of young petit-spot basalts erupted on the northwest Pacific plate, where a clearly defined Gutenberg discontinuity exists. Our results reveal a specific linkage between the Gutenberg discontinuity beneath the normal oceanic regions and the recycling of ancient subducted crust and carbonate through the deep Earth.

Gene Modification and Immunological Analyses for the Development of Immunotherapy Utilizing T Cells Redirected with Antigen-Specific Receptors.

Cancer immunotherapy has been developed and established as a new treatment modality. Recently, adoptive transfer therapy using T cells redirected with antigen-specific antitumor receptors, such as T-cell receptor (TCR) and chimeric antigen receptor (CAR), has demonstrated clinical benefits even in patients with refractory malignancies. To advance this treatment modality, both generation of gene-modified T cells and evaluation of their reactivity with high quality in vitro are required. To achieve this, it is important to establish the ways (1) to generate optimal viral particle for T-cell transduction, (2) to transduce antitumor receptors into T cells and expand redirected T cells efficiently, and (3) to assess the functionality of antigen-specific gene-modified T cells precisely. Here, we summarize established protocols to generate and analyze antitumor receptor-transduced T cells. These procedures help to further assess characteristics of gene-modified T cells, resulting in promotion of translational research for cancer immunotherapy.

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials.

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials.

Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87.

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPß chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.

Phase II clinical trial of adoptive cell therapy for patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and low-dose interleukin-2.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown significant clinical benefit, but is limited by toxicities due to a requirement for post-infusion interleukin-2 (IL-2), for which high dose is standard. To assess a modified TIL protocol using lower dose IL-2, we performed a single institution phase II protocol in unresectable, metastatic melanoma. The primary endpoint was response rate. Secondary endpoints were safety and assessment of immune correlates following TIL infusion. Twelve metastatic melanoma patients were treated with non-myeloablative lymphodepleting chemotherapy, TIL, and low-dose subcutaneous IL-2 (125,000 IU/kg/day, maximum 9-10 doses over 2 weeks). All but one patient had previously progressed after treatment with immune checkpoint inhibitors. No unexpected adverse events were observed, and patients received an average of 6.8 doses of IL-2. By RECIST v1.1, two patients experienced a partial response, one patient had an unconfirmed partial response, and six had stable disease. Biomarker assessment confirmed an increase in IL-15 levels following lymphodepleting chemotherapy as expected and a lack of peripheral regulatory T-cell expansion following protocol treatment. Interrogation of the TIL infusion product and monitoring of the peripheral blood following infusion suggested engraftment of TIL. In one responding patient, a population of T cells expressing a T-cell receptor Vß chain that was dominant in the infusion product was present at a high percentage in peripheral blood more than 2 years after TIL infusion. This study shows that this protocol of low-dose IL-2 following adoptive cell transfer of TIL is feasible and clinically active. (ClinicalTrials.gov identifier NCT01883323.).