Salvage therapy expands highly cytotoxic and metabolically fit resilient CD8+ T cells via ME1 up-regulation.

Patients with advanced cancers who either do not experience initial response to or progress while on immune checkpoint inhibitors (ICIs) receive salvage radiotherapy to reduce tumor burden and tumor-related symptoms. Occasionally, some patients experience substantial global tumor regression with a rebound of cytotoxic CD8+ T cells. We have termed the rebound of cytotoxic CD8+ T cells in response to salvage therapy as T cell resilience and examined the underlying mechanisms of resilience. Resilient T cells are enriched for CX3CR1+ CD8+ T cells with low mitochondrial membrane potential, accumulate less reactive oxygen species (ROS), and express more malic enzyme 1 (ME1). ME1 overexpression enhanced the cytotoxicity and expansion of effector CD8+ T cells partially via the type I interferon pathway. ME1 also increased mitochondrial respiration while maintaining the redox state balance. ME1 increased the cytotoxicity of peripheral lymphocytes from patients with advanced cancers. Thus, preserved resilient T cells in patients rebound after salvage therapy and ME1 enhances their resiliency.

A Novel PD-L1 Antibody Promotes Antitumor Function of Peripheral Cytotoxic Lymphocytes after Radical Nephrectomy in Patients with Renal Cell Carcinoma.

The intrinsic and acquired resistance to PD-1/PD-L1 immune checkpoint blockade is an important challenge for patients and clinicians because no reliable tool has been developed to predict individualized response to immunotherapy. In this study, we demonstrate the translational relevance of an ex vivo functional assay that measures the tumor cell killing ability of patient-derived CD8 T and NK cells (referred to as "cytotoxic lymphocytes," or CLs) isolated from the peripheral blood of patients with renal cell carcinoma. Patient-derived PBMCs were isolated before and after nephrectomy from patients with renal cell carcinoma. We compared the efficacy of U.S. Food and Drug Administration (FDA)-approved PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, atezolizumab) and a newly developed PD-L1 inhibitor (H1A Ab) in eliciting cytotoxic function. CL activity was improved at 3 mo after radical nephrectomy compared with baseline, and it was associated with higher circulating levels of tumor-reactive effector CD8 T cells (CD11ahighCX3CR1+GZMB+). Treatment of PBMCs with FDA-approved PD-1/PD-L1 inhibitors enhanced tumor cell killing activity of CLs, but a differential response was observed at the individual-patient level. H1A demonstrated superior efficacy in promoting CL activity compared with FDA-approved PD-1/PD-L1 inhibitors. PBMC immunophenotyping by mass cytometry revealed enrichment of effector CD8 T and NK cells in H1A-treated PBMCs and immunosuppressive regulatory T cells in atezolizumab-treated samples. Our study lays the ground for future investigation of the therapeutic value of H1A as a next-generation immune checkpoint inhibitor and the potential of measuring CTL activity in PBMCs as a tool to predict individual response to immune checkpoint inhibitors in patients with advanced renal cell carcinoma.

A Novel Humanized PD-1/PD-L1 Mouse Model Permits Direct Comparison of Antitumor Immunity Generated by Food and Drug Administration-Approved PD-1 and PD-L1 Inhibitors.

Seven different anti-PD-1 and PD-L1 mAbs are now widely used in the United States to treat a variety of cancer types, but no clinical trials have compared them directly. Furthermore, because many of these Abs do not cross-react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by Food and Drug Administration-approved Abs. To do this, we performed an in vivo T cell priming assay in which anti-PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor Ag (OVA), followed by subsequent B16-OVA tumor challenge. Surprisingly, both control and Ab-treated mice formed an equally robust OVA-specific T cell response at the time of priming. Despite this, anti-PD-1/L1-treated mice exhibited significantly better tumor rejection versus controls, with avelumab generating the best protection. To determine what could be mediating this, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the avelumab-treated mice, the same phenotype of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of Food and Drug Administration-approved anti-PD-1/L1 mAbs and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for optimizing and better utilizing anti-PD-1/L1 therapeutics.

Resilient T-cell responses in patients with advanced cancers.

Although cancer burden in patients with advanced disease results in many failed prior therapies, some patients still achieve durable responses to immunotherapy implying that remnant and resilient cytotoxic T cells are present in these responders. Since patients with more resilient T cells are likely to benefit from immunotherapy, it will be important to determine how resilient T cells in patients can be identified and to define the mechanisms by which tumor-reactive resilient T cells can be generated. In this review, we summarized recent advances in research on resilient T cells in patients with advanced cancers and proposed future research directions. From there, we expect to leverage this knowledge to generate or expand the resilient T cells in patients who do not respond to initial immunotherapy and convert them into responders.

NKG7 Is a T-cell-Intrinsic Therapeutic Target for Improving Antitumor Cytotoxicity and Cancer Immunotherapy.

Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti-PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell-mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti-PD-1 or anti-PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen-specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell-intrinsic therapeutic target for enhancing cancer immunotherapy.See related article by Li et al., p. 154.

Chimeric Antigen Receptor T Cells Targeting NKG2D-Ligands Show Robust Efficacy Against Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia.

CAR T cell approaches to effectively target AML and T-ALL without off-tumor effects on healthy myeloid or T cell compartments respectively are an unmet medical need. NKG2D-ligands are a promising target given their absence on healthy cells and surface expression in a wide range of malignancies. NKG2D-ligand expression has been reported in a substantial group of patients with AML along with evidence for prognostic significance. However, reports regarding the prevalence and density of NKG2D-ligand expression in AML vary and detailed studies to define whether low level expression is sufficient to trigger NKG2D-ligand directed CART cell responses are lacking. NKG2D ligand expression in T-ALL has not previously been interrogated. Here we report that NKG2D-ligands are expressed in T-ALL cell lines and primary T-ALL. We confirm that NKG2D-ligands are frequently surface expressed in primary AML, albeit at relatively low levels. Utilizing CAR T cells incorporating the natural immune receptor NKG2D as the antigen binding domain, we demonstrate striking in vitro activity of CAR T cells targeting NKG2D-ligands against AML and T-ALL cell lines and show that even low-level ligand expression in primary AML targets results in robust NKG2D-CAR activity. We found that NKG2D-ligand expression can be selectively enhanced in low-expressing AML cell lines and primary AML blasts via pharmacologic HDAC inhibition. Such pharmacologic NKG2D-ligand induction results in enhanced NKG2D-CAR anti-leukemic activity without affecting healthy PBMC, thereby providing rationale for the combination of HDAC-inhibitors with NKG2D-CAR T cell therapy as a potential strategy to achieve clinical NKG2D-CAR T cell efficacy in AML.

Phase I Trial of Autologous CAR T Cells Targeting NKG2D Ligands in Patients with AML/MDS and Multiple Myeloma.

NKG2D ligands are widely expressed in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. We conducted a phase I dose-escalation study to evaluate the safety and feasibility of a single infusion of NKG2D-chimeric antigen receptor (CAR) T cells, without lymphodepleting conditioning in subjects with acute myeloid leukemia/myelodysplastic syndrome or relapsed/refractory multiple myeloma. Autologous T cells were transfected with a γ-retroviral vector encoding a CAR fusing human NKG2D with the CD3ζ signaling domain. Four dose levels (1 × 106-3 × 107 total viable T cells) were evaluated. Twelve subjects were infused [7 acute myeloid leukemia (AML) and 5 multiple myeloma]. NKG2D-CAR products demonstrated a median 75% vector-driven NKG2D expression on CD3+ T cells. No dose-limiting toxicities, cytokine release syndrome, or CAR T cell-related neurotoxicity was observed. No significant autoimmune reactions were noted, and none of the ≥ grade 3 adverse events were attributable to NKG2D-CAR T cells. At the single injection of low cell doses used in this trial, no objective tumor responses were observed. However, hematologic parameters transiently improved in one subject with AML at the highest dose, and cases of disease stability without further therapy or on subsequent treatments were noted. At 24 hours, the cytokine RANTES increased a median of 1.9-fold among all subjects and 5.8-fold among six AML patients. Consistent with preclinical studies, NKG2D-CAR T cell-expansion and persistence were limited. Manufactured NKG2D-CAR T cells exhibited functional activity against autologous tumor cells in vitro, but modifications to enhance CAR T-cell expansion and target density may be needed to boost clinical activity.

Paradox-driven adventures in the development of cancer immunology and immunotherapy.

After more than one hundred years of documented trials, immunotherapy has become a standard of care in the treatment of human cancer. Much of the knowledge that led to recent breakthroughs seems quite logical from today's point of view. However, what we now cite as facts were originally considered paradoxes, meaning something contrary to expectations or perceived opinion at the time. In order to make gains in the field of immunotherapy, one had to be willing to confront ideas and concepts that seemed to contradict one another, and reconcile how each could be true. This is what led to new knowledge and advances. Here, we highlight some of these paradoxes and the milestone discoveries that followed, each one critical for our understanding of immune checkpoint pathways. By outlining some of the steps that we took and the challenges that we overcame, we hope to inspire and encourage future generations of researchers to confront the paradoxes that still permeate the field.