Common Gamma Chain Research Articles

Use of Humanized Mice to Study the Pathogenesis of Autoimmune and Inflammatory Diseases.

Animal models of disease have been used extensively by the research community for the past several decades to better understand the pathogenesis of different diseases and assess the efficacy and toxicity of different therapeutic agents. Retrospective analyses of numerous preclinical intervention studies using mouse models of acute and chronic inflammatory diseases reveal a generalized failure to translate promising interventions or therapeutics into clinically effective treatments in patients. Although several possible reasons have been suggested to account for this generalized failure to translate therapeutic efficacy from the laboratory bench to the patient's bedside, it is becoming increasingly apparent that the mouse immune system is substantially different from the human. Indeed, it is well known that >80 major differences exist between mouse and human immunology; all of which contribute to significant differences in immune system development, activation, and responses to challenges in innate and adaptive immunity. This inconvenient reality has prompted investigators to attempt to humanize the mouse immune system to address important human-specific questions that are impossible to study in patients. The successful long-term engraftment of human hematolymphoid cells in mice would provide investigators with a relatively inexpensive small animal model to study clinically relevant mechanisms and facilitate the evaluation of human-specific therapies in vivo. The discovery that targeted mutation of the IL-2 receptor common gamma chain in lymphopenic mice allows for the long-term engraftment of functional human immune cells has advanced greatly our ability to humanize the mouse immune system. The objective of this review is to present a brief overview of the recent advances that have been made in the development and use of humanized mice with special emphasis on autoimmune and chronic inflammatory diseases. In addition, we discuss the use of these unique mouse models to define the human-specific immunopathological mechanisms responsible for the induction and perpetuation of chronic gut inflammation.

Mild Heat Treatment Primes Human CD34(+) Cord Blood Cells for Migration Toward SDF-1α and Enhances Engraftment in an NSG Mouse Model.

Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short-term exposure of CD34(+) CB cells to 39.5°C would enhance their response to stromal-derived factor-1 (SDF-1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34(+) CB that migrated toward SDF-1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34(+) cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl-β-cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application-wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34(+) CB cells in sublethally irradiated non-obese diabetic severe combined immunodeficiency IL-2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients.

441. Direct Comparison of EF1α and PGK Promoters Reveals Superior Performance of the PGK Promoter for Expression of the Common Gamma Chain in a Canine Model of In Vivo Foamy Virus Gene Therapy for Severe Combined Immunodeficiency

Foamy viruses (FV) are ideal vectors for in vivo gene therapy because FV infection is non-pathogenic in people, FV are more resistant to serum inactivation than lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and FV exhibit favorable proviral integration patterns compared to gammaretroviruses. In vivo gene therapy with a FV vector expressing the human common gamma chain (γC) under the short EF1α promoter results in a functional immune reconstitution in X-SCID dogs.

Interleukin-15 receptor α signaling constrains the development of IL-17-producing γδ T cells (HEM2P.232)

Abstract The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of interleukin (IL)-15 and its unique receptor, IL-15Rα, in the development of IL-17-producing γδ (γδ-17) T cells. Phenotypic analysis showed that CD44high γδ-17 cells expressed IL-15Rα and the common gamma chain (CD132) yet lacked the IL-2/15Rβ chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice but not IL-15 KO mice. γδ-17 cells were also increased in the peripheral lymph nodes of transgenic knock-in mice where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. Finally, an analysis of neonatal thymi revealed that the CD44low precursors of γδ-17 cells were increased in newborn mice lacking IL-15Rα. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny with long-term effects on their peripheral homeostasis in the adult.

563. Rapid and Nuclease-Free Generation of Gene-Repaired Human iPS Cells from IL2RG-Defective Fibroblasts

Recent developments of artificial nucleases, such as TALENs and CRISPR/Cas9, have made gene repair therapy of inherited diseases more realistic. However, off-target chromosomal breaks resulting in imprecise DNA repair by the nucleases are still significant concerns from safety aspects, which is the most critical issue of clinical gene therapy. Furthermore, prolonged culture during induction and manipulation of induced pluripotent stem cells (iPSCs) has been shown to increase point mutations and copy number variations in the iPSC genome, raising additional safety concerns for therapeutic applications of iPSCs. In order to minimize cell culture for iPSC induction and achieve efficient gene repair without using nucleases, we examined a strategy of consecutive iPSC induction and gene repair without cell cloning between the two processes. For this purpose, we utilized Sendai virus vector (SeV)-mediated iPSC induction and helper-dependent adenoviral vector (HDAdV)-mediated gene targeting.To mimic somatic cells from patients of X-linked severe combined immunodeficiency, we firstly knocked out exon 1 of the common gamma chain of human interleukin-2 receptor (IL2RG) locus by utilizing TALENs, and subsequently derived fibroblast-like cells. Next, these cells were subject to iPSC induction by using the SeV expressing Yamanaka factors, SeVdp(KOSM). Instead of isolating and characterizing individual SeV-iPS colonies before gene targeting, iPSC-like colonies were grown as a cell population. Twenty-two to 26 days after SeV infection, the cells were transduced with a HDAdV targeting the IL2RG locus. This HDAdV was designed to introduce the PGK-neo cassette into intron 4 and to repair the mutation of exon 1. Cells were subject to G418/FIAU selection to isolate gene-targeted SeV-iPS colonies. Correct targeting at the IL2RG locus by the vector in G418/FIAU double-resistant colonies was verified by PCR analyses. From two independent experiments, the gene targeting frequencies were similar to those in authentic human iPSCs at 1.6 – 1.8 × 10-5 per cell and 60 – 75% of chromosomal integration (G418/FIAU-resistance), among them 60 – 80% of gene-targeted colonies had the exon 1 mutation repaired. At the time of HDAdV infection, the SeV-iPSCs already showed characteristics of pluripotency, revealed by bisulfite sequencing of the NANOG locus and TRA-1-60 immunostaining. The pluripotency was also maintained in gene-targeted iPSC colonies. From the start of iPSC induction (infection with SeV) to isolation of gene-repaired iPSC colonies, it took only 40 days. We are currently analyzing the expression of IL2RG in differentiated T cells derived from the gene-repaired iPSCs.These results suggest that, by combining efficient methods for iPSC induction and gene targeting, gene repair therapy using patients’ somatic cells becomes more realistic.

Optimization and validation of processes related to the GMP compliant manufacture of genetically modified CAR expressing NK-92 cells

Emerging evidences show immunotherapy by enhancing our own immune system has become an attractive approach for cancer treatment due to the rapid drug resistance of chemotherapy. Fusion cytokines derived from GM-CSF and common g-chain interleukins have potent gain-of-function properties to alter host immune response.Wehave previously demonstrated that coupling ofGM-CSF at the N-terminus of common g-chain interleukins leads to meaningful gain-of function activity in interleukin-responsive lymphomyeloid cells. Recently, wehave generated a newly bioengineered fusokine GIFT4 that is derived from GM-CSF and IL-4. Here we tested the bioactivity of the novel fusion cytokine GIFT4 as a potential immunotherapeutic agent against caner. We observed that GIFT4 directly elicits anti-tumor adaptive B-cell immune response and consequent T cell immunity in vitro and in vivo. Murine GIFT4 expression suppresses melanoma tumor growth in immunologically intact mouse and administration of GIFT4 protein inhibits murine melanoma growth in vivo. GIFT4-B cell primed cytotoxic T cells frommelanoma patients specifically kill humanmelanoma cells in vitro and in immunedeficientNSGmice.Thus,GIFT4defines a novel engineered cytokine that mediates endogenous expansion of B-cells with potent anti-tumor effector function. We propose that GIFT4 protein could serve as a novel immunotherapeutic agent and defines a previously unrecognized potential of B-cells for personalized cancer cell immunotherapy. We expect that GIFT4 as an anti-melanomaagentwill provideanovel strategyandopens anewavenue forhumanBcellbased immunotherapy against melanoma. We propose that GIFT4-B cells could serve as a potent immunotherapeutic agent and define a previously unrecognized potential of B-cells for personalized cancer cell immunotherapy.

Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1 associated myelopathy/ tropical spastic paraparesis. (P6.299)

Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1 associated myelopathy/ tropical spastic paraparesis. (P6.299)

Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes by activated T-cells with various differentiation status in vitro

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common γ-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vivo using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+ - cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common Γ-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal “surrogate” cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important in the construction of a general model of self-maintenance and differentiation of immune cells.

Sa1772 Ilc-Like Lymphomas in Refractory Celiac Disease Type II Respond to Multiple Common γ-Chain Cytokines and Are Sensitive to JAK-Inhibitors

Sa1772 Ilc-Like Lymphomas in Refractory Celiac Disease Type II Respond to Multiple Common γ-Chain Cytokines and Are Sensitive to JAK-Inhibitors

Lymphoid Regeneration from Gene-Corrected SCID-X1 Subject-Derived iPSCs

X-linked Severe Combined Immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted life span of less than 1 year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ), leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic subject-specific mutant and gene-corrected iPSC lines. While the subject-derived mutant iPSCs have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSCs can additionally generate mature NK cells and T cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 subjects.

Requirement for innate immunity and CD90⁺ NK1.1⁻ lymphocytes to treat established melanoma with chemo-immunotherapy.

We sought to define cellular immune mechanisms of synergy between tumor-antigen-targeted monoclonal antibodies and chemotherapy. Established B16 melanoma in mice was treated with cytotoxic doses of cyclophosphamide in combination with an antibody targeting tyrosinase-related protein 1 (αTRP1), a native melanoma differentiation antigen. We find that Fcγ receptors are required for efficacy, showing that antitumor activity of combination therapy is immune mediated. Rag1(-/-) mice deficient in adaptive immunity are able to clear tumors, and thus innate immunity is sufficient for efficacy. Furthermore, previously treated wild-type mice are not significantly protected against tumor reinduction, as compared with mice inoculated with irradiated B16 alone, consistent with a primarily innate immune mechanism of action of chemo-immunotherapy. In contrast, mice deficient in both classical natural killer (NK) lymphocytes and nonclassical innate lymphocytes (ILC) due to deletion of the IL2 receptor common gamma chain IL2γc(-/-)) are refractory to chemo-immunotherapy. Classical NK lymphocytes are not critical for treatment, as depletion of NK1.1⁺ cells does not impair antitumor effect. Depletion of CD90⁺NK1.1⁻ lymphocytes, however, both diminishes therapeutic benefit and decreases accumulation of macrophages within the tumor. Tumor clearance during combination chemo-immunotherapy with monoclonal antibodies against native antigen is mediated by the innate immune system. We highlight a novel potential role for CD90⁺NK1.1⁻ ILCs in chemo-immunotherapy.

The role of IL-21 in immunity and cancer

Interleukin-21 (IL-21), produced predominantly by CD4+ T cells and natural killer T (NKT) cells, is a newly discovered member of the common γ-chain family of cytokines. It has been implicated in many immunological processes and has been linked to autoimmune diseases, allergies and other inflammatory diseases. In recent years, the role for IL-21 in the pathogenesis of cancer has also been extensively studied. In this review, we will discuss recent advances concerning the role of IL-21 in immunological processes and the pathogenesis of cancer.

The role of soluble common gamma chain in autoimmune disease.

The common gamma chain (γc) is the central signaling unit for a number of cytokine receptors collectively known as the γc cytokine receptor family. γc is critical for ligand binding and signaling by γc cytokines. γc cytokine signaling had been thought to be mainly regulated by cytokine-specific receptor α chain expression levels with little or no effect by γc surface levels because γc expression was presumed to remain unchanged during T-cell activation and development. The extent of γc cytokine responses is thought to be regulated by cytokine specific receptor subunits and not by the γc receptor. In contrast to this prevailing view, we have recently reported that γc itself actively regulates γc cytokine responses. Interestingly, γc exerted its regulatory effects not only as a conventional membrane receptor protein but also as a secreted protein whose expression was upregulated upon T-cell stimulation. Here we will review how a soluble form of γc, which is generated by alternative splicing, regulates γc cytokine signaling and plays a role in controlling immune activation related to autoimmune disease.

Severe combined imunodeficiency caused by defect of the common gamma chain of the interleukin 2 receptor

Results The mutation detected was a defect of the common gamma chain of the interleukin 2 receptor (IL2Rg). Even though genetic counseling advised otherwise the patient’s mother got pregnant during follow-up and as no compatible donor was found we chose to wait birth and verify compatibility. Genetic evaluation of the newborn revealed the absence of the IL2Rg gene defect in blood cord and a matching HLA. Cord stem cell transplantation was scheduled afterwards.

JAK3 Mutants Transform Hematopoietic Cells through JAK1 Activation Causing T-Cell Acute Lymphoblastic Leukemia in a Bone Marrow Transplant Mouse Model

▪JAK3 is a cytosolic tyrosine kinase that associates with the common gamma chain in different cytokine receptors, in which the JAK1 kinase is another essential signaling protein. Large scale sequencing efforts recently identified mutations in the IL7R, JAK1 or JAK3 genes in about 25% of T-cell acute lymphoblastic leukemia (T-ALL) cases, with JAK3 being the most frequently (15% of T-ALL cases) mutated gene in this pathway. To determine if all mutations in JAK3 are true oncogenic mutations, we generated expression plasmids for 16 JAK3 mutants (M511I, A572T, A573V, R657Q, R657W, V674A, V678M, V678L, R775C, L857Q, Q865E, L875H, P906S, R925S, E958K, E1106G) and determined the in vitro and in vivo transforming properties, as well as their sensitivity to JAK kinase inhibitors.For 12 of 16 mutants expression in the IL3-dependent Ba/F3 cell line resulted in transformation to IL3 independent growth. Similarly, expression of the transforming mutants in IL7-receptor reconstituted 293T cells confirmed their ligand independent activation, while this was not observed for the non-transforming mutants. These 4 non-transforming mutants are likely to be passenger mutations, illustrating that results from sequencing always need to be confirmed by functional assays to distinguish driver mutations from passenger mutations. Most JAK3 mutants, except JAK3 L857Q and JAK3 L875H, were dependent on JAK1 kinase activity for their transforming capacities. In agreement with this, we observed that Ba/F3 cells transformed by the JAK1 dependent JAK3 mutants could be inhibited by ruxolitinib, a JAK1/JAK2 selective inhibitor, while the JAK3 L857Q and JAK3 L875H transformed cells were significantly less sensitive to ruxolitinib treatment. As expected, all JAK3 mutants were sensitive to the JAK3 selective inhibitor tofacitinib, except for JAK3 L875H, which showed resistance to all inhibitors tested.To determine the in vivo oncogenic properties of the JAK3 mutants, we expressed selected JAK3 mutants (M511I, A573V, L857Q, V674A and R657Q) in mouse hematopoietic cells through viral transduction. Mice transplanted with cells expressing JAK3 M511I, A573V or V674A showed a gradual increase of the WBC count and developed a T-ALL like disease within 120 to 200 days. In contrast, mice transplanted with cells expressing JAK3 L857Q or R657Q showed a lower increase in WBC count, and did present with severe splenomegaly and lymphadenopathy. Expression of JAK3 L857Q caused severe thymus hyperplasia, while the JAK3 R657Q mutant caused B-cell leukemia, illustrating that different JAK3 mutants seem to have variable oncogenic characteristics.Mice transplanted with cells expressing JAK3 M511I were treated with the JAK3 selective inhibitor tofacitinib and disease progression was followed by white blood cell count measurements. Treatment of the animals for 5 weeks with tofacitinib (oral gavage, 40 mg/kg/day) significantly decreased the disease progression compared to placebo treated mice. Moreover, we observed severe apoptosis of the leukemia cells in spleen and thymus in tofacitinib treated animals and not in placebo treated mice. However, tofacitnib treatment could not eradicate all leukemia cells, and the mice progressed when treatment was stopped.In conclusion, JAK3 is recurrently mutated in T-ALL patients, and we demonstrate that most JAK3 mutants are transforming proteins using In vitro and in vivo experiments. Our results show that JAK1 is an essential kinase for most JAK3 mutants, and that the majority of JAK3 mutants are sensitive to JAK3 and JAK1 specific inhibitors such as tofacitinib and ruxolitinib. However, some JAK3 mutants do show resistance to these inhibitors, which will need to be taken into account when trials are initiated for the treatment of ALL patients with JAK specific inhibitors. DisclosuresNo relevant conflicts of interest to declare.

TNFR2 Is Expressed Preferentially By Late Differentiated CD8 T-Cells and Can be Triggered By TNFR2-Specific Ligand to Induce Cell Death of Recently Activated Antigen-Specific T Cells: A Possible Role of TNFR2 in T-Cell Deflation

Recent evidence suggests that Tumor Necrosis Factor (TNF) can selectively kill antigen-specific autoreactive CD8+ T-cells through engagement with TNF Receptor 2 (TNFR2) (1). Within the immune system, TNFR2 expression is restricted to subsets of T-cells, a profile which is in marked contrast to the ubiquitous pattern of expression of TNFR1. However, the spectrum and physiological significance of TNFR2 expression by CD8+ T-cell subpopulations is unknown.In this study we analysed the expression of TNFR2 by CD8 T-cell subsets isolated from normal healthy donors by flow cytometry. In addition, in order to understand the physiological significance of TNFR2 expression on recently activated T cells, we further studied expression on CMV-specific CD8 T-cells which expanded in stem cell transplant patients in response to episodes of CMV reactivation. The expression of TNFR2 was compared to that of other common gamma chain receptors including IL2R and IL7R, and to the expression of a receptor for inflammatory cytokine IL6.TNFR2 expression was found to increase during differentiation of CD8+ T cells. In particular, TNFR2 expression was seen on 6.5% of naïve, 14.6% of central memory, 37.9% of effector memory and 45.2% of CD45RA-revertant effector memory (TEMRA) CD8+ T cells. In contrast, common gamma chain cytokine receptor expression was skewed towards less differentiated T-cell subsets. For example, IL-7R was expressed by 63% of central memory populations but only 18.4% of the TEMRA subset. Comparable expression of IL2R was 12.1% on TCM and 2% on TEMRA. Of interest, IL-6 receptor expression was predominantly expressed by naïve CD8 T-cells (69.5%). In support of these results, we went on to show that expression of TNFR2 was inducible on primary T cells following activation with anti-CD3 and IL-2 in vitro. Healthy CMV seropositive donors had a larger median number of CD8+ T cells expressing TNFR2 (53%) in comparison to CMV seronegative donors (15%), (p<0.0001), consistent with the known accumulation of differentiated T-cells within CMV seropositive individuals.The expression of TNFR2 was then examined on CMV-specific CD8 T-cells which were undergoing acute expansion in response to viremia in six haemopoietic stem cell transplant patients. The expansion of CMV-specific CD8 T-cells was accompanied by an increase in the intensity of TNFR2 expression which later decreased during the retraction of antigen-specific T-cells during resolution of viremia. In order to explore the functional significance of TNFR2 expression, T-cells isolated from healthy donors were treated with recombinant TNFR2-specific ligand. This induced cell loss ranging from 13% to 60% of all CD8 T-cells in relation to untreated control cells, with selective depletion of the TNFR2+ population. A similar proportion of CMV-specific T-cells from transplant patients were eliminated by ex vivo stimulation of TNFR2.In conclusion our work shows that TNFR2 expression increases during differentiation of CD8+ T cells. In addition, we were able to utilize virus-specific T cells from SCT patients to show that expression is increased during the acute response to stimulation with antigen. We also provide evidence that TNFR2 activation can lead to the partial elimination of antigen-specific CMV-specific T-cells and it may thus play an important role in the ‘deflation’ of a pathogen-specific T-cell immune response following resolution of infection. These data suggest that TNFR2 expression may act as a ligand to signal activation-induced cell death in late differentiated populations of CD8+ T cells. Further investigations are required to assess the molecular pathways of TNFR2 signalling that are activated following receptor ligation in vivoand whether or not these are disrupted in disorders associated with chronic CD8+ T cell lymphproliferation.(1) L. Ban et al, PNAS 2008, 105: 3644 DisclosuresNo relevant conflicts of interest to declare.

Robust Therapeutic Expression of the Common Gamma Chain with the Human Pgk Promoter Using Foamy Virus in Vivo Gene Therapy in a Canine Model of Severe Combined Immunodeficiency

In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy for 3 primary reasons: (1) FV are non-pathogenic in humans, (2) they exhibit enhanced serum stability as compared to lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and (3) FV integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of CD3+ lymphocytes in canine X-SCID, which undergo T cell receptor rearrangement and exhibit a functional signaling response to T cell activating mitogens (Burtner CR, Beard BC, Kennedy DR, et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy, including different promoters, using injections of vectors marked with different fluorophores.Preliminary data indicated that ex vivo transduction of canine CD34+ cells with a FV vector expressing human γC and a fluorescent reporter under the human phosphoglycerate kinase (PGK) promoter resulted in higher transduction efficiencies and increased mean fluorescence intensity, compared to that of an identical vector containing the EF1α promoter. We therefore performed a head-to-head comparison of the two promoters by simultaneously injecting X-SCID pups with equal titers of 2 therapeutic, human γC-encoding FV vectors that differed only in the promoter used to drive human γC expression and in the fluorophore color to distinguish gene-marked cells (GFP and mCherry). Each dog received 4 x 108 infectious units of each FV vector. A significant population of gene-marked lymphocytes appeared in the PGK arm 42 days post in vivo gene therapy, which continued to expand over the next two months of follow-up (Fig 1A). By 84 days post injection, lymphocyte gene marking in the competitive PGK arm reached 60% in both dogs. For comparison, this robust level of lymphocyte gene marking was achieved in only 2 of 5 dogs after 122 and 160 days, respectively, in our previous EF1α virus treated cohort. In contrast, the EF1α arm peaked at 42 days after in vivo gene therapy and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the by the EF1α arm, suggesting competition within the expanding T cell niche. The expansion of gene-marked lymphocytes was followed by the development of CD3+ T cells, leading to a therapeutic level of CD3+ cells (1000 cells/μl of blood) in both dogs (Fig 1B). Additionally, our data indicate low but persistent gene marking in other blood cells, including granulocytes and B cells, with B cell marking in one animal exceeding 2% in the PGK arm. Our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. These data also highlight the utility of the in vivo approach to explore key parameters of vector design in competitive repopulation experiments that may be useful for other diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

­Impact of Porcine-Human Mixed Hematopoietic Chimerism on Human NK Cell Response to Porcine Cells

Mixed hematopoietic chimerism permits durable tolerance of T, B and NK cells to xenoantigens in a rat to mouse bone marrow transplant model. However, it is unclear whether tolerance of human NK cells to pig xenoantigens can be induced by mixed hematopoietic chimerism. We assessed the tolerance of human NK cells towards pig cells in a humanized mouse model with established pig and human mixed xenogeneic chimerism. Pig and human mixed chimeras (MCs) were generated by injection of pig bone marrow cells to irradiated pig cytokine (IL3, GMCSF and SCF) transgenic NOD-scid common gamma chain knockout (NSG) mice followed by injection of human fetal liver CD34+ cells 3 day later. In the control group, only human CD34+ cells were transplanted. 12 weeks post-transplant, hydrodynamic injection of plasmid encoding human Flt3L followed by injection of three rounds of recombinant IL15/IL-15 receptor alpha Fc complex was given to promote human NK cell reconstitution. The control non–mixed chimeric group (Non-MC) received the same treatment without pig cells. 12 days following induction of human NK cell reconstitution, human NK cells from both MC and Non-MC mice were isolated from the spleen and their cytotoxic responses in vitro to pig cells were determined in a chromium release assay. In addition, the presence of pig cells in various tissues of the chimeric mice was studied.While human NK cells were usually undetectable in peripheral blood prior to the injection of human Flt3L plasmid and IL-15/IL-15 receptor alpha-Fc complex, they were detected by 5 days post-injection of IL-15 protein. 12 days post-induction of human NK cell reconstitution, pig cells remained detectable in peripheral blood, spleen, bone marrow and liver in the chimeric mice together with human NK cells. The co-existence of human NK cells and pig cells suggested that human NK cells in MCs might be tolerant to pig cells. Consistent with this notion, cytotoxicity assays showed that human NK cells from MCs demonstrated decreased killing of pig PBMC blasts compared to NK cells from Non-MC mice. Importantly, killing of K562 cells by NK cells from MCs mice was also decreased compared to that of Non-MC mice, suggesting that human NK cell tolerance to pig cells induced by mixed chimerism was associated with global hyporesponsiveness, as we have previously observed in a rat-to-mouse bone marrow transplantation model. Moreover, higher percentages of CD56highCD16low and lower percentages of CD56lowCD16high human NK cell subsets were observed in bone marrow of chimeric mice than in non-chimeric mice, indicating that the development of human NK cells in bone marrow might be altered by the presence of pig cells. In summary, our data suggest that mixed xenogeneic chimerism may induce tolerance of human NK cells towards porcine cells, but the tolerance may be associated with global hyporesponsiveness. DisclosuresNo relevant conflicts of interest to declare.

Human regulatory T-cell development is dictated by Interleukin-2 and -15 expressed in a non-overlapping pattern in the thymus

Thymus-derived FOXP3-expressing regulatory T-cells (tTregs) are master orchestrators of physiological and pathological immune responses, thus constituting ideal targets for the treatment of autoimmunity. Despite their clinical importance, the developmental program governing their differentiation in the human thymus remains poorly understood.Here, we investigated the role of common gamma-chain cytokines in human tTreg differentiation, by performing gain- and loss-of-function experiments in 3D and 2D postnatal thymic cultures. We identified IL-2 and IL-15 as key molecular determinants in this process and excluded a major function for IL-4, IL-7 and IL-21. Mechanistically, IL-2 and IL-15 were equally able to drive tTreg precursor differentiation into FOXP3+ cells, and promote tTreg proliferation and survival. Both cytokines also increased the expression levels of molecules associated with effector function within FOXP3+ subsets, supporting their involvement in tTreg functional maturation. Furthermore, we revealed that IL-2 and IL-15 are expressed in a non-overlapping pattern in the human thymus, with the former produced mainly by mature αβ and γδ thymocytes and the latter by monocyte/macrophages and B lymphocytes.Our results identify core mechanisms dictating human tTreg development, with IL-2 and IL-15 defining specific niches required for tTreg lineage stabilization and differentiation, with implications for their therapeutic targeting in autoimmune conditions.

Concise review: engineering the fusion of cytokines for the modulation of immune cellular responses in cancer and autoimmune disorders.

As our understanding of the basic precepts of immunobiology continue to advance at a rapid pace, translating such discoveries into meaningful therapies for patients has proved challenging. This is especially apparent in the use of cytokine-based immunotherapies for cancer. Unanticipated and serious side effects, as well as low objective response rates seen in clinical trials, have dealt setbacks to the field. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and common γ-chain (γ-c) interleukins are cytokines that have been used as stand-alone immunotherapies with moderate success. Our group has found that the fusion of GM-CSF to members of γ-c interleukins results in the generation of novel proteins with unique signaling properties and unheralded biological effects. These fusion proteins, termed GIFT (GM-CSF interleukin fusion transgenes) fusokines, are the result of combining GM-CSF and a γ-c interleukin into a single, bifunctional polypeptide. In our experience, GIFT fusokines often confer immune cells with a gain of function that cannot be explained by the mere sum of their constituent moieties. They act as bispecific ligands, coupling activated GM-CSF and interleukin receptors together to drive unique downstream signaling events. The synergy that arises from these fusions has shown great promise in its ability to modulate the immune response and overcome maladaptive biological processes that underlie diseases such as cancer and autoimmune conditions. In this review, we discuss the ways in which the GIFT fusokines are able to alter the immune response, particularly in disease states, with a special emphasis on how these novel molecules may be translated into effective therapies in the clinical setting.