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Introduction Immune checkpoint inhibitors (ICI) have significantly improved the prognosis of non-small cell lung cancer (NSCLC). However, only a limited proportion of patients can benefit from this therapy, and clinically useful predictive biomarkers remain to be elucidated. Methods Blood was collected from 189 patients with NSCLC before and six weeks after the initiation of the ICI treatment (anti-PD-1 or anti-PD-L1 antibody). Soluble PD-1 (sPD-1) and PD-L1 (sPD-L1) in plasma before and after treatment were analyzed for evaluation of their clinical significance. Results The Cox regression analysis demonstrated that higher sPD-L1 levels before treatment significantly predicted unfavorable progression-free survival (PFS; HR 15.4, 95%CI 1.10–86.7, P = 0.009) and overall survival (OS; HR 11.4, 95%CI 1.19–52.3, P = 0.007) in NSCLC patients treated with ICI monotherapy (n = 122), but not in those treated with ICI combined with chemotherapy (n = 67: P = 0.729 and P = 0.155, respectively). In addition, higher sPD-1 levels after treatment were significantly associated with better OS (HR 0.24, 95%CI 0.06–0.91, P = 0.037) in patients treated with anti-PD-1 monotherapy, whereas higher sPD-L1 levels after treatment were significantly associated with worse PFS (HR 6.09, 95%CI 1.42–21.0, P = 0.008) and OS (HR 42.6, 95%CI 6.83–226, P

Neoantigens derived from tumor-specific genetic mutations might be suitable targets for cancer immunotherapy because of their high immunogenicity. In the current study, we evaluated the immunogenicity of 10 driver mutations that are frequently expressed in various cancers using peripheral blood mononuclear cells from healthy donors (n = 25). Of the 10 synthetic peptides (27-mer) derived from these mutations, the six peptides from KRAS-G12D, KRAS-G12R, KRAS-G13D, NRAS-Q61R, PIK3CA-H1047R, and C-Kit-D816V induced T cell responses, suggesting that frequent driver mutations are not always less immunogenic. In particular, immune responses to PIK3CA-H1047R, C-Kit-D816V, KRAS-G13D, and NRAS-Q61R were observed in more than 10% of the donors. All six peptides induced human leukocyte antigen (HLA) class II-restricted CD4+ T cell responses, notably, PIK3CA-H1047R contained at least two different CD4+ T cell epitopes restricted to different HLA class II alleles. In addition, PIK3CA-H1047R and C-Kit-D816V induced antigen-specific CD8+ T cells as well, indicating that they might contain both HLA class I- and class II-restricted epitopes. Since the identified neoantigens might be shared by patients with various types of cancers and are not easily lost due to immune escape, they have the potential to be promising off-the-shelf cancer immunotherapy targets in patients with the corresponding mutations.

SummaryHow hematopoietic stem cells (HSCs) produce particular lineages is insufficiently understood. We searched for key factors that direct HSC to lymphopoiesis. Comparing gene expression profiles for HSCs and early lymphoid progenitors revealed that Satb1, a global chromatin regulator, was markedly induced with lymphoid lineage specification. HSCs from Satb1-deficient mice were defective in lymphopoietic activity in culture and failed to reconstitute T lymphopoiesis in wild-type recipients. Furthermore, Satb1 transduction of HSCs and embryonic stem cells robustly promoted their differentiation toward lymphocytes. Whereas genes that encode Ikaros, E2A, and Notch1 were unaffected, many genes involved in lineage decisions were regulated by Satb1. Satb1 expression was reduced in aged HSCs with compromised lymphopoietic potential, but forced Satb1 expression partly restored that potential. Thus, Satb1 governs the initiating process central to the replenishing of lymphoid lineages. Such activity in lymphoid cell generation may be of clinical importance and useful to overcome immunosenescence.

IL-5 is involved in a number of immune responses such as helminth infection and allergy. IL-5 also plays roles in innate immunity by maintaining B-1 B cells and mucosal IgA production. However, the identity of IL-5–producing cells has not been unambiguously characterized. In this report, we describe the generation of an IL-5 reporter mouse and identify IL-5–producing non-T lymphoid cells that reside in the intestine, peritoneal cavity, and lungs in naive mice. They share many characteristics with natural helper cells, nuocytes, and Ih2 cells, including surface Ags and responsiveness to cytokines. However, these phenotypes do not completely overlap with any particular one of these cell types. Innate non-T IL-5–producing cells localized most abundantly in the lung and proliferated and upregulated IL-5 production in response to IL-25 and IL-33. IL-33 was more effective than IL-25. These cells contribute to maintaining sufficient numbers of lung eosinophils and are important for eosinophil recruitment mediated by IL-25 and IL-33. Given that eosinophils are shown to possess antitumor activity, we studied lung tumor metastasis and showed that innate IL-5–producing cells were increased in response to tumor invasion, and their regulation of eosinophils is critical to suppress tumor metastasis. Genetic blockade or neutralization of IL-5 impaired eosinophil recruitment into the lung and resulted in increased tumor metastasis. Conversely, exogenous IL-5 treatment resulted in suppressed tumor metastasis and augmented eosinophil infiltration. These newly identified innate IL-5–producing cells thus play a role in tumor surveillance through lung eosinophils and may contribute to development of novel immunotherapies for cancer.

The survival and fate of blood cell precursors is dependent on their communication with stromal cells of various types within bone marrow. Monoclonal antibodies have proven to be powerful tools for identifying molecules responsible for such interactions and we now describe one that selectively blocks B lymphopoiesis. The BF/32 antibody inhibited the establishment, but not the maintenance of long-term bone marrow cultures capable of lymphocyte production. However, there was no obvious effect on lymphocyte-stromal cell adhesion or responsiveness of pre-B cells to intereleukin-7. Furthermore, the reagent had no influence on myeloid precursors or myeloid bone marrow cultures. Injection of adult mice with BF/32 reduced B lineage precursors within bone marrow, but spared mature B cells. Moreover, the reagent did not alter responsiveness of mature B cells to activating stimuli. The 60 kDa protein recognized by this antibody was widely expressed on lymphocytes. Amino terminal protein sequencing and transfection experiments identified it as the murine homologue of ICAM-2 (CD102).

Immune cells express multiple Toll-like receptors (TLRs) that are concomitantly activated by a variety of pathogen products. Although there is presumably a need to coordinate the expression and function of TLRs in individual cells, little is known about the mechanisms governing this process. We show that a protein associated with TLR4 (PRAT4A) is required for multiple TLR responses. PRAT4A resides in the endoplasmic reticulum, and PRAT4A knockdown inhibited trafficking of TLR1 and TLR4 to the cell surface and ligand-induced trafficking of TLR9 to lysosomes. Other cell-surface molecules were expressed normally on immunocytes from PRAT4A−/− mice. There was impaired cytokine production to TLR ligands, except to the TLR3 ligand poly(I:C), and to whole bacteria. Activation of antigen-specific T helper type 1 responses were also defective. Moreover, PRAT4A−/− bone marrow chimeric mice were resistant to lipopolysaccharide-induced sepsis. These results suggest that PRAT4A regulates the subcellular distribution and response of multiple TLRs and is required for both innate and adaptive immune responses.

Multiple subsets of the bone marrow contain T cell precursors, but it remains unclear which is most likely to replenish the adult thymus. Therefore, RAG-1+ early lymphoid progenitors (RAG-1+ ELP), and CD62L/L-selectin+ progenitors (LSP), as well as common lymphoid progenitors from C57BL6-Thy1.1-RAG-1/GFP mouse bone marrow were directly compared in transplantation assays. The two c-Kithigh populations vigorously regenerated the thymus and were superior to common lymphoid progenitors in magnitude and frequency of thymic reconstitution. Regeneration was much faster than the 22 days described for transplanted stem cells, and RAG-1+ ELP produced small numbers of lymphocytes within 13 days. As previously reported, LSP were biased to a T cell fate, but this was not the case for RAG-1+ ELP. Although RAG-1+ ELP and LSP had reduced myeloid potential, they were both effective progenitors for T lymphocytes and NK cells. The LSP subset overlapped with and included most RAG-1+ ELP and many RAG-1−TdT+ ELP. LSP and RAG-1+ ELP were both present in the peripheral circulation, but RAG-1+ ELP had no exact counterpart among immature thymocytes. The most primitive of thymocytes were similar to Lin−c-KithighL-selectin+TdT+RAG-1− progenitors present in the marrow, suggesting that this population is normally important for sustaining the adult thymus.

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.

The Src homology (SH)2-containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP(-/-) animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP(-/-) mice. Lineage-negative marrow (Lin(-)) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP(-/-) mice. Furthermore, culture supernatants of SHIP(-/-) Lin(-) cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP(-/-) Lin(-) cells contained more IL-6 transcripts than wild-type Lin(-) cells, and neutralizing anti-IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP(-/-) Lin(-) cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin(-) bone marrow cells reproduced the phenotype of SHIP(-/-) bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.

Adiponectin, an adipocyte-derived hormone, is attracting considerable interest as a potential drug for diabetes and obesity. Originally cloned from human s.c. fat, the protein is also found in bone marrow fat cells and has an inhibitory effect on adipocyte differentiation. The aim of the present study is to explore possible influences on lymphohematopoiesis. Recombinant adiponectin strongly inhibited B lymphopoiesis in long-term bone marrow cultures, but only when stromal cells were present and only when cultures were initiated with the earliest category of lymphocyte precursors. Cyclooxygenase inhibitors abrogated the response of early lymphoid progenitors to adiponectin in stromal cell-containing cultures. Furthermore, PGE2, a major product of cyclooxygenase-2 activity, had a direct inhibitory influence on purified hematopoietic cells, suggesting a possible mechanism of adiponectin action in culture. In contrast to lymphopoiesis, myelopoiesis was slightly enhanced in adiponectin-treated bone marrow cultures, and even when cultures were initiated with single lymphomyeloid progenitors. Finally, human B lymphopoiesis was also sensitive to adiponectin in stromal cell cocultures. These results suggest that adiponectin can negatively and selectively influence lymphopoiesis through induction of PG synthesis. They also indicate ways that adipocytes in bone marrow can contribute to regulation of blood cell formation.

RAG1/GFP knockin mice were exploited to isolate and characterize fetal lymphoid progenitors. CD11b and IL-7Rα are expressed in a developmental stage-dependent fashion, revealing how substantial numbers of early lymphoid progenitors were discarded or neglected in previous studies. The myeloerythroid potential of fetal progenitors in clonal assays declined in synchrony with activation of the RAG1 locus but was not completely extinguished. Lymphoid differentiation corresponded to patterns of gene expression previously found for adult marrow, but no fraction of fetal liver was enriched with respect to B + T progenitors. Also, unlike adults, fetal lymphoid progenitors transiently expressed endothelial cell markers. These findings help to reconcile discrepancies in previous reports and suggest that the fetal immune system arises via unique mechanisms.

The earliest progenitors of lymphocytes are extremely rare and typically present among very complex populations of hematopoietic cells. Additionally, it is difficult to know how cells with any given set of characteristics are developmentally related to stem cells and maturing lymphoid precursors. However, it is now possible to divide bone marrow into progressively smaller fractions and exploit well-defined culture systems to determine which ones contain cells that can turn into lymphocytes. Analysis of steroid hormone sensitive cells and use of two-step cultures is providing additional information about the most likely differentiation pathways for B and natural killer cell lineage lymphocytes. A newly identified category of early lymphoid progenitors can now be sorted to high purity from RAG1/GFP knock in mice. Furthermore, the same experimental model makes it possible to image lymphoid progenitors in fetal and adult hematopoietic tissues.

The human interleukin-5 receptor (hIL-5R) consists of a unique alpha subunit (hIL-5Ralpha) and a common beta subunit (betac) that activate two Janus kinases (JAK1 and JAK2) and a signal transducer and activator of transcription (STAT5). The precise stoichiometry of the hIL-5R subunits and the role of JAK kinases used in IL-5 signaling were investigated. We analyzed the interaction between hIL-5Ralpha and betac by immunoprecipitation using anti-hIL-5Ralpha and anti-betac monoclonal antibodies. The binding of JAK1 and JAK2 to each hIL-5R subunit was also evaluated in the hIL-5-responsive cell line, TF-h5Ralpha. It was observed that IL-5 stimulation induced the recruitment of betac to hIL-5Ralpha, although in the absence of IL-5 the subunits remain independent. In the absence of IL-5, JAK2 and JAK1 were associated with hIL-5Ralpha and betac, respectively. IL-5 stimulation resulted in tyrosine phosphorylation of JAK2, JAK1, betac, and STAT5. Moreover, IL-5-induced dimerization of IL-5R subunits caused JAK2 activation and betac phosphorylation even in the absence of JAK1 activation. Furthermore, tyrosine phosphorylation of JAK1 was dependent on the activation of JAK2. Detailed study of the C-terminal truncated cytoplasmic domain of hIL-5Ralpha revealed that the cytoplasmic stretch at position 346-387, containing the proline-rich region, is necessary for JAK2 binding. These observations suggest that activation of hIL-5Ralpha-associated JAK2 is indispensable for the IL-5 signaling event.

B-1 cells represent a sub-fraction of B lymphocytes that participate in T cell-independent antibody production and contribute to innate immunity. While the production of B-1 cells is favored during the fetal waves of lymphopoiesis, it has been unclear when and how that differentiation option is specified. To clarify this, lymphoid and hematopoietic progenitors of fetal liver (FL) and adult bone marrow (ABM) were examined for the B cell differentiation potential. Mouse common lymphoid progenitors (CLPs) and more primitive KSL fraction of FL and ABM were transferred to SCID mice and donor-derived B cell subsets were analyzed 4 weeks later. CLPs were also cultured on ST2 stromal cells for 6 days prior to transplantation. While Lin- IL-7Rα+ CLPs from ABM differentiated to B-1, B-2 and marginal zone B (MZB) cells, equivalent cells from d15 FL differentiated mostly to B-1a cells. We found that fetal CLPs had less ability to colonize the bone marrow than adult CLPs. However, the fetal/adult difference was already present when progenitors were cultured in an identical condition before transplantation. More primitive KSL fraction of FL could generate the same broad spectrum of B cells typical of adults, including splenic MZB cells. In conclusion, we argue that FL and ABM-CLPs are intrinsically different regarding B-1/B-2 fates and the difference is acquired just before or coincident with the acquisition of IL-7Rα expression.

IL-5 was originally defined as a T-cell-derived cytokine that triggers activated B cells for terminal differentiation into antibody-secreting plasma cells, at least in mice. Concurrently, IL-5 was recognized as the major maturation and differentiation factor for eosinophils in mice and humans. Over-expression of IL-5 significantly increases eosinophil numbers and antibody levels in vivo. Conversely, mice lacking a functional gene for IL-5 or the IL-5 receptor alpha chain (IL-5Ralpha) display a number of developmental and functional impairments in B-cell and eosinophil lineages. In addition to the Janus kinase-signal transducer and activator of transcription pathway, the tyrosine kinases Lyn and Btk (Bruton agammaglobulinemia tyrosine kinase) are involved, and Ras GTPase-extracellular signal-regulated kinase (Ras-ERK) signals are important for IL-5-dependent cell proliferation and survival. IL-5 critically regulates expression of genes involved in proliferation, cell survival and maturation and effector functions of B cells and eosinophils. Thus, IL-5 plays a pivotal role in innate and acquired immune responses and eosinophilia. In humans, the biologic effects of IL-5 are best characterized for eosinophils. The recent expansion in our understanding of the mechanisms of eosinophil development and activation in the context of IL-5 has led to advances in therapeutic options. A new therapy currently in clinical trials uses humanized mAbs against IL-5 or the IL-5R.

It has long been known that lymphopoiesis is transiently suppressed during pregnancy, which can be experimentally simulated by estrogen treatment. We now confirm with Rag1/GFP reporter mice that early lymphoid progenitors in the lineage marker− c-kithigh ScaI+, hematopoietic stem cell-enriched fraction of bone marrow are particularly depressed in these circumstances. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little is known regarding mechanisms. We have now identified soluble Frizzled-related protein (sFRP)1 as an estrogen-inducible gene in stromal cells, whose expression corresponded to inability to support lymphopoiesis. Bone-lining stromal cells express sFRP1, and the transcripts were elevated by pregnancy or estrogen injection. Estrogen receptor-α was essential for both lymphoid suppression and induction of the sFRP family. SFRP1 has been mainly described as an antagonist for complex Wnt signals. However, we found that sFRP1, like Wnt3a, stabilized β-catenin and blocked early lymphoid progression. Myeloerythroid progenitors were less affected by sFRP1 in culture, which was similar to estrogen with respect to lineage specificity. Hematopoietic stem cells expressed various Frizzled receptors, which markedly declined as they differentiated to lymphoid lineage. Thus, hormonal control of early lymphopoiesis in adults might partly relate to sFRP1 levels.

The earliest progenitors of lymphocytes are extremely rare and typically present among very complex populations of hematopoietic cells. Additionally, it is difficult to know how cells with any given set of characteristics are developmentally related to stem cells and maturing lymphoid precursors. However, it is now possible to divide bone marrow into progressively smaller fractions and exploit well-defined culture systems to determine which ones contain cells that can turn into lymphocytes. Analysis of steroid hormone sensitive cells and use of two-step cultures is providing additional information about the most likely differentiation pathways for B and natural killer cell lineage lymphocytes. A newly identified category of early lymphoid progenitors can now be sorted to high purity from RAG1/GFP knock in mice. Furthermore, the same experimental model makes it possible to image lymphoid progenitors in fetal and adult hematopoietic tissues.

Recent studies have demonstrated that lineage marker-negative (Lin(-)) c-kit(Lo) Flk-2/Flt3(+) IL-7R(+) Sca-1(Lo) CD27(+) Ly-6C(-) Thy-1(-)CD43(+) CD16/32(Lo/-) terminal deoxynucleotidyl transferase (TdT)(+) cells in murine bone marrow are functional lymphocyte precursors. However, it has not been clear if this is an obligate intermediate step for transit of multipotential hematopoietic stem cells to natural killer (NK) cells. We have now used serum-free, stromal cell-free cultures to determine that NK progenitors are enriched among an estrogen-regulated, c-kit(Lo) subset of the Lin(-) fraction. However, several experimental approaches suggested that this population is heterogeneous and likely represents a stage where B and NK lineages diverge. Although most B-cell precursors were directly sensitive to estrogen in culture, much of the NK-cell precursor activity in that fraction was hormone resistant. B-lineage potential was largely associated with interleukin 7 receptor alpha (IL-7R(alpha)) expression and was selectively driven in culture by IL-7. In contrast, many NK precursors did not display detectable amounts of this receptor and their maturation was selectively supported by IL-15. Finally, single-cell experiments showed that the Lin(-) c-kit(Lo) fraction contains a mixture of B/NK, B-restricted, and NK-restricted progenitors. Two-step culture experiments revealed that NK precursors become hormone resistant on or before acquisition of CD122, signaling commitment to the NK lineage. CD45R is preferentially, but not exclusively, expressed on maturing B-lineage cells. Production of these 2 blood cell types is regulated in bone marrow by common and then independent mechanisms that can now be studied with greater precision.

Sex steroids negatively regulate B lymphopoiesis in adult mice. Paradoxically, lymphocytes arise during fetal life, when estrogen levels are high and maternal lymphopoiesis is suppressed. Here we demonstrate that embryonic B lymphopoiesis was unaffected by estrogen, but sensitive to glucocorticoids. Both fetal and adult precursors contained glucocorticoid receptor transcripts, but only adult precursors expressed estrogen receptor α and β together with the androgen receptor. Fetal hematopoietic cells did not efficiently acquire functional estrogen receptors after transplantation to irradiated adult mice. Sex steroid receptors were also expressed in a stage- and developmental age-dependent fashion in human precursors. A developmental switch in responsiveness of hematopoietic cells to sex steroids may be essential for formation of the immune system.

Recently, a collection of surface markers was exploited to isolate viable Lin− TdT+ cells from murine bone marrow. These early pro-B cells were enriched for B-lineage lymphocyte precursor activity measured by short-term culture and had little responsiveness to myeloid growth factors. Early precursors can be propagated with remarkably high cloning frequencies in stromal cell–free, serum-free cultures, permitting this analysis of direct regulatory factors. Expression of the interleukin-7 receptor (IL-7Rα) chain marks functional precursors and IL-7 is necessary for progression beyond the CD45RA+ CD19− stage. Efficient survival and differentiation were only observed when stem cell factor and Flt-3 ligand were also present. IL-7–responsive CD19+precursors are estrogen resistant. However, B-lineage differentiation was selectively abrogated when highly purified Lin− precursors were treated with hormone in the absence of stromal cells. In addition, early stages of B lymphopoiesis were arrested by limitin, a new interferon (IFN)–like cytokine as well as IFN-α, IFN-γ, or transforming growth factor β (TGF-β), but not by epidermal growth factor (EGF). Lin− TdT+early pro-B cells are shown here to be CD27+AA4.1+/−Ki-67+ Ly-6C−Ly-6A/Sca-1Lo/−Thy-1−CD43+CD4+/−CD16/32Lo/−CD44Hi and similar in some respects to the “common lymphoid progenitors” (CLP) identified by others. Although early pro-B cells have lost myeloid differentiation potential, transplantation experiments described here reveal that at least some can generate T lymphocytes. Of particular importance is the demonstration that a pivotal early stage of lymphopoiesis is directly sensitive to negative regulation by hormones and cytokines.

Formation of the pre-BCR complex is a critical check point during B cell development and induces the transition of pro-B to pre-B cells. CD79b (Igbeta) is a signaling component in the pre-BCR complex, since differentiation to the pre-B phenotype is induced by cross-linking the CD79b expressed on developmentally arrested pro-B cells from recombination-activating gene (RAG)-2-deficient mice. Bruton's tyrosine kinase (BTK) plays important roles in B cell development. However, its molecular mechanisms in early B cell development are not fully understood. To examine whether BTK functions in CD79b-mediated signaling for the pro-B/pre-B transition, we utilized RAG2/BTK double-knockout (DKO) mice. Pro-B cells from RAG2/BTK-DKO mice did not differentiate into pre-B cells following CD79b cross-linking, although tyrosine phosphorylation of cellular proteins including Erk1/2 and phospholipase C-gamma2 was induced in the same manner as RAG2-KO mice. BTK is phosphorylated after cross-linking of CD79b on RAG2-deficient pro-B cells. These findings suggest that BTK-dependent pathways downstream of CD79b are critical for the pro-B/pre-B transition and BTK-independent signaling pathways are also activated via the pre-BCR complex.

We have examined phosphorylation mediated by cross-talk between growth signal pathways induced by IL-2 and IL-5. To analyze the phosphorylation process in the same cells, we established two sublines, T88-Mbeta1, which is a subline of a murine IL-5-dependent cell line, T88-M, by introduction of the human IL-2 receptor beta chain (IL-2Rbeta), and secondly CTLL-5Ralphabeta, which is a subline of a murine IL-2-dependent cell line, CTLL-2, by introduction of the murine IL-5 receptor alpha chain (IL-5Ralpha) and IL-5 receptor beta chain (IL-5Rbeta, betac) genes. Both T88-Mbeta1 and CTLL-5Ralphabeta expressed high-affinity receptors for IL-2 and IL-5, and proliferated in response to both factors. Tyrosine phosphorylation of IL-2Rbeta was induced by stimulation of T88-Mbeta1 with not only IL-2 but also IL-5. Anti-IL-2Rbeta-directed immune complexes from T88-Mbeta1 stimulated with IL-5 as well as with IL-2 contained an activated tyrosine kinase. However, stimulation with IL-5 but not IL-2 induced the tyrosine phosphorylation of IL-5Rbeta, betac, suggesting that IL-2 does not activate a tyrosine kinase which efficiently catalyzes the IL-5Rbeta molecule in response to IL-5. On the other hand, the detection of JAK1 and the other common set of phosphotyrosine-containing proteins after stimulation with either IL-5 or IL-2 suggests the existence of the same tyrosine phosphorylation pathways.

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.

It has long been known that lymphopoiesis is transiently suppressed during pregnancy, and that this can be experimentally simulated by treatment with estrogen. Indeed, sensitivity to estrogen made it possible to identify very primitive lymphoid cells in the Lin- c-kitHi Sca-1+ fraction that is enriched with hematopoietic stem cells (HSC) and multipotent progenitors of mouse bone marrow. However, analyses showing how the earliest events in lymphopoiesis is suppressed in those circumstances have not been performed. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little has been known regarding mechanisms. In the present study, we performed high resolution analysis using RAG-1/GFP reporter mice and confirmed that early lymphoid progenitors (ELP) in the Lin-c-kitHi Sca-1+ fraction are particularly depressed in pregnancy or after estrogen injection. The Lin- c-kitLo fraction containing common lymphoid progenitors (CLP)/pro-lymphocytes was very sensitive to estrogen in stroma-free serum-free culture, but earlier stages of lymphopoiesis seemed to be blocked in vivo. Thus, we focused on identifying mechanisms involving bone marrow environment. Two independent strategies with macroarrays or differential display-PCR were used to isolate stromal cell genes that were both estrogen regulated and associated with inability to support B lymphopoiesis. We have identified secreted frizzled related protein 1 (sFRP1) as an estrogen inducible gene, and its expression corresponded to inability to support lymphopoiesis. This member of the complex Wnt family has been previously described as both an agonist and an antagonist of canonical, β-catenin dependent signaling. We found that sFRP1, like recombinant Wnt3a, stimulated the canonical pathway in the Lin- c-kitHi Sca-1+ fraction, and blocked progression in the B lymphocyte lineage even when exogenous Wnt ligands were not provided. The suppressive effects of sFRP1 and Wnt3a on B lymphopoiesis were canceled out when both were present simultaneously. To better understand the vulnerability of early progenitors to the Wnt signaling, we examined the expression pattern of Frizzled receptors. Interestingly, the HSC-enriched fraction (Lin- c-kitHi Sca-1+ IL-7Rα-RAG-1/GFP-) highly expressed 7 out of 9 Frizzled receptors, which markedly declined as they differentiated to ELP (Lin- c-kitHi Sca-1+ IL-7Rα- RAG-1/GFP+) and CLP (Lin- c-kitLo Sca-1Lo/- IL-7Rα+). Myelo-erythroid progenitors were less affected by sFRP1 in culture, suggesting that it is similar to estrogen with respect to lineage specificity. Bone-lining stromal cells express sFRP1 and the transcripts were elevated in bone marrow by pregnancy or estrogen injection. In summary, these observations implicate sFRP1 as a possible mediator in hormone regulation of the earliest events in lymphopoiesis.

SummaryToll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression also extended to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen-mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.

Although eosinophils play an essential role in allergic inflammation, their role has recently been under controversy. Epidemic studies suggest that hypereosinophilia induced by parasite infection could suppress subsequent Ag sensitization, although the mechanism has not been fully clarified. In this study, we investigated whether eosinophils could suppress the Ag-specific immune response in the airway. BALB/c mice were sensitized and airway challenged with OVA. Systemic hypereosinophilia was induced by delivery of an IL-5-producing plasmid. IL-5 gene delivery suppressed the Ag-specific proliferation and cytokine production of CD4+ T cells in the spleen. IL-5 gene delivery before OVA sensitization significantly suppressed airway eosinophilia and hyperresponsiveness provoked by subsequent OVA airway challenge, while delivery during the OVA challenge did not suppress them. This IL-5-induced immune suppression was abolished in eosinophil-ablated mice, suggesting an essential role of eosinophils. IL-5 treatment increased the production of TGF-β1 in the spleen, and we demonstrated that the main cellular source of TGF-β1 production was eosinophils, using eosinophil-ablated mice and depletion study. TGF-β1, but not IL-5 itself, suppressed the Ag-specific immune response of CD4+ T cells in vitro. Furthermore, IL-5 treatment enhanced phosphorylation of Smad2 in CD4+ T cells. Finally, a TGF-β type I receptor kinase inhibitor restored this IL-5-induced immune suppression both in vitro and in vivo. These results suggest that IL-5-induced hypereosinophilia could suppress sensitization to Ag via a TGF-β-dependent mechanism, thus suppressed allergic airway inflammation. Therefore, hypereosinophilia could reveal an immunosuppressive effect in the early stage of Ag-induced immune response.