Epigenomic-Guided Mass Cytometry Profiling Reveals Disease-Specific Features of Exhausted CD8 T Cells.

Exhausted CD8 T (Tex) cells are immunotherapy targets in chronic infection and cancer, but a comprehensive assessment of Tex cell diversity in human disease is lacking. Here, we developed a transcriptomic- and epigenetic-guided mass cytometry approach to define core exhaustion-specific genes and disease-induced changes in Tex cells in HIV and human cancer. Single-cell proteomic profiling identified 9 distinct Tex cell clusters using phenotypic, functional, transcription factor, and inhibitory receptor co-expression patterns. An exhaustion severity metric was developed and integrated with high-dimensional phenotypes to define Tex cell clusters that were present in healthy subjects, common across chronic infection and cancer or enriched in either disease, linked to disease severity, and changed with HIV therapy. Combinatorial patterns of immunotherapy targets on different Tex cell clusters were also defined. This approach and associated datasets present a resource for investigating human Tex cell biology, with implications for immune monitoring and immunomodulation in chronic infections, autoimmunity, and cancer.

Neoadjuvant Gene-Mediated Cytotoxic Immunotherapy for Non-Small-Cell Lung Cancer: Safety and Immunologic Activity.

Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies.

CAR T Cell Therapy for Solid Tumors.

The field of cancer immunotherapy has been re-energized by the application of chimeric antigen receptor (CAR) T cell therapy in cancers. These CAR T cells are engineered to express synthetic receptors that redirect polyclonal T cells to surface antigens for subsequent tumor elimination. Many CARs are designed with elements that augment T cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematologic malignancies (e.g., CD19 CARs in leukemias). However, this success has yet to be extrapolated to solid tumors, and the reasons for this are being actively investigated. We characterize some of the challenges that CAR T cells have to surmount in the solid tumor microenvironment and new approaches that are being considered to overcome these hurdles.

Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection.

Using a model of lethal oral infection with Toxoplasma gondii, we examined the fate of both induced and natural regulatory T (Treg) cells in the face of strong inflammatory responses occurring in a tolerogenic-prone environment. We found that during highly T helper 1 (Th1) cell-polarized mucosal immune responses, Treg cell numbers collapsed via multiple pathways, including blockade of Treg cell induction and disruption of endogenous Treg cell homeostasis. In particular, shutdown of interleukin 2 (IL-2) in the highly Th1 cell-polarized environment triggered by infection directly contributes to Treg cell incapacity to suppress effector responses and eventually leads to immunopathogenesis. Furthermore, we found that environmental cues provided by both local dendritic cells and effector T cells can induce the expression of T-bet transcription factor and IFN-gamma by Treg cells. These data reveal a mechanism for Th1 cell pathogenicity that extends beyond their proinflammatory program to limit Treg cell survival.

HDAC5 controls the functions of Foxp3(+) T-regulatory and CD8(+) T cells.

Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.

Ikaros imposes a barrier to CD8+ T cell differentiation by restricting autocrine IL-2 production.

Naive CD4(+) T cells require signals from the TCR and CD28 to produce IL-2, expand, and differentiate. However, these same signals are not sufficient to induce autocrine IL-2 production by naive CD8(+) T cells, which require cytokines provided by other cell types to drive their differentiation. The basis for failed autocrine IL-2 production by activated CD8(+) cells is unclear. We find that Ikaros, a transcriptional repressor that silences IL-2 in anergic CD4(+) T cells, also restricts autocrine IL-2 production by CD8(+) T cells. We find that CD8(+) T cell activation in vitro in the absence of exogenous cytokines and CD4 help leads to marked induction of Ikaros, a known repressor of the Il2 gene. Naive murine CD8 T cells haplo-insufficient for Ikzf1 failed to upregulate Ikaros, produced autocrine IL-2, and differentiated in an IL-2-dependent manner into IFN-γ-producing CTLs in response to TCR/CD28 stimulation alone. Furthermore, Ikzf1 haplo-insufficient CD8(+) T cells were more effective at controlling Listeria infection and B16 melanoma growth in vivo, and they could provide help to neighboring, non-IL-2-producing cells to differentiate into IFN-γ-producing effectors. Therefore, by repressing autocrine IL-2 production, Ikaros ensures that naive CD8(+) T cells remain dependent on licensing by APCs and CD4(+) T cells, and it may therefore act as a cell-intrinsic safeguard against inappropriate CTL differentiation and immunopathology.

Management of significant and widespread, acute subcutaneous emphysema: should we manage surgically or conservatively?

BACKGROUND: Subcutaneous emphysema of a limb after acute injury is classically associated with gas gangrene. Delayed management can result in amputation and death. Typically caused by a clostridial infection, patients are unwell, with rapidly spreading clinical signs, abnormal laboratory results, and cultures positive. There are reports of widespread subcutaneous emphysema of a limb in well-appearing patients, with blood parameters within normal limits; however, the optimum management of this type of case is unclear. OBJECTIVE: Our objectives were to present 4 new cases of acute subcutaneous emphysema in well-appearing patients managed with early surgery, review the literature, and discuss the management decisions in cases of acute subcutaneous emphysema in clinically well patients. CASE REPORTS: Here we present a case series of 4 patients, all with penetrating injuries to the upper limb resulting in widespread subcutaneous emphysema within 24 h of injury. Mean age was 33 years. All were fit and well, with the exception of one with type 1 diabetes, no cardiorespiratory compromise, and no significant derangement of laboratory investigations. X-ray studies showed widespread gas within the soft tissues. All were treated aggressively with immediate surgical fasciotomy of the upper limb, thorough debridement, and washout as required. Gram stains revealed pus cells (polymorphonuclear leucocytes) in all, but organisms in only one case (Gram-positive cocci and bacilli). Prolonged culture grew organisms in all. All patients had a second washout and closure plus 6 weeks of antibiotics. All survived and had fully functioning limbs. Why should an emergency physician be aware of this? We recommend having a low threshold for rapid referral to an appropriate surgical speciality, allowing prompt and radical surgical management of this type of presentation, even in the presence of a well patient.

Nivolumab monotherapy or combination with ipilimumab with or without cobimetinib in previously treated patients with pancreatic adenocarcinoma (CheckMate 032).

BACKGROUND: Pancreatic cancer is one of the deadliest cancer types and represents a major unmet medical need. CheckMate 032 investigated safety and efficacy of nivolumab monotherapy and nivolumab plus ipilimumab with/without cobimetinib in advanced/metastatic solid tumors, including pancreatic cancer. METHODS: In the original pancreatic cancer cohort, previously treated patients (≥1 prior regimen) with advanced/metastatic pancreatic adenocarcinoma were assigned to nivolumab 3 mg/kg every 2 weeks (monotherapy arm) or nivolumab 1 mg/kg and ipilimumab 1 mg/kg or 3 mg/kg every 3 weeks for four doses, followed by nivolumab 3 mg/kg every 2 weeks (combination arm). A subsequent modified pancreatic cohort (one or two prior regimens) received nivolumab 3 mg/kg every 2 weeks, ipilimumab 1 mg/kg every 6 weeks, and cobimetinib 60 mg orally once daily for 21 days on and 7 days off (triplet arm). The primary endpoint was investigator-assessed objective response rate (ORR). Secondary endpoints were investigator-assessed progression-free survival (PFS), PFS rate, overall survival (OS), OS rate, safety, and tolerability. Additionally, ORR, PFS, and duration of response were assessed by blinded independent central review (BICR) in the triplet arm. RESULTS: 18 patients received nivolumab monotherapy, 21 received nivolumab plus ipilimumab, and 30 received nivolumab plus ipilimumab plus cobimetinib. In the triplet arm, partial responses were observed in two patients per investigator (ORR 6.7% (95% CI 0.8% to 22.1%)) and in three patients per BICR (ORR 10% (95% CI 2.1% to 26.5%)); no responses were observed in the other arms. Median (95% CI) PFS per investigator was 1.4 (1.3 to 2.0), 1.4 (1.2 to 2.7), and 3.0 (1.5 to 4.1) months for the monotherapy, nivolumab plus ipilimumab, and triplet arms, respectively. Median (95% CI) OS was 5.1 (2.0 to 9.0) months, 4.0 (1.9 to 5.6) months, and 6.2 (3.9 to 11.4) months, respectively. Most treatment-related adverse events were grade 2 or less. CONCLUSIONS: Nivolumab with or without ipilimumab did not elicit objective responses in previously treated patients with advanced pancreatic adenocarcinoma, although three confirmed partial responses and manageable safety were observed with cobimetinib-containing triplet therapy. The small sample size and differences in baseline disease-specific characteristics between arms limit interpretation of these results.

Pericyte requirement for anti-leak action of angiopoietin-1 and vascular remodeling in sustained inflammation.

Blood vessel leakiness is an early, transient event in acute inflammation but can also persist as vessels undergo remodeling in sustained inflammation. Angiopoietin/Tie2 signaling can reduce the leakiness through changes in endothelial cells. The role of pericytes in this action has been unknown. We used the selective PDGF-B-blocking oligonucleotide aptamer AX102 to determine whether disruption of pericyte-endothelial crosstalk alters vascular leakiness or remodeling in the airways of mice under four different conditions: i) baseline, ii) acute inflammation induced by bradykinin, iii) sustained inflammation after 7-day infection by the respiratory pathogen Mycoplasma pulmonis, or iv) leakage after bradykinin challenge in the presence of vascular stabilization by the angiopoietin-1 (Ang1) mimic COMP-Ang1 for 7 days. AX102 reduced pericyte coverage but did not alter the leakage of microspheres from tracheal blood vessels at baseline or after bradykinin; however, AX102 exaggerated leakage at 7 days after M. pulmonis infection and increased vascular remodeling and disease severity at 14 days. AX102 also abolished the antileakage effect of COMP-Ang1 at 7 days. Together, these findings show that pericyte contributions to endothelial stability have greater dependence on PDGF-B during the development of sustained inflammation, when pericyte dynamics accompany vascular remodeling, than under baseline conditions or in acute inflammation. The findings also show that the antileakage action of Ang1 requires PDGF-dependent actions of pericytes in maintaining endothelial stability.

The use of a revision femoral stem to manage a distal femoral periprosthetic fracture in a well-fixed total knee arthroplasty.

Managing very distal femoral periprosthetic fracture above a total knee arthroplasty (TKA) is a difficult problem. When a cruciate sacrificing TKA is used, bone stock around the implant is compromised and, therefore, can limit fixation options. We present technique using the revision system femoral stem for the PFC Sigma TKA (Depuy; Leeds, England) to stabilize this particular type of fracture.

Angiopoietin/Tie2 signaling transforms capillaries into venules primed for leukocyte trafficking in airway inflammation.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor in tumors, but less is known about what drives vascular remodeling in inflammation, where plasma leakage and leukocyte influx are prominent features. In chronic airway inflammation in mice infected by the bacterium Mycoplasma pulmonis (M. pulmonis), the segment of the microvasculature that supports leukocyte adhesion and migration expands through remodeling of capillaries into vessels with features of venules. Here, we report that the angiopoietin/Tie2 pathway is an essential driving force for capillary remodeling into venules in M. pulmonis-infected mouse airways. Similar to M. pulmonis infection, systemic overexpression of angiopoietin-1 (Ang1) resulted in remodeling of airway capillaries into venular-like vessels that expressed venous markers like P-selectin, ICAM-1, and EphB4 and were sites of leukocyte adhesion during lipopolysaccharide-induced acute inflammation. Ang1 and Ang2 protein increased in M. pulmonis-infected mouse airways but came from different cellular sources: Ang1 was expressed in infiltrating neutrophils and Ang2 in endothelial cells. Indeed, systemic administration of soluble Tie2 inhibited capillary remodeling, induction of venous markers, and leukocyte influx in M. pulmonis-infected mouse airways. Together, these findings suggest that blockade of the Ang/Tie2 pathway may represent a therapeutic approach in airway inflammation.

Broken guidewire retrieval from the hip joint: A case report.

Hardware breakage during orthopaedic surgery especially closed intramedullary nailing is a nightmare for orthopaedic surgeons. During hip fracture surgery a mechanical failure of the guidewire or the reamer poses an additional risk of intrapelvic migration and neurovascular or visceral injury which can lead to devastating complications and litigation. We report a case of removal of the broken guidewire using a cannulated reamer & discectomy forceps and recommend some suggestions for prevention of this catastrophe.

Addition of anti-TIM3 or anti-TIGIT Antibodies to anti-PD1 Blockade Augments Human T cell Adoptive Cell Transfer.

PD1 blockade to reinvigorate T cells has become part of standard of care for patients with NSCLC across disease stages. However, the majority of patients still do not respond. One potential mechanism of resistance is increased expression of other checkpoint inhibitory molecules on T cells leading to their suppression; however, this phenomenon has not been well studied in tumor-reactive, human T cells. The purpose of this study was to evaluate this compensatory mechanism in a novel model using human effector T cells infiltrating and reactive against human lung cancer. Immunodeficient mice with flank tumors established from a human lung cancer cell line expressing the NYESO1 antigen were treated with activated human T cells expressing a TCR reactive to NYESO1 (Ly95) with or without anti-PD1 alone and with combinations of anti-PD1 plus anti-TIM3 or anti-TIGIT. A month later, the effect on tumor growth and the phenotype and ex vivo function of the TILs were analyzed. Anti-PD1 and Ly95 T cells led to greater tumor control than Ly95 T cells alone; however, tumors continued to grow. The ex-vivo function of PD1-blocked Ly95 TILs was suppressed and was associated with increased T cell expression of TIM3/TIGIT. Administering combinatorial blockade of PD1+ TIM3 or PD1+ TIGIT with Ly95 T cells led to greater tumor control than blocking PD1 alone. In our model, PD1 blockade was suboptimally therapeutic alone. The effect of TIM3 and TIGIT was upregulated on T cells in response to PD1 blockade and anti-tumor activity could be enhanced when these inhibitory receptors were also blocked with antibodies in combination with anti-PD1 therapy.

A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) delivers inhibitory signals to activated T cells. CTLA4 is constitutively expressed on regulatory CD4(+) T cells (Tregs), but its role in these cells remains unclear. CTLA4 blockade has been shown to induce antitumor immunity. In this study, we examined the effects of anti-CTLA4 antibody on the endogenous CD4(+) T cells in cancer patients. We show that CTLA4 blockade induces an increase not only in the number of activated effector CD4(+) T cells, but also in the number of CD4(+) FoxP3(+) Tregs. Although the effects were dose-dependent, CD4(+) FoxP3(+) regulatory T cells could be expanded at lower antibody doses. In contrast, expansion of effector T cells was seen only at the highest dose level studied. Moreover, these expanded CD4(+) FoxP3(+) regulatory T cells are induced to proliferate with treatment and possess suppressor function. Our results demonstrate that treatment with anti-CTLA4 antibody does not deplete human CD4(+) FoxP3(+) Tregs in vivo, but rather may mediate its effects through the activation of effector T cells. Our results also suggest that CTLA4 may inhibit Treg proliferation similar to its role on effector T cells. This study is registered at http://www.clinicaltrials.gov/ct2/show/NCT00064129, registry number NCT00064129.

Rapid vascular regrowth in tumors after reversal of VEGF inhibition.

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

Function of Human Tumor-Infiltrating Lymphocytes in Early-Stage Non-Small Cell Lung Cancer.

Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non-small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeß7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

Phenotypic and functional analysis of malignant mesothelioma tumor-infiltrating lymphocytes.

Given the growing interest and promising preliminary results of immunotherapy in malignant pleural mesothelioma (MPM), it has become important to more fully understand the immune landscape in this tumor. This may be especially relevant in deciding who might benefit most from checkpoint blockade or agonist antibody therapy. Since the phenotype of tumor infiltrating lymphocytes (TILs) in MPM has not been fully described and their function has not been carefully assessed, we collected fresh tumor and blood from 22 patients undergoing surgical resection and analysed single cell suspensions by flow cytometry. The functionality of TILs was assessed by measurement of cytokine expression (IFN-γ) following overnight stimulation ex vivo. Results showed low numbers of CD8+ TILs whose function was either moderately or severely suppressed. The degree of TIL hypofunction did not correlate with the presence of co-existing macrophages or neutrophils, nor with expression of the inhibitory receptors PD-1, CD39 and CTLA-4. Hypofunction was associated with higher numbers of CD4 regulatory T cells (Tregs) and with expression of the inhibitory receptor TIGIT. On the other hand, presence of tissue-resident memory (Trm) cells and expression of TIM-3 on CD8+ cells were positively associated with cytokine production. However, Trm function was partially suppressed when the transcription factor Eomesodermin (Eomes) was co-expressed. Understanding the function of TILs in malignant mesothelioma may have clinical implications for immunotherapy, especially in choosing the best immunotherapy targets. Our data suggests that Treg cell blocking agents or TIGIT inhibitor antibodies might be especially valuable in these patients.

Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer.

Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs). The TAMs coexpressed M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors. Functionally, TAMs did not primarily suppress tumor-specific effector T cell responses, whereas tumor monocytes tended to be more T cell inhibitory. TAMs expressing relevant MHC class I/tumor peptide complexes were able to activate cognate effector T cells. Mechanistically, programmed death-ligand 1 (PD-L1) expressed on bystander TAMs, as opposed to PD-L1 expressed on tumor cells, did not inhibit interactions between tumor-specific T cells and tumor targets. TAM-derived PD-L1 exerted a regulatory role only during the interaction of TAMs presenting relevant peptides with cognate effector T cells and thus may limit excessive activation of T cells and protect TAMs from killing by these T cells. These results suggest that the function of TAMs as primarily immunosuppressive cells might not fully apply to early-stage human lung cancer and might explain why some patients with strong PD-L1 positivity fail to respond to PD-L1 therapy.

Augmentation of CAR T-cell Trafficking and Antitumor Efficacy by Blocking Protein Kinase A Localization.

Antitumor treatments based on the infusion of T cells expressing chimeric antigen receptors (CAR T cells) are still relatively ineffective for solid tumors, due to the presence of immunosuppressive mediators [such as prostaglandin E2 (PGE2) and adenosine] and poor T-cell trafficking. PGE2 and adenosine activate protein kinase A (PKA), which then inhibits T-cell receptor (TCR) activation. This inhibition process requires PKA to localize to the immune synapse via binding to the membrane protein ezrin. We generated CAR T cells that expressed a small peptide called the "regulatory subunit I anchoring disruptor" (RIAD) that inhibits the association of PKA with ezrin, thus blunting the negative effects of PKA on TCR activation. After exposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showed increased TCR signaling, released more cytokines, and showed enhanced killing of tumor cells compared with CAR T cells. When injected into tumor-bearing mice, the antitumor efficacy of murine and human CAR-RIAD T cells was enhanced compared with that of CAR T cells, due to resistance to tumor-induced hypofunction and increased T-cell infiltration of established tumors. Subsequent in vitro assays showed that both mouse and human CAR-RIAD cells migrated more efficiently than CAR cells did in response to the chemokine CXCL10 and also had better adhesion to various matrices. Thus, the intracellular addition of the RIAD peptide to adoptively transferred CAR T cells augments their efficacy by increasing their effector function and by improving trafficking into tumor sites. This treatment strategy, therefore, shows potential clinical application for treating solid tumors. Cancer Immunol Res; 4(6); 541-51. ©2016 AACR.