Potentiating adoptive cell therapy using synthetic IL-9 receptors.

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

BCG vaccination induces enhanced frequencies of memory T cells and altered plasma levels of common γc cytokines in elderly individuals.

BCG vaccination is known to induce innate immune memory, which confers protection against heterologous infections. However, the effect of BCG vaccination on the conventional adaptive immune cells subsets is not well characterized. We investigated the impact of BCG vaccination on the frequencies of T cell subsets and common gamma c (γc) cytokines in a group of healthy elderly individuals (age 60-80 years) at one month post vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrate that BCG vaccination induced enhanced frequencies of central (p<0.0001) and effector memory (p<0.0001) CD4+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001), stem cell memory (p = 0.0001) CD4+ T cells and regulatory T cells. In addition, BCG vaccination induced enhanced frequencies of central (p = 0.0008), effector (p<0.0001) and terminal effector memory (p<0.0001) CD8+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001) and stem cell memory (p = 0.0034) CD8+T cells. BCG vaccination also induced enhanced plasma levels of IL-7 (p<0.0001) and IL-15 (p = 0.0020) but diminished levels of IL-2 (p = 0.0033) and IL-21 (p = 0.0020). Thus, BCG vaccination was associated with enhanced memory T cell subsets as well as memory enhancing γc cytokines in elderly individuals, suggesting its ability to induce non-specific adaptive immune responses.

A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells.

The use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαßγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rßγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule's in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

Case Report: A Novel IL2RG Frame-Restoring Rescue Mutation Mimics Early T Cell Engraftment Following Haploidentical Hematopoietic Stem Cell Transplantation in a Patient With X-SCID.

Mutations of the interleukin 2 receptor γ chain (IL2RG) result in the most common form of severe combined immunodeficiency (SCID), which is characterized by severe and persistent infections starting in early life with an absence of T cells and natural killer cells, normal or elevated B cell counts and hypogammaglobulinemia. SCID is commonly fatal within the first year of life, unless the immune system is reconstituted by hematopoietic stem cell transplantation (HSCT) or gene therapy. We herein describe a male infant with X-linked severe combined immunodeficiency (X-SCID) diagnosed at 5 months of age. Genetic testing revealed a novel C to G missense mutation in exon 1 resulting in a 3' splice site disruption with premature stop codon and aberrant IL2 receptor signaling. Following the diagnosis of X-SCID, the patient subsequently underwent a TCRαß/CD19-depleted haploidentical HSCT. Post transplantation the patient presented with early CD8+ T cell recovery with the majority of T cells (>99%) being non-donor T cells. Genetic analysis of CD4+ and CD8+ T cells revealed a spontaneous 14 nucleotide insertion at the mutation site resulting in a novel splice site and restoring the reading frame although defective IL2RG function was still demonstrated. In conclusion, our findings describe a spontaneous second-site mutation in IL2RG as a novel cause of somatic mosaicism and early T cell recovery following haploidentical HSCT.

Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

Pathology of macrophage activation syndrome in humanized NSGS mice.

"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.

The JAK1 Selective Inhibitor ABT 317 Blocks Signaling Through Interferon-γ and Common γ Chain Cytokine Receptors to Reverse Autoimmune Diabetes in NOD Mice.

Cytokines that signal through the JAK-STAT pathway, such as interferon-γ (IFN-γ) and common γ chain cytokines, contribute to the destruction of insulin-secreting ß cells by CD8+ T cells in type 1 diabetes (T1D). We previously showed that JAK1/JAK2 inhibitors reversed autoimmune insulitis in non-obese diabetic (NOD) mice and also blocked IFN-γ mediated MHC class I upregulation on ß cells. Blocking interferons on their own does not prevent diabetes in knockout NOD mice, so we tested whether JAK inhibitor action on signaling downstream of common γ chain cytokines, including IL-2, IL-7 IL-15, and IL-21, may also affect the progression of diabetes in NOD mice. Common γ chain cytokines activate JAK1 and JAK3 to regulate T cell proliferation. We used a JAK1-selective inhibitor, ABT 317, to better understand the specific role of JAK1 signaling in autoimmune diabetes. ABT 317 reduced IL-21, IL-2, IL-15 and IL-7 signaling in T cells and IFN-γ signaling in ß cells, but ABT 317 did not affect GM-CSF signaling in granulocytes. When given in vivo to NOD mice, ABT 317 reduced CD8+ T cell proliferation as well as the number of KLRG+ effector and CD44hiCD62Llo effector memory CD8+ T cells in spleen. ABT 317 also prevented MHC class I upregulation on ß cells. Newly diagnosed diabetes was reversed in 94% NOD mice treated twice daily with ABT 317 while still on treatment at 40 days and 44% remained normoglycemic after a further 60 days from discontinuing the drug. Our results indicate that ABT 317 blocks common γ chain cytokines in lymphocytes and interferons in lymphocytes and ß cells and are thus more effective against diabetes pathogenesis than IFN-γ receptor deficiency alone. Our studies suggest use of this class of drug for the treatment of type 1 diabetes.

Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9.

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

Establishment of Humanized Mice from Peripheral Blood Mononuclear Cells or Cord Blood CD34+ Hematopoietic Stem Cells for Immune-Oncology Studies Evaluating New Therapeutic Agents.

The clinical success of immune checkpoint modulators and the development of next-generation immune-oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdcscid Il2rgtm1Sug ) and triple-transgenic NOG-EXL [Prkdcscid Il2rgtm1Sug Tg (SV40/HTLV-IL3, CSF2)], which expresses human IL-3 and GM-CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post-transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T-cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC-HIS model can be limited due to onset of xenogeneic graft-versus-host disease (xGVHD) within 3 to 5 weeks post-implantation. Host deficiency in MHC class I, as occurs in beta-2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood-derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC-HIS and PBMC-HIS mouse models are provided. In addition, the co-engraftment and growth kinetics of patient-derived and cell line-derived xenograft tumors in humanized mice and recovery of tumor-infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors. Basic Protocol 1: Establishment of patient-derived xenograft tumors in CD34+ hematopoietic stem cell-humanized mice Basic Protocol 2: Establishment of patient-derived xenograft tumors in peripheral blood mononuclear cell-humanized mice Support Protocol 1: Flow cytometry assessment of humanization in mice Support Protocol 2: Flow cytometry assessment of tumor-infiltrating lymphocytes in tumor-bearing humanized mouse models.

CD4+ T-Cells With High Common γ Chain Expression and Disturbed Cytokine Production Are Enriched in Children With Type-1 Diabetes.

The common gamma chain (γc) contributes to the formation of different cytokine receptors [e.g., IL-2 receptor (IL-2R), IL-7R, and IL-15R], which are important for generation of self-reactive T-cells in autoimmune diseases, like in type 1 diabetes (T1D). Whereas, the roles of membrane and soluble IL-2Rα and IL-7Rα variants in T1D disease pathogenesis are well-described, effects of γc expression and availability for dependent receptors remain elusive. We investigated expression of the γc and dependent receptors on T-cells and soluble γc concentrations in serum from patients with T1D (n = 34) and healthy controls (n = 27). Effector T-cell cytokines as well as IL-2, IL-7, and IL-15 induced STAT5 phosphorylation were analyzed to determine functional implications of differential γc expression of CD4+ T-cell subsets classified by t-distributed Stochastic Neighbor Embedding (t-SNE) analyses. We found increased γc and IL-7Rα expression of CD4+ T-cells from T1D patients as compared to controls. t-SNE analyses assigned differential expression to subsets of memory T-cells co-expressing γc and IL-7Rα. Whereas, γc expression was positively correlated with IL-2Rα in memory T-cells from healthy controls, no dependency was found for patients with T1D. Similarly, the effector T-cell cytokine, IL-21, correlated inversely with γc expression in healthy controls, but not in T1D patients. Finally, T1D patients with high γc expression had increased proportions of IL-2 sensitive pSTAT5+ effector T-cells. These results indicated aberrantly high γc expression of T-cells from T1D patients with implications on dependent cytokine receptor signaling and effector T-cell cytokine production.

A synonymous splice site mutation in IL2RG gene causes late-onset combined immunodeficiency.

X-Linked severe combined immunodeficiency (X-SCID) is a severe form of primary immunodeficiency characterized by absence of T cells and NK cells. X-SCID is caused by a loss-of-function mutation in the IL2RG gene that encodes common gamma chain (γc), which plays an essential role in lymphocyte development. We report the first case of hypomorphic X-SCID caused by a synonymous mutation in the IL2RG gene leading to a splice anomaly, in a family including two patients with diffuse cutaneous warts, recurrent molluscum contagiosum, and mild respiratory infections. The mutation caused aberrant splicing of IL2RG mRNA, subsequently resulted in reduced γc expression. The leaky production of normally spliced IL2RG mRNA produced undamaged protein; thus, T cells and NK cells were generated in the patients. Functional assays of the patients' T cells and NK cells revealed diminished cytokine response in the T cells and absent cytokine response in the NK cells. In addition, the TCR repertoire in these patients was limited. These data suggest that a fine balance between aberrant splicing and leaky production of normally spliced IL2RG mRNA resulted in late-onset combined immunodeficiency in these patients.

HER2 CAR-T Cells Eradicate Uveal Melanoma and T-cell Therapy-Resistant Human Melanoma in IL2 Transgenic NOD/SCID IL2 Receptor Knockout Mice.

Chimeric antigen receptors (CAR) can transmit signals akin to those from activated T-cell receptors when bound to a cell surface target. CAR-expressing T cells against CD19 can cause curative effects in leukemia and lymphoma and is approved for clinical use. However, no CAR-T therapy is currently approved for use in solid tumors. We hypothesize that the resistance of solid tumors to CAR-T can be overcome by similar means as those used to reactivate tumor-infiltrating T lymphocytes (TIL), for example, by cytokines or immune checkpoint blockade. Here we demonstrate that CAR-T cells directed against HER2 can kill uveal and cutaneous melanoma cells in vitro and in vivo. Curative effects in vivo were only observed in xenografts grown in a NOD/SCID IL2 receptor gamma (NOG) knockout mouse strain transgenic for human IL2. The effect was target-specific, as CRISPR/Cas9-mediated disruption of HER2 in the melanoma cells abrogated the killing effect of the CAR-T cells. The CAR-T cells were also able to kill melanoma cells from patients resistant to adoptive T-cell transfer (ACT) of autologous TILs. Thus, CAR-T therapy represents an option for patients that do not respond to immunotherapy with ACT of TIL or immune checkpoint blockade. In addition, our data highlight the use of IL2 transgenic NOG mice as models to prove efficacy of CAR-T-cell products, possibly even in a personalized manner. SIGNIFICANCE: These findings demonstrate that a novel humanized mouse model can help clinical translation of CAR-T cells against uveal and cutaneous melanoma that do not respond to TIL therapy or immune checkpoint blockade.

Arginine methylation controls the strength of γc-family cytokine signaling in T cell maintenance.

The methylation of arginine residues in proteins is a post-translational modification that contributes to a wide range of biological processes. Many cytokines involved in T cell development and activation utilize the common cytokine receptor γ-chain (γc) and the kinase JAK3 for signal transduction, but the regulatory mechanism that underlies the expression of these factors remains unclear. Here we found that the arginine methyltransferase PRMT5 was essential for the maintenance of invariant natural killer T cells (iNKT cells), CD4+ T cells and CD8+ T cells. T cell-specific deletion of Prmt5 led to a marked reduction in signaling via γc-family cytokines and a substantial loss of thymic iNKT cells, as well as a decreased number of peripheral CD4+ T cells and CD8+ T cells. PRMT5 induced the symmetric dimethylation of Sm proteins that promoted the splicing of pre-mRNA encoding γc and JAK3, and this critically contributed to the expression of γc and JAK3. Thus, arginine methylation regulates strength of signaling via γc-family cytokines by facilitating the expression of signal-transducing components.

Adoptive transfer of xenoantigen­stimulated T cell receptor Vß­restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice.

Polyclonal expansion of human regulatory T cells (Tregs) prevents xenogeneic rejection by suppressing effector T cell responses in vitro and in vivo. However, a major limitation to using polyclonally expanded Tregs is that they may cause pan­immunosuppressive effects. The present study was conducted to compare the ability of ex vivo expanded human xenoantigen­stimulated Tregs (Xeno­Treg) and polyclonal Tregs (Poly­Treg) to protect islet xenografts from rejection in NOD­SCID interleukin (IL)­2 receptor (IL2r)γ­/­ mice. Human cluster of differentiation (CD)4+CD25+CD127lo Tregs, expanded either by stimulating with porcine peripheral blood mononuclear cells (PBMCs) or anti­CD3/CD28 beads, were characterized by immune cell phenotyping, T cell receptor (TCR) Vß CDR3 spectratyping and performing suppressive activity assays in vitro. The efficiency of adoptively transferred ex vivo human Tregs was evaluated in vivo using neonatal porcine islet cell clusters (NICC) transplanted into NOD­SCID IL­2rγ­/­ mice, which received human PBMCs with or without Xeno­Treg or Poly­Treg. Xeno­Treg, which expressed increased levels of human leukocyte antigen­DR and secreted higher levels of IL­10, demonstrated enhanced suppressive capacity in a pig­human mixed lymphocyte reaction. Spectratypes of TCR Vß4, Vß10, Vß18 and Vß20 in Xeno­Treg showed restriction and expanded clones at sizes of 205, 441, 332 and 196 respectively, compared to those of Poly­Treg. Reconstitution of mice with human PBMCs and Poly­Treg resulted in NICC xenograft rejection at 63 days. Adoptive transfer with human PBMCs and Xeno­Treg prolonged islet xenograft survival beyond 84 days, with grafts containing intact insulin­secreting cells surrounded by a small number of human CD45+ cells. This study demonstrated that adoptive transfer of ex vivo expanded human Xeno­Treg may potently prevent islet xenograft rejection in humanized NOD­SCID IL2rγ­/­ mice compared with Poly­Treg. These findings suggested that adoptive Treg therapy may be used for immunomodulation in islet xenotransplantation by minimizing systemic immunosuppression.

RORγt limits the amount of the cytokine receptor γc through the prosurvival factor Bcl-xL in developing thymocytes.

The cytokine receptor subunit γc provides critical signals for T cell survival and differentiation. We investigated the molecular mechanism that controls the cell surface abundance of γc during T cell development in the thymus. We found that the amount of γc was low on CD4+CD8+ double-positive (DP) thymocytes before their positive selection to become mature T cells. The transcription factor RORγt was abundant in immature DP thymocytes, and its loss resulted in an increase in the abundance of surface γc, specifically on preselection DP cells. Rather than directly repressing expression of the gene encoding γc, RORγt acted through the antiapoptotic protein Bcl-xL to reduce the abundance of surface γc, which resulted in decreased cytokine signaling and was associated with inhibition of cell metabolism and mitochondrial biogenesis. Accordingly, overexpression of Bcl-xL in RORγt-deficient thymocytes restored the amount of surface γc to that present on normal preselection DP cells. Together, these data highlight a previously unappreciated role for RORγt and Bcl-xL in limiting γc abundance at the cell surface and reveal a signaling circuit in which survival factors control cytokine signaling by limiting the abundance and surface distribution of a receptor subunit shared by several cytokines.

Long-term antigen exposure irreversibly modifies metabolic requirements for T cell function.

Energy metabolism is essential for T cell function. However, how persistent antigenic stimulation affects T cell metabolism is unknown. Here, we report that long-term in vivo antigenic exposure induced a specific deficit in numerous metabolic enzymes. Accordingly, T cells exhibited low basal glycolytic flux and limited respiratory capacity. Strikingly, blockade of inhibitory receptor PD-1 stimulated the production of IFNγ in chronic T cells, but failed to shift their metabolism towards aerobic glycolysis, as observed in effector T cells. Instead, chronic T cells appeared to rely on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) to produce ATP for IFNγ synthesis. Check-point blockade, however, increased mitochondrial production of superoxide and reduced viability and effector function. Thus, in the absence of a glycolytic switch, PD-1-mediated inhibition appears essential for limiting oxidative metabolism linked to effector function in chronic T cells, thereby promoting survival and functional fitness.

B-cell differentiation and IL-21 response in IL2RG/JAK3 SCID patients after hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplant (HSCT) typically results in donor T-cell engraftment and function in patients with severe combined immunodeficiency (SCID), but humoral immunity, particularly when using donors other than matched siblings, is variable. B-cell function after HSCT for SCID depends on the genetic cause, the use of pre-HSCT conditioning, and whether donor B-cell chimerism is achieved. Patients with defects in IL2RG or JAK3 undergoing HSCT without conditioning often have poor B-cell function post-HSCT, perhaps as a result of impairment of IL-21 signaling in host-derived B cells. To investigate the effect of pre-HSCT conditioning on B-cell function, and the relationship of in vitro B-cell function to clinical humoral immune status, we analyzed 48 patients with IL2RG/JAK3 SCID who were older than 2 years after HSCT with donors other than matched siblings. T follicular helper cells (TFH) developed in these patients with kinetics similar to healthy young children; thus, poor B-cell function could not be attributed to a failure of TFH development. In vitro differentiation of B cells into plasmablasts and immunoglobulin secretion in response to IL-21 strongly correlated with the use of conditioning, donor B-cell engraftment, freedom from immunoglobulin replacement, and response to tetanus vaccine. Patients receiving immunoglobulin replacement who had normal serum immunoglobulin M showed poor response to IL-21 in vitro, similar to those with low serum IgM. In vitro response of B cells to IL-21 may predict clinically relevant humoral immune function in patients with IL2RG/JAK3 SCID after HSCT.

Regulation of T cell proliferation with drug-responsive microRNA switches.

As molecular and cellular therapies advance in the clinic, the role of genetic regulation is becoming increasingly important for controlling therapeutic potency and safety. The emerging field of mammalian synthetic biology provides promising tools for the construction of regulatory platforms that can intervene with endogenous pathways and control cell behavior. Recent work has highlighted the development of synthetic biological systems that integrate sensing of molecular signals to regulated therapeutic function in various disease settings. However, the toxicity and limited dosing of currently available molecular inducers have largely inhibited translation to clinical settings. In this work, we developed synthetic microRNA-based genetic systems that are controlled by the pharmaceutical drug leucovorin, which is readily available and safe for prolonged administration in clinical settings. We designed microRNA switches to target endogenous cytokine receptor subunits (IL-2Rß and γc) that mediate various signaling pathways in T cells. We demonstrate the function of these control systems by effectively regulating T cell proliferation with the drug input. Each control system produced unique functional responses, and combinatorial targeting of multiple receptor subunits exhibited greater repression of cell growth. This work highlights the potential use of drug-responsive genetic control systems to improve the management and safety of cellular therapeutics.