Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Multipotent adult progenitor cells induce regulatory T cells and promote their suppressive phenotype via TGFß and monocyte-dependent mechanisms.

Dysregulation of the immune system can initiate chronic inflammatory responses that exacerbate disease pathology. Multipotent adult progenitor cells (MAPC cells), an adult adherent bone-marrow derived stromal cell, have been observed to promote the resolution of uncontrolled inflammatory responses in a variety of clinical conditions including acute ischemic stroke, acute myocardial infarction (AMI), graft vs host disease (GvHD), and acute respiratory distress syndrome (ARDS). One of the proposed mechanisms by which MAPC cells modulate immune responses is via the induction of regulatory T cells (Tregs), however, the mechanism(s) involved remains to be fully elucidated. Herein, we demonstrate that, in an in vitro setting, MAPC cells increase Treg frequencies by promoting Treg proliferation and CD4+ T cell differentiation into Tregs. Moreover, MAPC cell-induced Tregs (miTregs) have a more suppressive phenotype characterized by increased expression of CTLA-4, HLA-DR, and PD-L1 and T cell suppression capacity. MAPC cells also promoted Treg activation by inducing CD45RA+ CD45RO+ transitional Tregs. Additionally, we identify transforming growth factor beta (TGFß) as an essential factor for Treg induction secreted by MAPC cells. Furthermore, inhibition of indoleamine 2, 3-dioxygenase (IDO) resulted in decreased Treg induction by MAPC cells demonstrating IDO involvement. Our studies also show that CD14+ monocytes play a critical role in Treg induction by MAPC cells. Our study describes MAPC cell dependent Treg phenotypic changes and provides evidence of potential mechanisms by which MAPC cells promote Treg differentiation.

Graft-versus-host disease depletes plasmacytoid dendritic cell progenitors to impair tolerance induction.

Graft-versus-host disease (GVHD) causes failed reconstitution of donor plasmacytoid dendritic cells (pDCs) that are critical for immune protection and tolerance. We used both murine and human systems to uncover the mechanisms whereby GVHD induces donor pDC defects. GVHD depleted Flt3-expressing donor multipotent progenitors (MPPs) that sustained pDCs, leading to impaired generation of pDCs. MPP loss was associated with decreased amounts of MPP-producing hematopoietic stem cells (HSCs) and oxidative stress-induced death of proliferating MPPs. Additionally, alloreactive T cells produced GM-CSF to inhibit MPP expression of Tcf4, the transcription factor essential for pDC development, subverting MPP production of pDCs. GM-CSF did not affect the maturation of pDC precursors. Notably, enhanced recovery of donor pDCs upon adoptive transfer early after allogeneic HSC transplantation repressed GVHD and restored the de novo generation of donor pDCs in recipient mice. pDCs suppressed the proliferation and expansion of activated autologous T cells via a type I IFN signaling-dependent mechanism. They also produced PD-L1 and LILRB4 to inhibit T cell production of IFN-γ. We thus demonstrate that GVHD impairs the reconstitution of tolerogenic donor pDCs by depleting DC progenitors rather than by preventing pDC maturation. MPPs are an important target to effectively bolster pDC reconstitution for controlling GVHD.

Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis.

Achieving immunoregulation via in vivo expansion of Foxp3+ regulatory CD4+ T cells (Treg) remains challenging. We have shown that mobilization confers to multipotent hematopoietic progenitors (MPPs) the capacity to enhance Treg proliferation. Transcriptomic analysis of Tregs co-cultured with MPPs revealed enhanced expression of genes stabilizing the suppressive function of Tregs as well as the activation of IL-1ß-driven pathways. Adoptive transfer of only 25,000 MPPs effectively reduced the development of experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for multiple sclerosis (MS). Production of the pathogenic cytokines IL-17 and GM-CSF by spinal cord-derived CD4+ T-cells in MPP-protected recipients was reduced while Treg expansion was enhanced. Treg depletion once protection by MPPs was established, triggered disease relapse to the same level as in EAE mice without MPP injection. The key role of IL-1ß was further confirmed in vivo by the lack of protection against EAE in recipients of IL-1ß-deficient MPPs. Mobilized MPPs may thus be worth considering for cell therapy of MS either per se or for enrichment of HSC grafts in autologous bone marrow transplantation already implemented in patients with severe refractory multiple sclerosis.

Mobilized Multipotent Hematopoietic Progenitors Promote Expansion and Survival of Allogeneic Tregs and Protect Against Graft Versus Host Disease.

Allogeneic Hematopoietic Stem Cell Transplantation (Allo-HSCT) is routinely performed with peripheral blood stem cells (PBSCs) mobilized by injection of G-CSF, a growth factor which not only modulates normal hematopoiesis but also induces diverse immature regulatory cells. Based on our previous evidence that G-CSF-mobilized multipotent hematopoietic progenitors (MPP) can increase survival and proliferation of natural regulatory T cells (Tregs) in autoimmune disorders, we addressed the question how these cells come into play in mice and humans in an alloimmune setting. Using a C57BL/6 mouse model, we demonstrate that mobilized MPP enhance the immunosuppressant effect exerted by Tregs, against alloreactive T lymphocytes, both in vitro and in vivo. They do so by migrating to sites of allopriming, interacting with donor Tregs and increasing their numbers, thus reducing the lethality of graft-versus-host disease (GVHD). Protection correlates likewise with increased allospecific Treg counts. Furthermore, we provide evidence for a phenotypically similar MPP population in humans, where it shares the capacity to promote selective Treg expansion in vitro. We postulate that G-CSF-mobilized MPPs might become a valuable cellular therapy to expand donor Tregs in vivo and prevent GVHD, thereby making allo-HSCT safer for the treatment of leukemia patients.

Potential of breastmilk in stem cell research.

Breastmilk is a dynamic, multi-faceted, and complex fluid containing a plethora of biochemical and cellular components that execute developmental effects or differentiation program, providing nourishment and immunity to newborns. Recently, it was reported that breastmilk contains a heterogeneous population of naïve cells, including pluripotent stem cells, multipotent stem cells, immune cells, and non-immune cells. The stem cells derived from breastmilk possess immune privilege and non-tumorigenic properties. Thus, breastmilk may represent an ideal source of stem cells collected by non-perceive procedure than other available sources. Thus, this "maternally originating natural regenerative medicine" may have innumerable applications in clinical biology, cosmetics, and pharmacokinetics. This review describes the efficient integrated cellular system of mammary glands, the impressive stem cell hierarchy of breastmilk, and their possible implications in translational research and therapeutics.

Effect of Activated Immunocompetent Cells on the Number of Multipotent Stromal Cells in Bone Marrow Transplants of CBA and CBA/N Mice in a Short Time after Polyvinylpyrrolidone Administration to Animals.

One hour after polyvinylpyrrolidone administration, the content of multipotent stromal cells in the spleen of CBA and CBA/N mice increased almost equally (by 2.5 and 2.9 times, respectively), but in 24 h, the effectiveness of multipotent stromal cell cloning in the spleen of CBA/N mice decreased almost to the control level, whereas in CBA mice, the number of multipotent stromal cells continued to increase. Serum concentration of IL-5, TNFα, and IL-2 in both lines was elevated in 1 h after polyvinylpyrrolidone administration, which is likely to reflect activation of the innate immunity. One day after polyvinylpyrrolidone administration, the number of multipotent stromal cells in bone marrow transplants in the CBA/N→CBA/N and CBA→CBA/N groups remained practically unchanged, while in groups CBA→CBA and CBA/N→CBA it was equally increased (by 3.6 and 3.4 times, respectively). Thus, the number of multipotent stromal cells in bone marrow transplants after 1 day was increased only in groups where recipients (CBA mice) were capable of responding to polyvinylpyrrolidone administration, i.e. the number of stromal cells by this term, was apparently determined by the presence of activated immunocompetent cells. These findings also indicate that activation of the stromal tissue dur ing immune response can have a two-phasic pattern: the first phase (1 h after antigen adminis tration) can be determined by activation of innate immunity receptors (in multipotent stromal cells or other cells) observed in CBA and CBA/N mice, and the second phase occurs during further development of the immune response (that was observed in CBA mice, but not in CBA/N mice due to absence of CD+B-1a lymphocytes). The findings attest to close interactions between the stromal tissue and the immune system.

Accumulation of Multipotent Hematopoietic Progenitors in Peripheral Lymphoid Organs of Mice Over-expressing Interleukin-7 and Flt3-Ligand.

Interleukin-7 (IL-7) and Flt3-ligand (FL) are two cytokines important for the generation of B cells, as manifested by the impaired B cell development in mice deficient for either cytokine or their respective receptors and by the complete block in B cell differentiation in the absence of both cytokines. IL-7 is an important survival and proliferation factor for B cell progenitors, whereas FL acts on several early developmental stages, prior to B cell commitment. We have generated mice constitutively over-expressing both IL-7 and FL. These double transgenic mice develop splenomegaly and lymphadenopathy characterized by tremendously enlarged lymph nodes even in young animals. Lymphoid, myeloid and dendritic cell numbers are increased compared to mice over-expressing either of the two cytokines alone and the effect on their expansion is synergistic, rather than additive. B cell progenitors, early progenitors with myeloid and lymphoid potential (EPLM), common lymphoid progenitors (CLP) and lineage-, Sca1+, kit+ (LSK) cells are all increased not only in the bone marrow but also in peripheral blood, spleen and even lymph nodes. When transplanted into irradiated wild-type mice, lymph node cells show long-term multilineage reconstitution, further confirming the presence of functional hematopoietic progenitors therein. Our double transgenic mouse model shows that sustained and combined over-expression of IL-7 and FL leads to a massive expansion of most bone marrow hematopoietic progenitors and to their associated presence in peripheral lymphoid organs where they reside and potentially differentiate further, thus leading to the synergistic increase in mature lymphoid and myeloid cell numbers. The present study provides further in vivo evidence for the concerted action of IL-7 and FL on lymphopoiesis and suggests that extramedullary niches, including those in lymph nodes, can support the survival and maintenance of hematopoietic progenitors that under physiological conditions develop exclusively in the bone marrow.

The role of monocytosis and neutrophilia in atherosclerosis.

Monocytosis and neutrophilia are frequent events in atherosclerosis. These phenomena arise from the increased proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) and HSPC mobilization from the bone marrow to other immune organs and circulation. High cholesterol and inflammatory signals promote HSPC proliferation and preferential differentiation to the myeloid precursors (i.e., myelopoiesis) that than give rise to pro-inflammatory immune cells. These cells accumulate in the plaques thereby enhancing vascular inflammation and contributing to further lesion progression. Studies in animal models of atherosclerosis showed that manipulation with HSPC proliferation and differentiation through the activation of LXR-dependent mechanisms and restoration of cholesterol efflux may have a significant therapeutic potential.

Pretreatment with Antiviral Preparation Kagocel Normalizes the Content of Bone Marrow Multipotent Stromal Cells and TNFα in Blood Serum of CBA Mice Disturbed by Administration of S. typhimurium Antigen Complex In Vivo and Maintains High Concentration of IL-10 and Th1 Cytokines.

Pretreatment with the active substance of antiviral preparation Kagocel, inductor of type I endogenous IFN, in a daily therapeutic dose (30 µg/mouse) 3 h prior to administration of S. typhimurium antigens to CBA mice reduced the number of bone marrow multipotent stromal cell (significantly increased by 3.2 times on the next day after antigen injection) to the initial level. Thus, activation of the stromal tissue induced by administration of the bacterial antigen was blocked. In addition, preliminary administration of Kagocel modulated the cytokine profile of the blood serum affected by S. typhimurium antigens: reduced 1.6-fold elevated concentration a proinflammatory cytokine TNFα to the control level (in 4 h after antigen injection) and maintained this level in 20 h after antigen administration. Kagocel also maintained the concentration of anti-inflammatory cytokine IL-10 at the level surpassing the normal by 1.6 times and high concentrations of Th1 cytokines (IL-2, IFNγ, and IL-12). These results suggest that Kagocel can reduce the immune response to bacterial antigens (similar to type I IFN [7]), which can contribute to its therapeutic and preventive effects in addition to its well documented antiviral activity and then this preparation can be used for the therapy of diseases accompanied by excessive or chronic inflammation.

Multipotent mesenchymal stem cells in lung fibrosis.

RATIONALE: Stem cells have been identified in the human lung; however, their role in lung disease is not clear. We aimed to isolate mesenchymal stem cells (MSC) from human lung tissue and to study their in vitro properties. METHODS: MSC were cultured from lung tissue obtained from patients with fibrotic lung diseases (n = 17), from emphysema (n = 12), and normal lungs (n = 3). Immunofluorescence stainings were used to characterize MSC. The effect of MSC-conditioned media (MSC-CM) on fibroblast proliferation and on lung epithelial wound repair was studied. RESULTS: Expression of CD44, CD90, and CD105 characterized the cells as MSC. Moreover, the cells stained positive for the pluripotency markers Oct3/4 and Nanog. Positive co-stainings of chemokine receptor type 4 (CXCR4) with CD44, CD90 or CD105 indicated the cells are of bone marrow origin. MSC-CM significantly inhibited the proliferation of lung fibroblasts by 29% (p = 0.0001). Lung epithelial repair was markedly increased in the presence of MSC-CM (+ 32%). Significantly more MSC were obtained from fibrotic lungs than from emphysema or control lungs. CONCLUSIONS: Our study demonstrates enhanced numbers of MSC in fibrotic lung tissue as compared to emphysema and normal lung. The cells inhibit the proliferation of fibroblasts and enhance epithelial repair in vitro. Further in vivo studies are needed to elucidate their potential role in the treatment of lung fibrosis.

NLR2 and TLR3, TLR4, TLR5 Ligands, Injected In Vivo, Improve after 1 h the Efficiency of Cloning and Proliferative Activity of Bone Marrow Multipotent Stromal Cells and Reduce the Content of Osteogenic Multipotent Stromal Cells in CBA Mice.

Ligands NLR2 (muramyldipeptide) and TLR (bacterial LPS, flagellin, CpG-dinucleotide, and Poly I:C) and S. typhimurium antigenic complex by 1.5-3-fold increase the efficiency of cloning and content of multipotent stromal cells (MSC) in the bone marrow of CBA mice as soon as 1 h postinjection. The counts of large colonies (150-500 cells) increased by 2.5-3.3 times in comparison with intact bone marrow cultures at the expense of a decrease in the number of smaller colonies, which attests to enhanced proliferation of stromal cells in the colonies. The efficiency of cloning and hence, MSC content in the femoral bone decreased by 1.2-1.9 times after 3 h and increased again after 24 h to the level 1.3-1.5 times higher than the level 1 h postinjection (LPS, Poly I:C, and S. typhimurium antigenic complex). The dynamics of bone marrow MSC cloning efficiency after 1-3 h corresponded to the dynamics of serum cytokine concentrations during the same period. However, the levels of serum cytokines after 24 h in general were similar to those in intact mice or were lower. The concentrations of osteogenic multipotent stromal cells in the bone marrow decreased 2-3-fold after 3 h and thus persisted by 24 h postinjection. Twofold (at 24-h interval) and a single injection of S. typhimurium antigenic complex to mice led to a significant increase of cloning efficiency, observed as early as just 1 h postinjection (1.9 and 1.5 times, respectively). The same picture was observed for serum cytokines. On the whole, injections of TLR and NLR ligands and of S. typhimurium antigenic complex led to stromal tissue activation within 1 h postinjection, this activation consisting in a significant increase of the efficiency of cloning and of MSC count in the bone marrow, and also in an increase in their proliferative activity and a decrease (after 3 h) of osteogenic MSC concentration.

Counts of MSC in the Bone Marrow of Young and Old CBA Mice after a Single Exposure to Osteogenic Stimuli (Curettage, BMP-2 Injection) or Antigens (S. typhimurium Antigenic Complex) and in Heterotopic Bone Marrow Transplants.

The efficiency of cloning and the content of multipotent stromal cells (MSC) in the femoral bone marrow of intact CBA mice was 1.5 times less in old mice (24-36 months) than in young ones (2-3 months). The concentration of osteogenic MSC was higher in old vs. young mice (42±3 vs. 22±2%, respectively). Changes in the total counts of MSC and concentrations of osteogenic MSC in response to osteogenic (curettage, BMP-2) and immunogenic stimuli (S. typhimurium antigenic complex) were similar in young and old mice in comparison with intact controls of respective age. The counts of the total pool of bone marrow MSC and pool of osteogenic MSC in response to osteogenic stimuli were 1.5-2 times less in old vs. young mice. This difference seemed to be a result of age-specific decrease of their bone marrow count but not of age-specific decrease of the MSC functional activity, this leading to a decrease in the transplantability of bone marrow stromal tissue of old mice. Comparison of transplantations "old donor - young recipient" vs. "young donor - young recipient" demonstrated a decrease in the count of nuclear cells (1.8 times), size of bone capsule (2-fold), efficiency of MSC cloning (1.6 times), count of MSC per transplant (2.9 times), and count of osteogenic MSC per transplant (3.3 times). The concentrations of osteogenic MSC in transplants from young and old donors leveled in young recipients, that is, seemed to be regulated by the host. Serum concentrations of IL-10 and TNF-α in intact old mice were at least 2.9 and 2 times higher than in young animals, while the concentrations of almost all the rest studied cytokines (IL-2, IL-5, GM-CSF, IFN-γ, IL-4, IL-12) were lower. Presumably, the decrease in the content of bone marrow MSC and in transplantability of bone marrow stromal tissue in old mice were caused by exhaustion of the MSC pool as a result of age-specific chronic inflammation. These data indicated a close relationship between age-specific changes in the stromal tissue and immune system.

Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8+ T Cell Terminal Differentiation and Loss of Multipotency.

Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8+ T cells during viral infection revealed increased H3K27me3 deposition at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8+ memory T cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro-memory genes in effector T cells.

Development of a Functional Biomarker for Use in Cell-Based Therapy Studies in Seropositive Rheumatoid Arthritis.

UNLABELLED: Cell-based therapy has potential therapeutic value in autoimmune diseases such as rheumatoid arthritis (RA). In RA, reduction of disease activity has been associated with improvement in the function of regulatory T cells (Treg) and attenuated responses of proinflammatory effector T cells (Teff). Mesenchymal stem cells (MSCs) and related multipotent adult progenitor cells (MAPC) have strong anti-inflammatory and immunomodulatory properties and may be able to "reset" the immune system to a pre-RA state. MAPC are MSC-like cells that are slightly earlier in lineage, have greater expansion capacity, and can be used as "off-the-shelf" therapy. Assessment of cell-based therapy to treat arthritis and related diseases is limited by the lack of available biological correlates that can be measured early on and indicate treatment response. We set out to develop a functional measure that could be used ex vivo as a biomarker of response. We were able to demonstrate that MAPC products could inhibit Teff responses from patients with active RA and that Treg from RA patients suppressed Teff. This assay used ex vivo can be used with MAPC or Treg alone or in combination and reflects the overall level of Teff suppression. Use of a novel functional biomarker as an exploratory endpoint in trials of cell-based therapy should be of value to detect biological outcomes at a point prior to the time that clinical response might be observed. SIGNIFICANCE: Therapy with mesenchymal stem cells and related multipotent adult progenitor cells is immune modifying in a variety of diseases. There is interest in using cell-based therapy in rheumatoid arthritis (RA) to induce tolerance and "reset" the immune system to its pre-RA state. In a clinical trial, it should be known as soon as possible if there is a chance of response. A biomarker has been developed that permits measurement of the effects of cell-based therapy on effector T cell function.

Immunophenotype of mouse cerebral hemispheres-derived neural precursor cells.

Postnatally isolated neural precursor cells (piNPCs) from mouse cerebral tissue have been studied in cell-based therapeutic approaches for Experimental Autoimmune Encephalomyelitis (EAE). Transplantation experiments in EAE rodents revealed that piNPCs manage to integrate into the host tissue and ameliorate clinical symptoms. When cultured in vitro, mouse cerebral piNPCs form neurospheres consisting of immature cells positive for polysialylated neural adhesion molecule (PSA-NCAM) that differentiate mainly towards glial cells, but also neurons. Herein, we have characterized piNPCs immunophenotype, with flow cytometry. NPCs were positive for CD24, CD44, and CD133 though negative for CD15, CD184 and CD49d. This immunophenotype, determined for the first time, among cells isolated from neonates might be useful for the identification of NPC population aiming at the development of transplantation protocols.

Enrichment for Repopulating Cells and Identification of Differentiation Markers in the Bovine Mammary Gland.

Elucidating cell hierarchy in the mammary gland is fundamental for understanding the mechanisms governing its normal development and malignant transformation. There is relatively little information on cell hierarchy in the bovine mammary gland, despite its agricultural potential and relevance to breast cancer research. Challenges in bovine-to-mouse xenotransplantation and difficulties obtaining bovine-compatible antibodies hinder the study of mammary stem-cell dynamics in this species. In-vitro indications of distinct bovine mammary epithelial cell populations, sorted according to CD24 and CD49f expression, have been provided. Here, we successfully transplanted these bovine populations into the cleared fat pads of immunocompromised mice, providing in-vivo evidence for the multipotency and self-renewal capabilities of cells that are at the top of the cell hierarchy (termed mammary repopulating units). Additional outgrowths from transplantation, composed exclusively of myoepithelial cells, were indicative of unipotent basal stem cells or committed progenitors. Sorting luminal cells according to E-cadherin revealed three distinct populations: luminal progenitors, and early- and late-differentiating cells. Finally, miR-200c expression was negatively correlated with differentiation levels in both the luminal and basal branches of the bovine mammary cell hierarchy. Together, these experiments provide further evidence for the presence of a regenerative entity in the bovine mammary gland and for the multistage differentiation process within the luminal lineage.

Hematopoietic stem and multipotent progenitor cells produce IL-17, IL-21 and other cytokines in response to TLR signals associated with late apoptotic products and augment memory Th17 and Tc17 cells in the bone marrow of normal and lupus mice.

We studied effects of early and late apoptotic (necroptotic) cell products, related damage associated alarmins and TLR agonists, on hematopoietic stem and progenitor cells (HSPC). Surprisingly, normal HSPC themselves produced IL-17 and IL-21 after 1½days of stimulation, and the best stimulators were TLR 7/8 agonist; HMGB1-DNA; TLR 9 agonist, and necroptotic B cells. The stimulated HSPC expressed additional cytokines/mediators, directly causing rapid expansion of IL-17(+) memory CD4 T (Th17), and CD8 T (Tc17) cells, and antigen-experienced IL-17(+) T cells with "naïve" phenotype. In lupus marrow, HSPC were spontaneously pre-stimulated by endogenous signals to produce IL-17 and IL-21. In contrast to HSPC, megakaryocyte progenitors (MKP) did not produce IL-17, and unlike HSPC, they could process and present particulate apoptotic autoantigens to augment autoimmune memory Th17 response. Thus abnormally stimulated primitive hematopoietic progenitors augment expansion of IL-17 producing immune and autoimmune memory T cells in the bone marrow, which may affect central tolerance.