IFNα induces CCR5 in CD4+ T cells of HIV patients causing pathogenic elevation.

BACKGROUND: Among people living with HIV, elite controllers (ECs) maintain an undetectable viral load, even without receiving anti-HIV therapy. In non-EC patients, this therapy leads to marked improvement, including in immune parameters, but unlike ECs, non-EC patients still require ongoing treatment and experience co-morbidities. In-depth, comprehensive immune analyses comparing EC and treated non-EC patients may reveal subtle, consistent differences. This comparison could clarify whether elevated circulating interferon-alpha (IFNα) promotes widespread immune cell alterations and persists post-therapy, furthering understanding of why non-EC patients continue to need treatment. METHODS: Levels of IFNα in HIV-infected EC and treated non-EC patients were compared, along with blood immune cell subset distribution and phenotype, and functional capacities in some cases. In addition, we assessed mechanisms potentially associated with IFNα overload. RESULTS: Treatment of non-EC patients results in restoration of IFNα control, followed by marked improvement in distribution numbers, phenotypic profiles of blood immune cells, and functional capacity. These changes still do not lead to EC status, however, and IFNα can induce these changes in normal immune cell counterparts in vitro. Hypothesizing that persistent alterations could arise from inalterable effects of IFNα at infection onset, we verified an IFNα-related mechanism. The protein induces the HIV coreceptor CCR5, boosting HIV infection and reducing the effects of anti-HIV therapies. EC patients may avoid elevated IFNα following on infection with a lower inoculum of HIV or because of some unidentified genetic factor. CONCLUSIONS: Early control of IFNα is essential for better prognosis of HIV-infected patients.

The treatment for HIV, known as antiretroviral therapy (ART), does not cure HIV but enables individuals to live longer, healthier lives. In this study, we compared immune responses between elite controllers (ECs), who control their HIV infection without any treatment, and ART-treated and untreated patients. We demonstrate that IFNα, a small protein crucial in controlling immune system, is excessively produced at the onset of HIV infection and at levels that persist, resulting in poor HIV control without therapy. We show a mechanism for lack of control of HIV by IFNα. While inhibiting HIV, IFNα also simultaneously increases the HIV co-receptor, CCR5, thereby facilitating virus entry into the target cell. This is avoided by ECs which we hypothesize is associated with a lower infectious inoculum of HIV.

Early elevated IFNα is a key mediator of HIV pathogenesis.

BACKGROUND: A complete understanding of the different steps of HIV replication and an effective drug combination have led to modern antiretroviral regimens that block HIV replication for decades, but these therapies are not curative and must be taken for life. "Elite controllers" (ECs) is a term for the 0.5% of HIV-infected persons requiring no antiretroviral therapy, whose status may point the way toward a functional HIV cure. Defining the mechanisms of this control may be key to understanding how to replicate this functional cure in others. METHODS: In ECs and untreated non-EC patients, we compared IFNα serum concentration, distribution of immune cell subsets, and frequency of cell markers associated with immune dysfunction. We also investigated the effect of an elevated dose of IFNα on distinct subsets within dendritic cells, natural killer cells, and CD4+ and CD8 + T cells. RESULTS: Serum IFNα was undetectable in ECs, but all immune cell subsets from untreated non-EC patients were structurally and functionally impaired. We also show that the altered phenotype and function of these cell subsets in non-EC patients can be recapitulated when cells are stimulated in vitro with high-dose IFNα. CONCLUSIONS: Elevated IFNα is a key mediator of HIV pathogenesis.

Currently, HIV infection is not curable, but infected individuals can manage their condition by taking daily doses of antiretroviral therapy. Some individuals, known as elite controllers (ECs), control their infection without antiretroviral treatment, and studying how their immune system responds to HIV exposure could lead to a potential cure for others. Here, we compare immune cell responses between ECs and untreated non-ECs. We find that IFNα, a small protein with an important role in controlling white blood cell activity, is produced in excess in immune cells from non-ECs compared with ECs during early infection. This insight provides an important clue for the future development of a targeted cure for HIV.

The CEPH aging cohort and biobank: a valuable collection of biological samples from exceptionally long-lived French individuals and their offspring for longevity studies.

The increasing aging of the human population is currently and for the coming decades a major public health issue in many countries, requiring the implementation of global public health policies promoting healthy and successful aging. Individuals are not equal in the face of aging and some can present exceptional healthspan and/or lifespan, which are notably influenced by both genetic and environmental factors. Research and studies on human aging, healthy aging and longevity should rely in particular on cohorts of long-lived individuals, also including biological samples allowing studies on the biology of aging and longevity. In this manuscript, we provide for the first time a complete description of the CEPH (Centre d'Etude du Polymophisme Humain) Aging cohort, an exceptional cohort recruited during the 90s to 2000s, including more than 1700 French long-lived individuals (≥ 90 years old) born between 1875 and 1916 as well as for some of them their siblings and offspring. Among the participants, 1265 were centenarians, including 255 semi-supercentenarians ([105-110] years old) and 25 supercentenarians (≥ 110 years old). The available anthropometric, epidemiologic and clinical data for the cohort participants are described and especially the collection of blood-derived biological samples associated with the cohort which includes DNA, cryopreserved cells and cell lines, plasma, and serum. This biological collection from the first cohort of centenarians in the world is an inestimable resource for ongoing and future molecular, cellular, and functional studies aimed at deciphering the mechanisms of human (successful) aging and longevity.

IFNα induces CCR5 in CD4+ T-cells, causing its anti- HIV inefficiency and its subsequent pathogenic elevation, partially controlled by anti-HIV therapy.

Like EC, we find that ART-treated patients control serum IFNα concentration and show few immune cell alterations enabling a healthy but fragile medical status. However, treatment interruption leads to elevated IFNα reflecting virus production indicating that like EC, ART does not achieve a virological cure. The immune system becomes overwhelmed by multiple immune cell abnormalities as found in untreated patients. These are chiefly mediated by elevated IFNα inducing signaling checkpoints abnormalities, including PD1, in cytotoxic immune cells. Importantly, during acute infection, elevated IFNα correlated with HIV load and we found that IFNα enhances CCR5, the HIV coreceptor in CD4+ T-cells, impairing its anti-viral response and accounting for the pathogenic vicious cycle: HIV → IFNα ↗ → infected CD4+ T-cells ↗ →HIV ↗. This study opens immunotherapeutic perspectives showing the need to control IFNα in order to convert ART patients into EC.

Early Elevated IFNα Identified as the Key Mediator of HIV Pathogenesis and its low level a Hallmark of Elite Controllers.

Advances in HIV therapy came from understanding its replication. Further progress toward "functional cure" -no therapy needed as found in Elite Controllers (EC)- may come from insights in pathogenesis and avoidance by EC. Here we show that all immune cells from HIV-infected persons are impaired in non-EC, but not in EC. Since HIV infects few cell types, these results suggest an additional mediator of pathogenesis. We identify that mediator as elevated pathogenic IFNα, controlled by EC likely by their preserved potent NK-cells and later by other killer cells. Since the earliest days of infection predict outcome genetic or chance events must be key to EC, and since we found no unique immune parameter at the onset, we suggest a chance infection with a lower HIV inoculum. These results offer an additional approach toward functional cure: a judicious targeting of IFNα for all non-EC patients.

Cytotoxic activity of peripheral blood mononuclear cells in patients with endometriosis: A cross-sectional study.

Background: Endometriosis is believed to be associated with dysfunction of the lymphocyte population and cytotoxicity of natural killer (NK) cells, induced by the production of interleukin-2 (IL-2). Objective: This study aimed to investigate T lymphocytes and NK cell activity in the peripheral blood mononuclear cells (PBMCs) of women with endometriosis. Materials and Methods: PBMCs were obtained from the peripheral venous blood samples of 14 women with and without endometriosis (n = 7 for each group). Then, the PBMCs were co-cultured for 4 days and were treated with recombinant IL-2 for cytotoxic activity toward target cells (Daudi and K562 cells). The cytotoxicity activity was determined using the 51 chromium release assay before and after stimulation. Flow cytometry measurement was used to examine the expression of T lymphocytes and NK cells before and after being treated with IL-2. Results: The concentration of CD3+CD28+ (co-stimulatory) was significantly lower in the endometriosis group (65.62 ± 5.38) compared to in its counterpart (50.24 ± 4.22) (p = 0.04) before stimulation. However, no significant differences were observed in any other T lymphocytes and NK cells. It was also found that there was a significant increase of CD3-CD28+ after treatment with IL-2 only in the healthy control but not in women with endometriosis. Conclusion: Increased expression of CD160 and decreased CD28 play a role in inhibiting NK cell activation and T cell response in women with endometriosis.

Microenvironment tailors nTreg structure and function.

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFß, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts.

Membrane expression of NK receptors CD160 and CD158k contributes to delineate a unique CD4+ T-lymphocyte subset in normal and mycosis fungoides skin.

CD160 is a GPI-anchored Ig-like receptor identified by the BY55 mAb on human circulating CD56dim+ NK cells and TCRγδ lymphocytes. In addition, while most intestinal T lymphocytes express it, only a minor circulating CD4+ or CD8+ T lymphocyte subset is CD160+. Here we describe a population of CD4+ CD160+ human blood T lymphocytes of circulating cutaneous T cells. These rare T lymphocytes represent 2.1 ± 1.9% of the circulating CD3+ CD4+ T cells, coexpress CD8αα, CD244, and perforin but lack CD28 expression, a phenotype corresponding to effector memory cytotoxic T-lymphocytes. Functional studies further confirmed their cytotoxic potential. These cells lack αEß7 integrin and CCR7 expression but do express skin-addressing molecules CLA, and CCR4. In normal human skin, CD4+ CD160+ cells represent 34.6 ± 14.7% of the CD4+ T lymphocytes extracted by collagenase treatment. These T cells coexpress CLA (81 ± 13.6%), CCR4 (62.3 ± 15.9%), and some CD8αα (19.6 ± 13%) or CCR7 (24.4 ± 11.7%) expression. Cutaneous T-cell lymphoma cells express the natural killer receptor KIR3DL2 (CD158k) used as a tumor marker. Not only we confirmed the expression of this marker in the blood and/or skin of mycosis fungoides patients but we also show for the first time CD158k expression (often associated with CD160) on cutaneous CD4+ T cells from healthy individuals (25.3 ± 15%). Therefore, CD4+ CD160+ T cells expressing CD158k might represent specialized cutaneous lymphocytes devoted to immune surveillance, from which could originate cutaneous T-cell lymphomas such as mycosis fungoides.

Engagement of IL-1 receptor accessory protein (IL-1RAcP) with the monoclonal antibody AY19 provides co-activating signals and prolongs the CD2-induced proliferation of peripheral blood lymphocytes.

IL-1 receptor accessory protein (IL-1RAcP) is the second subunit required to form a functional receptor complex for IL-1α and ß, IL-1F6, IL-1F8, IL1-F9 and IL-33. While it does not directly interact with the cytokines, IL-1RAcP is necessary to mediate signal transduction. We previously reported a monoclonal antibody with an unknown specificity, termed AY19, that was capable to induce a significant increase in the size of CFU-GM colonies when added to cultures of human cord blood CD34(+) hematopoietic progenitors. Here we demonstrate that AY19 mAb recognizes IL1-RAcP. We show that this adaptor molecule is significantly present on peripheral blood monocytes and lymphocytes including CD4(+) and CD8(+) T lymphocytes, B and NK cells. Interestingly, its expression is found increased on CD127(low)CD4(+)CD25(high) T cells when compared to CD127(low)CD4(+)CD25(-) T cell subset, suggesting that the level of IL-1RAcP membrane expression could allow to distinguish within CD127(low)CD4(+) T lymphocytes the CD25(high) T regulatory subset from conventional CD25(-) T lymphocytes. Functional studies reveal that addition of AY19 mAb enhances the proliferation of peripheral blood mononuclear cells (PBMC) obtained with mitogenic concentrations of PMA. Interestingly, we found that although AY19 mAb does not increase the optimal PBMC proliferation induced by a mitogenic pair of anti-CD2 mAbs it prolongs their time of proliferation. Thus, these results indicate that the anti-IL-1RAcP mAb AY19 exhibits unique functional properties by triggering co-stimulatory signals in lymphocytes.

Identification of a novel CD160+ CD4+ T-lymphocyte subset in the skin: a possible role for CD160 in skin inflammation.

CD160 is a glycosylphosphatidylinositol-anchored cell surface molecule expressed by human circulating cytotoxic lymphocytes that correspond to the majority of natural killer cell (NK) expressing CD56(dim), TCRgammadelta lymphocytes, and to a minor CD8 T-cell subset. CD160 engagement by major histocompatibility complex class I molecules triggers by itself both cytotoxic function and cytokine production in NK lymphocytes, whereas it provides co-activating signals to TCR-induced proliferation in T CD8+ lymphocytes. In this study, we analyzed by immunohistochemistry the phenotype of lymphocytes infiltrating normal skin and inflammatory skin lesions of atopic dermatitis, contact dermatitis, and psoriasis. We identified a minor original subset of CD4+ CD160+ T cells infiltrating inflammatory lesions. We found that this lymphocyte subset localization is not restricted to the skin, as we demonstrated that CD160 transcripts could be induced in IL-2 or IL-15-activated CD4+ peripheral blood lymphocytes. Finally, we report that CD160 acts as a co-activator receptor for CD3-induced proliferation of CD4+ CD160+ T cells isolated from inflammatory skin lesions. Thus, we hypothesize that the unique CD4+ CD160+ lymphocyte subset plays a role in the pathogenesis of skin inflammation.

The co-expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160-mediated cytolytic effector function.

Within human CD8+ T lymphocytes, the CD27-CD45RAhigh or CD56+ phenotypes contribute to precisely define the cells with CTL effector function. Novel markers were demonstrated to correlate with CTL properties, such as the 2B4 (CD244) receptor, a member of the CD2 subset of the immunoglobulin superfamily or the glycosylphosphatidylinositol-anchored CD160 receptor. We performed a study of these markers to further define the population of effectors with CTL functions. Here we show that cytotoxic subpopulations defined by surface markers CD160, CD56 and CD57 are mostly contained in the 2B4+CD8+ T cell population. Expression of CD160 identifies two populations in the 2B4+ population. The 2B4+CD160+ subset expresses a bona fide CTL phenotype. The co-expression of 2B4 and CD160 defines T cells containing high amounts of perforin and granzyme B. During CTL ontogeny, an up-regulation of 2B4 and CD160 is observed from a naive to a terminally differentiated phenotype. Finally, we demonstrated that CD160 triggering failed to induce cytotoxicity per se, but costimulated CD3-redirected killing. We conclude that the co-expression of 2B4 and CD160 defines a CD8+ T lymphocyte subpopulation with high CTL activity.

Significance of circulating T-cell clones in Sezary syndrome.

Identification of malignant Sézary cells by T-cell receptor (TCR) clonality studies is routinely used for the diagnosis of Sézary syndrome, but T-cell clones expressed in a single patient have never been accurately characterized. We previously reported that CD158k expression delineates Sézary syndrome malignant cells, and, more recently, we identified vimentin at the surface membranes of Sézary cells and normal activated lymphocytes. In the present study, T-cell clones from 13 patients with Sézary syndrome were identified by immunoscopy and further characterized in the blood according to their TCR Vbeta, CD158k, and vimentin cell-surface expression. We found in most patients a unique malignant T-cell clone that coexpressed CD158k and vimentin and that, when patients were tested, was also present in the skin. However, in some patients we detected the presence of a nonmalignant circulating clone expressing high amounts of vimentin and lacking CD158k. These results indicate that clonal expansion may originate from circulating malignant and nonmalignant CD4(+) T cell populations in patients with Sézary syndrome. Identification of the malignant cells in Sézary syndrome cannot be achieved by T-cell clonality studies or by TCR Vbeta monoclonal antibody (mAb) analysis alone; it also relies on CD158k phenotyping.

Circulating natural killer lymphocytes are potential cytotoxic effectors against autologous malignant cells in sezary syndrome patients.

Patients with advanced cutaneous T cell lymphoma (CTCL) exhibit profound defects in cell-mediated immunity. Although it has been suggested that Sezary syndrome (SS) patients have a decreased natural killer (NK) lymphocyte activity, nothing has been reported concerning the sensitivity of Sezary cells to NK lymphocyte-mediated cytotoxicity. Peripheral blood NK cells from healthy donors were tested against Sezary tumoral cell lines as well as against freshly isolated Sezary cells. Further, we studied their ability to exhibit antibody -dependent cell-mediated cytotoxicity using either the murine anti-CD158k/KIR3DL2 monoclonal antibody (moAb) AZ158 that specifically recognizes Sezary cells, or the anti-CD52 monoclonal antibody alemtuzumab. The results show that Sezary cell lines are susceptible to NK lymphocyte lysis. More importantly, we found that freshly isolated malignant cells are killed either by IL-2 activated allogeneic NK lymphocytes or when the tumor lymphocyte targets are incubated with an anti-MHC class I F(ab)'2 antibody. Further, anti-KIR3DL2 and anti-CD52 moAb can enhance the NK lysis. Finally, we report that NK lymphocytes isolated from SS patients are potentially cytotoxic lymphocytes against autologous malignant Sezary cells. These findings indicate that antitumor-mediated NK lymphocyte cytotoxic activity can be triggered in patients with CTCL and raise the possibility of developing novel therapeutic strategies by stimulating their innate immunity.

Soluble CD100 functions on human monocytes and immature dendritic cells require plexin C1 and plexin B1, respectively.

CD100 represents the first semaphorin described in the immune system. It is expressed as a 300-kDa homodimer at the surface of most hematopoietic cells, but is also found in a soluble form following a proteolytic cleavage upon cell activation. We herein established that soluble CD100 (sCD100) impaired the migration of human monocytes and immature dendritic cells (DCs), but not of mature DCs. Performing competition assays, we identified plexin C1 (VESPR/CD232) as being involved in sCD100-mediated effects on human monocytes. Interestingly, we observed a complete down-regulation of plexin C1 expression during the in vitro differentiation process of monocytes to immature DCs, while concomitantly the surface expression of plexin B1 was induced. The latter receptor then binds sCD100 on immature DCs, mediating its inhibitory effect on cell migration. Finally, we showed that sCD100 modulated the cytokine production from monocytes and immature DCs. Together these results suggest that sCD100 plays a critical role in the regulation of antigen-presenting cell migration and functions via a tightly regulated process of receptor expression.

Cutting edge: engagement of CD160 by its HLA-C physiological ligand triggers a unique cytokine profile secretion in the cytotoxic peripheral blood NK cell subset.

CD160 is an Ig-like activating NK cell receptor expressed on the majority of circulating NK cells. This population corresponds to the nonproliferating, highly cytolytic, CD56dimCD16+ subset. CD160 engagement by HLA-C molecules mediates cytotoxic function. In this study, we report that upon specific activation by the physiological ligand HLA-C, or Ab cross-linking, CD160+ peripheral blood NK cells produce IFN-gamma, TNF-alpha, and IL-6. This unique CD160-mediated cytokine production differs from the one observed after CD16 engagement whose expression is also restricted to the CD56dim cytotoxic NK cell subset. As already reported for the CD160-mediated cytotoxic effector function, CD160-mediated cytokine production by peripheral blood-NK cells is negatively controlled by the killer Ig-like receptor CD158b. Thus, the CD160 receptor represents a unique triggering surface molecule expressed by cytotoxic NK cells that participates in the inflammatory response and determines the type of subsequent specific immunity.

CD158k/KIR3DL2 is a new phenotypic marker of Sezary cells: relevance for the diagnosis and follow-up of Sezary syndrome.

CD158k molecules belong to the family of killer cell immunoglobulin-like receptors (KIR) that are expressed on a minor population of circulating NK and CD8+ T lymphocytes. Here, we report a strong positive correlation between the percentage of CD158k+ blood lymphocytes analyzed by flow cytometry and the percentage of atypical circulating cells (Sezary cells) determined by cytomorphology in a large group of patients with Sezary syndrome. Moreover, we show that circulating CD4+CD158k+ lymphocytes correspond to the malignant clonal cell population. Our findings suggest that the CD158k marker could be a useful tool for the evaluation of the circulating tumoral burden and the follow-up of patients with Sezary syndrome.

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (alphaIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41(-) cells from embryoid bodies converted to CD41(+) hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (beta3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41(+), whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34(+). Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.

Requirement for mitogen-activated protein kinase activation in the response of embryonic stem cell-derived hematopoietic cells to thrombopoietin in vitro.

Enforced expression of c-mpl in embryonic stem (ES) cells inactivated for this gene results in protein expression in all the ES cell progeny, producing cells that do not belong to the megakaryocytic lineage and are responsive to PEG-rhuMGDF, a truncated form of human thrombopoietin (TPO) conjugated to polyethylene glycol. These include a primitive cell called BL-CFC, thought to represent the equivalent of the hemangioblast, and all myeloid progenitor cells. In this model, PEG-rhuMGDF was able to potentiate the stimulating effects of other growth factors, including vascular endothelial growth factor, on BL-CFC and a combination of cytokines on the growth of granulocyte macrophage-colony-forming units. The importance of the C-terminal domain of Mpl and of mitogen-activated protein kinase (MAPK) activation in TPO-dependent megakaryocytic differentiation has been well studied in vitro. Here, the role of this domain and the involvement of MAPK in upstream and nonmegakaryocytic cells are examined by using 2 truncated mutants of Mpl (Delta34, deletion of residues 71 to 121 in the C-terminal domain; and Delta3, deletion of residues 71-94) and specific inhibitors of the MAPK pathway. The 2 deleted regions support different functions, mediated by different signals. Residues 71 to 121 were required for PEG-rhuMGDF-dependent growth of BL-CFC, for megakaryocytic and other myeloid progenitors, and for megakaryocyte polyploidization. These responses were mediated by the ERK1-ERK2 MAPK pathway. In contrast, the only function of the sequence comprising residues 71 to 94 was to mediate the synergistic effects of PEG-rhuMGDF with other hematopoietic growth factors. This function is not mediated by MAPK activation.