CD300f signalling induces inhibitory human monocytes/macrophages.

The CD300 glycoproteins are a family of related leucocyte surface molecules that regulate the immune response via their paired triggering and inhibitory receptors. Here we studied CD300f, an apoptotic cell receptor, and how it modulates the function of human monocytes and macrophages. We showed that CD300f signalling by crosslinking with anti-CD300f mAb (DCR-2) suppressed monocytes causing upregulation of the inhibitory molecule, CD274 (PD-L1) and their inhibition of T cell proliferation. Furthermore, CD300f signalling drove macrophages preferentially towards M2-type with upregulation of CD274, which was further enhanced by IL-4. CD300f signalling activates the PI3K/Akt pathway in monocytes. Inhibition of PI3K/Akt signalling resulting from CD300f crosslinking leads to downregulation of CD274 expression on monocytes. These findings highlight the potential use of CD300f blockade in cancer immune therapy to target immune suppressive macrophages in the tumour microenvironment, a known resistance mechanism to PD-1/PD-L1 checkpoint inhibitors.

Anti-Mouse CD83 Monoclonal Antibody Targeting Mature Dendritic Cells Provides Protection Against Collagen Induced Arthritis.

Antibodies targeting the activation marker CD83 can achieve immune suppression by targeting antigen-presenting mature dendritic cells (DC). This study investigated the immunosuppressive mechanisms of anti-CD83 antibody treatment in mice and tested its efficacy in a model of autoimmune rheumatoid arthritis. A rat anti-mouse CD83 IgG2a monoclonal antibody, DCR-5, was developed and functionally tested in mixed leukocyte reactions, demonstrating depletion of CD83+ conventional (c)DC, induction of regulatory DC (DCreg), and suppression of allogeneic T cell proliferation. DCR-5 injection into mice caused partial splenic cDC depletion for 2-4 days (mostly CD8+ and CD83+ cDC affected) with a concomitant increase in DCreg and regulatory T cells (Treg). Mice with collagen induced arthritis (CIA) treated with 2 or 6 mg/kg DCR-5 at baseline and every three days thereafter until euthanasia at day 36 exhibited significantly reduced arthritic paw scores and joint pathology compared to isotype control or untreated mice. While both doses reduced anti-collagen antibodies, only 6 mg/kg achieved significance. Treatment with 10 mg/kg DCR-5 was ineffective. Immunohistological staining of spleens at the end of CIA model with CD11c, CD83, and FoxP3 showed greater DC depletion and Treg induction in 6 mg/kg compared to 10 mg/kg DCR-5 treated mice. In conclusion, DCR-5 conferred protection from arthritis by targeting CD83, resulting in selective depletion of mature cDC and subsequent increases in DCreg and Treg. This highlights the potential for anti-CD83 antibodies as a targeted therapy for autoimmune diseases.

Dihydrotestosterone (DHT) Enhances Wound Healing of Major Burn Injury by Accelerating Resolution of Inflammation in Mice.

Androgens have been known to inhibit cutaneous wound healing in men and male mice. However, in children with major burn injuries, a synthetic androgen was reported clinically to improve wound healing. The aim of this study is to investigate the role of dihydrotestosterone (DHT) as a new therapeutic approach in treating major burn injury. In the present study, mice received systemic androgen treatment post major burn injury. Wound healing rate and body weight were monitored over 21 days. The serum level of inflammatory cytokines/chemokines were measured using multiplex immunoassays. In addition, splenocyte enumeration was performed by flow cytometry. Healing phases of inflammation, re-epithelialization, cell proliferation and collagen deposition were also examined. In results, DHT treated mice lost less weight and displayed accelerated wound healing but has no impact on hypermetabolism. Mice, after burn injury, displayed acute systemic inflammatory responses over 21 days. DHT treatment shortened the systemic inflammatory response with reduced splenic weight and monocyte numbers on day 14 and 21. DHT treatment also reduced wound infiltrating macrophage numbers. In conclusion, DHT treatment facilitates local wound healing by accelerating the resolution of inflammation, but not through alterations of post-burn hypermetabolic response.

Targeting CD83 in mantle cell lymphoma with anti-human CD83 antibody.

OBJECTIVES: Effective antibody-drug conjugates (ADCs) provide potent targeted cancer therapies. CD83 is expressed on activated immune cells including B cells and is a therapeutic target for Hodgkin lymphoma. Our objective was to determine CD83 expression on non-Hodgkin lymphoma (NHL) and its therapeutic potential to treat mantle cell lymphoma (MCL) which is currently an incurable NHL. METHODS: We analysed CD83 expression on MCL cell lines and the lymph node/bone marrow biopsies of MCL patients. We tested the killing effect of CD83 ADC in vitro and in an in vivo xenograft MCL mouse model. RESULTS: CD83 is expressed on MCL, and its upregulation is correlated with the nuclear factor κB (NF-κB) activation. CD83 ADC kills MCL in vitro and in vivo. Doxorubicin and cyclophosphamide (CP), which are included in the current treatment regimen for MCL, enhance the NF-κB activity and increase CD83 expression on MCL cell lines. The combination of CD83 ADC with doxorubicin and CP has synergistic killing effect of MCL. CONCLUSION: This study provides evidence that a novel immunotherapeutic agent CD83 ADC, in combination with chemotherapy, has the potential to enhance the efficacy of current treatments for MCL.

Targeting CD300f to enhance hematopoietic stem cell transplantation in acute myeloid leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly reduces the rate of relapse in acute myeloid leukemia (AML) but comes at the cost of significant treatment-related mortality. Despite the reduction in relapse overall, it remains common, especially in high-risk groups. The outcomes for patients who relapse after transplant remains very poor. A large proportion of the morbidity that prevents most patients from accessing allo-HSCT is due to toxic nonspecific conditioning agents that are required to remove recipient hematopoietic stem and progenitor cells (HSPCs), allowing for successful donor engraftment. CD300f is expressed evenly across HSPC subtypes. CD300f has transcription and protein expression equivalent to CD33 on AML. We have developed an anti-CD300f antibody that efficiently internalizes into target cells. We have generated a highly potent anti-CD300f antibody-drug conjugate (ADC) with a pyrrolobenzodiazepine warhead that selectively depletes AML cell lines and colony forming units in vitro. The ADC synergizes with fludarabine, making it a natural combination to use in a minimal toxicity conditioning regimen. Our ADC prolongs the survival of mice engrafted with human cell lines and depletes primary human AML engrafted with a single injection. In a humanized mouse model, a single injection of the ADC depletes CD34+ HSPCs and CD34+CD38-CD90+ hematopoietic stem cells. This work establishes an anti-CD300f ADC as an attractive potential therapeutic that, if validated in transplant models using a larger cohort of primary AML samples, will reduce relapse rate and toxicity for patients with AML undergoing allo-HSCT.

Is Hematopoietic Stem Cell Transplantation Required to Unleash the Full Potential of Immunotherapy in Acute Myeloid Leukemia?

From monoclonal antibodies (mAbs) to Chimeric Antigen Receptor (CAR) T cells, immunotherapies have enhanced the efficacy of treatments against B cell malignancies. The same has not been true for Acute Myeloid Leukemia (AML). Hematologic toxicity has limited the potential of modern immunotherapies for AML at preclinical and clinical levels. Gemtuzumab Ozogamicin has demonstrated hematologic toxicity, but the challenge of preserving normal hematopoiesis has become more apparent with the development of increasingly potent immunotherapies. To date, no single surface molecule has been identified that is able to differentiate AML from Hematopoietic Stem and Progenitor Cells (HSPC). Attempts have been made to spare hematopoiesis by targeting molecules expressed only on later myeloid progenitors as well as AML or using toxins that selectively kill AML over HSPC. Other strategies include targeting aberrantly expressed lymphoid molecules or only targeting monocyte-associated proteins in AML with monocytic differentiation. Recently, some groups have accepted that stem cell transplantation is required to access potent AML immunotherapy and envision it as a rescue to avoid severe hematologic toxicity. Whether it will ever be possible to differentiate AML from HSPC using surface molecules is unclear. Unless true specific AML surface targets are discovered, stem cell transplantation could be required to harness the true potential of immunotherapy in AML.

Distinguishing human peripheral blood CD16+ myeloid cells based on phenotypic characteristics.

Myeloid lineage cells present in human peripheral blood include dendritic cells (DC) and monocytes. The DC are identified phenotypically as HLA-DR+ cells that lack major cell surface lineage markers for T cells (CD3), B cells (CD19, CD20), NK cells (CD56), red blood cells (CD235a), hematopoietic stem cells (CD34), and Mo that express CD14. Both DC and Mo can be phenotypically divided into subsets. DC are divided into plasmacytoid DC, which are CD11c- , CD304+ , CD85g+ , and myeloid DC that are CD11c+ . The CD11c+ DC are readily classified as CD1c+ DC and CD141+ DC. Monocytes are broadly divided into the CD14+ CD16- (classical) and CD14dim CD16+ subsets (nonclassical). A population of myeloid-derived cells that have DC characteristics, that is, HLA-DR+ and lacking lineage markers including CD14, but express CD16 are generally clustered with CD14dim CD16+ monocytes. We used high-dimensional clustering analyses of fluorescence and mass cytometry data, to delineate CD14+ monocytes, CD14dim CD16+ monocytes (CD16+ Mo), and CD14- CD16+ DC (CD16+ DC). We sought to identify the functional and kinetic relationship of CD16+ DC to CD16+ Mo. We demonstrate that differentiation of CD16+ DC and CD16+ Mo during activation with IFNγ in vitro and as a result of an allo-hematopoietic cell transplant (HCT) in vivo resulted in distinct populations. Recovery of blood CD16+ DC in both auto- and allo-(HCT) patients after myeloablative conditioning showed similar reconstitution and activation kinetics to CD16+ Mo. Finally, we show that expression of the cell surface markers CD300c, CCR5, and CLEC5a can distinguish the cell populations phenotypically paving the way for functional differentiation as new reagents become available.

CD300f epitopes are specific targets for acute myeloid leukemia with monocytic differentiation.

Antibody-based therapy in acute myeloid leukemia (AML) has been marred by significant hematologic toxicity due to targeting of both hematopoietic stem and progenitor cells (HSPCs). Achieving greater success with therapeutic antibodies requires careful characterization of the potential target molecules on AML. One potential target is CD300f, which is an immunoregulatory molecule expressed predominantly on myeloid lineage cells. To confirm the value of CD300f as a leukemic target, we showed that CD300f antibodies bind to AML from 85% of patient samples. While one CD300f monoclonal antibody (mAb) reportedly did not bind healthy hematopoietic stem cells, transcriptomic analysis found that CD300f transcripts are expressed by healthy HSPC. Several CD300f protein isoforms exist as a result of alternative splicing. Importantly for antibody targeting, the extracellular region of CD300f can be present with or without the exon 4-encoded sequence. This results in CD300f isoforms that are differentially bound by CD300f-specific antibodies. Furthermore, binding of one mAb, DCR-2, to CD300f exposes a structural epitope recognized by a second CD300f mAb, UP-D2. Detailed analysis of publicly available transcriptomic data indicated that CD34+ HSPC expressed fewer CD300f transcripts that lacked exon 4 compared to AML with monocytic differentiation. Analysis of a small cohort of AML cells revealed that the UP-D2 conformational binding site could be induced in cells from AML patients with monocytic differentiation but not those from other AML or HSPC. This provides the opportunity to develop an antibody-based strategy to target AMLs with monocytic differentiation but not healthy CD34+ HSPCs. This would be a major step forward in developing effective anti-AML therapeutic antibodies with reduced hematologic toxicity.

CD83: Activation Marker for Antigen Presenting Cells and Its Therapeutic Potential.

CD83 is a member of the immunoglobulin (Ig) superfamily and is expressed in membrane bound or soluble forms. Membrane CD83 (mCD83) can be detected on a variety of activated immune cells, although it is most highly and stably expressed by mature dendritic cells (DC). mCD83 regulates maturation, activation and homeostasis. Soluble CD83 (sCD83), which is elevated in the serum of patients with autoimmune disease and some hematological malignancies is reported to have an immune suppressive function. While CD83 is emerging as a promising immune modulator with therapeutic potential, some important aspects such as its ligand/s, intracellular signaling pathways and modulators of its expression are unclear. In this review we discuss the recent biological findings and the potential clinical value of CD83 based therapeutics in various conditions including autoimmune disease, graft-vs.-host disease, transplantation and hematological malignancies.

Examination of CD302 as a potential therapeutic target for acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with ~20,000 new cases yearly. The disease develops in people of all ages, but is more prominent in the elderly, who due to limited treatment options, have poor overall survival rates. Monoclonal antibodies (mAb) targeting specific cell surface molecules have proven to be safe and effective in different haematological malignancies. However, AML target molecules are currently limited so discovery of new targets would be highly beneficial to patients. We examined the C-type lectin receptor CD302 as a potential therapeutic target for AML due to its selective expression in myeloid immune populations. In a cohort of 33 AML patients with varied morphological and karyotypic classifications, 88% were found to express CD302 on the surface of blasts and 80% on the surface of CD34+ CD38- population enriched with leukemic stem cells. A mAb targeting human CD302 was effective in mediating antibody dependent cell cytotoxicity and was internalised, making it amenable to toxin conjugation. Targeting CD302 with antibody limited in vivo engraftment of the leukemic cell line HL-60 in NOD/SCID mice. While CD302 was expressed in a hepatic cell line, HepG2, this molecule was not detected on the surface of HepG2, nor could HepG2 be killed using a CD302 antibody-drug conjugate. Expression was however found on the surface of haematopoietic stem cells suggesting that targeting CD302 would be most effective prior to haematopoietic transplantation. These studies provide the foundation for examining CD302 as a potential therapeutic target for AML.

On the Other Side: Manipulating the Immune Checkpoint Landscape of Dendritic Cells to Enhance Cancer Immunotherapy.

Monoclonal antibodies targeting co-inhibitory immune checkpoint molecules have been successful in clinical trials of both solid and hematological malignancies as acknowledged by the 2018 Nobel Prize in Medicine, however improving clinical response rates is now key to expanding their efficacy in areas of unmet medical need. Antibodies to checkpoint inhibitors target molecules on either T cells or tumor cells to stimulate T cells or remove tumor mediated immunosuppression, respectively. However, many of the well-characterized T cell immune checkpoint receptors have their ligands on antigen presenting cells or exert direct effects on those cells. Dendritic cells are the most powerful antigen presenting cells; they possess the ability to elicit antigen-specific responses and have important roles in regulation of immune tolerance. Despite their theoretical benefits in cancer immunotherapy, the translation of DC therapies into the clinic is yet to be fully realized and combining DC-based immunotherapy with immune checkpoint inhibitors is an attractive strategy. This combination takes advantage of the antigen presenting capability of DC to maximize specific immune responses to tumor antigens whilst removing tumor-associated immune inhibitory mechanisms with immune checkpoint inhibition. Here we review the expression and functional effects of immune checkpoint molecules on DC and identify rational combinations for DC vaccination to enhance antigen-specific T cell responses, cytokine production, and promotion of long-lasting immunological memory.

The cell surface phenotype of human dendritic cells.

Dendritic cells (DC) are bone marrow derived leucocytes that are part of the mononuclear phagocytic system. These are surveillance cells found in all tissues and, as specialised antigen presenting cells, direct immune responses. Membrane molecules on the DC surface form a landscape that defines them as leucocytes and part of the mononuclear phagocytic system, interacts with their environment and directs interactions with other cells. This review describes the DC surface landscape, reflects on the different molecules confirmed to be on their surface and how they provide the basis for manipulation and translation of the potent functions of these cells into new diagnostics and immune therapies for the clinic.

CD83 is a new potential biomarker and therapeutic target for Hodgkin lymphoma.

Chemotherapy and hematopoietic stem cell transplantation are effective treatments for most Hodgkin lymphoma patients, however there remains a need for better tumor-specific target therapy in Hodgkin lymphoma patients with refractory or relapsed disease. Herein, we demonstrate that membrane CD83 is a diagnostic and therapeutic target, highly expressed in Hodgkin lymphoma cell lines and Hodgkin and Reed-Sternberg cells in 29/35 (82.9%) Hodgkin lymphoma patient lymph node biopsies. CD83 from Hodgkin lymphoma tumor cells was able to trogocytose to surrounding T cells and, interestingly, the trogocytosing CD83+T cells expressed significantly more programmed death-1 compared to CD83-T cells. Hodgkin lymphoma tumor cells secreted soluble CD83 that inhibited T-cell proliferation, and anti-CD83 antibody partially reversed the inhibitory effect. High levels of soluble CD83 were detected in Hodgkin lymphoma patient sera, which returned to normal in patients who had good clinical responses to chemotherapy confirmed by positron emission tomography scans. We generated a human anti-human CD83 antibody, 3C12C, and its toxin monomethyl auristatin E conjugate, that killed CD83 positive Hodgkin lymphoma cells but not CD83 negative cells. The 3C12C antibody was tested in dose escalation studies in non-human primates. No toxicity was observed, but there was evidence of CD83 positive target cell depletion. These data establish CD83 as a potential biomarker and therapeutic target in Hodgkin lymphoma.

Disruption of Serinc1, which facilitates serine-derived lipid synthesis, fails to alter macrophage function, lymphocyte proliferation or autoimmune disease susceptibility.

During immune cell activation, serine-derived lipids such as phosphatidylserine and sphingolipids contribute to the formation of protein signaling complexes within the plasma membrane. Altering lipid composition in the cell membrane can subsequently affect immune cell function and the development of autoimmune disease. Serine incorporator 1 (SERINC1) is a putative carrier protein that facilitates synthesis of serine-derived lipids. To determine if SERINC1 has a role in immune cell function and the development of autoimmunity, we characterized a mouse strain in which a retroviral insertion abolishes expression of the Serinc1 transcript. Expression analyses indicated that the Serinc1 transcript is readily detectable and expressed at relatively high levels in wildtype macrophages and lymphocytes. The ablation of Serinc1 expression in these immune cells, however, did not significantly alter serine-derived lipid composition or affect macrophage function and lymphocyte proliferation. Analyses of Serinc1-deficient mice also indicated that systemic ablation of Serinc1 expression did not affect viability, fertility or autoimmune disease susceptibility. These results suggest that Serinc1 is dispensable for certain immune cell functions and does not contribute to previously reported links between lipid composition in immune cells and autoimmunity.

The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83+-Activated T Cell Population.

CD83 is a member of the Ig gene superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding characteristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, monocytes, and monocyte-derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases.

CMRF-56(+) blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses.

There are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56(+) BDC, we showed that transfection of hCMRF-56(+) BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56(+) BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56(+) BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8(+) T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56(+) BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56(+) BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.

Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration.

C-type lectin receptors play important roles in immune cell interactions with the environment. We described CD302 as the simplest, single domain, type I C-type lectin receptor and showed it was expressed mainly on the myeloid phagocytes in human blood. CD302 colocalized with podosomes and lamellopodia structures, so we hypothesized that it played a role in cell adhesion or migration. In this study, we used mouse models to obtain further insights into CD302 expression and its potential immunological function. Mouse CD302 transcripts were, as in humans, highest in the liver, followed by lungs, lymph nodes (LN), spleen, and bone marrow. In liver, CD302 was expressed by hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. A detailed analysis of CD302 transcription in mouse immune cells revealed highest expression by myeloid cells, particularly macrophages, granulocytes, and myeloid dendritic cells (mDC). Interestingly, 2.5-fold more CD302 was found in migratory compared with resident mDC populations and higher CD302 expression in mouse M1 versus M2 macrophages was also noteworthy. CD302 knockout (CD302KO) mice were generated. Studies on the relevant immune cell populations revealed a decrease in the frequency and numbers of migratory mDC within CD302KO LN compared with wild-type LN. In vitro studies showed CD302KO and wild-type DC had an equivalent capacity to undergo maturation, prime T cells, uptake Ags, and migrate toward the CCL19/CCL21 chemokines. Nevertheless, CD302KO migratory DC exhibited reduced in vivo migration into LN, confirming a functional role for CD302 in mDC migration.

A CD2 high-expressing stress-resistant human plasmacytoid dendritic-cell subset.

Human plasmacytoid dendritic cells (pDCs) were considered to be a phenotypically and functionally homogeneous cell population; however, recent analyses indicate potential heterogeneity. This is of major interest, given their importance in the induction of anti-viral responses and their role in creating immunologically permissive environments for human malignancies. For this reason, we investigated the possible presence of human pDC subsets in blood and bone marrow, using unbiased cell phenotype clustering and functional studies. This defined two major functionally distinct human pDC subsets, distinguished by differential expression of CD2. The CD2(hi) and CD2(lo) pDCs represent discontinuous subsets, each with hallmark pDC functionality, including interferon-alpha production. The rarer CD2(hi) pDC subset demonstrated a significant survival advantage over CD2(lo) pDC during stress and upon exposure to glucocorticoids (GCs), which was associated with higher expression of the anti-apoptotic molecule BCL2. The differential sensitivity of these two human pDC subsets to GCs is demonstrated in vivo by a relative increase in CD2(hi) pDC in multiple myeloma patients treated with GCs. Hence, the selective apoptosis of CD2(lo) pDC during stress represents a novel mechanism for the control of innate responses.

Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes.

A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying "natural" alleles in the human population is to engineer "artificial" alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis.

TRAF2 regulates peripheral CD8(+) T-cell and NKT-cell homeostasis by modulating sensitivity to IL-15.

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8(+) T-cell and NKT-cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8(+) T-cell subsets. IL-15-dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8(+) CD44(hi) CD122(+) T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8(+) CD44(hi) T cells exhibited impaired dose-dependent proliferation to exogenous IL-15. In contrast, TRAF2TKO CD8(+) T cells proliferated normally to anti-CD3 and TRAF2TKO CD8(+) CD44(hi) T cells exhibited normal proliferation to exogenous IL-2. TRAF2TKO CD8(+) T cells expressed normal levels of IL-15-associated receptors and possessed functional IL-15-mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8(+) CD44(hi) CD122(+) and NKT cells was mechanistically linked to an inability to respond to IL-15. The reduced CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell populations in TRAF2TKO mice were rescued in the presence of high dose IL-15 by IL-15/IL-15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell homeostasis by modulating sensitivity to T-cell intrinsic growth factors such as IL-15.