Conventional T-cell Research Articles

IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors

Chimeric antigen receptor (CAR)-redirected Tcell therapy often fails to control tumors in the long term due to selecting cancer cells that downregulated or lost CAR targeted antigen. To reprogram the functional capacities specifically of engineered CAR Tcells, we inserted IL12 into the extracellular moiety of a CD28-ζ CAR; both the CAR endodomain and IL12 were functionally active, as indicated by antigen-redirected effector functions and STAT4 phosphorylation, respectively. The IL12-CAR reprogrammed CD8+ Tcells toward a so far not recognized natural killer (NK) cell-like signature and a CD94+CD56+CD62Lhigh phenotype closely similar, but not identical, to NK and cytokine induced killer (CIK) cells. In contrast to conventional CAR Tcells, IL12-CAR Tcells acquired antigen-independent, human leukocyte antigen E (HLA-E) restricted cytotoxic capacities eliminating antigen-negative cancer cells in addition to eliminating cancer cells with CAR cognate antigen. Simultaneous signaling through both the CAR endodomain and IL12 were required for inducing maximal NK-like cytotoxicity; adding IL12 to conventional CAR Tcells was not sufficient. Antigen-negative tumors were attacked by IL12-CAR Tcells, but not by conventional CAR Tcells. Overall, we present a prototype of a new family of CARs that augments tumor recognition and elimination through expanded functional capacities by an appropriate cytokine integrated into the CAR exodomain.

Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels reaching up to 43 colors at the single-cell level. However, as panel size and complexity increase, so too does the detail involved in designing and optimizing successful high-quality panels fit for downstream high-dimensional data analysis. In contrast to conventional flow cytometers, full-spectrum flow cytometers measure the entire emission spectrum of each fluorophore across all lasers. This allows for fluorophores with very similar emission maxima but unique overall spectral fingerprints to be used in conjunction, enabling relatively straightforward design of larger panels. Although a protocol for best practices in full-spectrum flow cytometry panel design has been published, there is still a knowledge gap in going from the theoretically designed panel to the necessary steps required for panel optimization. Here, we aim to guide users through the theory of optimizing a high-dimensional full-spectrum flow cytometry panel for immunophenotyping using comprehensive step-by-step protocols. These protocols can also be used to troubleshoot panels when issues arise. A practical application of this approach is exemplified with a 24-color panel designed for identification of conventional T-cell subsets in human peripheral blood. © 2021 Malaghan Institute of Medical Research, Cytek Biosciences. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and evaluation of optimal spectral reference controls Support Protocol 1: Antibody titration Support Protocol 2: Changing instrument settings Basic Protocol 2: Unmixing evaluation of fully stained sample Basic Protocol 3: Evaluation of marker resolution Support Protocol 3: Managing heterogeneous autofluorescence Basic Protocol 4: Assessment of data quality using expert gating and dimensionality reduction algorithms.

CT-080: A Tumor-Agnostic TCR-Mimic CAR-T cell Specific for NDC80 Targets Multiple Hematological Malignancies

Context Target identification for CAR-T cell therapies, especially shared targets among different malignancies, remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through TCR-mimic antibodies. Objective and Design We hypothesized that mass spectrometry (MS)-based analysis of the presented HLA ligands of multiple cancer cell lines can be utilized to identify a shared, tumor-associated HLA ligand, which could then be targeted by TCR-mimic CAR-T cells specific for this particular HLA ligand. Results MS of HLA ligands from eight hematological and non-hematological cancer cell lines identified a shared, non-immunogenic, HLA-A*02 restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene, which was later identified in >90% (20/22) of all A*02-positive cell lines tested. A TCR-mimic scFv was prepared against this epitope, termed “NDC80-C.” CAR-T cells transduced with the NDC80-C, directed against the ALNEQIARL:HLA-A*02 complex on cancer cells, demonstrated high sensitivity and specificity for recognizing and killing multiple cancer types with a high preference for hematological malignancies (e.g., AML, ALL, DLBCL, and ALCL). In contrast, healthy leukocytes, activated B and T-cells, or hematopoietic stem cells from A*02-positive and -negative donors were not lysed. NDC80-C CAR-T cells suppressed colony formation of primary AML cells but not of healthy stem cells. Additionally, NDC80-C CAR-T cells were efficacious in mouse models against human mesothelioma and leukemia. Conclusions Our study demonstrates how MS can inform the design of tumor-agnostic TCR-mimic therapeutic platforms that target structures that are currently not druggable by small molecules, conventional CAR-T cells, T-cells, or antibodies. This strategy lays the groundwork for a potential antibody platform therapy or CAR-T cell with efficacy against highly proliferative A*02-positive cancer cells, independent of the respective cancer type.

TRAIL produced by SAM-1-activated CD4+ and CD8+ subgroup T cells induces apoptosis in human tumor cells through upregulation of death receptors

Bacterial superantigens potently activate conventional T-cells to induce massive cytokine production and mediate tumor cell death. To engineer superantigens for immunotherapy against tumors in clinic, we previously generated SAM-1, a staphylococcal enterotoxins C2 (SEC2) mutant, that exhibited significantly reduced toxicity but maintained the superantigen activity in animal models. This present study aimed to investigate whether SAM-1 activates T cells and induces apoptosis in human tumor cells. We found that SAM-1 induced the maturation of dendritic cells (DCs) with upregulating expression of the surface markers CD80, CD86 and HLA-DR, which secreted high levels of IL-12p70 by activating TLR2-NF-κB signaling pathways. SAM-1 could activate human CD4+ subgroup T cells and CD8+ subgroup T cells in the presence of mature dendritic cells (DCs), leading to the productions of cytokines TRAIL, IL-2, IFN-γ and TNF-α. We observed that TRAIL mediated the apoptosis and S-phase and G2/M-phase arrest in HGC-27 tumor cells via binding to upregulated death receptors DR4 and DR5. Using shRNA knockdown in HGC-27 cells or constitutive overexpression in ES2 cells for DR4 and DR5, we demonstrated the vital requirement of DR4 and DR5 in apoptosis of tumor cells in response to TRAIL secreted from SAM-1-activated T cells. Collectively, our results will facilitate better understanding of SAM-1-based immunotherapies for cancer.

Allotransplantation Is Associated With Exacerbation of CD8 T-Cell Senescence: The Particular Place of the Innate CD8 T-Cell Component.

Immunosenescence is a physiological process that is associated with changes in the immune system, particularly among CD8 T-cells. Recent studies have hypothesized that senescent CD8 T-cells are produced with chronologic age by chronic stimulation, leading to the acquisition of hallmarks of innate-like T-cells. While conventional CD8 T-cells are quite well characterized, CD8 T-cells sharing features of NK cells and memory CD8 T-cells, are a newly described immune cell population. They can be distinguished from conventional CD8 T-cells by their combined expression of panKIR/NKG2A and Eomesodermin (E), a unique phenotype closely associated with IFN-γ production in response to innate stimulation. Here, we first provided new evidence in favor of the innate character of panKIR/NKG2A(+) E(+) CD8 T-cells in normal subjects, documenting their position at an intermediate level in the innateness gradient in terms of both innate IFN-γ production and diminished mitochondrial mass. We also revealed that CD8 E(+) panKIR/NKG2A(+) T-cells, hereafter referred to as Innate E(+) CD8 T-cells, exhibit increased senescent (CD27(-) CD28(-)) phenotype, compared to their conventional memory counterparts. Surprisingly, this phenomenon was not dependent on age. Given that inflammation related to chronic viral infection is known to induce NK-like marker expression and a senescence phenotype among CD8 T-cells, we hypothesized that innate E(+) CD8 T-cells will be preferentially associated with exacerbated cellular senescence in response to chronic alloantigen exposure or CMV infection. Accordingly, in a pilot cohort of stable kidney allotransplant recipients, we observed an increased frequency of the Innate E(+) CD8 T-cell subset, together with an exacerbated senescent phenotype. Importantly, this phenotype cannot be explained by age alone, in clear contrast to their conventional memory counterparts. The senescent phenotype in CD8 T-cells was further increased in cytomegalovirus (CMV) positive serology transplant recipients, suggesting that transplantation and CMV, rather than aging by itself, may promote an exacerbated senescent phenotype of innate CD8 T-cells. In conclusion, we proposed that kidney transplantation, via the setting of inflammatory stimuli of alloantigen exposure and CMV infection, may exogenously age the CD8 T-cell compartment, especially its innate component. The physiopathological consequences of this change in the immune system remain to be elucidated.

Anti-tumor effects of vascular endothelial growth factor/vascular endothelial growth factor receptor binding domain-modified chimeric antigen receptor T cells

Background aimsThe vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) signaling pathway plays an important role in angiogenesis and lymphangiogenesis, which are closely related to tumor cell growth, survival, tissue infiltration and metastasis. Blocking/interfering with the interaction between VEGF and VEGFR to inhibit angiogenesis/lymphangiogenesis has become an important means of tumor therapy. MethodsHere the authors designed a novel chimeric antigen receptor (CAR) lentiviral vector expressing the VEGF-C domain targeting both VEGFR-2 and VEGFR-3 (VEGFR-2/3 CAR) and then transduced CD3-positive T cells with VEGFR-2/3 CAR lentivirus. ResultsAfter co-culturing with target cells, VEGFR-2/3 CAR T cells showed potent cytotoxicity against both VEGFR-2- and VEGFR-3-positive breast cancer cells, with increased simultaneous secretion of interferon gamma, tumor necrosis factor alpha and interleukin-2 cytokines. Moreover, CAR T cells were able to destroy the tubular structures formed by human umbilical vein endothelial cells and significantly inhibit the growth, infiltration and metastasis of orthotopic mammary xenograft tumors in a female BALB/c nude mice model. ConclusionsThe authors’ results indicate that VEGFR-2/3 CAR T cells targeting both VEGFR-2 and VEGFR-3 have significant anti-tumor activity, which expands the application of conventional CAR T-cell therapy.

Liposomal Alpha-Galactosylceramide Ameliorates Graft-Versus-Host Disease with Remaining Intensified GVL Gained By Dose-Reduction of Posttransplant Cyclophosphamaide after Haploidentical HSCT

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment for many hematological malignancies. Clinical studies have shown that HSCT from haplo-identical donors with posttransplant cyclophosphamide (PTCy) can widely provide alternative donor sources and is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD). However, on the other hands, it is concerned that the immune suppressive effect by PTCy also might diminish graft-versus-leukemia (GVL) effect and increase the relapse rate in patients with high tumor burden at transplant. Indeed, recent clinical studies suggested that a certain extent of dose-reduction of PTCy could sufficiently prevent GVHD, but whether the dose-reduction can lead to enhance GVL effect has not been clarified. In this study, we investigated the effects of dose-reduction of PTCy on GVHD and GVL by using murine haploidentical BMT model. Also, we explored the immune-modulating impacts of liposomal alpha-galactosylceramide (liposomal a-galcer; RGI2001) on reduced PTCy. Liposomal a-galcer is a synthetic glycolipid, a ligand stimulating invariant natural killer T (NKT) cell in CD1d restricted manner, which is shown to prevent GVHD via expansion of regulatory T cell (Treg) in murine experiments. First, we tested the effects of dose-reduction of PTCy on GVL effect, we transplanted from C57BL/6J donor to B6D2F1 recipients bearing with luciferase-expressing P815 leukemia cells. The impact of PTCy dose-reduction on GVL was evaluated by using in vivo bioluminescence system. Lethally irradiated B6D2F1 mice were transplanted with 5x106 T cell depleted (TCD) BM, 10x106 whole splenic cells from C57BL/6J donor mice and 5x105 luciferase-expressing P815 leukemia cells. PTCy or control vehicle was administered at day3 after BMT. As for PTCy, 50mg/kg of cyclophosphamide (Cy) has already shown as standard dose to sufficiently suppress GVHD, thus we tested effect of 25mg/kg of Cy comparing with 50mg/kg of Cy. In vivo imaging system (IVIS) was followed weekly since day10 after BMT to evaluate tumor burden. A trend toward lower levels of radiance was measured by IVIS in reduced-dose group than full-dose group through the experimental period, suggesting reduced-dose of Cy contribute to leave alloagressive T cell activity and result in the enhancement of GVL effect. However, the benefit of GVL enhancement in reduced PTCy group was counterbalanced by the deterioration of GVHD when we used higher dose of total body irradiation (TBI) for the aggressive GVHD-prone experimental setting. To overcome the negative impact of PTCy-reduction on GVHD, we next tested the immune-modulating effect of liposomal a-galcer on this system. Liposomal a-galcer 1mg/kg or control vehicle was administered at day3, 5, 7, after BMT. Our data indicated that reduced PTCy plus liposomal a-galcer significantly improved acute GVHD in overall survival (vs control group, p < 0.01). In comparison with those treated with PTCy plus liposomal a-galcer, the others kept on losing weight and worsening GVHD score to the end beyond the third week without recovery. Importantly, liposomal a-galcer did not influence the effect of GVL when we evaluated tumor-bearing recipents with IVIS, suggesting that add-on of liposomal a-galcer improved GVHD with remaining intensified GVL effect which was gained by dose-reduction of PTCy. Flowcytometric analysis of splenic cells at day 14 showed that %Treg of CD4+ Tcell was significantly higher in reduced PTCy plus liposomal a-galcer group than control group (5.30 vs 2.75, p < 0.05) and reduced PTCy alone group (5.30 vs 2.57, p < 0.05). Additionally, in PTCy plus liposomal a-galcer group, both Treg/CD4+ conventional Tcell (Tcon) ratio and Treg/CD8+ Tcell ratio were significantly higher than control group (p < 0.05) and reduced PTCy alone group (p < 0.05), respectively. Analysis of MLN cells also showed the selective increase of Treg in PTCy plus liposomal a-galcer group more than in other groups. Analysis of splenic cells and MLN cells at day28 showed similar trends with at day14. Taken together, these data demonstarated that liposomal a-galcer could efficiently compensate the immune-suppressive effect after reduced PTCy by preferentially promoting Treg recovery with remaining GVL. Our data might provide the important information for developing new strategy of haploidentical HSCT with PTCy for patient with high-tumor burden. DisclosuresMaeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.

Efficacy of CRISPR-Cas9 CAR T-Cell Therapy vs. Blinatumomab in the Treatment of Acute Lymphoblastic Leukemia

Introduction: With immunotherapy drugs such as Blinatumomab (Blincyto), the current overall survival rate of relapsed/refractory B Lymphocyte Acute Lymphoblastic Leukemia (B-ALL) is 7.8 months in adults. However, with CRISPR-Cas9 gene editing, CAR T-cell therapy may extend survival. By knocking out T-cell receptors (TCRs) and HLA class I receptors on T-cells, CRISPR-Cas9 edited CAR T-cells could become a universal treatment alternative for B-ALL. This study compares the efficacy of CRISPR-Cas9 edited CAR T-cell therapy, a new era of CAR T-cell therapy, to the treatment of relapsed/ refractory B-ALL with Blinatumomab in mice. Methods: A cohort of 30 NSG mice receiving xenografts from an 18-24 year old patient with relapsed B-ALL will be randomly assigned to one of three groups, where they will receive treatment with: (1) mock transduced T-cells, (2) Blincyto, or (3) CRISPR-Cas9 edited TRAC- and B2M- knockout CAR T-cells. Another cohort of 30 NSG mice, receiving human skin xenografts from a healthy donor, will also be assigned to one of the groups and treated accordingly. Tumour shrinkage will be analyzed through in vivo antibody fluorescent imaging in the first cohort. Graft Vs Host Disease (GvHD) development will be assessed in the second cohort. Results: It is expected that mice treated with CRISPR-Cas9 edited CAR T-cells will be more effective at eliminating tumours. Compared to mice treated with Blincyto or mock-transfused T-cells, these mice will exhibit the highest overall response rates, complete response rates and tumour shrinkage rates, and the lowest relapse rates. It is also expected that these mice will not develop GvHD. Discussion: The use of CRISPR-Cas9 edited CAR T-cells in the treatment of B-ALL reveals a new facet to cancer therapeutics. The efficacy of this treatment will be demonstrated through greater tumour shrinkage rates and higher overall response rates than treatment with Blincyto, the current standard treatment drug for B-ALL. Conclusion: Current treatments involving Blinatumomab or conventional CAR T-cell therapy are expensive and therefore inaccessible to many. These results could demonstrate that CRISPR-Cas9 edited CAR T-cell therapy is viable as a safe, universal and more affordable alternative for relapsed/refractory B-ALL treatment.

Issue Highlights — May 2021

Issue Highlights — May 2021

Haploidentical age-adapted myeloablative transplant and regulatory and effector T cells for acute myeloid leukemia

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day −4 followed by 1 × 106/kg donor conventional T cells on day −1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor–type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #NCT03977103.

Spontaneously Resolved Atopic Dermatitis Shows Melanocyte and Immune Cell Activation Distinct From Healthy Control Skin.

Atopic dermatitis (AD) typically starts in infancy or early childhood, showing spontaneous remission in a subset of patients, while others develop lifelong disease. Despite an increased understanding of AD, factors guiding its natural course are only insufficiently elucidated. We thus performed suction blistering in skin of adult patients with stable, spontaneous remission from previous moderate-to-severe AD during childhood. Samples were compared to healthy controls without personal or familial history of atopy, and to chronic, active AD lesions. Skin cells and tissue fluid obtained were used for single-cell RNA sequencing and proteomic multiplex assays, respectively. We found overall cell composition and proteomic profiles of spontaneously healed AD to be comparable to healthy control skin, without upregulation of typical AD activity markers (e.g., IL13, S100As, and KRT16). Among all cell types in spontaneously healed AD, melanocytes harbored the largest numbers of differentially expressed genes in comparison to healthy controls, with upregulation of potentially anti-inflammatory markers such as PLA2G7. Conventional T-cells also showed increases in regulatory markers, and a general skewing toward a more Th1-like phenotype. By contrast, gene expression of regulatory T-cells and keratinocytes was essentially indistinguishable from healthy skin. Melanocytes and conventional T-cells might thus contribute a specific regulatory milieu in spontaneously healed AD skin.

Befriending the Hostile Tumor Microenvironment in CAR T-Cell Therapy

T-cells genetically engineered to express a chimeric antigen receptor (CAR) have shown remarkable results in patients with B-cell malignancies, including B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and mantle cell lymphoma, with some promising efficacy in patients with multiple myeloma. However, the efficacy of CAR T-cell therapy is still hampered by local immunosuppression and significant toxicities, notably cytokine release syndrome (CRS) and neurotoxicity. The tumor microenvironment (TME) has been identified to play a major role in preventing durable responses to immunotherapy in both solid and hematologic malignancies, with this role exaggerated in solid tumors. The TME comprises a diverse set of components, including a heterogeneous population of various cells and acellular elements that collectively contribute towards the interplay of pro-immune and immunosuppressive signaling. In particular, macrophages, myeloid-derived suppressor cells, regulatory T-cells, and cell-free factors such as cytokines are major contributors to local immunosuppression in the TME of patients treated with CAR T-cells. In order to create a more favorable niche for CAR T-cell function, armored CAR T-cells and other combinatorial approaches are being explored for potential improved outcomes compared to conventional CAR T-cell products. While these strategies may potentiate CAR T-cell function and efficacy, they may paradoxically increase the risk of adverse events due to increased pro-inflammatory signaling. Herein, we discuss the mechanisms by which the TME antagonizes CAR T-cells and how innovative immunotherapy strategies are being developed to address this roadblock. Furthermore, we offer perspective on how these novel approaches may affect the risk of adverse events, in order to identify ways to overcome these barriers and expand the clinical benefits of this treatment modality in patients with diverse cancers. Precise immunomodulation to allow for improved tumor control while simultaneously mitigating the toxicities seen with current generation CAR T-cells is integral for the future application of more effective CAR T-cells against other malignancies.

279 - HLA-Haploidentical Transplantation with Regulatory and Conventional T-Cells Adoptive Immunotherapy in Pediatric Patients with High-Risk Acute Leukemia

279 - HLA-Haploidentical Transplantation with Regulatory and Conventional T-Cells Adoptive Immunotherapy in Pediatric Patients with High-Risk Acute Leukemia

The burgeoning role of MR1-restricted T-cells in infection, cancer and autoimmune disease.

MR1 is a ubiquitously expressed, monomorphic antigen presenting molecule that has been largely preserved throughout mammalian evolution. The primary role of MR1 is to present conserved microbial metabolites to highly abundant mucosal-associated invariant T (MAIT) cells. The role of MAIT cells and other MR1-restricted T cells (MR1T) has been recently extended to immunomodulation during cancer. MR1Ts have also been implicated in autoimmune disease. The highly conserved nature of MR1 across the human population is in stark contrast to the MHC molecules recognised by conventional αβ T-cells, therefore MR1Ts may form fertile ground for the development of pan-population T-cell immunotherapeutics for a wide range of important morbidities.

Functional Characterization of Ly49+CD8 T-Cells in Both Normal Condition and During Anti-Viral Response.

The role of Ly49+CD8 T-cells in the immune system is not clear. Previously, several papers suggested Ly49+CD8 T-cells as immunosuppressors, while multiple studies also suggested their role as potent participants of the immune response. The mechanism of Ly49 expression on CD8 T-cells is also not clear. We investigated phenotype, functions, and regulation of Ly49 expression on murine CD8 T-cells in both normal state and during LCMV infection. CD8 T-cells express different Ly49 receptors compared with NK-cells. In intact mice, Ly49+CD8 T-cells have a phenotype similar to resting central memory CD8 T-cells and do not show impaired proliferation and cytokine production. Conventional CD8 T-cells upregulate Ly49 receptors during TCR-induced stimulation, and IL-2, as well as IL-15, affect it. At the same time, Ly49+CD8 T-cells change the Ly49 expression profile dramatically upon re-stimulation downregulating inhibitory and upregulating activating Ly49 receptors. We observed the expression of Ly49 receptors on the virus-specific CD8 T-cells during LCMV infection, especially marked in the early stages, and participation of Ly49+CD8 T-cells in the anti-viral response. Thus, CD8 T-cells acquire Ly49 receptors during the T-cell activation and show dynamic regulation of Ly49 receptors during stimulation.

NKTR-358: A novel regulatory T-cell stimulator that selectively stimulates expansion and suppressive function of regulatory T cells for the treatment of autoimmune and inflammatory diseases

Impaired interleukin-2 (IL-2) production and regulatory T-cell dysfunction have been implicated as immunological mechanisms central to the pathogenesis of multiple autoimmune and inflammatory diseases. NKTR-358, a novel regulatory T-cell stimulator, is an investigational therapeutic that selectively restores regulatory T-cell homeostasis in these diseases. We investigated NKTR-358's selectivity for regulatory T-cells, receptor-binding properties, ex vivo and in vivo pharmacodynamics, ability to suppress conventional T-cell proliferation in mice and non-human primates, and functional activity in a murine model of systemic lupus erythematosus. In vitro, NKTR-358 demonstrated decreased affinity for IL-2Rα, IL-2Rβ, and IL-2Rαβ compared with recombinant human IL-2 (rhIL-2). A single dose of NKTR-358 in cynomolgus monkeys produced a greater than 15-fold increase in regulatory T-cells, and the increase lasted until day 14, while daily rhIL-2 administration for 5 days only elicited a 3-fold increase, which lasted until day 7. Repeated dosing of NKTR-358 over 6 months in cynomolgus monkeys elicited cyclical, robust increases in regulatory T-cells with no loss in drug activity over the course of treatment. Regulatory T-cells isolated from NKTR-358-treated mice displayed a sustained, higher suppression of conventional T-cell proliferation than regulatory T-cells isolated from vehicle-treated mice. NKTR-358 treatment in a mouse model (MRL/MpJ-Faslpr) of systemic lupus erythematosus for 12 weeks maintained elevated regulatory T-cells for the treatment duration and ameliorated disease progression. Together, these results suggest that NKTR-358 has the ability to elicit sustained and preferential proliferation and activation of regulatory T-cells without corresponding effects on conventional T-cells, with improved pharmacokinetics compared with rhIL-2.

Phenotypical and Functional Characterization of Cytotoxic Unconventional T-Cells.

During the last two decades, the immunology field has been focused on the study of conventional T-cells, leading to important advances in identification of specific subsets involved in promoting and suppressing immune response in patients with cancer, autoimmune disease or transplanted patients. In these recent years, the research on unconventional subset of CD4-CD8- double-negative T-cells is growing. DNTs are a unique subset of T-cells characterized by being CD4-CD8-CD3+ and express either ß or α T-cell receptors (TCR). In this chapter, we describe the methods used to phenotypically characterize and isolate these cells in order to study their functional profile.

Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry.

Although many flow cytometers can analyze 30-50 parameters, it is still challenging to develop a 40+ color panel for the phenotyping of immune cells using fluorochrome conjugated antibodies due to limitations in the availability of spectrally unique fluorochromes that can be excited by the commonly used laser lines (UV, Violet, Blue, Green/Yellow-green, and Red). Spectral flowcytometry is capable of differentiating fluorochromes with significant overlap in the emission spectra, enabling the use of spectrally similar fluorochrome pairs such as Brilliant Blue 515 and FITC in a single panel. We have developed a 43 color panel to characterize most of the immune subsets within the peripheral immune system, including conventional T cells, unconventional T cells such as invariant natural killer T cells (iNKT), Gamma delta (γδ) T-cell subsets (TCR Vδ2, TCR Vγ9) and mucosal-associated invariant T cells (MAIT), B-cell subsets, natural killer (NK) cells, plasmacytoid dendritic cells, dendritic cell subsets, hematopoietic progenitor cells, basophils, and innate lymphoid cell (ILC) subsets (CD117, CRTH2). The panel includes surface markers to analyze activation (CD38, HLA-DR, ICOS/CD278), differentiation (CD45RA, CD27, CD28, CD57), expression of cytokine and chemokine receptors (CD25, CD127, CCR10, CCR6, CCR4, CXCR3, CXCR5, CRTH2/CD294), and co-inhibitory molecules and exhaustion (PD-1, CD223/LAG-3, TIGIT), which enables a deep characterization of PBMCs from peripheral blood. Cells were analyzed on a 5-laser Cytek Aurora and data analysis was done using FlowJo. This panel can help to make a thorough interpretation of immune system, specifically when specimen quantity is low. The panel has not been completely optimized but would rather act as a guide toward the development of a workflow for optimized multicolor immunophenotyping panel.

Deep phenotypical characterization of human CD3+ CD56+ T cells by mass cytometry.

CD56+ T cells are a group of pro-inflammatory CD3+ lymphocytes with characteristics of natural killer cells, being involved in antimicrobial immune defense. Here, we performed deep phenotypic profiling of CD3+ CD56+ cells in peripheral blood of normal human donors and individuals sensitized to birch-pollen or/and house dust mite by high-dimensional mass cytometry combined with manual and computational data analysis. A co-regulation between major conventional T-cell subsets and their respective CD3+ CD56+ cell counterparts appeared restricted to CD8+ , MAIT, and TCRγδ+ T-cell compartments. Interestingly, we find a co-regulation of several CD3+ CD56+ cell subsets in allergic but not in healthy individuals. Moreover, using FlowSOM, we distinguished a variety of CD56+ T-cell phenotypes demonstrating a hitherto underestimated heterogeneity among these cells. The novel CD3+ CD56+ subset description comprises phenotypes superimposed with naive, memory, type 1, 2, and 17 differentiation stages, in part represented by a phenotypical continuum. Frequencies of two out of 19 CD3+ CD56+ FlowSOM clusters were significantly diminished in allergic individuals, demonstrating less frequent presence of cells with cytolytic, presumably protective, capacity in these donors consistent with defective expansion or their recruitment to the affected tissue. Our results contribute to defining specific cell populations to be targeted during therapy for allergic conditions.

Induction of Antigen-Independent Proliferation of Regulatory T-Cells by TNF Superfamily Ligands OX40L and GITRL.

TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.