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Disruption in homeostasis of interleukin-15 (IL-15) is linked to poor maternal and fetal outcomes during pregnancy. The only cells described to respond to IL-15 at the early maternal-fetal interface have been natural killer (NK) cells. We now show a novel population of macrophages, evident in several organs but enriched in the uterus of mice and humans, expressing the β chain of the IL-15 receptor complex (CD122) and responding to IL-15. CD122+ macrophages (CD122+Macs) are morphologic, phenotypic, and transcriptomic macrophages that can derive from bone marrow monocytes. CD122+Macs develop in the uterus and placenta with kinetics that mirror interferon (IFN) activity at the maternal-fetal interface. Macrophage colony-stimulating factor (M-CSF) permits macrophages to express CD122, and IFNs are sufficient to drive expression of CD122 on macrophages. Neither Type-I nor Type-II IFNs are required to generate CD122+Macs, however. In response to IL-15, CD122+Macs activate the ERK signaling cascade and enhance production of proinflammatory cytokines after stimulation with the Toll-like receptor 9 agonist CpG. Finally, we provide evidence of human cells that phenocopy murine CD122+Macs in secretory phase endometrium during the implantation window and in first-trimester uterine decidua. Our data support a model wherein IFNs local to the maternal-fetal interface direct novel IL-15-responsive macrophages with the potential to mediate IL-15 signals critical for optimal outcomes of pregnancy.

Objective Human T cell lymphotropic virus 1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neurological disease. Chronic activation of CD8+ T cells, as evidenced by increased spontaneous lymphoproliferation and HTLV‐1‐specific cytotoxic T cells, has been demonstrated in HAM/TSP patients. Since IL‐2 and IL‐15 stimulate memory CD8+ T cell activity, these cytokines have been implicated in the immunopathogenesis of HAM/TSP. In this phase I trial, we evaluated the safety, pharmacokinetics, and ability of Hu‐Mikβ1, a humanized monoclonal antibody directed toward the IL‐2/IL‐15 receptor β‐chain (IL‐2/IL‐15Rβ: CD122), to saturate CD122 and regulate abnormal immune responses in patients with HAM/TSP by inhibition of IL‐15 action. Methods Hu‐Mikβ1 was administered intravenously at doses of 0.5 mg/kg, 1.0 mg/kg, or 1.5 mg/kg in a total of nine HAM/TSP patients. Five doses of Hu‐Mikβ1 were administered at 3‐week intervals. The clinical response was evaluated using standardized scales. Viral and immunologic outcome measures were examined including HTLV‐1 proviral load, T cell phenotypic analysis and spontaneous lymphoproliferation in HAM/TSP patients. Results There was no significant toxicity associated with Hu‐Mikβ1 administration in HAM/TSP patients. Saturation of CD122 by Hu‐Mikβ1 was achieved in five out of nine HAM/TSP patients. Administration of Hu‐Mikβ1 was associated with inhibition of aberrant CD8+ T cell function including spontaneous lymphoproliferation and degranulation and IFN‐γ expression, especially in HAM/TSP patients that achieved CD122 saturation. Interpretation The treatment with Hu‐Mikβ1 had a number of immunological effects on HAM/TSP patients although no clinical efficacy was observed. We also did not see any dose‐related toxicity.

Zhang et al. identify human IL-2Rβ deficiency as a cause of severe immune dysregulation. The hypomorphic gene mutations reveal variable IL-2Rβ expression and function between different lymphocyte subsets as a means of selectively modulating immune responses., Interleukin-2, which conveys essential signals for immunity, operates through a heterotrimeric receptor. Here we identify human interleukin-2 receptor (IL-2R) β chain (IL2RB) gene defects as a cause of life-threatening immune dysregulation. We report three homozygous mutations in the IL2RB gene of eight individuals from four consanguineous families that cause disease by distinct mechanisms. Nearly all patients presented with autoantibodies, hypergammaglobulinemia, bowel inflammation, dermatological abnormalities, lymphadenopathy, and cytomegalovirus disease. Patient T lymphocytes lacked surface expression of IL-2Rβ and were unable to respond to IL-2 stimulation. By contrast, natural killer cells retained partial IL-2Rβ expression and function. IL-2Rβ loss of function was recapitulated in a recombinant system in which IL2RB mutations caused reduced surface expression and IL-2 binding. Stem cell transplant ameliorated clinical symptoms in one patient; forced expression of wild-type IL-2Rβ also increased the IL-2 responsiveness of patient T lymphocytes in vitro. Insights from these patients can inform the development of IL-2–based therapeutics for immunological diseases and cancer.

Regulatory T cells (Treg) are essential to maintain immune homeostasis and prevent autoimmune disorders. While the function and molecular regulation of Foxp3+CD4+ Tregs are well established, much of CD8+ Treg biology remains to be revealed. Here, we will review the heterogenous subsets of CD8+ T cells have been named “CD8+ Treg” and mainly focus on CD122hiLy49+CD8+ Tregs present in naïve mice. CD122hiLy49+CD8+ Tregs, which depends on transcription factor Helios and homeostatic cytokine IL-15, have been established as a non-redundant regulator of germinal center (GC) reaction. Recently, we have demonstrated that TGF-β (Transforming growth factor-β) and transcription factor Eomes (Eomesodermin) are essential for the function and homeostasis of CD8+ Tregs. In addition, we will discuss several open questions regarding the differentiation, function and true identity of CD8+ Tregs as well as a brief comparison between two regulatory T cell subsets critical to control GC reaction, namely CD4+ TFR (follicular regulatory T cells) and CD8+ Tregs.

Invariant NKT (iNKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature iNKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for iNKT cell generation, cytokines are proposed to contribute to iNKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic iNKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of iNKT cells, surprisingly, premature IL-2Rβ expression on immature iNKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control iNKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic iNKT cell differentiation.

Background Triggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer’s disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells. Results Here, we demonstrated for the first time that CD3−CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice. Conclusions Collectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rβ. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8+ T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8+ T cell responses.

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.

Lymphopenia-induced homeostatic proliferation (LIP) is a critical mechanism for restoring T cell immunity upon lymphodepleting insults or infections. LIP is primarily driven by homeostatic cytokines, such as IL-7 and IL-15, but not all T cells respond with the same efficiency to homeostatic proliferative cues. Although CD8 T cells vigorously proliferate under lymphopenic conditions, naive CD4 T cells are substantially impaired in their response to homeostatic cytokines, and they fail to fully expand. In this study, we show that the availability of IL-2Rβ (CD122), which is a receptor subunit shared by IL-2 and IL-15, affects both the cytokine responsiveness and the LIP of naive CD4 T cells in the mouse. The enumeration of surface IL-2Rβ molecules on murine naive CD4 and naive CD8 T cells revealed a 5-fold difference in IL-2Rβ abundance. Notably, it was the limited availability of IL-2Rβ that impaired CD4 T cell responsiveness to IL-15 and suppressed their LIP. As such, forced IL-2Rβ expression on CD4 T cells by transgenesis bestowed IL-15 responsiveness onto naive CD4 T cells, which thus acquired the ability to undergo robust LIP. Collectively, these results identify IL-2Rβ availability as a new regulatory mechanism to control cytokine responsiveness and the homeostatic proliferation of murine CD4 T cells.

Interleukin-2 (IL-2) has both pro- and anti-inflammatory properties that have been harnessed clinically and that are used experimentally to modulate leukocyte subsets in vivo. In mice, the bioavailability and half-life of IL-2 in vivo can be increased by complexing recombinant IL-2 with different clones of anti-IL-2 monoclonal antibodies that differentially target the cytokine to cells expressing different kinds of IL-2 receptors. While the impacts of systemic IL-2: anti-IL-2 antibody complex (IL-2C) administration are well-defined in the spleen and peripheral lymph nodes, how immune cells in the gut and gut-associated lymphoid tissues respond to IL-2C is not well characterized. Here, we analyze how major leukocyte populations in these tissues respond to IL-2C. We find that IL-2C targeting cells expressing IL-2 receptor beta cause an acute decrease in cellularity of Peyer’s Patches while cell numbers in the lamina propria and intraepithelial lymphocytes are unaffected. Cell contraction in Peyer’s Patches is associated with the apoptosis of multiple B cell subsets. Our results are important to consider for understanding off-target impacts of IL-2C regimes in experimental models and for considering how IL-2 may contribute to the etiology or severity of gut-associated conditions such as Crohn’s Disease.

PROBLEM To investigate risk factors that can help identify the possibility of pregnancy loss in threatened late miscarriage (TLM) patients with and without spontaneous uterine contractions. METHOD OF STUDY Amniotic immune biomarkers (IL2β receptor, IL6, IL8, IL10, IL1β, and TNFα) were assayed, and "sludge" was assessed. Patients without intrauterine infections were treated and followed up until delivery, and pregnancy outcomes were recorded. The two groups were compared for the differences in biomarker levels and "sludge," and the independent associations of biomarkers, "sludge," and other maternal factors with late miscarriage were investigated. RESULTS The amniotic levels of IL2βR, IL8, and TNFα were higher in the group with contractions (P

BackgroundProtein tyrosine phosphatases (PTPs) play critical roles in human autoimmunity. Previous studies found that PTPN2 may be the key regulatory factor in the T-cell-mediated immune response. PTPN2 regulates the Janus kinase/signal transducers and activators of transcription pathway by inhibiting signalling via the interleukin (IL)-2 receptor (CD122). An association between genetic variations in PTPN2 and CD122 with ocular Behcet’s disease (BD) has not yet been addressed and was therefore the purpose of this study.MethodsA two-stage case–control study was performed in 906 patients with ocular BD and 2178 healthy controls. Genotyping analysis of 11 single nucleotide polymorphisms was carried out. The expression of PTPN2 in peripheral blood mononuclear cells (PBMCs) was quantified by real-time PCR and cytokine production was measured by ELISA.ResultsThe frequency of the GG genotype of PTPN2-rs7234029 was significantly lower in patients with ocular BD (p=1.94×10−5, pc=8.34×10−4, OR=0.466). Stratification according to gender showed that rs7234029 was significantly associated with BD in men. A stratified analysis according to the main clinical features showed that rs7234029 was significantly associated with genital ulcers, skin lesions and a positive pathergy test. No association could be detected between BD and CD122 gene polymorphisms. Functional studies showed that rs7234029 GG genotype carriers had a higher PNPT2 mRNA expression level than those which carrying the AA or AG genotype, and a decreased secretion of IL-17 and tumour necrosis factor-alpha was seen by PBMCs from GG carriers. No significant difference could be detected concerning IL-1β or IL-6 production by stimulated PBMCs between the different genotype groups.ConclusionsThis study shows that a PTPN2-rs7234029 polymorphism is associated with ocular BD and is strongly influenced by gender. In addition, our results suggest that the genetic association with PTPN2 may involve the regulation of PTPN2 mRNA expression and cytokine secretion.

Autosomal recessive mutations in IL2RB shape immunity and peripheral immune tolerance., How the IL-2 receptor β-chain specifically shapes immunity has remained enigmatic. In this issue of JEM, Zhang et al. (https://doi.org/10.1084/jem.20182304) and Fernandez et al. (https://doi.org/10.1084/jem.20182015) independently report the first observations of autosomal recessive mutations in IL2RB, revealing a requirement for IL2RB in immunity and peripheral immune tolerance.

International audience; Interleukin (IL)-15 is a pleiotropic cytokine, which is structurally close to IL-2 and shares with it the IL-2 β and γ receptor (R) subunits. By promoting the activation and proliferation of NK, NK-T and CD8+ T cells, IL-15 plays important roles in innate and adaptative immunity. Moreover, the association of high levels of IL-15 expression with inflammatory and autoimmune diseases has led to the development of various antagonistic approaches targeting IL- 15. This study is an original approach aimed at discovering small-molecule inhibitors impeding IL-15/IL-15R interaction. A pharmacophore and docking-based virtual screening of compound libraries led to the selection of 240 high-scoring compounds, 36 of which were found to bind IL-15, to inhibit the binding of IL-15 to the IL-2Rβ chain and/or the proliferation of IL-15-dependent cells. One of them was selected as a hit, optimized by a structure-activity relationship approach, leading to the first small-molecule IL-15 inhibitor with sub-micromolar activity.

CD8 T cells play a crucial role in immune responses to virus infections and tumors. Naïve CD8 T lymphocytes after TCR stimulation undergo differentiation into CTLs and memory cells, which are essential sources of IFN-γ. We investigated IFN-γ production by CD8 T cell subsets found in nonimmune mice. A minor fraction of in vitro TCR-stimulated CD8 T cells produce IFN-γ, and it is regulated at the transcriptional level. Antigen inexperienced C57BL/6 mice present the coexistence of 2 populations. The main population exhibits a CD44lowCD122low profile, which is compatible with naïve lymphocytes. The minor expresses a phenotype of immunologic memory, CD44hiCD122hi. Both subsets are able to produce IL-2 in response to TCR activation, but only the memory-like population is responsible for IFN-γ production. Similar to memory CD8 T cells, CD44hiCD8+ T cells also present a higher level of the transcriptional factor Eomes and a lower level of T-bet (Tbx21) mRNA than CD44lowCD8+ T cells. The presence of the CD44hiCD8+ T cell population in nonimmune OT-I transgenic mice reveals that the population is generated independently of antigenic stimulation. CpG methylation is an efficient epigenetic mechanism for gene silencing. DNA methylation at posttranscriptional CpG sites in the Ifng promoter is higher in CD44lowCD8+ T cells than in CD44hiCD8+ T cells. Thus, memory-like CD8 T cells have a distinct epigenetic pattern in the Ifng promoter and can rapidly produce IFN-γ in response to TCR stimulation.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memoryCD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM)and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that,following a critical number of cell divisions, memory precursors down regulate CCR7 and upregulate IL-2Rb, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM. Refereed/Peer-reviewed

This study aimed to explore the associations between polymorphisms of a very important pharmacogene, ADRB2, two inflammation-related genes, IL33 and IL2RB, and the risk of lung cancer.Six polymorphisms of ADRB2, IL33, and IL2RB were genotyped in 300 lung cancer patients and 300 healthy controls using MassARRAY. The relationship between genotypes and lung cancer risk was evaluated using chi-square tests.The minor allele of rs1042711 was a risk allele for lung cancer, whereas the minor alleles of rs7025417 and rs5756523 had protective effects against lung cancer (p＜0.05). The CT genotype of rs1042711 and the GT genotype of rs1560642 were associated with increased risk of lung cancer, whereas the CC and AA genotypes of rs7025417 and the CT and CC genotypes of rs5756523 were associated with decreased disease risk (p 0.05). Genetic model analysis shows that rs1042711 and rs1560642 were associated with increased risk of lung cancer; whereas rs7025417, rs5756523, and rs2284033 were associated with decreased disease risk (p 0.05). Stratification analysis showed that rs1042711 and rs1560642 were associated with increased risk of lung cancer in nonsmokers and smokers, respectively, whereas rs7025417 and rs5756523 were associated with decreased disease risk in both subgroups (p＜0.05).Our results shed new light on the association between polymorphisms of ADRB2, IL33, and IL2RB and the risk of lung cancer.

Background Inflammation is crucial for lung cancer development. Variants of multiple genes in inflammation pathways may lead to susceptibility to lung cancer. In the present study, we aimed to assess the influence of polymorphisms in inflammation-related genes ( IL2RA and IL2RB ) on lung cancer risk. Methods A total of 507 patients with lung cancer and 503 healthy controls were genotyped for seven polymorphisms of IL2RA and IL2RB using the Agena MassARRAY platform. We evaluated the relationship of the genotypes with lung cancer susceptibility using odds ratio (OR), 95% confidence interval (95% CI) and chi square test. Results We found that IL2RA rs12722498 was significantly associated with a decreased risk of lung cancer in dominant ( p = 0.040, OR = 0.71, 95% CI = 0.51–0.98), additive ( p = 0.016, OR = 0.68, 95% CI = 0.50–0.93) and allele ( p = 0.019, OR = 0.69, 95% CI = 0.51–0.94) models. After stratification analysis, the results showed that IL2RA rs12569923 (non-smokers), IL2RA rs791588 (≤60 years old, non-drinkers, BMI 2 ), IL2RA rs12722498 (≤60 years old, non-drinkers, BMI 2 , female) and IL2RB rs2281089 (female, stage) significantly decreased the risk of lung cancer. Additionally, the haplotypes of rs12569923 and rs791588 in IL2RA had strong relationships with lung cancer in the subgroups of BMI 2 , age ≤ 60 years old, non-smokers and non-drinkers. Conclusion Our results showed that the IL2RA and IL2RB polymorphisms were associated with lung cancer risk in the Chinese Han population, which suggests roles for IL2RA and IL2RB polymorphisms in lung cancer.

A transplanted organ is always rejected in the absence of any immunosuppressive treatment due to vigorous alloimmunity. However, continuously global immunosuppression with a conventional immunosuppressant may result in severe side effects, including nephrotoxicity, tumors and infections. Tregs have been widely used to inhibit allograft rejection, especially in animal models. However, it's well accepted that administration of Tregs alone is not satisfactory in immune-competent wild-type animals. Therefore, it's imperative to promote Treg therapies under the cover of other approaches, including costimulatory blockade. In the present study, we demonstrated that administration of in vitro-expanded CD8+CD122+PD-1+ Tregs synergized with costimulatory blockade of CD40/CD154, but not B7/CD28, to prolong skin allograft survival in wild-type mice and to reduce cellular infiltration in skin allografts as well. Treg treatment and blockade of CD40/CD154, but not B7/CD28, also exhibited an additive effect on suppression of T cell proliferation in vitro and pro-inflammatory cytokine expression in skin allografts. Importantly, blocking B7/CD28, but not CD40/CD154, costimulation decreased the number of transferred CD8+CD122+PD-1+ Tregs and their expression of IL-10 in recipient mice. Furthermore, it's B7/CD28, but not CD40/CD154, costimulatory blockade that dramatically reduced IL-10 production by CD8+CD122+PD-1+ Tregs in vitro, suggesting that B7/CD28, but not CD40/CD154, costimulation is critical for their production of IL-10. Indeed, infusion of IL-10-deficient CD8+CD122+PD-1+ Tregs failed to synergize with anti-CD154 Ab treatment to further prolong allograft survival. Our data may explain why blocking B7/CD28 costimulatory pathway does not boost IL-10-dependent Treg suppression of alloimmunity. Thus, these findings could be implicated in clinical organ transplantation.

Leishmaniasis is a disease caused by intracellular protozoan parasites of the genus Leishmania. In endemic areas, only a portion of exposed subjects develops cutaneous leishmaniasis (CL), suggesting that the genetic inheritance of the host plays a vital role in both resistance and susceptibility to the disease. Interleukin-2 (IL-2) is a cytokine that plays a central role in the regulation of the immune response in infection through the axis IL-2/IL-2R (receptor) complex, triggering a series of intracellular events, among which the signaling of Janus kinase/signal transducers and activators of transcription (JAK-STAT). The present study aimed at verifying the possible relationship between single nucleotide polymorphism (s) (SNP s) in the genes IL-2, IL-2RB, and JAK3 in subjects with CL caused by Leishmania guyanensis in the city of Manaus, state of Amazonas, Brazil. 820 patients with CL and 850 healthy subjects (control group) coming from the same endemic areas as the patients were examined. The SNPs -2425G/A (rs4833248) and -330 T/G (rs2069762), located in the IL-2 gene promoter region, seem to influence the expression of the gene and the SNP +10558G/A (rs1003694) and +13295T/C (rs3212760) located in the 3rd intron of the IL-2RB gene and the 13th intron of the JAK3 gene, respectively, were studied by PCR-RFLP. Genotypes and alleles frequencies were obtained by direct counting. For the comparison between the two groups, the χ2 test with OR (odds ratio) and the 95% confidence interval (CI) were used. Similar genotypes and alleles frequencies for the different SNPs were observed in both patients with CL and healthy controls. Comparison of genotypic and allelic frequency between patients with CL and healthy subjects did not show any difference. These polymorphisms do not predict susceptibility to, or protection against the development of CL caused by L. guyanensis in the Amazonas. © 2019 de Araújo Santos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The bone marrow (BM) is not only a reservoir of hematopoietic stem cells but a repository of immunological memory cells. Further characterizing BM-resident memory T cells would be helpful to reveal the complicated relationship between the BM and immunological memory. In this study, we identified CD122high stem cell antigen-1 (Sca-1) high B cell lymphoma 2 (Bcl-2) high CD4+ stem cell-like memory T cells (TSCMs) as a distinct memory T cell subset, which preferentially reside in the BM, where they respond vigorously to blood-borne antigens. Interestingly, the natural CD4+ TSCMs homing to the BM colocalized with VCAM-1+ IL-15+ IL-7+ CXCL-12+ stromal cells. Furthermore, compared to spleen-resident CD4+ TSCMs, BM-resident TSCMs induced the production of high-affinity antibodies against influenza by B lymphocytes more efficiently. Taken together, these observations indicate that the BM provides an appropriate microenvironment for the survival of CD4+ TSCMs, which broadens our knowledge regarding the memory maintenance of antigen-specific CD4+ T lymphocytes.

BackgroundAnti-programmed death-ligand 1 (αPD-L1) immunotherapy is approved to treat bladder cancer (BC) but is effective in + antitumor T cells known to improve αPD-L1 efficacy. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses and that IL-2c effects could differ from, and thus complement αPD-L1.MethodsWe studied mechanisms of IL-2c and αPD-L1 efficacy using PD-L1+ mouse BC cell lines MB49 and MBT-2 in orthotopic (bladder) and metastatic (lung) sites.ResultsIL-2c reduced orthotopic tumor burden and extended survival in MB49 and MBT-2 BC models, similar to αPD-L1. Using antibody-mediated cell depletions and genetically T cell-deficient mice, we unexpectedly found that CD8+ T cells were not necessary for IL-2c efficacy against tumors in bladder, whereas γδ T cells, not reported to contribute to αPD-L1 efficacy, were indispensable for IL-2c efficacy there. αPD-L1 responsiveness in bladder required conventional T cells as expected, but not γδ T cells, altogether defining distinct mechanisms for IL-2c and αPD-L1 efficacy. γδ T cells did not improve IL-2c treatment of subcutaneously challenged BC or orthotopic (peritoneal) ovarian cancer, consistent with tissue-specific and/or tumor-specific γδ T cell contributions to IL-2c efficacy. IL-2c significantly altered bladder intratumoral γδ T cell content, activation status, and specific γδ T cell subsets with antitumor or protumor effector functions. Neither IL-2c nor αPD-L1 alone treated lung metastatic MB49 or MBT-2 BC, but their combination improved survival in both models. Combination treatment efficacy in lungs required CD8+ T cells but not γδ T cells.ConclusionsMechanistic insights into differential IL-2c and αPD-L1 treatment and tissue-dependent effects could help develop rational combination treatment strategies to improve treatment efficacy in distinct cancers. These studies also provide insights into γδ T cell contributions to immunotherapy in bladder and engagement of adaptive immunity by IL-2c plus αPD-L1 to treat refractory lung metastases.

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 β-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.

While the functional plasticity of memory CD4(+) T cells has been studied extensively, less is known about this property in memory CD8(+) T cells. Here, we report the direct measurement of plasticity by paired daughter analysis of effector and memory OT-I CD8(+) T cells primed in vivo with ovalbumin. Naïve, effector, and memory OT-I cells were isolated and activated in single-cell culture; then, after the first division, their daughter cells were transferred to new cultures with and without IL-4; expression of IFN-γ and IL-4 mRNAs was measured 5 days later in the resultant subclones. Approximately 40% of clonogenic memory CD8(+) T cells were bipotential in this assay, giving rise to an IL-4(-) subclone in the absence of IL-4 and an IL-4(+) subclone in the presence of IL-4. The frequency of bipotential cells was lower among memory cells than naïve cells but markedly higher than among 8-day effectors. Separation based on high or low expression of CD62L, CD122, CD127, or Ly6C did not identify a phenotypic marker of the bipotential cells. Functional plasticity in memory CD8(+) T-cell populations can therefore reflect modulation at the level of a single memory cell and its progeny.

CD8+ T cells play a cardinal feature in response to alloantigens and are able to generate effector/memory T cells independently from CD4+ T cells. To investigate the impact of aging on CD8 T cells, we used a fully mismatched mouse skin transplant model. Our findings showed a prolonged allograft survival in older recipients associated with a significant increase of CD4+ and CD8+ CD44high CD62Llow effector/memory T cells and a reduced systemic IFNγ production. When reconstituting young CBA Rag-1 mice that lack mature T and B cells with old CD8+ T cells expressing clonal anti-H2K T cell receptor (TCR) alloreactive for MHC I, graft survival was significantly prolonged and comparable to those receiving young CD8+ T cells. Moreover, our data showed that reduced systemic IFNγ levels observed in old recipients had been linked to a compromised expression of the IL-2R β subunit (CD122) by old CD8+ T cells. In addition, we observed an impaired IFNγ production on IL-2 receptor activation. At the same time, gene profiling analysis of old CD8 T cells demonstrated reduced chemokine ligand-3 and CD40L expression that resulted in compromised CD8+ T cell/dendritic cell communication, leading to impaired migratory and phagocytic activity of CD11c cells.Collectively, our study demonstrated that aging delays allograft rejection. CD8 T cells play a critical role in this process linked to a compromised production of IFNγ, in addition to a defective IL-2 receptor signaling machinery and a defective communication between CD8 T cells and dendritic cells.

CD101 exerts negative-costimulatory effects in vitro, but its function in vivo remains poorly defined. CD101 is abundantly expressed on lymphoid and myeloid cells in intestinal tissues, but absent from naive splenic T cells. Here, we assessed the impact of CD101 on the course of inflammatory bowel disease (IBD). Using a T-cell transfer model of chronic colitis, we found that in recipients of naive T cells from CD101(+/+) donors up to 30% of the recovered lymphocytes expressed CD101, correlating with an increased interleukin (IL)-2-mediated FoxP3 expression. Transfer of CD101(-/-) T cells caused more severe colitis and was associated with an expansion of IL-17-producing T cells and an enhanced expression of IL-2Rα/β independently of FoxP3. The co-transfer of naive and regulatory T cells (Treg) protected most effectively from colitis, when both donor and recipient mice expressed CD101. Although the expression of CD101 on T cells was sufficient for Treg-function and the inhibition of T-cell proliferation, sustained IL-10 production required additional CD101 expression by myeloid cells. Finally, in patients with IBD a reduced CD101 expression on peripheral and intestinal monocytes and CD4(+) T cells correlated with enhanced IL-17 production and disease activity. Thus, CD101 deficiency is a novel marker for progressive colitis and potential target for therapeutic intervention.

While IL-2 can potently activate both NK and T cells, its short in vivo half-life, severe toxicity, and propensity to amplify Treg cells are major barriers that prevent IL-2 from being widely used for cancer therapy. In this study, we construct a recombinant IL-2 immunocytokine comprising a tumor-targeting antibody (Ab) and a super mutant IL-2 (sumIL-2) with decreased CD25 binding and increased CD122 binding. The Ab-sumIL2 significantly enhances antitumor activity through tumor targeting and specific binding to cytotoxic T lymphocytes (CTLs). We also observe that pre-existing CTLs within the tumor are sufficient and essential for sumIL-2 therapy. This next-generation IL-2 can also overcome targeted therapy-associated resistance. In addition, preoperative sumIL-2 treatment extends survival much longer than standard adjuvant therapy. Finally, Ab-sumIL2 overcomes resistance to immune checkpoint blockade through concurrent immunotherapies. Therefore, this next-generation IL-2 reduces toxicity while increasing TILs that potentiate combined cancer therapies.

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.

The destruction of β-cells in type 1 diabetes (T1D) progresses silently until only a minor fraction of the β-cells remain. A late acting therapy leading to the prevention of further β-cell killing would therefore be desirable. CD122, the β chain of the interleukin-2 receptor, is highly expressed on natural killer (NK) cells and on a subpopulation of CD8 T cells. In this study, we have treated non-obese diabetic (NOD) mice with a depleting antibody against CD122. The treatment protected from diabetes, even when initiated just before disease onset. The degree of leukocyte infiltration into islets was unaffected by the treatment, further supporting effectiveness late in the disease process. It effectively removed all NK cells from the spleen, pancreas and pancreatic lymph nodes and abolished NK cell activity. Interestingly, despite the lack of CD122 expression on CD8 T cells in the pancreas, the overall frequency of CD8 cells decreased in this organ, whereas it was unaffected in the spleen. T cells were also still capable to respond against a foreign antigen. Conclusively, targeting of CD122(+) cells could represent a novel treatment strategy against T1D.

CD4(+) CD44(v.low) cells are peripheral precursor T cells that inhibit lymphopenia by generating a large CD4(+) T cell pool containing balanced numbers of naïve, memory, and regulatory Foxp3(+) cells with a diverse TCR repertoire. Recent thymic emigrants (RTE) and stem cell-like memory T cells (T(SCM)) can also replenish a T cell pool. In this study we formally test whether CD44(v.low) cells are the same population as RTE and T(SCM). Our data show that, in contrast to RTE, CD44(v.low) cells express high levels of CD45RB and low levels of CD24. Moreover, CD44(v.low) cells isolated from mice devoid of RTE retain their capacity to repopulate lymphopenic mice with naïve and memory cells and Foxp3(+) Tregs. In addition, CD44(v.low) cells do not express IL-2Rβ, Sca-1, and CXCR3, the phenotypic hallmarks of T(SCM). Overall, these data demonstrate that CD44(v.low) cells are neither RTE nor T(SCM).

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1⁻TIGIT⁺, and PD-1⁺TIGIT⁻ CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.

A network of mechanisms operates to maintain tolerance in the gut mucosa. Although CD103 marks many lymphoid cells within the gut, its direct functional role in intestinal tolerance is poorly understood. CD103 may be part of a redundant pathway, as CD103−/− mice do not exhibit autoimmunity. To reduce such redundancy, CD103−/− mice were crossed to mice (designated Y3) whose T cells expressed a mutant IL-2Rβ–chain that lowers IL-2R signaling. Unlike overtly healthy Y3 mice, all Y3/CD103−/− mice rapidly developed severe colitis. The large intestine of these mice contained an increase in CD4+ Th1 and Th17 effector cells and a reduced ratio of regulatory T cells (Tregs). Importantly, colitis was effectively prevented by the transfer of wild-type Tregs into Y3/CD103−/− mice. Impaired intestinal tolerance was not attributed to an obvious lack of CD103-dependent gene regulation or intestinal homing/retention by Tregs nor a lack of functional activities typically associated with CD103+ dendritic cells, such as peripherally induced Treg development or imprinting CCR9 and α4β7 homing molecules on Tregs and T effector cells. Transcriptome analysis of Tregs was consistent with altered homeostasis due to impaired IL-2Rβ–dependent signaling with minimal dysregulation added by the absence of CD103. Rather, the absence of CD103 functioned to alter the localization of the cells within the gut microenvironment that may alter Treg homeostasis. Thus, IL-2Rβ–dependent signaling and CD103 normally cooperate through distinctive processes to promote Treg homeostasis and immune tolerance.

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 α subunit (IL-2Rα 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-γ, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2Rβγ 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.

The cytokine interleukin-2 (IL-2) is critical for the functions of regulatory T cells (T regs ). The contribution of polymorphisms in the gene encoding the IL-2 receptor α subunit ( IL2RA ), which are associated with type 1 diabetes, is difficult to determine because autoimmunity depends on variations in multiple genes, where the contribution of any one gene product is small. We investigated the mechanisms whereby a modest reduction in IL-2R signaling selectively in T lymphocytes influenced the development of diabetes in the NOD mouse model. The sensitivity of IL-2R signaling was reduced by about two- to threefold in T regs from mice that coexpressed wild-type IL-2Rβ and a mutant subunit (IL-2Rβ Y3 ) with reduced signaling (designated NOD-Y3). Male and female NOD-Y3 mice exhibited accelerated diabetes onset due to intrinsic effects on multiple activities in T regs . Bone marrow chimera and adoptive transfer experiments demonstrated that IL-2Rβ Y3 T regs resulted in impaired homeostasis of lymphoid-residing central T regs and inefficient development of highly activated effector T regs and that they were less suppressive. Pancreatic IL-2Rβ Y3 T regs showed impaired development into IL-10–secreting effector T regs . The pancreatic lymph nodes and pancreases of NOD-Y3 mice had increased numbers of antigen-experienced CD4 + effector T cells, which was largely due to impaired T regs , because adoptively transferred pancreatic autoantigen–specific CD4 + Foxp3 − T cells from NOD-Y3 mice did not accelerate diabetes in NOD.SCID recipients. Our study indicates that the primary defect associated with chronic, mildly reduced IL-2R signaling is due to impaired T regs that cannot effectively produce and maintain highly functional tissue-seeking effector T reg subsets.

IL2 is an immunostimulatory cytokine for key immune cells including T cells and natural killer (NK) cells. Systemic IL2 supplementation could enhance NK-mediated immunity in a variety of diseases ranging from neoplasms to viral infection. However, its systemic use is restricted by its serious side effects and limited efficacy due to activation of T regulatory cells (Tregs). IL2 signaling is mediated through interactions with a multi-subunit receptor complex containing IL2Rα, IL2Rβ, and IL2Rγ. Adult natural killer (NK) cells express only IL2Rβ and IL2Rγ subunits and are therefore relatively insensitive to IL2. To overcome these limitations, we created a novel chimeric IL2-IL2Rβ fusion protein of IL2 and its receptor IL2Rβ joined via a peptide linker (CIRB). NK92 cells expressing CIRB (NK92CIRB) were highly activated and expanded indefinitely without exogenous IL2. When compared with an IL2-secreting NK92 cell line, NK92CIRB were more activated, cytotoxic, and resistant to growth inhibition. Direct contact with cancer cells enhanced the cytotoxic character of NK92CIRB cells, which displayed superior in vivo antitumor effects in mice. Overall, our results showed how tethering IL2 to its receptor IL2Rβ eliminates the need for IL2Rα and IL2Rβ, offering a new tool to selectively activate and empower immune therapy. Cancer Res; 77(21); 5938–51. ©2017 AACR.

Mounting evidence has shown that naturally occurring CD8+CD122+ T cells are regulatory T cells (Tregs) that suppress both autoimmunity and alloimmunity. We have previously shown that CD8+CD122+PD-1+ Tregs not only suppress allograft rejection, but also are more potent in suppression than conventional CD4+CD25+ Tregs. However, the mechanisms underlying their suppression of alloimmunity are not well understood. In an adoptive T-cell transfer model of mice lacking lymphocytes, we found that suppression of skin allograft rejection by CD8+CD122+PD-1+ Tregs was mostly dependent on their expression of Fas ligand as either lacking Fas ligand or blocking it with antibodies largely abolished their suppression of allograft rejection mediated by transferred T cells. Their suppression was also mostly reversed when effector T cells lacked Fas receptor. Indeed, these FasL+ Tregs induced T cell apoptosis in vitro in a Fas/FasL-dependent manner. However, their suppression of T cell proliferation in vitro was dependent on IL-10, but not FasL expression. Furthermore, adoptive transfer of CD8+CD122+PD-1+ Tregs significantly extended allograft survival even in wild-type mice if Tregs lacked Fas receptor or if recipients received recombinant IL-15, as these two measures synergistically expanded adoptively-transferred Tregs in recipients. Thus, this study may have important implications for Treg therapies in clinical transplantation.

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-2Rβ/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.

Introduction. Impaired maternal immune tolerance resulting in systemic inflammation plays a pivotal role in the pathogenesis of preeclampsia. Phenotypical changes of monocytes and neutrophil granulocytes have already been studied in preeclampsia, and some studies also included T lymphocyte activation markers; however, the results are controversial and a comprehensive analysis of activation markers is lacking. The characteristics of cellular adhesion molecules in preeclampsia are yet to be described.Material and Methods. Peripheral blood samples of 18 preeclamptic patients and 20 healthy pregnant women in the third trimester were evaluated using flow cytometry to characterize the cell surface expression of T lymphocyte activation markers and selectins.Results. We found an elevated ratio of HLA-DR and CD122-, CD62E-, and CD62L-expressing cells among the CD4+ T lymphocytes in PE in comparison to healthy pregnancy. No alterations were found in the prevalence of CD69-, CD25-, and CD62P-expressing lymphocytes and CD11c-expressing monocytes.Conclusions. Our findings support the role of activated T lymphocytes and specific cell adhesion molecules in the pathogenesis of preeclampsia.

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαβγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαβγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαβγ to a greater extent than for IL2Rβγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rβγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action.

If Bcl11b activity is compromised, CD4+CD8+ double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8+ single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11bF/S826GCD4cre, Bcl11bF/+CD4cre and Bcl11b+/S826G mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαβhighCD44highCD122high innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.

T follicular helper (Tfh) cells contribute to the establishment of humoral immunity by controlling the delivery of helper signals to activated B cells; however, Tfh development must be restrained, as aberrant accumulation of these cells is associated with positive selection of self-reactive germinal center B cells and autoimmunity in both humans and mice. Here, we show that TGF-β signaling in T cells prevented Tfh cell accumulation, self-reactive B cell activation, and autoantibody production. Using mice with either T cell–specific loss or constitutive activation of TGF-β signaling, we demonstrated that TGF-β signaling is required for the thymic maturation of CD44+CD122+Ly49+CD8+ regulatory T cells (Tregs), which induce Tfh apoptosis and thus regulate this cell population. Moreover, peripheral Tfh cells escaping TGF-β control were resistant to apoptosis, exhibited high levels of the antiapoptotic protein BCL2, and remained refractory to regulation by CD8+ Tregs. The unrestrained accumulation of Tfh cells in the absence of TGF-β was dependent on T cell receptor engagement and required B cells. Together, these data indicate that TGF-β signaling restrains Tfh cell accumulation and B cell–associated autoimmunity and thereby controls self-tolerance.

Despite extensive studies on CD4(+)CD25(+) regulatory T cells (Tregs) during the past decade, the progress on their clinical translation remains stagnant. Mounting evidence suggests that naturally occurring CD8(+)CD122(+) T cells are also Tregs with the capacity to inhibit T-cell responses and suppress autoimmunity as well as alloimmunity. In fact, they are memory-like Tregs that resemble a central memory T cell (TCM) phenotype. The mechanisms underlying their suppression are still not well understood, although they may include IL-10 production. We have recently demonstrated that programmed death-1 (PD-1) expression distinguishes between regulatory and memory CD8(+)CD122(+) T cells and that CD8(+)CD122(+) Tregs undergo faster homeostatic proliferation and are more potent in the suppression of allograft rejection than conventional CD4(+)CD25(+) Tregs. These findings may open a new line of investigation for accelerating effective Treg therapies in the clinic. In this review, we summarize the significant progress in this promising field of CD8(+)CD122(+) Treg research and discuss their phenotypes, suppressive roles in autoimmunity and alloimmunity, functional requirements, mechanisms of action and potential applications in the clinic.

Aims: This study aimed to examine the efficacy of a nanocarrier (polyethyleneimine [PEI]-superparamagnetic iron oxide nanoparticle [SPIO]), composed of a core of iron oxide and a shell of PEI, in the systemic delivery of therapeutic siRNA to experimental arthritic joints. Materials & methods: PEI-SPIO/siRNA nanoparticles were synthesized and characterized in vitro. Nanoparticles were administered intravenously to arthritic rats to analyze cellular uptake, tissue distribution and the therapeutic effect of a siRNA against the IL-2/-15 receptor β chain (IL-2/IL-15Rβ). Results: PEI-SPIOs loaded with siRNA displayed negligible cytotoxicity, improved siRNA stability, efficient uptake by macrophages and the ability to induce specific gene silencing in vitro. PEI-SPIO-delivered siRNA accumulated easily in inflamed joints and was efficiently taken up by joint macrophages and T cells. Although IL-2/IL-15Rβ siRNA-loaded PEI-SPIOs alone were efficacious in the treatment of experimental arthritis, combination therapy with both PEI-SPIO/IL-2/IL-15Rβ siRNA and a magnetic field displayed an additive anti-inflammatory effect. Conclusion: PEI-functionalized SPIOs can be employed for systemic siRNA delivery in rheumatoid arthritis and enhanced therapeutic benefit can be achieved by the use of an external magnetic field. Original submitted 1 November 2013; Revised submitted 12 December 2013

Type 1 diabetes (T1D) shows ~ 40% concordance rate in monozygotic twins (MZ) suggesting a role for environmental factors and/or epigenetic modifications in the etiology of the disease. The aim of our study was to dissect the contribution of epigenetic factors, particularly, DNA methylation (DNAm), to the incomplete penetrance of T1D. We performed DNAm profiling in lymphocyte cell lines from 3 monozygotic (MZ) twin pairs discordant for T1D and 6 MZ twin pairs concordant for the disease using HumanMethylation27 BeadChip. This assay assesses the methylation state of 27,578 CpG sites, mostly located within proximal promoter regions. We identified 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. Functional annotation of the genes with distinct CpG methylation profiles in T1D samples showed differential DNAm of immune response and defense response pathways between affected and unaffected twins. Integration of DNAm data with GWAS data mapped several known T1D associated genes, HLA, INS, IL-2RB, CD226, which showed significant differences in DNAm between affected and unaffected of twins. Our findings suggest that abnormalities of DNA methylation patterns, known to regulate gene transcription, may be involved in the pathogenesis of T1D.

Naive T cell responses are eroded with aging. We and others have recently shown that unimmunized old mice lose ≥70% of Ag-specific CD8 T cell precursors and that many of the remaining precursors acquire a virtual (central) memory (VM; CD44hiCD62Lhi) phenotype. In this study, we demonstrate that unimmunized TCR transgenic (TCRTg) mice also undergo massive VM conversion with age, exhibiting rapid effector function upon both TCR and cytokine triggering. Age-related VM conversion in TCRTg mice directly depended on replacement of the original TCRTg specificity by endogenous TCRα rearrangements, indicating that TCR signals must be critical in VM conversion. Importantly, we found that VM conversion had adverse functional effects in both old wild-type and old TCRTg mice; that is, old VM, but not old true naive, T cells exhibited blunted TCR-mediated, but not IL-15–mediated, proliferation. This selective proliferative senescence correlated with increased apoptosis in old VM cells in response to peptide, but decreased apoptosis in response to homeostatic cytokines IL-7 and IL-15. Our results identify TCR as the key factor in differential maintenance and function of Ag-specific precursors in unimmunized mice with aging, and they demonstrate that two separate age-related defects—drastic reduction in true naive T cell precursors and impaired proliferative capacity of their VM cousins—combine to reduce naive T cell responses with aging.

Therapeutic responses following adoptive transfer of T cells correlate to levels of long-term T cell persistence. Lymphodepletion and exogenous γc cytokine administration can improve T cell persistence following adoptive transfer, but their effects are not uniform and toxicities are significant. To overcome these limitations, we designed a chimeric γc cytokine receptor (CγCR) composed of Interleukin-7 (IL-7) tethered to IL-7Rα/CD127 that confers exogenous cytokine independent, cell intrinsic, STAT5 cytokine signals. We additionally show that this design is modular in that the IL-2Rβ/CD122 cytoplasmic chain can be exchanged for that of IL-7Rα/CD127, enhancing Shc activity. When expressed in central memory-derived primary human CD8(+) CTL (T(E/CM)), these CγCRs signal according to their corresponding wild-type counterparts to support exogenous cytokine independent viability and homeostatic proliferation, while retaining full effector function. In vivo studies demonstrate that both CγCR-CD127(+) and CγCR-CD122(+) CD8(+) T((E/CM)) engraft in mice and persist in an absence of exogenous cytokine administration. Engrafted CγCR-CD127(+) CD8(+) T(E/CM) preferentially retain central memory marker expression in vivo demonstrating a dichotomy between CD127 versus CD122 signaling. Together, these results suggest that expression of CγCR in therapeutic T cells may aid in the in vivo persistence of these cells, particularly under conditions of limiting homeostatic cytokines.

Hepatitis C virus (HCV) infection typically results in chronic disease with HCV outpacing antiviral immune responses. Here we asked whether innate immune responses are induced in healthcare workers who are exposed to small amounts of HCV, but do not develop systemic infection and acute liver disease. Twelve healthcare workers with accidental percutaneous exposure to HCV-infected blood were prospectively studied up to 6 months for phenotype and function of NKT and NK cells, kinetics of serum chemokines, and vigor and specificity of HCV-specific T cell responses. Eleven healthcare workers tested negative for HCV RNA and HCV-antibodies. All but one of these aviremic cases displayed NKT cell activation, increased serum chemokines levels, and NK cell responses with increased CD122, NKp44, NKp46 and NKG2A expression, cytotoxicity (as determined by TRAIL and CD107a expression) and IFN-γ production. This multifunctional NK cell response appeared a month earlier than in the one health care worker who developed high-level viremia, and it differed from the impaired IFN-γ production, which is typical for NK cells in chronic HCV infection. The magnitude of NKT cell activation and NK cell cytotoxicity correlated with the magnitude of the subsequent HCV-specific T cell response. T cell responses targeted nonstructural HCV sequences that require translation of viral RNA, which suggests that transient or locally contained HCV replication occurred without detectable systemic viremia. Collectively, these results demonstrate that exposure to small amounts of HCV induces innate immune responses, which correlate with the subsequent HCV-specific T cell response and may contribute to antiviral immunity.