Search

IL‐2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti‐immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL‐2, Mutakine‐6 (MK‐6), with reduced IL‐2Rα–binding capability. MK‐6 induced comparable cell growth potential toward IL‐2Rβγ–positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL‐2, in vivo administration of MK‐6 produced minimal adverse effects. Using CT26 and B16F10‐syngeneic tumor models, we found MK‐6 was highly efficacious on tumor regression. Serum albumin conjugation to MK‐6 prolonged in vivo half‐life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor‐infiltrating leukocytes analysis revealed that albumin‐fused MK‐6 increased the ratio of effector CD8+ T cells to CD4+ Treg cells. These results demonstrated that MK‐6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation., Albumin‐conjugated IL‐2 mutein, MK‐6, exerts a potent antitumor effect in the CT26 tumor model.

Background and aims The relationship between inflammation and lipid metabolism is complex and bidirectional. Lymphocyte-driven inflammation has been shown to modulate both atherosclerotic plaque development and cholesterol levels, but the mechanisms are incompletely understood. Methods The cardiometabolic effects of IL-2Rβγ signalling in atherosclerotic Apoe−/− mice were investigated by treatment with an agonistic IL-2Rβγ-targeting IL-2/anti-IL-2 complex or a monoclonal anti-CD122 (IL-2Rβ) blocking antibody. Results Administration of IL-2Rβγ agonistic IL-2/anti-IL-2 complexes to Apoe−/− mice augmented opposing arms of the adaptive immune system. Expansion of effector/memory T cells and increased levels of circulating pro-inflammatory cytokines were observed along with elevated levels of regulatory T cells and IL-10. Notably, IL-2/anti-IL-2 treatment did not affect plaque size but decreased levels of plasma cholesterol. The cholesterol lowering effect of IL-2Rβγ agonism was not affected by anti-CD8 or anti-NK1.1 depleting antibody treatment but was contingent on the presence of adaptive immunity. Expression of multiple liver X receptor (LXR)-related genes, including Pltp and Srebp1c in the liver, was decreased by IL-2/anti-IL-2 treatment. Although IL-2Rβγ agonism lowered cholesterol levels, blocking IL-2Rβγ signalling using an anti-CD122 monoclonal antibody did not impact cholesterol levels or plaque burden in Apoe−/− mice. Conclusions Elevated IL-2Rβγ signalling results in activation of both inflammatory and regulatory lymphocytes with a net zero effect on atherosclerosis and decreased plasma cholesterol levels. Changes in cholesterol levels were associated with reductions in hepatic LXR-related gene expression. Further studies are needed to investigate the clinical significance of IL-2 mediated modulation of hepatic LXR signalling in inflammatory disorders.

The prognostic value of current multigene assays for breast cancer is limited to hormone receptor-positive, human epidermal growth factor receptor 2-negative early breast cancer. Despite the prognostic significance of immune response-related genes in breast cancer, immune gene signatures have not been incorporated into most multigene assays. Here, using public gene expression microarray datasets, we classified breast cancer patients into three risk groups according to clinical risk and proliferation risk. We then developed the immune prognostic index based on expression of five immune response-related genes (TRAT1, IL2RB, CTLA4, IGHM and IL21R) and lymph node status to predict the risk of recurrence in the clinical and proliferation high-risk (CPH) group. The 10-year probability of disease-free survival (DFS) or distant metastasis-free survival (DMFS) of patients classified as high risk according to the immune prognostic index was significantly lower than those of patients classified as intermediate or low risk. Multivariate analysis revealed that the index is an independent prognostic factor for DFS or DMFS. Moreover, the C-index revealed that it is superior to clinicopathological variables for predicting prognosis. Its prognostic significance was also validated in independent datasets. The immune prognostic index identified low-risk patients among patients classified as CPH, regardless of the molecular subtype of breast cancer, and may overcome the limitations of current multigene assays.

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-2Rβ and a common γ chain (γC). In this study, the full-length cDNA sequences of IL-15 and IL-2Rβ were identified in grass carp (Ctenopharyngodon idella, Ci) and their expression profiles analysed. The CiIL-15 and CiIL-2Rβ were shown to be broadly expressed in tissues, with the highest levels detected in the spleen. Moreover, the CiIL-15 and CiIL-2Rβ 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 IL2 receptor (IL2R) is an attractive cancer immunotherapy target that controls immunosuppressive T regulatory cells (Treg) and antitumor T cells. Here we used IL2Rβ-selective IL2/anti-IL2 complexes (IL2c) to stimulate effector T cells preferentially in the orthotopic mouse ID8agg ovarian cancer model. Despite strong tumor rejection, IL2c unexpectedly lowered the tumor microenvironmental CD8+/Treg ratio. IL2c reduced tumor microenvironmental Treg suppression and induced a fragile Treg phenotype, helping explain improved efficacy despite numerically increased Tregs without affecting Treg in draining lymph nodes. IL2c also reduced Treg-mediated, high-affinity IL2R signaling needed for optimal Treg functions, a likely mechanism for reduced Treg suppression. Effector T-cell IL2R signaling was simultaneously improved, suggesting that IL2c inhibits Treg functions without hindering effector T cells, a limitation of most Treg depletion agents. Anti-PD-L1 antibody did not treat ID8agg, but adding IL2c generated complete tumor regressions and protective immune memory not achieved by either monotherapy. Similar anti-PD-L1 augmentation of IL2c and degradation of Treg functions were seen in subcutaneous B16 melanoma. Thus, IL2c is a multifunctional immunotherapy agent that stimulates immunity, reduces immunosuppression in a site-specific manner, and combines with other immunotherapies to treat distinct tumors in distinct anatomic compartments. Significance: These findings present CD122-targeted IL2 complexes as an advancement in cancer immunotherapy, as they reduce Treg immunosuppression, improve anticancer immunity, and boost PD-L1 immune checkpoint blockade efficacy in distinct tumors and anatomic locations.

Long‐term subjection to shift work increases the risk of cancer. The purpose of the present study was to explore the mechanism by which chronic circadian disruption impairs natural killer (NK) cell immunosurveillance. Mice were subjected to light‐dark reverse every 4 days for 12 weeks to disrupt normal circadian rhythm. NK cell development and function were evaluated by flow cytometry. The mRNA and protein levels of period 1 (per1) and per2 were suppressed, while circadian locomotor output cycle kaput (CLOCK) was increased in the shifted mice, indicating successful generation of the circadian rhythm disruption mouse model. Chronic shift‐lag promoted NK cell ageing, which is likely due to the reduction in Ly49 family receptor expression in shifted NK. We further studied the effects of circadian rhythm disruption on NK cell function. Chronic shift‐lag inhibited NK cell secretion of granular CD107a and interferon gamma. Moreover, chronic shift‐lag attenuated the clearance of MHC‐I–deficient tumour cells by NK cells in vivo and promoted lung metastasis of B16F10 melanomas. Furthermore, chronic shift‐lag reduced NK cell killing function, which may be due to the suppression of Eomes transcription factor expression, which inhibiting the transcription of CD122. In conclusion, our findings suggest that chronic circadian disruption attenuates NK cell cytolytic activity by decreasing the expression of CD122.

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.

IL-2R signaling is essential for regulatory T cell (Treg) function. However, the precise contribution of IL-2 during Treg thymic development, peripheral homeostasis and lineage stability remains unclear. Here we show that IL-2R signaling is required by thymic Tregs at an early step for expansion and survival, and a later step for functional maturation. Using inducible, conditional deletion of CD25 in peripheral Tregs, we also find that IL-2R signaling is indispensable for Treg homeostasis, whereas Treg lineage stability is largely IL-2-independent. CD25 knockout peripheral Tregs have increased apoptosis, oxidative stress, signs of mitochondrial dysfunction, and reduced transcription of key enzymes of lipid and cholesterol biosynthetic pathways. A divergent IL-2R transcriptional signature is noted for thymic Tregs versus peripheral Tregs. These data indicate that IL-2R signaling in the thymus and the periphery leads to distinctive effects on Treg function, while peripheral Treg survival depends on a non-conventional mechanism of metabolic regulation., Interleukin-2 (IL-2) signaling is required for regulatory T (Treg) cell differentiation in the thymus, but its function in peripheral Tregs is still unclear. Here the authors show, using inducible deletion of IL-2 receptor subunit CD25, that IL-2 signaling is essential for maintaining peripheral Treg homeostasis, but dispensable for lineage stability.

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.

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.

Introduction. Acute lung injury (ALI) induced by sepsis is a process related to inflammatory reactions, which involves lung cell apoptosis and production of inflammatory cytokine. Here, lipopolysaccharide (LPS) was applied to stimulate the mouse or human normal lung epithelial cell line (BEAS-2B) to construct a sepsis model in vivo and in vitro, and we also investigated the effect of miR-497-5p on sepsis-induced ALI. Material and Methods. Before LPS treatment, miR-497-5p antagomir was injected intravenously into mice to inhibit miR-497-5p expression in vivo. Similarly, miR-497-5p was knocked down in BEAS-2B cells. Luciferase reporter assay was applied to predict and confirm the miR-497-5p target gene. Cell viability, apoptosis, the levels of miR-497-5p, IL2RB, SP1, inflammatory cytokine, and lung injury were assessed. Results. In BEAS-2B cells, a significant increase of apoptosis and inflammatory cytokine was shown after LPS stimulation. In septic mice, increased inflammatory cytokine production and apoptosis in lung cells and pulmonary morphological abnormalities were shown. The miR-497-5p inhibitor transfection showed antiapoptotic and anti-inflammatory effects on BEAS-2B cells upon LPS stimulation. In septic mice, the miR-497-5p antagomir injection also alleviated ALI, apoptosis, and inflammation caused by sepsis. The downregulation of IL2RB in BEAS-2B cells reversed the protective effects of the miR-497-5p inhibitor against ALI. Conclusion. In conclusion, downregulation of miR-497-5p reduced ALI caused by sepsis through targeting IL2RB, indicating the potential effect of miR-497-5p for improving ALI caused by sepsis.

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection(1). Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, unlike PD-1 monotherapy, dramatically changes the differentiation program of the PD-1(+)TCF-1(+) stem-like CD8(+) T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8(+) T cells that resemble highly functional effector CD8(+) T cells seen after an acute viral infection. The generation of these qualitatively superior CD8(+) T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1(+)TCF-1(+) stem-like CD8(+) T cells, also referred to as precursors of exhausted CD8(+) T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8(+) T cells emerging from the stem-like CD8(+) T cells after combination therapy expressed increased levels of the high affinity IL-2 trimeric (CD25, CD122, CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 plays an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind CD25 but still binds CD122/CD132 almost completely abrogated the synergistic effects seen after PD-1 + IL-2 combination therapy. There is currently considerable interest in PD-1 + IL-2 combination therapy for patients with cancer(2,3) and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

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.

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

Abdominal aortic aneurysm (AAA) is potentially life threatening and characterized by immune-inflammatory cell infiltration and extracellular matrix degradation. Currently, pharmacotherapy mainly aims to control risk factors without reversion of the dilated aorta. This study analyzed the immune-inflammatory response and identified the immune-related hub genes of AAA.Gene Expression Omnibus datasets (GSE57691, GSE47472 and GSE7084) were downloaded. After identification of GSE57691 differentially expressed genes (DEGs), weighted gene co-expression network analysis of the DEGs was performed. Through enrichment analysis of each module and screening in Immunology Database and Analysis Portal, immune-related hub genes were identified via protein-protein interaction (PPI) network construction and lasso regression. CIBERSORT was utilized to analyze AAA immune infiltration. The correlations between the immune-related hub genes and infiltrating immune cells were investigated. Receiver operating characteristic (ROC) curve analysis was performed to determine immune-related hub gene cutoff values, which were validated in GSE47472 and GSE7084.In GSE57691, 1,018 DEGs were identified. Five modules were identified in the co-expression network. The blue and green modules were found to be related to immune-inflammatory responses, and 61 immune-related genes were identified. PPI and lasso regression analyses identified FOS, IL-6 and IL2RB as AAA immune-related hub genes. CIBERSORT analysis indicated significantly increased infiltration of naive B cells, memory activated CD4 T cells, follicular helper T cells, monocytes and M1 macrophages and significantly decreased infiltration of M2 macrophages in AAA compared with normal samples. IL2RB was more strongly associated with immune infiltration in AAA than were FOS and IL6. The IL2RB area under the ROC curve (AUC) value was 0.9 in both the training and validation set, demonstrating its strong, stable diagnostic value in AAA.AAA and normal samples had different immune infiltration statuses. IL2RB was identified as an immune-related hub gene and a potential hub gene with significant diagnostic value in AAA.

NKTR-255 is a PEG conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans or cis presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naive and memory OVA-specific CD8+ T cells (OT-I) and NK cells in mice. NKTR-255 induced a 2.5- and 2.0-fold expansion of CD8+ T and NK cells, respectively, in WT mice. In adoptive transfer studies, proliferation of naive and memory WT OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα-/- mice, suggesting trans presentation was not utilized by NKTR-255. Interestingly, naive IL-15Rα-/- OT-I cells had deficient responses to NKTR-255, while memory IL-15Rα-/- OT-I cell responses were partially impaired, suggesting that naive CD8+ T cells are more dependent on cis presentation of NKTR-255 than memory CD8+ T cells. In bone marrow chimera studies, IL-15Rα-/- and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells, showing that conversion to IL-15Rβ agonist biases the response toward NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis presentation and act as an IL-15Rαβ agonist on CD8+ T cells.

The expression of IL-2RA and IL-2RB was correlated with breast cancer (BC) progression. However, there is no literature investigating the association of IL-2RA and IL-2RB polymorphisms with BC predisposition among Chinese Han Women. Seven SNPs in IL-2RA and IL-2RB were genotyped by Agena MassARRAY platform among 553 BC patients and 550 healthy controls. Odds ratios (OR) and 95% confidence interval (CI) adjusted for age were calculated for the effect of IL-2RA and IL-2RB variants on BC susceptibility. IL-2RA rs12722498 was a protective factor for BC occurrence (OR = 0.70, p = 0.019), especially in subjects with age ≤ 52 years (OR = 0.55, p = 0.004). IL-2RA rs12569923 (OR = 9.07, p = 0.033), IL-2RB rs2281089 (OR = 0.67, p = 0.043) and rs9607418 (OR = 0.59, p = 0.012) were related to the incidence of estrogen receptor positive (ER +) BC. IL-2RB rs3218264 (OR = 1.38, p = 0.010) and rs9607418 (OR = 0.56, p = 0.009) were associated with the risk of developing progesterone receptor positive (PR +) BC. Rs2281089 (OR = 1.54, p = 0.012) and rs1573673 (OR = 0.72, p = 0.035) were correlated to Ki-67 level. Moreover, IL-2RB rs2281089 (OR = 0.72, p = 0.022) showed a reduced risk of BC metastasis, and IL-2RA rs12722498 (OR = 0.54, p = 0.030) had a lower frequency in BC patients with tumor size 2 cm. Our study identified the potential effect of genetic variations in IL-2RA and IL-2RB on BC susceptibility and/or BC clinicopathologic indicators among Chinese Han Women.

Wilms' tumor 1 (WT1) is a transcription factor which plays a major role in cell proliferation, differentiation, survival, and apoptosis. WT1 was first identified as a tumor suppressor gene in Wilms' tumor. However, overexpression of WT1 has been detected in several types of malignancy including some types of leukemia. To investigate the molecular mechanism underlying WT1-mediated leukemogenesis, lentiviral-based siRNA was employed as a tool to suppress WT1 expression in the myeloid leukemia cell line, K562. Successfully, both WT1 RNA and protein levels were downregulated in the leukemia cells. The silencing of WT1 resulted in significant growth inhibition in WT1-siRNA-treated cells for 40 ± 7.0%, 44 ± 9.5%, and 88 ± 9.1% at 48, 72, and 96 hours posttransduction as compared with the control cells, respectively. By using apoptosis detection assays (caspase-3/7 activity and Annexin V-FITC/PI assays), WT1 silencing induced a higher degree of early and late apoptosis in siRNA-treated K562 as compared with the control cells. Interestingly, the expression of survival signaling genes, IL-2, IL-2RB, and IL-2RG, was also suppressed after WT1-siRNA treatment. In addition, the WT1 silencing also inhibited the S phase of the cell cycle and induced cell death. Our results indicated that WT1 silencing by siRNA can suppress cellular proliferation, induce apoptosis, and reduce S phase fraction of K562 cells. Moreover, transcriptional modulation of IL-2, IL-2RB, and IL2-2RG expression by WT1 was likely involved in this phenotypic change. Overall, this study confirmed the oncogenic role of WT1 in myeloid leukemia and discovered the new target genes of WT1 which are likely involved in WT1-mediated leukemogenesis.

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.

The mechanism exhausting CD8

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

Interleukin-2 (IL-2) is an important immunomodulatory cytokine that primarily promotes the activation, proliferation, and differentiation of CD4+ T helper subsets and CD4+ T regulatory cells. In our previous studies, IL-2 and IL-2 receptor beta (IL-2Rβ) genes of flounder (

Engaging in exercise while undergoing radiotherapy (RT) has been reported to be safe and achievable. The impact of exercise training (ET) on RT efficiency is however largely unknown. Our study aims to investigate the interactions between ET and RT on prostate cancer growth. Athymic mice received a subcutaneous injection of PPC-1 cells and were randomly assigned to either cancer control, cancer ET, cancer RT, or cancer RT combined with ET (CaRT-ET). Mice were sacrificed 24 days post-injection. All three intervention groups had reduced tumor size, the most important decrease being observed in CaRT-ET mice. Apoptotic marker cleaved caspase-3 was not modified by ET, but enhanced with RT. Importantly, this increase was the highest when the two strategies were combined. Furthermore, NK1.1 staining and gene expression of natural killer (NK) cell receptors Klrk1 and Il2rβ were not affected by ET alone but were increased with RT, this effect being potentiated when combined with ET. Overall, our study shows that (a) ET enhances RT efficiency by potentiating NK cell infiltration, and (b) while ET alone and ET combined with RT both reduce tumor growth, the mechanisms mediating these effects are different.

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions., The metabolic regulator protein family, mTOR, regulate natural killer (NK) cell development and function, but the underlying mechanism is unclear. Here, the authors show that Raptor/mTORC1 and Rictor/mTORC2 form a feedback crosstalk network to variegate cytokine and cellular signaling and modulate NK maturation and effector functions.

Regulation of intracellular signaling pathways in lymphocytes is critical for cell homeostasis and immune response. Interleukin-2 (IL-2), a key regulator of lymphocytes, signals following receptor-ligand engagement and subsequent recruitment and activation of effector proteins including JAKs and STATs. Lymphocytes can also be regulated by the central nervous system through the β2 adrenergic receptor (β2AR) pathway which can affect cell trafficking, proliferation, differentiation, and cytokine production. The cross-talk between these two signaling pathways represents an important mechanism that has yet to be fully elucidated. The present study provides evidence for communication between the IL-2 receptor (IL-2R) and β2AR. Treatment of human lymphoid cell lines with the β2AR agonist isoproterenol (ISO) alone increased cAMP levels and mediated a stimulatory response by activating AKT and ERK to promote cell viability. Interestingly, ISO activation of β2AR also induced threonine phosphorylation of the IL-2Rβ. In contrast, ISO treatment prior to IL-2 stimulation produced an inhibitory signal that disrupted IL-2 induced activation of the JAK/STAT, MEK/ERK, and PI3K pathways by inhibiting the formation of the IL-2R beta–gamma chain complex, and subsequently cell proliferation. Moreover, γc-family cytokines-mediated STAT5 activation was also inhibited by ISO. These results suggest a molecular mechanism by which β2AR signaling can both stimulate and suppress lymphocyte responses and thus explain how certain therapeutic agents, such as vasodilators, may impact immune responsiveness.

Interrupting T cell costimulatory signals as a strategy to control undesired immune responses, such as occur in autoimmunity or transplantation, has the potential to alleviate many of the unwanted side effects associated with current immunosuppressive therapies. Belatacept, a high-affinity version of CTLA4-Ig that blocks ligand ligation to CD28, has been approved for use in kidney transplant recipients. Despite the long-term benefits associated with its use, such as improved renal function and lower cardiovascular risk, a subset of patients treated with belatacept experience elevated rates of acute T cell-mediated rejection, tempering enthusiasm for its use. Here we demonstrate that costimulation-independent T cell alloreactivity relies on signaling through CD122, the shared IL-2 and IL-15 receptor β-chain. Combined costimulatory and CD122 blockade improved survival of transplanted tissue in mice and nonhuman primates by controlling proliferation and effector function of CD8+ T cells. The high-affinity IL-2 receptor was dispensable for memory CD8+ T cell responses, whereas signaling through CD122 as a component of the high-affinity IL-15 receptor was critical for costimulation-independent memory CD8+ T cell recall, distinguishing specific roles for IL-2 and IL-15 in T cell activation. These studies outline a novel approach for clinical optimization of costimulatory blockade strategies in transplantation by targeting CD122.

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.

Background: Constitutional immunity shaped by exposure to endemic infectious diseases and parasitic worms in Sub-Saharan Africa may play a role in the etiology of breast cancer among African American (AA) women. Methods: A total of 149,514 gene variants in 433 genes across 45 immune pathways were analyzed in the AMBER consortium among 3,663 breast cancer cases and 4,687 controls. Gene-based pathway analyses were conducted using the adaptive rank truncated product statistic for overall breast cancer risk, and risk by estrogen receptor (ER) status. Unconditional logistic regression analysis was used to estimate ORs and 95% confidence intervals (CIs) for single variants. Results: The top pathways were Interleukin binding (P = 0.01), Biocarta TNFR2 (P = 0.005), and positive regulation of cytokine production (P = 0.024) for overall, ER+, and ER− cancers, respectively. The most significant gene was IL2RB (P = 0.001) for overall cancer, with rs228952 being the top variant identified (OR = 0.85; 95% CI, 0.79–0.92). Only BCL3 contained a significant variant for ER+ breast cancer. Variants in IL2RB, TLR6, IL8, PRKDC, and MAP3K1 were associated with ER− disease. The only genes showing heterogeneity between ER− and ER+ cancers were TRAF1, MAP3K1, and MAPK3 (P ≤ 0.02). We also noted genes associated with autoimmune and atopic disorders. Conclusions: Findings from this study suggest that genetic variants in immune pathways are relevant to breast cancer susceptibility among AA women, both for ER+ and ER− breast cancers. Impact: Results from this study extend our understanding of how inherited genetic variation in immune pathways is relevant to breast cancer susceptibility. Cancer Epidemiol Biomarkers Prev; 27(3); 321–30. ©2018 AACR.

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.

The development of CD1d-restricted invariant natural killer T (iNKT) cells, a population that is critical for both innate and adaptive immunity, is regulated by multiple transcription factors, but the molecular mechanisms underlying how the transcriptional activation of these factors are regulated during iNKT development remain largely unknown. We found that the histone acetyltransferase general control non-derepressible 5 (GCN5) is essential for iNKT cell development during the maturation stage. GCN5 deficiency blocked iNKT cell development in a cell-intrinsic manner. At the molecular level, GCN5 is a specific lysine acetyltransferase of early growth responsive gene 2 (EGR2), a transcription factor required for iNKT cell development. GCN5-mediated acetylation positively regulated EGR2 transcriptional activity, and both genetic and pharmacological GCN5 suppression specifically inhibited the transcription of EGR2 target genes in iNKT cells, including Runx1, promyelocytic leukemia zinc finger protein (PLZF), interleukin (IL)-2Rb, and T-bet. Therefore, our study revealed GCN5-mediated EGR2 acetylation as a molecular mechanism that regulates iNKT development.

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.

A T cell sleeper agent against stress Considerable changes in cellular metabolism occur when T cells transition from a resting to an activated state. One side effect of this process is an increase in reactive oxygen species (ROS). These molecules potentiate T cell receptor (TCR) signaling but can also result in detrimental oxidative stress (see the Perspective by Su and Dutta). Yue et al. describe one mechanism by which T cells can resolve this contradiction. Using mice with a T cell-specific deficiency in Schlafen 2 (SLFN2), they found that this protein binds to and protects transfer RNAs from oxidative stress-induced cleavage by the ribonuclease angiogenin. This process is downstream of ROS generation, which allows activated T cells to maintain protein synthesis despite the ROS that would otherwise inhibit translation. Science , this issue p. eaba4220 ; see also p. 683

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