Oxygen Sensing by T Cells Establishes an Immunologically Tolerant Metastatic Niche.

Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T-cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4(+)-regulatory T (Treg) cell induction, and restrain CD8(+) T cell effector function. Tumor colonization is accompanied by PHD-protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. T-cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. PAPERCLIP.

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

The utility of intraoperative marrow margin frozen section in extremity bone sarcoma resection.

BACKGROUND AND OBJECTIVES: Intraoperative frozen section analysis is commonly used to evaluate marrow margins during extremity bone sarcoma resections, but its efficacy in the era of magnetic resonance imaging is debated. This study aimed to compare the accuracy of intraoperative frozen section assessment with final pathology, assess its correlation with gross intraoperative margin assessment, and evaluate its impact on surgical decision making. METHODS: Consecutive patients undergoing extremity bone sarcoma resections from 2010 to 2022 at a single sarcoma center were included. Intraoperative frozen section and gross margin assessments were compared to final pathology using positive predictive values (PPV) and negative predictive values (NPV). Changes in surgical decisions due to positive intraoperative margins were recorded. RESULTS: Of 166 intraoperative frozen section marrow margins, four were indeterminant/positive, with two false positive/indeterminant findings and two false negatives compared to final pathology. Gross intraoperative assessment had no false positives and two false negatives. Frozen section analysis yielded a PPV of 50% (95% confidence interval [CI]: 16%-84%) and NPV of 98.8% (95% CI: 97%-100%), while gross assessment had a PPV of 100% (95% CI: 16%-100%) and NPV of 98.8% (95% CI: 97%-100%). Positive frozen section margins led to additional resections in three of four cases. CONCLUSIONS: Intraoperative frozen section analysis did not offer added clinical value beyond gross margin assessment in extremity bone sarcoma resections. It exhibited a low PPV and led to unnecessary additional resections. Gross intraoperative assessment proved adequate for margin evaluation, potentially saving time and resources.

The Role of Routine Pathologic Assessment After Pediatric Osteochondroma Excision.

BACKGROUND: Osteochondromas are benign osseous lesions often excised for pain, growth abnormalities, and aesthetic concerns. While characteristic clinical and radiographic features leave little diagnostic ambiguity in most cases of osteochondroma, pathologic analysis to confirm the diagnosis and screen for malignancy is routinely performed following surgical excision. The purpose of this study was to determine the clinical and economic value of routine pathologic analysis after osteochondroma excision in a pediatric population. METHODS: A retrospective review of clinical records from 2 pediatric orthopaedic hospitals (St. Louis Children's Hospital and Shriner's Hospital for Children, St. Louis) identified 426 osteochondroma lesions surgically resected from 201 patients. Patients with solitary and multiple lesions were included. Clinical, radiographic, and surgical data were recorded for each resection surgery. Pathologic reports were evaluated. Costs incurred for routine pathologic assessment was also noted. RESULTS: Totally, 132 patients were treated with surgical resection of a solitary osteochondroma lesion, while an additional 291 lesions were resected from 69 patients with multiple lesions. Average age at the time of surgical resection was 13.0 years (2.1 to 17.9). The most common anatomic locations of excised lesions included the distal femur (110, 25.8%), proximal tibia/fibula (95, 22.3%), and distal radius/ulna (58, 13.6%). All resected specimens were sent for pathologic analysis. The average size of the resected lesions was 19.9 mm 3 (0.02 to 385.0 mm 3 ). In all cases, the histologic diagnosis confirmed benign osteochondroma. The total charges of pathologic analysis including processing and interpretation fees was ∼$755.00 for each lesion assessed, for a total cohort charge of $321,630. CONCLUSION: We propose that in most cases of pediatric osteochondroma excision procedures, postoperative histologic analysis is not strictly indicated as it rarely, if ever, alters diagnosis or management. We suggest using a "gross only" analysis in these cases. However, we do believe that with preoperative diagnostic ambiguity, or if patients present with concerning features such as rapidly expansile lesions or cortical destruction, have axial skeleton or pelvic involvement, or enlarged cartilaginous caps, full histologic evaluation of the excised lesions will continue to be prudent. LEVEL OF EVIDENCE: Level IV-case series.

Ionic immune suppression within the tumour microenvironment limits T cell effector function.

Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.

Pilot Study Analysis of Serum Cytokines to Differentiate Pediatric Septic Arthritis and Transient Synovitis.

BACKGROUND: In pediatric patients, the presentation of the nontraumatic acutely painful joint/limb poses a diagnostic dilemma due to the similarity of presentations of the most likely diagnoses [septic arthritis (SA), transient synovitis (TS), osteomyelitis]. Current tools employed to differentiate these diagnoses rely on nonspecific inflammatory markers, radiologic imaging, and arthrocentesis. Diagnostic algorithms utilizing these clinical, radiographic, and biochemical parameters have produced conflicting results. The purpose of this study was to identify a serum-based inflammatory signature which can differentiate SA from TS in pediatric patients. METHODS: Serum samples were collected from 22 pediatric patients presenting with joint/extremity pain whose working diagnosis included SA or TS. Each sample was analyzed for serum abundance of 72 distinct biomarkers and cytokines using enzyme linked immunosorbent assay based arrays. Linear discriminant analysis was performed to identify a combinatorial biomarker panel to predict a diagnosis of SA or TS. Efficacy of the biomarker panel was compared with definitive diagnoses as based on laboratory tests, arthrocentesis results, and clinical scenario. RESULTS: At the time of presentation: (1) mean erythrocyte sedimentation rate in the SA group was 56.6 mm/h and 12.4 mm/h in the TS group (P<0.001), (2) mean C-reactive protein was 55.9 mg/dL in the SA group and 13.7 mg/dL in the TS group (P=0.12), and (3) mean white blood cell was 10.9 k/mm3 in the SA group and 11.0 k/mm3 in the TS group (P=0.95). A combined panel of 72 biomarkers was examined using discriminant analysis to identify a limited set of predictors which could accurately predict whether a patient was diagnosed with SA or TS. A diagnostic algorithm consisting of transforming growth factor alpha, interleukin (IL)-7, IL-33, and IL-28A serum concentration correctly classified 20 of the 22 cases with a sensitivity and specificity of 90.9% (95% confidence interval: 73.9%-100.0%). CONCLUSION: This study identifies a novel serum-based 4-cytokine panel that accurately differentiates SA from TS in pediatric patients with joint/limb pain. LEVEL OF EVIDENCE: Level II-diagnostic study.

BACH2 represses effector programs to stabilize T(reg)-mediated immune homeostasis.

Through their functional diversification, distinct lineages of CD4(+) T cells can act to either drive or constrain immune-mediated pathology. Transcription factors are critical in the generation of cellular diversity, and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage commitment. Genetic polymorphisms within a single locus encoding the transcription factor BACH2 are associated with numerous autoimmune and allergic diseases including asthma, Crohn's disease, coeliac disease, vitiligo, multiple sclerosis and type 1 diabetes. Although these associations point to a shared mechanism underlying susceptibility to diverse immune-mediated diseases, a function for BACH2 in the maintenance of immune homeostasis has not been established. Here, by studying mice in which the Bach2 gene is disrupted, we define BACH2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programs of multiple effector lineages in CD4(+) T cells. BACH2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg-cell-dependent. Assessment of the genome-wide function of BACH2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, BACH2 constrained full effector differentiation within TH1, TH2 and TH17 cell lineages. These findings identify BACH2 as a key regulator of CD4(+) T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity.

miR-155 augments CD8+ T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic γc cytokines.

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic γc cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.

Reprogramming antitumor immunity.

Regenerative medicine holds great promise in replacing tissues and organs lost to degenerative disease and injury. Application of the principles of cellular reprogramming for the treatment of cancer, however, is not well established. Here, we present an overview of cellular reprogramming techniques used in regenerative medicine, and within this context, envision how the scope of regenerative medicine may be expanded to treat metastatic cancer by revitalizing an exhausted and senescent immune system.

Cancer-associated fibroblast-derived Dickkopf-1 suppresses NK cell cytotoxicity in breast cancer.

Breast cancer is poorly immunogenic, hence able to evade T cell recognition and respond poorly to immune checkpoint blockade. Breast cancer cells can also evade NK cell-mediated immune surveillance, but the mechanism remains enigmatic. Dickkopf-1 (DKK1) is a Wnt/b-catenin inhibitor, whose levels are increased in breast cancer patients and correlate with reduced overall survival. DKK1 is expressed by cancer-associated fibroblasts (CAFs) in orthotopic breast tumors and patient samples, and at higher levels by bone cells. While bone-derived DKK1 contributes to the systemic elevation of DKK1 in tumor-bearing mice, CAFs represent the primary source of DKK1 at the tumor site. Systemic or bone-specific DKK1 targeting reduces primary tumor growth. Intriguingly, specific deletion of CAF-derived DKK1 also limits breast cancer progression, regardless of its elevated levels in circulation and in the bone. DKK1 does not support tumor proliferation directly but rather suppresses the activation and tumoricidal activity of NK cells. Importantly, increased DKK1 levels and reduced number of cytotoxic NK cells are detected in breast cancer patients with progressive bone metastases compared to those with stable disease. Our findings indicate that DKK1 creates a tumor-supporting environment through the suppression of NK cells in breast cancer.

Mapping the Course of Recovery Following Limb-Salvage Surgery for Soft-Tissue Sarcoma of the Extremities.

BACKGROUND: Despite the goal of an acceptable functional result, the surgical treatment of soft-tissue sarcoma can portend a prolonged course of recovery. More comprehensive data on the expected course of recovery following extremity sarcoma surgery are needed to help to inform physicians and patients. The purpose of the present study was to describe the typical course of functional recovery following limb-salvage resection of a soft-tissue sarcoma and to identify factors associated with a delayed postoperative course of recovery. METHODS: A retrospective review of a prospectively maintained institutional database was performed for all patients undergoing surgical treatment with limb salvage of a soft-tissue sarcoma of the extremities or pelvis with at least 1 year of follow-up after the definitive surgical procedure. All patients were required to have preoperative functional outcomes recorded for either the Toronto Extremity Salvage Score (TESS) or the Musculoskeletal Tumor Society (MSTS) score and functional outcome measures at 1 year postoperatively. The primary outcome measures were time to recovery and maximal functional improvement. RESULTS: In this study, 916 patients met inclusion criteria following surgical resection of a soft-tissue sarcoma of the extremities. The median follow-up was 74 months. Patients typically achieved a return to their baseline preoperative level of function for all functional outcome measures by 1 to 2 years and achieved maximal functional recovery by 2 years postoperatively. Older age, female sex, deep tumor location, larger tumor size, pelvic location, osseous resection, motor nerve resection, free and/or rotational soft-tissue coverage, and postoperative complications were independently associated with worse TESS and/or MSTS scores (p ≤ 0.05). Tumor recurrence was associated with worse functional outcomes scores. An analysis was performed to determine which patients had a prolonged course of recovery (i.e., were considered to still be recovering). Older age, female sex, larger tumor size, osseous resection, and motor nerve resection were associated with a delayed course of recovery (p ≤ 0.04). Complications and tumor recurrence were associated with delayed functional recovery across all domains. CONCLUSIONS: Most patients will achieve maximal recovery by 2 to 3 years following surgical resection for soft-tissue sarcoma of the extremities. Older age, female sex, larger tumor size, osseous resection, motor nerve resection, postoperative complications, and tumor recurrence portend poorer functional outcomes and a delayed course of recovery. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect.

Multiple myeloma (MM) patients who receive killer cell Ig-like receptor (KIR) ligand-mismatched, T cell-depleted, allogeneic transplantation may have a reduced risk of relapse compared with patients who receive KIR ligand-matched grafts, suggesting the importance of this signaling axis in the natural killer (NK) cell-versus-MM effect. Expanding on this concept, IPH2101 (1-7F9), an anti-inhibitory KIR mAb, enhances NK-cell function against autologous MM cells by blocking the engagement of inhibitory KIR with cognate ligands, promoting immune complex formation and NK-cell cytotoxicity specifically against MM cell targets but not normal cells. IPH2101 prevents negative regulatory signals by inhibitory KIR, whereas lenalidomide augments NK-cell function and also appears to up-regulate ligands for activating NK-cell receptors on MM cells. Lenalidomide and a murine anti-inhibitory NK-cell receptor Ab mediate in vivo rejection of a lenalidomide-resistant tumor. These mechanistic, preclinical data support the use of a combination of IPH2101 and lenalidomide in a phase 2 trial for MM.

The Effect of Social Deprivation on Fracture-Healing and Patient-Reported Outcomes Following Intramedullary Nailing of Tibial Shaft Fractures.

BACKGROUND: Social deprivation is a state marked by limited access to resources due to poverty, discrimination, or other marginalizing factors. We investigated the links between social deprivation and orthopaedic trauma, including patient-reported outcomes, radiographic healing, and complication rates following intramedullary nailing of tibial shaft fractures. METHODS: We retrospectively reviewed 229 patients who underwent intramedullary nailing of tibial shaft fractures at our Level-I trauma center. The Area Deprivation Index (ADI), a validated proxy for social deprivation, was used to group patients into the most deprived tercile (MDT), the intermediate deprived tercile (IDT), and the least deprived tercile (LDT) for outcome comparison. The Patient-Reported Outcomes Measurement Information System (PROMIS) was used to measure the domains of Physical Function (PF), Pain Interference (PI), Anxiety, and Depression, and radiographic healing was assessed with the Radiographic Union Scale in Tibial fractures (RUST) system. RESULTS: On univariate analyses, patients from the MDT reported worse PF, PI, Anxiety, and Depression scores than those from the LDT within the first year of postoperative follow-up. On multivariable regression analysis, PROMIS score outcomes were influenced by age, race, and smoking status, but not by social deprivation tercile. Furthermore, residing in the MDT was associated with a 31% increase in time to radiographic union compared with the LDT (ß = 0.27; p = 0.01). CONCLUSIONS: Following intramedullary nailing of tibial shaft fractures, social deprivation is associated with slower fracture-healing and potentially influences short-term PROMIS scores. These results warrant further investigation in additional patient populations with orthopaedic trauma and highlight the importance of developing interventions to reduce inequities faced by patients from low-resource settings. LEVEL OF EVIDENCE: Prognostic Level III . See Instructions for Authors for a complete description of levels of evidence.

In-Hospital Morbidity and Mortality With Delays in Femoral Shaft Fracture Fixation.

OBJECTIVES: To investigate trends in the timing of femur fracture fixation in trauma centers in the United States, identify predictors for delayed treatment, and analyze the association of timing of fixation with in-hospital morbidity and mortality using data from the National Trauma Data Bank. METHODS: Patients with femoral shaft fractures treated from 2007 to 2015 were identified from the National Trauma Data Bank and grouped by timing of femur fixation: <24, 24-48 hours, and >48 hours after hospital presentation. The primary outcome measure was in-hospital postoperative mortality rate. Secondary outcomes included complication rates, hospital length of stay (LOS), days spent in the intensive care unit LOS (ICU LOS), and days on a ventilator. RESULTS: Among the 108,825 unilateral femoral shaft fractures identified, 74.2% was fixed within 24 hours, 16.5% between 24 and 48 hours, and 9.4% >48 hours. The mortality rate was 1.6% overall for the group. When fixation was delayed >48 hours, patients were at risk of significantly higher mortality rate [odds ratio (OR) 3.60; 95% confidence interval (CI), 3.13-4.14], longer LOS (OR 2.14; CI 2.06-2.22), longer intensive care unit LOS (OR 3.92; CI 3.66-4.20), more days on a ventilator (OR 5.38; CI 4.89-5.91), and more postoperative complications (OR 2.05; CI 1.94-2.17; P < 0.0001). CONCLUSIONS: Our study confirms that delayed fixation of femoral shaft fractures is associated with increased patient morbidity and mortality. Patients who underwent fixation >48 hours after presentation were at the greatest risk of increased morbidity and mortality. Although some patients require optimization/resuscitation before fracture fixation, efforts should be made to expedite operative fixation. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Effective Treatment of Established Bone Metastases Can Be Achieved by Combinatorial Osteoclast Blockade and Depletion of Granulocytic Subsets.

Osteoclast (OC) blockade has been successful in reducing tumor growth in bone in preclinical settings, but antiresorptive drugs, such as zoledronic acid (ZA), fail to improve the overall survival rate of patients with bone metastasis despite ameliorating skeletal complications. To address this unmet clinical need, we interrogated what other cells modulated tumor growth in bone in addition to OCs. Because myeloid-derived suppressor cells (MDSC)-heterogeneous populations expressing CD11b, Ly6C, and Ly6G markers-originate in the bone marrow and promote tumor progression, we hypothesized that their accumulation hinders ZA antitumor effects. By using a murine model of bone metastasis insensitive to OC blockade, we assessed the antitumor effect of MDSC depletion using anti-Gr1 in mice bearing skeletal lung [Lewis lung carcinoma (LLC)], melanoma (B16-F10), and mammary (4T1) tumors. Differently from soft tissue tumors, anti-Gr1 did not reduce bone metastases and led to the paradoxical accumulation of bone marrow-resident CD11b+Ly6CintLy6Gint cells that differentiated into OCs when cultured in vitro Anti-Gr1-mediated depletion of Ly6G+ granulocytic MDSCs combined with ZA-induced OC blockade reduced growth of established skeletal metastases compared with each agent alone. CD15+ granulocytic populations were increased in patients with breast cancer with progressive bone disease after antiresorptive treatment compared with those with stable bone disease. We provide evidence that antiresorptive therapies fail to reduce bone metastases in the presence of elevated granulocytic populations and that effective treatment of established skeletal metastases requires combinatorial depletion of granulocytes and OC blockade.

Novel "Elements" of Immune Suppression within the Tumor Microenvironment.

Adaptive evolution has prompted immune cells to use a wide variety of inhibitory signals, many of which are usurped by tumor cells to evade immune surveillance. Although tumor immunologists often focus on genes and proteins as mediators of immune function, here we highlight two elements from the periodic table-oxygen and potassium-that suppress the immune system in previously unappreciated ways. While both are key to the maintenance of T-cell function and tissue homeostasis, they are exploited by tumors to suppress immuno-surveillance and promote metastatic spread. We discuss the temporal and spatial roles of these elements within the tumor microenvironment and explore possible therapeutic interventions for effective and promising anticancer therapies. Cancer Immunol Res; 5(6); 426-33. ©2017 AACR.

Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy.

Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR-modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.

The transcription factor BACH2 promotes tumor immunosuppression.

The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy.

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.