A Chimeric IL-7Rα/IL-2Rß Receptor Promotes the Differentiation of T Cell Progenitors into B Cells and Type 2 Innate Lymphoid Cells.

IL-7 and IL-2 are evolutionarily related cytokines that play critical roles in the development and expansion of immune cells. Although both IL-7R and IL-2R activate similar signaling molecules, whether their signals have specific or overlapping functions during lymphocyte differentiation remains unclear. To address this question, we generated IL-7R α-chain (IL-7Rα)/IL-2R ß-chain (IL-24ß) (72R) knock-in mice expressing a chimeric receptor consisting of the extracellular domain of IL-7Rα and the intracellular domain of IL-2Rß under the control of the endogenous IL-7Rα promoter. Notably, this 72R receptor induced higher levels of STAT5 and Akt phosphorylation in T cells. In the periphery of 72R mice, the number of T cells, B cells, and type 2 innate lymphoid cells (ILC2s) was increased, whereas early T cell progenitors and double-negative 2 thymocytes were reduced in the thymus. In addition, cell proliferation and Notch signaling were impaired in the early thymocytes of 72R mice, leading to their differentiation into thymic B cells. Interestingly, ILC2s were increased in the thymus of 72R mice. Early T cell progenitors from 72R mice, but not from wild-type mice, differentiated into NK cells and ILC2-like cells when cocultured with a thymic stromal cell line. Thus, this study indicates that the chimeric 72R receptor transduces more robust signals than the authentic IL-7Rα, thereby inducing the alternative differentiation of T cell progenitors into other cell lineages. This suggests that cytokine receptors may provide instructive signals for cell fate decisions.

Bridging therapy-induced phenotypes and genetic immune dysregulation to study interleukin-2-induced immunotoxicology.

Interleukin-2 (IL-2) holds promise for the treatment of cancer and autoimmune diseases, but its high-dose usage is associated with systemic immunotoxicity. Differential IL-2 receptor (IL-2R) regulation might impact function of cells upon IL-2 stimulation, possibly inducing cellular changes similar to patients with hypomorphic IL2RB mutations, presenting with multiorgan autoimmunity. Here, we show that sustained high-dose IL-2 stimulation of human lymphocytes drastically reduces IL-2Rß surface expression especially on T cells, resulting in impaired IL-2R signaling which correlates with high IL-2Rα baseline expression. IL-2R signaling in NK cells is maintained. CD4+ T cells, especially regulatory T cells are more broadly affected than CD8+ T cells, consistent with lineage-specific differences in IL-2 responsiveness. Given the resemblance of cellular characteristics of high-dose IL-2-stimulated cells and cells from patients with IL-2Rß defects, impact of continuous IL-2 stimulation on IL-2R signaling should be considered in the onset of clinical adverse events during IL-2 therapy.

CD122 is an activation marker ensuring proper proliferation of T cells in teleost.

As one of subunits for interleukin-2 receptor (IL-2R), CD122 can bind to IL-2 and then activate downstream signal transduction to participate in adaptive immune response. Although CD122 has been identified and investigated from several teleost species, studies on its function at T-cell level are still scarce for lack of specific antibodies. In this study, a typical CD122 in Nile tilapia (Oreochromis niloticus) was characterized by bioinformatics analysis, cloned to produce retrovirus infected NIH/3T3 cells for mouse immunization. After cell fusion and screening, we successfully developed a mouse anti-tilapia CD122 monoclonal antibody (mAb), which could specifically recognize CD122 and identify CD122-producing T cells of tilapia. Using the mAb to detect, CD122 was found to widely distribute in immune-related tissues, and significantly elevate post Edwardsiella piscicida infection or T-cell activation. More importantly, the expansion of CD122+ T cells and up-regulation of CD122 occurred both in total T cells and T-cell subsets during T-cell activation upon in vitro stimulation or in vivo infection. These results indicate that CD122 can be used as a T-cell activation marker in tilapia. Notably, CD122 mAb blocking blunted the activation of MAPK/Erk and mTORC1 pathways, and inhibited T-cell proliferation, suggesting a critical role of CD122 in ensuring proper proliferation of tilapia T cells. Therefore, this study enriches the knowledge of T-cell responses in fish and provides new evidence for understanding the evolution of lymphocyte-mediated adaptive immunity.

Galectin-1 fosters an immunosuppressive microenvironment in colorectal cancer by reprogramming CD8+ regulatory T cells.

Colorectal cancer (CRC) represents the third most common malignancy and the second leading cause of cancer-related deaths worldwide. Although immunotherapy has taken center stage in mainstream oncology, it has shown limited clinical efficacy in CRC, generating an urgent need for discovery of new biomarkers and potential therapeutic targets. Galectin-1 (Gal-1), an endogenous glycan-binding protein, induces tolerogenic programs and contributes to tumor cell evasion of immune responses. Here, we investigated the relevance of Gal-1 in CRC and explored its modulatory activity within the CD8+ regulatory T cell (Treg) compartment. Mice lacking Gal-1 (Lgals1-/- ) developed a lower number of tumors and showed a decreased frequency of a particular population of CD8+CD122+PD-1+ Tregs in the azoxymethane-dextran sodium sulfate model of colitis-associated CRC. Moreover, silencing of tumor-derived Gal-1 in the syngeneic CT26 CRC model resulted in reduced number and attenuated immunosuppressive capacity of CD8+CD122+PD-1+ Tregs, leading to slower tumor growth. Moreover, stromal Gal-1 also influenced the fitness of CD8+ Tregs, highlighting the contribution of both tumor and stromal-derived Gal-1 to this immunoregulatory effect. Finally, bioinformatic analysis of a colorectal adenocarcinoma from The Cancer Genome Atlas dataset revealed a particular signature characterized by high CD8+ Treg score and elevated Gal-1 expression, which delineates poor prognosis in human CRC. Our findings identify CD8+CD122+PD-1+ Tregs as a target of the immunoregulatory activity of Gal-1, suggesting a potential immunotherapeutic strategy for the treatment of CRC.

Activated T cells secrete an alternatively spliced form of common γ-chain that inhibits cytokine signaling and exacerbates inflammation.

The common γ-chain (γc) plays a central role in signaling by IL-2 and other γc-dependent cytokines. Here we report that activated T cells produce an alternatively spliced form of γc mRNA that results in protein expression and secretion of the γc extracellular domain. The soluble form of γc (sγc) is present in serum and directly binds to IL-2Rß and IL-7Rα proteins on T cells to inhibit cytokine signaling and promote inflammation. sγc suppressed IL-7 signaling to impair naive T cell survival during homeostasis and exacerbated Th17-cell-mediated inflammation by inhibiting IL-2 signaling upon T cell activation. Reciprocally, the severity of Th17-cell-mediated inflammatory diseases was markedly diminished in mice lacking sγc. Thus, sγc expression is a naturally occurring immunomodulator that regulates γc cytokine signaling and controls T cell activation and differentiation.

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Long-lived T-memory stem cells (TSCM ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA+ CCR7+ CD95+ phenotype. Significant heterogeneity within the TSCM population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 TSCM subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The TSCM CD122hi -expressing subset (versus CD122lo ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The TSCM CXCR3lo subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the TSCM CD122hi population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the TSCM CD122hi phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.

Characterisation of IL-15 and IL-2Rß in grass carp: IL-15 upregulates cytokines and transcription factors of type 1 immune response and NK cell activation.

Interleukin (IL) -15 belongs to the common cytokine receptor γ chain (γC) family and has diverse functions in regulating the development, proliferation and activation of NK and T cells. It activates a hetero-trimeric receptor complex consisting of IL-2Rα, IL-2Rß and a common γ chain (γC). In this study, the full-length cDNA sequences of IL-15 and IL-2Rß were identified in grass carp (Ctenopharyngodon idella, Ci) and their expression profiles analysed. The CiIL-15 and CiIL-2Rß were shown to be broadly expressed in tissues, with the highest levels detected in the spleen. Moreover, the CiIL-15 and CiIL-2Rß were modulated in primary head kidney leucocytes (HKLs) and splenocytes by immunostimulants and cytokines, and in the head kidney and spleen of fish after infection of Flavobacterium columnare and grass carp reovirus. The bioactivity of bacteria derived recombinant CiIL-15 protein was evaluated in the primary leucocytes. The CiIL-15 was shown to induce signature genes of type 1 immune response (IFN-γ and T-bet) and NK cell activation (perforin and Eomesa), whilst exhibiting inhibitory effects on the genes involved in the type 2 immune response (IL-4/13, IL-10 and Gata3). Our data suggest that IL-15 is a key regulator in promoting the type 1 immune response and NK cell activation in fish.

The Respiratory Environment Diverts the Development of Antiviral Memory CD8 T Cells.

Our understanding of memory CD8+ T cells has been largely derived from acute, systemic infection models. However, memory CD8+ T cells generated from mucosal infection exhibit unique properties and, following respiratory infection, are not maintained in the lung long term. To better understand how infection route modifies memory differentiation, we compared murine CD8+ T cell responses to a vesicular stomatitis virus (VSV) challenge generated intranasally (i.n.) or i.v. The i.n. infection resulted in greater peak expansion of VSV-specific CD8+ T cells. However, this numerical advantage was rapidly lost during the contraction phase of the immune response, resulting in memory CD8+ T cell numerical deficiencies when compared with i.v. infection. Interestingly, the antiviral CD8+ T cells generated in response to i.n. VSV exhibited a biased and sustained proportion of early effector cells (CD127loKLRG1lo) akin to the developmental program favored after i.n. influenza infection, suggesting that respiratory infection broadly favors an incomplete memory differentiation program. Correspondingly, i.n. VSV infection resulted in lower CD122 expression and eomesodermin levels by VSV-specific CD8+ T cells, further indicative of an inferior transition to bona fide memory. These results may be due to distinct (CD103+CD11b+) dendritic cell subsets in the i.n. versus i.v. T cell priming environments, which express molecules that regulate T cell signaling and the balance between tolerance and immunity. Therefore, we propose that distinct immunization routes modulate both the quality and quantity of antiviral effector and memory CD8+ T cells in response to an identical pathogen and should be considered in CD8+ T cell-based vaccine design.

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.

Immunological characteristics of Interleukin-2 receptor subunit beta (IL-2Rß) in flounder (Paralichtlys olivaceus): Implication for IL-2R function.

Interleukin-2 receptor subunit beta of flounder (Paralichthys olivace, fIL-2Rß) was annotated on the NCBI, its gene was cloned and characterized functionally in this study. And then the amino acids sequences and tertiary structure of fIL-2Rß were analyzed, respectively. RT-PCR and ImageJ analyzed showed that fIL-2Rß mRNA were expressed in the gill, spleen, kidney, intestines, liver, blood, muscle and skin, which showed high signals in spleen and blood. And then the recombinant protein of fIL-2Rß extracellular region and its polyclonal antibodies were produced, native fIL-2Rß molecules in flounder peripheral blood leukocytes (PBLs) were identified at 60.7 kDa by Mass spectrometry, which were in accordance with the molecular mass of full fIL-2Rß protein calculated on the predicted protein sequence. Then the IL-2Rß+ cell in T/B lymphocytes were characterized by Flow cytometry and indirect immunofluorescence assay, respectively. The results showed that the percentages of IL-2Rß+ leukocytes, IL-2Rß+/CD4+, IL-2Rß+/IgM+ lymphocytes were 18.4 ± 2.7%, 4.5 ± 0.8%, 4.3% ± 0.5 in PBLs, and were 13.6 ± 0.9%, 4.6 ± 1.1%, 6.1% ± 0.4 in spleen, similarly, the percentages of IL-2Rß+ leukocytes, IL-2Rß+/CD4+, IL-2Rß+/IgM+ lymphocytes were 9.4 ± 0.3%, 4.0 ± 0.5%, 5.7 ± 0.1% in head kidney, respectively. After KLH injection, compared with control group, the gene expression of IL-2, IL-2Rß, CD3, TCR, CD79b and IgM in spleen of flounder were up-regulated, respectively (p < 0.05). And the FCM results showed that the percentages of IL-2Rß+ leukocytes in PBLs were significantly increased post Keyhole limpet hemocyanin (KLH) injection, which peaked 23.9 ± 0.9% at 9th day (p < 0.05). To our knowledge, those results first reported that the characteristics of IL-2R and IL-2R + molecules were expressed on both B and T lymphocytes in fish. At the same time, this study lays a foundation for further exploring the interaction between IL-2 and IL-2R to promote cell proliferation and carrying out biological functions.

c-REL and IκBNS Govern Common and Independent Steps of Regulatory T Cell Development from Novel CD122-Expressing Pre-Precursors.

Foxp3-expressing regulatory T cells (Tregs) are essential regulators of immune homeostasis and, thus, are prime targets for therapeutic interventions of diseases such as cancer and autoimmunity. c-REL and IκBNS are important regulators of Foxp3 induction in Treg precursors upon γ-chain cytokine stimulation. In c-REL/IκBNS double-deficient mice, Treg numbers were dramatically reduced, indicating that together, c-REL and IκBNS are pivotal for Treg development. However, despite the highly reduced Treg compartment, double-deficient mice did not develop autoimmunity even when aged to more than 1 y, suggesting that c-REL and IκBNS are required for T cell effector function as well. Analyzing Treg development in more detail, we identified a CD122+ subset within the CD25-Foxp3- precursor population, which gave rise to classical CD25+Foxp3- Treg precursors. Importantly, c-REL, but not IκBNS, controlled the generation of classical CD25+Foxp3- precursors via direct binding to the Cd25 locus. Thus, we propose that CD4+GITR+CD122+CD25-Foxp3- cells represent a Treg pre-precursor population, whose transition into Treg precursors is mediated via c-REL.

Cutting Edge: Differential Fine-Tuning of IL-2- and IL-15-Dependent Functions by Targeting Their Common IL-2/15Rß/γc Receptor.

Interleukin 2 and IL-15 are two closely related cytokines, displaying important functions in the immune system. They share the heterodimeric CD122/CD132 receptor to deliver their signals within target cells. Their specificity of action is conferred by their α receptor chains, IL-2Rα and IL-15Rα. By combining an increased affinity for CD122 and an impaired recruitment of CD132, we have generated an original molecule named IL-2Rß/γ (CD122/CD132) inhibitor (BiG), targeting the CD122/CD132 receptor. BiG efficiently inhibited IL-15- and IL-2-dependent functions of primary cells, including CD8 T and NK cells, in vitro and in vivo. We also report a differential dynamic of action of these cytokines by highlighting a major role played by the IL-2Rα receptor. Interestingly, due to the presence of IL-2Rα, BiG had no impact on IL-2-dependent regulatory T cell proliferation. Thus, by acting as a fine switch in the immune system, BiG emphasizes the differential roles of these two cytokines.

IL2Rß-dependent signals drive terminal exhaustion and suppress memory development during chronic viral infection.

Exhaustion of CD8(+) T cells severely impedes the adaptive immune response to chronic viral infections. Despite major advances in our understanding of the molecular regulation of exhaustion, the cytokines that directly control this process during chronicity remain unknown. We demonstrate a direct impact of IL-2 and IL-15, two common gamma-chain-dependent cytokines, on CD8(+) T-cell exhaustion. Common to both cytokine receptors, the IL-2 receptor ß (IL2Rß) chain is selectively maintained on CD8(+) T cells during chronic lymphocytic choriomeningitis virus and hepatitis C virus infections. Its expression correlates with exhaustion severity and identifies terminally exhausted CD8(+) T cells both in mice and humans. Genetic ablation of the IL2Rß chain on CD8(+) T cells restrains inhibitory receptor induction, in particular 2B4 and Tim-3; precludes terminal differentiation of highly defective PD-1(hi) effectors; and rescues memory T-cell development and responsiveness to IL-7-dependent signals. Together, we ascribe a previously unexpected role to IL-2 and IL-15 as instigators of CD8(+) T-cell exhaustion during chronic viral infection.

A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease.

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.

A low interleukin-2 receptor signaling threshold supports the development and homeostasis of T regulatory cells.

Interleukin-2 receptor (IL-2R) signaling is essential for T regulatory (Treg) cell development and homeostasis. Here, we show that expression of IL-2Rbeta chains that lack tyrosine residues important for the association of the adaptor Shc and the transcription factor STAT5 in IL-2Rbeta-deficient mice resulted in production of a normal proportion of natural Treg cells that suppressed severe autoimmunity related with deficiency in IL-2 or IL-2R. These mutant IL-2Rbeta chains supported suboptimal and transient STAT5 activation that upregulate the transcription factor Foxp3 to normal amounts in natural, but not induced, Treg cells. Nevertheless, gene expression profiling revealed many targets in peripheral natural Treg cells that were IL-2 dependent and a substantial overlap between the Treg cell IL-2-dependent gene program and the Treg cell transcriptional signature. Collectively, these findings demonstrate that a critical, and perhaps minor, subset of IL-2-dependent targets is indexed to a low IL-2R signaling threshold and that a substantial proportion of the Treg cell gene program is regulated by IL-2.

Acetylation Modulates IL-2 Receptor Signaling in T Cells.

Ligand binding to the cognate cytokine receptors activates intracellular signaling by recruiting protein tyrosine kinases and other protein modification enzymes. However, the roles of protein modifications other than phosphorylation remain unclear. In this study, we examine a novel regulatory mechanism of Stat5, based on its acetylation. As for phosphorylation, IL-2 induces the acetylation of signaling molecules, including Stat5, in the murine T cell line CTLL-2. Stat5 is acetylated in the cytoplasm by CREB-binding protein (CBP). Acetylated Lys696 and Lys700 on Stat5 are critical indicators for limited proteolysis, which leads to the generation of a truncated form of Stat5. In turn, the truncated form of Stat5 prevents transcription of the full-length form of Stat5. We also demonstrate that CBP physically associates with the IL-2 receptor ß-chain. CBP, found in the nucleus in resting CTLL-2 cells, relocates to the cytoplasm after IL-2 stimulation in an MEK/ERK pathway-dependent manner. Thus, IL-2-mediated acetylation plays an important role in the modulation of cytokine signaling and T cell fate.

Characterization of Receptor-Associated Protein Complex Assembly in Interleukin (IL)-2- and IL-15-Activated T-Cell Lines.

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor ß subunit (IL-2Rß). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.

Glomerular common gamma chain confers B- and T-cell-independent protection against glomerulonephritis.

Crescentic glomerulonephritis is a life-threatening renal disease that has been extensively studied by the experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM-GN) model. Although T cells have a significant role in this model, athymic/nude mice and rats still develop severe renal disease. Here we further explored the contribution of intrinsic renal cells in the development of T-cell-independent GN lesions. Anti-GBM-GN was induced in three strains of immune-deficient mice (Rag2-/-, Rag2-/-Il2rg-/-, and Rag2-/-Il2rb-/-) that are devoid of either T/B cells or T/B/NK cells. The Rag2-/-Il2rg-/- or Rag2-/-Il2rb-/- mice harbor an additional deletion of either the common gamma chain (γC) or the interleukin-2 receptor ß subunit (IL-2Rß), respectively, impairing IL-15 signaling in particular. As expected, all these strains developed severe anti-GBM-GN. Additionally, bone marrow replenishment experiments allowed us to deduce a protective role for the glomerular-expressed γC during anti-GBM-GN. Given that IL-15 has been found highly expressed in nephritic kidneys despite the absence of lymphocytes, we then studied this cytokine in vitro on primary cultured podocytes from immune-deficient mice (Rag2-/-Il2rg-/- and Rag2-/-Il2rb-/-) compared to controls. IL-15 induced downstream activation of JAK1/3 and SYK in primary cultured podocytes. IL-15-dependent JAK/SYK induction was impaired in the absence of γC or IL-2Rß. We found γC largely induced on podocytes during human glomerulonephritis. Thus, renal lesions are indeed modulated by intrinsic glomerular cells through the γC/IL-2Rß receptor response, to date classically described only in immune cells.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.

IL-15 bound to the IL-15Rα-chain (IL-15Rα) is presented in trans to cells bearing the IL-2Rß-chain and common γ-chain. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor-ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR-HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to downregulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A(+) cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Coengagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across, inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15-dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.

In vivo maintenance of human regulatory T cells during CD25 blockade.

Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.