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Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, noninvasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T-cell–specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor-bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody–treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and nonresponders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor-draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy.Significance:These findings reveal differences in T-cell activation between responders and nonresponders early into anti-PD-1 treatment, which may impact many facets of immuno-oncology, including patient selection, management, and development of novel combinatorial approaches.

FACS analyses, expression of transporters and kinases in immune cells, tumor growth.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.

The JAK family of non-receptor tyrosine kinases includes four subtypes (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immuno(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor subtype selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here, we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent transphosphorylation and cytokine signaling, while appearing to act largely as “silent” ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented subtype selectivity.

Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, noninvasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T-cell–specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor-bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody–treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and nonresponders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor-draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy. Significance: These findings reveal differences in T-cell activation between responders and nonresponders early into anti-PD-1 treatment, which may impact many facets of immuno-oncology, including patient selection, management, and development of novel combinatorial approaches.

The molecular properties that characterize CD4+CD25+ regulatory T cells (TR cells) remain elusive. Absence of the transcription factor Scurfin (also known as forkhead box P3 and encoded by Foxp3) causes a rapidly fatal lymphoproliferative disease, similar to that seen in mice lacking cytolytic T lymphocyte-associated antigen 4 (CTLA-4). Here we show that Foxp3 is highly expressed by T(R) cells and is associated with T(R) cell activity and phenotype. Scurfin-deficient mice lack T(R) cells, whereas mice that overexpress Foxp3 possess more T(R) cells. In Foxp3-overexpressing mice, both CD4+CD25- and CD4-CD8+ T cells show suppressive activity and CD4+CD25- cells express glucocorticoid-induced tumor-necrosis factor receptor-related (GITR) protein. The forced expression of Foxp3 also delays disease in CTLA-4-/- mice, indicating that the Scurfin and CTLA-4 pathways may intersect and providing further insight into the T(R) cell lineage.

IPEX is a rare X-linked syndrome characterized by neonatal infantile insulin dependent diabetes mellitus, enteropathy, endocrinopathy, eczema and severe infections with variable immune dysfunction. Scurfy, an X-linked lethal disorder of immune regulation, is the murine equivalent of IPEX, providing a model for the human disease. The gene responsible for scurfy has recently been identified by positional cloning. The scurfy/IPEX gene is a member of a family of transcriptional regulators that have in common a forkhead winged-helix DNA binding domain. The cloning of the human ortholog of the mouse gene confirmed that FOXP3 codes for the immune specific DNA binding protein responsible for IPEX. Missense and nonsense mutations, microdeletions, elimination of the downstream stop codon and mutations of the polyadenylation signal have been identified in affected members of families with classic IPEX. It has been suggested that FOXP3 acts as a trascriptional repressor in vivo. Treatment with Cyclosporin A or FK506 improves the clinical course and HLA identical bone marrow transplantation appears to provide a permanent cure.

In the absence of the recently identified putative transcription factor scurfin, mice develop a lymphoproliferative disorder resulting in death by 3 wk of age from a pathology that resembles TGF-β or CTLA-4 knockout mice. In this report, we characterize mice that overexpress the scurfin protein and demonstrate that these animals have a dramatically depressed immune system. Mice transgenic for the Foxp3 gene (which encodes the scurfin protein) have fewer T cells than their littermate controls, and those T cells that remain have poor proliferative and cytolytic responses and make little IL-2 after stimulation through the TCR. Although thymic development appears normal in these mice, peripheral lymphoid organs, particularly lymph nodes, are relatively acellular. In a separate transgenic line, forced expression of the gene specifically in the thymus can alter thymic development; however, this does not appear to affect peripheral T cells and is unable to prevent disease in mice lacking a functional Foxp3 gene, indicating that the scurfin protein acts on peripheral T cells. The data indicate a critical role for the Foxp3 gene product in the function of the immune system, with both the number and functionality of peripheral T cells under the aegis of the scurfin protein.

Mice lacking CTLA-4 die at an age of 2–3 wk due to massive lymphoproliferation, leading to lymphocytic infiltration and destruction of major organs. The onset of the lymphoproliferative disease can be delayed by treatment with murine CTLA4Ig (mCTLA4Ig), starting day 12 after birth. In this study, we have characterized the T cells present in CTLA-4-deficient mice before and after mCTLA4Ig treatment. The T cells present in CTLA-4-deficient mice express the activation markers, CD69 and IL-2R; down-regulate the lymphoid homing receptor, CD62L; proliferate spontaneously in vitro and cannot be costimulated with anti-CD28 mAb consistent with a hyperactivated state. The T cells from CTLA-4-deficient mice survive longer in culture correlating with higher expression of the survival factor, Bcl-xL, in these cells. Most significantly, the CD4+ T cell subset present in CTLA-4-deficient mice secretes high levels of IL-4 and IL-5 upon TCR activation. Treatment of CTLA-4-deficient mice treated with mCTLA4Ig reverses the activation and hyperproliferative phenotype of the CTLA-4-deficient T cells and restores the costimulatory activity of anti-CD28 mAb. Furthermore, T cells from mCTLA4Ig-treated mice are not skewed toward a Th2 cytokine phenotype. Thus, CTLA-4 regulates CD28-dependent peripheral activation of CD4+ T cells. This process results in apoptosis-resistant, CD4+ T cells with a predominantly Th2 phenotype that may be involved in the lethal phenotype in these animals.

CTLA-4, a negative regulator of T cell function, was found to associate with the T cell receptor (TCR) complex ζ chain in primary T cells. The association of TCRζ with CTLA-4, reconstituted in 293 transfectants, was enhanced by p56lck-induced tyrosine phosphorylation. Coexpression of the CTLA-4–associated tyrosine phosphatase, SHP-2, resulted in dephosphorylation of TCRζ bound to CTLA-4 and abolished the p56lck-inducible TCRζ–CTLA-4 interaction. Thus, CTLA-4 inhibits TCR signal transduction by binding to TCRζ and inhibiting tyrosine phosphorylation after T cell activation. These findings have broad implications for the negative regulation of T cell function and T cell tolerance.

Specificity, memory, and self/nonself discrimination are the essential principles that underlie the acquired immune response. From birth through one's life, the immune system is continually responding to new environmental challenges (e.g. bacteria and viruses) and developing a specific, long-lasting immunity to those challenges. The first exposure to a pathogen results in the recruitment of multiple cell types, including macrophages, dendritic cells, and assorted leukocytes, that initiate an antigen nonspecific inflammatory response designed to attract T cells and B cells to the inflammatory sites including the draining lymph nodes. The foreign antigens are concentrated within the professional antigen-presenting cells (APCs) that process and present small antigenic peptides to CD4+ and CD8+ T cells in association with class II and class I major histocompatibility complex (MHC) molecules, respectively. The activated CD4+ T-cell receptor (TCR) alpha beta cells respond vigorously to the pathogen in an antigen-specific manner, liberating a barrage of cytokines that induce B cells to differentiate to antibody-secreting plasma cells and likewise cause CD8+ cells to differentiate into cytolytic effectors. The T cells and B cells expand in an evolving, highly specific manner to control the initial infection while developing long-term acquired immunity such that any further infection by that pathogen is virtually impossible. Thus TCR alpha beta T cells are the central lymphocyte in the immune system, providing specific pathogen recognition and long-term memory all within the context of distinguishing foreign from self antigens. Yet, during a primary immune response, there is a lag time of approximately 3 to 4 days before antigen-specific responses are evident, and TCR alpha beta responses do not peak until approximately day 7. Therefore, it is essential that other strategies are employed by the immune system in order to mount an aggressive early immune response.

C-reactive protein (CRP), an acute phase protein in human serum, is present on large granular lymphocytes (LGL). Anti-CRP inhibits natural killer (NK) cell-mediated lysis. Our current study shows that anti-CRP also inhibits antibody-dependent cell-mediated cytotoxicity (ADCC) of LGL. Calcium influx and protein kinase C (PKC) activation are the early signal transduction events in NK activation. In the conjugates formed between LGL and targets (NK or ADCC), 75-90% of LGL respond with a calcium influx. Addition of anti-CRP had no effect on the percentage of LGL which respond to target cell binding or on the magnitude of the calcium response of LGL. This was true for both NK and ADCC effector cells. Crosslinking anti-CRP with a secondary antibody did not alter this result. Next, the effect of PMA, a PKC activator, and calcium ionophore, A23187, on anti-CRP-mediated inhibition of cytotoxicity were studied. PMA alone reversed most of the inhibition of lysis seen with anti-CRP. Based on previous observations that anti-CRP inhibited target cell-stimulated release of lytic factors, the effect of anti-CRP on release of lytic factors stimulated by PMA and calcium ionophore was evaluated. Anti-CRP blocked the release of lytic factors stimulated by PMA and ionophore. Release of lytic factors involves the rearrangement of cytoskeletal element of NK cell toward the target cell. The effect of anti-CRP on cytoskeletal reorganization was studied. In conjugates formed between effector and target cells, the polarization of cytoskeleton at the contact site of NK and target cell was significantly reduced in the presence of anti-CRP. Although anti-CRP inhibits both ADCC and NK lytic mechanisms, it does not alter target cell-induced Ca2+ influx. CRP interacts with the secretory mechanisms involved in granule exocytosis since anti-CRP inhibits the cytoskeletal polarization and the release of lytic factors and PMA might reverse anti-CRP-mediated inhibition by activating alternative mechanisms of cytotoxicity in effectors.