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The milky spots (MS) are structures found in the omentum of humans and other vertebrates, representing a fraction of the lymphomyeloid tissue associated with the celom. They majorly consist of B lymphocytes, T lymphocytes, and macrophages. Also found in smaller quantities are mesothelial, stromal, dendritic, and rare mast cells. In an experimental model of Schistosoma mansoni infection, there is significant activation of the omentum and MS, which exhibit numerous eosinophils. Despite being described for many years, the complete profile of cells found in MS and their functions remains largely unexplored. Here, we evaluate the leukocyte populations of the MS in homeostasis and a murine model of S. mansoni infection. The histopathological characterization, phenotypic profile analysis, and characterization of the eosinophilic potential of progenitors and precursors comparing the MS with the spleen and bone marrow showed significant activation of MS in infected mice, with changes in the profile over the analyzed times, showing signs of migration and activation of eosinophils, with local eosinopoiesis and maintenance of the eosinophilic population. In naive mice, B1a and B1b cells make up only a small fraction of B lymphocytes. However, B1b cells expand significantly during infection, peaking at 60 days post-infection (DPI) before stabilizing by 90 DPI. B1a cells also increase initially but decrease over time. The behavior of MS differs from other primary and secondary lymphoid organs, acting as a central lymphoid organ in cavity immunity., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

CD45 + CD71 + erythroid cells generated through splenic extramedullary erythropoiesis have recently been found to suppress anti-infection and tumor immunity in neonates and adults with malignances. However, their role in tumor microenvironment has not been investigated. In the present study, we found that the number of CD45 + CD71 + erythroid cells was significantly elevated in hepatocellular carcinoma (HCC) tissues compared to that in paratumor region and circulation. Additionally, they were more abundant in HCC tissues compared to some immune suppressive cells as well as CD45 - CD71 + erythroid cells. CD45 + CD71 + erythroid cells suppressed T cells through generation of reactive oxygen species, IL-10, and TGF-β in a paracrine and cell-cell contact manner, and their suppressive effect was stronger than that of myeloid-derived suppressor cells. The abundance of CD45 + CD71 + erythroid cells in tumor tissue, as illustrated via immunofluorescence, predicted disease-free survival and overall survival, and its prognostic value was better than that of Cancer of the Liver Italian Program score. This study demonstrated that accumulation of intratumoral CD45 + CD71 + erythroid cells in HCC tissues could play a superior immunosuppressive role in tumor microenvironment and may serve as a valuable biomarker to predict recurrence of HCC., (Copyright © 2020. Published by Elsevier B.V.)

Dendritic cells (DC) play a key role in the adaptive immune response due to their ability to present antigens and stimulate naïve T cells. Many bacteria and viruses can efficiently target DC, resulting in impairment of their immunostimulatory function or elimination. Hence, the DC compartment requires replenishment following infection to ensure continued operational readiness of the adaptive immune system. Here, we investigated the molecular and cellular mechanisms of inflammation-induced DC generation. We found that infection with viral and bacterial pathogens as well as Toll-like receptor 9 (TLR9) ligation with CpG-oligodeoxynucleotide (CpG-ODN) expanded an erythropoietin (EPO)-dependent TER119 + CD11a + cell population in the spleen that had the capacity to differentiate into TER119 + CD11c high and TER119 - CD11c high cells both in vitro and in vivo . TER119 + CD11c high cells contributed to the conventional DC pool in the spleen and specifically increased in lymph nodes draining the site of local inflammation. Our results reveal a so far undescribed inflammatory EPO-dependent pathway of DC differentiation and establish a mechanistic link between innate immune recognition of potential immunosuppressive pathogens and the maintenance of the DC pool during and after infection., (Copyright © 2020 Einwächter, Heiseke, Schlitzer, Gasteiger, Adler, Voehringer, Manz, Ruzsics, Dölken, Koszinowski, Sparwasser, Reindl and Jordan.)

Purpose of Review: We reviewed recent progress on the role of sclerostin (SOST) and its effects on the immune system in order to summarize the current state of knowledge in osteoimmunology, in regard to hematopoiesis, lymphopoiesis, and inflammation., Recent Findings: Changes in sclerostin levels affect distinct niches within the bone marrow that support hematopoietic stem cells and B cell development. Sclerostin's regulation of adipogenesis could also be important for immune cell maintenance with age. Surprisingly, B cell development in the bone marrow is influenced by Sost produced by mesenchymal stem cells and osteoblasts, but not by osteocytes. Additionally, extramedullary hematopoiesis in the spleen and increased pro-inflammatory cytokine levels in the bone marrow are observed in global Sost -/- mice. In addition to changes in bone marrow density, sclerostin depletion affects B lymphopoiesis and myelopoiesis, as well as other changes within the bone marrow cavity that could affect hematopoiesis. It is therefore important to monitor for hematopoietic changes in patients receiving sclerostin-depleting therapies.

The murine epidermis harbors two immune cell lineages, Langerhans cells (LCs) and γδ T cells known as dendritic epidermal T cells (DETCs). LCs develop from both early yolk sac (YS) progenitors and fetal liver monocytes before locally self-renewing in the adult. For DETCs, the mechanisms of homeostatic maintenance and their hematopoietic origin are largely unknown. Here, we exploited multicolor fate mapping systems to reveal that DETCs slowly turn over at steady state. Like for LCs, homeostatic maintenance of DETCs is achieved by clonal expansion of tissue-resident cells assembled in proliferative units. The same mechanism, albeit accelerated, facilitates DETC replenishment upon injury. Hematopoietic lineage tracing uncovered that DETCs are established independently of definitive hematopoietic stem cells and instead originate from YS hematopoiesis, again reminiscent of LCs. DETCs thus resemble LCs concerning their maintenance, replenishment mechanisms, and hematopoietic development, suggesting that the epidermal microenvironment exerts a lineage-independent influence on the initial seeding and homeostatic maintenance of its resident immune cells., (© 2018 Gentek et al.)

Cancer progression is associated with alterations of intra- and extramedullary hematopoiesis to support a systemic tumor-promoting myeloid response. However, the functional specialty, mechanism, and clinical relevance of extramedullary hematopoiesis (EMH) remain unclear. Here, we showed that the heightened splenic myelopoiesis in tumor-bearing hosts was not only characterized by the accumulation of myeloid precursors, but also associated with profound functional alterations of splenic early hematopoietic stem/progenitor cells (HSPCs). With the distinct capability to produce and respond to granulocyte-macrophage CSF (GM-CSF), these splenic HSPCs were "primed" and committed to generating immunosuppressive myeloid cells. Mechanistically, the CCL2/CCR2 axis-dependent recruitment and the subsequent local education by the splenic stroma were critical for eliciting this splenic HSPC response. Selective abrogation of this splenic EMH was sufficient to synergistically enhance the therapeutic efficacy of immune checkpoint blockade. Clinically, patients with different types of solid tumors exhibited increased splenic HSPC levels associated with poor survival. These findings reveal a unique and important role of splenic hematopoiesis in tumor-associated myelopoiesis.

Aim: Rheumatoid arthritis (RA) is associated with an approximately two-fold elevated risk of cardiovascular (CV)-related mortality. Patients with RA present with systemic inflammation including raised circulating myeloid cells, but fail to display traditional CV risk-factors, particularly dyslipidaemia. We aimed to explore if increased circulating myeloid cells is associated with impaired atherosclerotic lesion regression or altered progression in RA., Methods and Results: Using flow cytometry, we noted prominent monocytosis, neutrophilia, and thrombocytosis in two mouse models of RA. This was due to enhanced proliferation of the haematopoietic stem and progenitor cells (HSPCs) in the bone marrow and the spleen. HSPCs expansion was associated with an increase in the cholesterol content, due to a down-regulation of cholesterol efflux genes, Apoe, Abca1, and Abcg1. The HSPCs also had enhanced expression of key myeloid promoting growth factor receptors. Systemic inflammation was found to cause defective cellular cholesterol metabolism. Increased myeloid cells in mice with RA were associated with a significant impairment in lesion regression, even though cholesterol levels were equivalent to non-arthritic mice. Lesions from arthritic mice exhibited a less stable phenotype as demonstrated by increased immune cell infiltration, lipid accumulation, and decreased collagen formation. In a progression model, we noted monocytosis, enhanced monocytes recruitment to lesions, and increased plaque macrophages. This was reversed with administration of reconstituted high-density lipoprotein (rHDL). Furthermore, RA patients have expanded CD16+ monocyte subsets and a down-regulation of ABCA1 and ABCG1., Conclusion: Rheumatoid arthritis impairs atherosclerotic regression and alters progression, which is associated with an expansion of myeloid cells and disturbed cellular cholesterol handling, independent of plasma cholesterol levels. Infusion of rHDL prevented enhanced myelopoiesis and monocyte entry into lesions. Targeting cellular cholesterol defects in people with RA, even if plasma cholesterol is within the normal range, may limit vascular disease.

The mammalian spleen is a peripheral lymphoid organ that plays a central role in host defense. Consequently, the lack of spleen is often associated with immunodeficiency and increased risk of overwhelming infections. Growing evidence suggests that non-hematopoietic stromal cells are central players in spleen development, organization, and immune functions. In addition to its immunological role, the spleen also provides a site for extramedullary hematopoiesis (EMH) in response to injuries. A deeper understanding of the biology of stromal cells is therefore essential to fully comprehend how these cells modulate the immune system during normal and pathological conditions. Here, we review the specificities of the different mouse spleen stromal cell subsets and complement the murine studies with human data when available., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Myeloid-derived suppressor cells (MDSCs) have a strong immunosuppressive character that allows them to regulate immune responses and hinder overt inflammatory responses. In cancer, this leads to tumor immune evasion and disease progression. MDSCs come in at least two forms: monocytic (Mo-MDSCs) and granulocytic (G-MDSCs). The classical definition of MDSCs as immature myeloid cells blocked from differentiating has been challenged by recent studies suggesting that Mo-MDSCs and G-MDSCs may represent monocytes and granulocytes that have acquired immunosuppressive properties. The molecular mechanism behind their generation and their true origins are now widely debated. In this review we discuss the different proposed mechanisms of the generation of both types of MDSCs, with a special focus on human MDSCs in cancer.

Background: The spleen has been implicated in the pathogenesis of immune-complex glomerulonephritis by initiating and resolving adaptive immune responses. Thus, we aimed to evaluate the role of the spleen in experimental nephrotoxic serum nephritis (NTS)., Methods: In order to accelerate the disease, animals were subjected to NTS by preimmunizing male C57BL/6J mice with rabbit IgG three days before injecting the rabbit anti-glomerular basement antiserum, or were immunized only. A group underwent splenectomy before NTS induction., Results: We observed enlargement of the spleen with a maximum at 14 days after NTS induction or immunization only. Splenectomized mice were found to develop albuminuria and renal histological changes comparable to sham-operated controls. Nevertheless, anaemia was aggravated in mice after splenectomy. During the course of NTS, we detected CD41+ megakaryocytes and Ter119+ erythroid precursor cells in the spleen of mice with NTS and of immunized mice. Ter119+Cxcr4+ cells and the binding partner Cxcl12 increased in the spleen, and decreased in the bone marrow. This was accompanied by a significant systemic increase of interferon-gamma in the serum., Conclusions: In summary, splenectomy does not influence the course of NTS per se, but is involved in concomitant anaemia. Extramedullary haematopoiesis in the spleen is probably facilitated through the migration of Cxcr4+ erythroid precursor cells from the bone marrow to the spleen via a Cxcl12 gradient and likely arises from the suppressive capacity of chronic inflammation on the bone marrow.

This review is dedicated to comparative analysis of the early stages of thymus ontogeny in fish, amphibians, and mammals. Morphological and molecular-genetic aspects of the formation of thymic stroma, colonization of this organ with T-cell progenitors, and interaction of different cell populations in the course of organogenesis are considered. Particular attention is given to the hematopoietic role of the thymus during embryogenesis and new data on the origin of T-cell progenitors. The hypothesis about the possible presence in the organ of a self-sustaining population of stem cells, formed regardless of fetal hematopoiesis areas, is discussed.

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques., (© 2015 Dutta et al.)

Extramedullary hematopoiesis (EMH) refers to the differentiation of hematopoietic stem cells (HSCs) into effector cells that occurs in compartments outside of the bone marrow. Previous studies linked pattern-recognition receptor (PRR)-expressing HSCs, EMH, and immune responses to microbial stimuli. However, whether EMH operates in broader immune contexts remains unknown. Here, we demonstrate a previously unrecognized role for thymic stromal lymphopoietin (TSLP) in promoting the population expansion of progenitor cells in the periphery and identify that TSLP-elicited progenitors differentiated into effector cells including macrophages, dendritic cells, and granulocytes and that these cells contributed to type 2 cytokine responses. The frequency of circulating progenitor cells was also increased in allergic patients with a gain-of-function polymorphism in TSLP, suggesting the TSLP-EMH pathway might operate in human disease. These data identify that TSLP-induced EMH contributes to the development of allergic inflammation and indicate that EMH is a conserved mechanism of innate immunity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.

Resolution of a variety of acute bacterial and parasitic infections critically relies on the stimulation of myelopoiesis leading in cases to extramedullary hematopoiesis. Here, we report the isolation of the earliest myeloid-restricted progenitors in acute infection with the rodent malaria parasite, Plasmodium chabaudi. The rapid disappearance of these infection-induced myeloid progenitors from the bone marrow (BM) equated with contraction of the functional myeloid potential in that organ. The loss of BM myelopoiesis was not affected by the complete genetic inactivation of toll-like receptor signaling. De-activation of IFN-γ signaling completely abrogated the contraction of BM myeloid progenitors. Radiation chimeras of Ifngr1-null and control BM revealed that IFN-γ signaling in an irradiation-resistant stromal compartment was crucial for the loss of early myeloid progenitors. Systemic IFN-γ triggered the secretion of C-C motif ligand chemokines CCL2 and CCL7 leading to the egress of early, myeloid-committed progenitors from the bone marrow mediated by their common receptor CCR2. The mobilization of myeloid progenitors initiated extramedullary myelopoiesis in the spleen in a CCR2-dependent manner resulting in augmented myelopoiesis during acute malaria. Consistent with the lack of splenic myelopoiesis in the absence of CCR2 we observed a significant persistence of parasitemia in malaria infected CCR2-deficient hosts. Our findings reveal how the activated immune system mobilizes early myeloid progenitors out of the BM thereby transiently establishing myelopoiesis in the spleen in order to contain and resolve the infection locally.

Background: Atherosclerotic lesions are believed to grow via the recruitment of bone marrow-derived monocytes. Among the known murine monocyte subsets, Ly-6C(high) monocytes are inflammatory, accumulate in lesions preferentially, and differentiate. Here, we hypothesized that the bone marrow outsources the production of Ly-6C(high) monocytes during atherosclerosis., Methods and Results: Using murine models of atherosclerosis and fate-mapping approaches, we show that hematopoietic stem and progenitor cells progressively relocate from the bone marrow to the splenic red pulp, where they encounter granulocyte macrophage colony-stimulating factor and interleukin-3, clonally expand, and differentiate to Ly-6C(high) monocytes. Monocytes born in such extramedullary niches intravasate, circulate, and accumulate abundantly in atheromata. On lesional infiltration, Ly-6C(high) monocytes secrete inflammatory cytokines, reactive oxygen species, and proteases. Eventually, they ingest lipids and become foam cells., Conclusions: Our findings indicate that extramedullary sites supplement the hematopoietic function of the bone marrow by producing circulating inflammatory cells that infiltrate atherosclerotic lesions., (© 2011 American Heart Association, Inc.)

Extramedullary hematopoiesis (EMH) occurs as a compensatory reaction to an underlying hematologic abnormality. EMH is most commonly seen in the liver and spleen but rarely has been reported in other locations, as well. On rare occasions, EMH may mimic a neoplasm in hematologically noncompromised patients. In this report, we present three cases of EMH in unusual locations, two in the presacral soft tissue and one in the synovial lining of the knee joint. The patients with presacral masses had no hematologic abnormality. In all patients with EMH, imaging plays an important role in both localization of the lesion and guidance for the biopsy; when imaging results are correlated with histological findings, the diagnosis of EMH can be confirmed.

Allergic inflammation is associated with increased generation and trafficking of inflammatory cells, especially eosinophils, to sites of inflammation. The effect of acute versus chronic airway allergen challenge on hematopoietic activity in the bone marrow (BM) and lungs was investigated using murine models of allergic airway inflammation. Acute allergen challenge induced proliferation of BM cells and significantly increased generation of eosinophil, but not multipotent, granulocyte-macrophage (GM), or B-lymphocyte progenitor cells. However, no hematopoietic activity was observed in the lungs. With chronic challenge, BM cells failed to proliferate, but exhibited increased capacity to generate multipotent as well as eosinophil, GM, and B-lymphocyte progenitors. In addition, increased generation of eosinophil- and GM-specific progenitors was observed in the lungs. Although no differences were observed in their ability to roll on BM endothelium in vitro or in vivo, CD34-enriched hematopoietic/stem progenitor cells (HSPCs) from chronic-, but not acute-, challenged mice demonstrated reduced migration across BM endothelial cells associated with decreased CXCR4 expression. Overall, these studies demonstrate that chronic allergen exposure can alter BM homing due to decreased transendothelial migration enabling noninteracting HSPCs to egress out of the BM and recruit to sites of inflammation such as the airways, resulting in extramedullary hematopoiesis.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that share a common property of suppressing immune responses. Several helminth and protozoan parasite species have developed efficient strategies to increase the rate of medullary or extramedullary myelopoiesis and to induce the expansion and accumulation of immature myeloid cells such as MDSC. In this review, we examine current knowledge on the factors mediating enhanced myelopoiesis and MDSC induction and recruitment during parasitic infections and how the MDSC phenotype and mechanism of immune modulation and suppression depends on the factors they encounter within the host. Finally, we place MDSC expansion in the context of the critical balance between parasite elimination and pathogenicity to the host and suggest attractive avenues for future research., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case., Case Presentation: Hematopoietic stem cells were found in a benign mixed mammary tumor of a 7-year-old female mongrel dog that presents a nodule in the left inguinal mammary gland. The patient did not have any hematological abnormalities. Cytological evaluation demonstrated two distinct cell populations, composed of either epithelial or mesenchymal cells, sometimes associated with a fibrillar acidophilic matrix, apart from megakaryocytes, osteoclasts, metarubricytes, prorubricytes, rubricytes, rubriblasts, promyelocytes, myeloblasts. Histological examination confirmed the presence of an active hematopoietic bone marrow within the bone tissue of a benign mammary mixed tumor., Conclusions: EMH is a rare condition described in veterinary medicine that can be associated with mammary mixed tumors. It's detection can be associated with several neoplastic and non-neoplastic mammary lesions, i.e. osteosarcomas, mixed tumors and bone metaplasia.

Para-aortic foci of chicken embryos at 6-7 days of development are considered to provide a microenvironment for haematopoietic stem cell proliferation and initial differentiation similar to that of fetal liver in mammals. Here, we have investigated the genes involved in this process by constructing and analysing a subtractive cDNA library from CD45(+) cells in para-aortic region. Among 394 analysed clones 99 distinct genes were identified by sequence homology search. Classification of the identified genes according to biological processes revealed that innate immunity-related genes are highly expressed at this stage. This can be explained by the presence of yolk sac-derived macrophages in the original tissue sample but also by the indiscriminate expression of multiple lineage-specific genes in haematopoietic stem cells and primitive progenitors. Differentially expressed genes related to transcription, signalling and lymphocyte functions are potential candidates involved in lineage commitment.

Introduction: Little is known about the sites and kinetics of thrombopoiesis following bone marrow transplant. The spleen is a site of hematopoiesis in a healthy mouse, and hematopoietic activity increases in response to stress. We hypothesized that the spleen is a major site of early post-transplant thrombopoiesis., Methods: We transplanted whole bone marrow (WBM) or lineage depleted progenitor subsets fractionated based on expression of c-kit and Sca-1 from transgenic mice expressing green fluorescent protein into lethally irradiated C57BL/6 recipients. We also transplanted whole bone marrow cells into healthy and splenectomized mice. Post-transplant megakaryopoiesis was assessed by measuring circulating platelet number, percent donor-derived platelets, bone marrow cellularity, splenic weight, megakaryocyte size, and megakaryocyte concentration from hour 3 to day 28 post transplant., Results: Following transplant, circulating donor-derived platelets were derived only from c-kit expressing subsets. Donor-derived platelets first appeared on post-transplant day five. Splenectomy reduced the number of these earliest circulating platelets. Splenic megakaryopoiesis increased dramatically from day 7-14 post-transplant. However, splenectomy accelerated platelet engraftment during this time frame., Conclusion: Overall, these results demonstrate that the first platelets are produced by c-kit expressing megakaryocyte progenitors in the bone marrow and spleen. After post-transplant day 5, the net effect of the spleen on thrombopoiesis is to slow engraftment due to immune effects or hypersplenism.

Loss of CD4 T cells is the hallmark of HIV infection. However, type I IFN-producing plasmacytoid dendritic cells may also be lost. This results in susceptibility to an opportunistic infection such as Pneumocystis pneumonia. In addition, regenerative bone marrow failure resulting in pancytopenia is another common problem in advanced stage AIDS. This may be linked to both the failing immune system and recurrent opportunistic infections. We generated lymphocyte-deficient type I IFN receptor-deficient mice (IFrag-/-) to study the effects on Pneumocystis infection of the lung. When IFrag-/- animals were infected with Pneumocystis they died between days 16 and 21 postinfection with minimal pneumonia but severe anemia due to complete bone marrow failure. This included the loss of uncommitted hemopoietic precursor cells. Bone marrow failure was prevented by the reconstitution of IFrag-/- mice with wild-type lymphocytes, especially B cells. T and B cells lacking type I IFN receptor signaling could only partially prevent bone marrow failure in response to Pneumocystis infection. However, the presence of T and B cells lacking type I IFN signaling resulted in compensatory extramedullary hemopoiesis in the liver and spleen. Lymphocyte support of the regenerative capacity of the bone marrow was provided by both type I IFN-dependent and -independent mechanisms that acted synergistically. Our findings point to the requirement of both type I IFNs and lymphocytes in the regenerative capabilities of the hemopoietic system under the pressure of Pneumocystis infection, but not during steady-state hemopoiesis. This may have implications in the management of pancytopenia in AIDS.

Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11b(hi)Ia(lo) DCs secreting high levels of TGF-beta, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. Furthermore, CD11b(hi)Ia(lo) DCs have the ability to potently suppress allo-DTH in vivo, indicating their preventive or therapeutic perspectives for some immunologic disorders. The inhibitory function of CD11b(hi)Ia(lo) DCs is mediated through NO but not through induction of regulatory T (Treg) cells or T-cell anergy. IL-10, which is secreted by endothelial splenic stroma, plays a critical role in the differentiation of the regulatory CD11b(hi)Ia(lo) DCs from HSCs. These results suggest that splenic microenvironment may physiologically induce regulatory DC differentiation in situ.

The study of dendritic cells (DC) has been hampered by the difficulty of isolating rare cells for analysis of their phenotype and function. Interpretation of the DC lineage has been largely influenced by studies on cell populations which can be readily isolated and amplified in the presence of cytokines. Long term cultures (LTC) from murine spleen have been shown to support continuous in vitro hematopoiesis of DC dependent on interaction with a stromal cell monolayer. LTC-DC represent a single, stable class of DC derived by constant turnover of spleen DC progenitors maintained within stroma. They represent a resident DC population in spleen. The functional characteristics of LTC-DC have been studied in terms of capacity to stimulate T cells and response to activation by environmental stimuli. LTC-DC have many morphological, phenotypic and functional properties reflecting an immature or partially mature, marginal zone-like CD4(-)CD8(-) splenic DC subset. They are highly endocytic and can process and present protein antigen to naive hen egg lysozyme (HEL)-specific MHC-II-restricted TCR-Tg CD4(+) T cells. They do not, however, induce T cell proliferation in a mixed lymphocyte reaction. LTC-DC do not respond in a typical fashion to common DC activators like LPS and CD40L. They upregulate MHC-I and CD80/CD86 but not MHC-II and CD40. They reflect an endogenous, immature DC subset in spleen with properties distinct from immature DC located in peripheral tissues.

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-alpha and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.

Distinct and redundant chemokines are responsible for organizing the extraordinarily diverse thymocyte populations into discrete microenvironments, from the arrival of immature precursors in the thymus to the migration of different mature cell types to the periphery. We propose that, by selectively mobilizing cells, chemokines can sort positively selected cells from negatively selected cells, and that chemokines make distinctions among CD8+ subpopulations that previously have not been recognized.

IL-17 is a novel, CD4+ T cell-restricted cytokine. In vivo, it stimulates hematopoiesis and causes neutrophilia consisting of mature granulocytes. In this study, we show that IL-17-mediated granulopoiesis requires G-CSF release and the presence or induction of the transmembrane form of stem cell factor (SCF) for optimal granulopoiesis. However, IL-17 also protects mice from G-CSF neutralization-induced neutropenia. G-CSF neutralization completely reversed IL-17-induced BM progenitor expansion, whereas splenic CFU-GM/CFU-granulocyte-erythrocyte-megakaryocyte-monocyte was only reduced by 50% in both Sl/Sld and littermate control mice. Thus, there remained a significant SCF/G-CSF-independent effect of IL-17 on splenic granulopoiesis, resulting in a preservation of mature circulating granulocytes. IL-17 is a cytokine that potentially interconnects lymphocytic and myeloid host defense and may have potential for therapeutic development.

We show that the mesenchymal cells that surround the 12-d mouse embryo thymus are necessary for T cell differentiation. Thus, epithelial lobes with attached mesenchyme generate all T cell populations in vitro, whereas lobes from which mesenchyme has been removed show poor lymphopoiesis with few cells progressing beyond the CD4(-)CD8(-) stage of development. Interestingly, thymic mesenchyme is derived from neural crest cells, and extirpation of the region of the neural crest involved results in impaired thymic development and craniofacial abnormalities similar to the group of clinical defects found in the DiGeorge syndrome. Previous studies have suggested an inductive effect of mesenchyme on thymic epithelial morphogenesis. However, we have found that mesenchyme-derived fibroblasts are still required for early T cell development in the presence of mature epithelial cells, and hence mesenchyme might have a direct role in lymphopoiesis. We provide an anatomical basis for the role of mesenchyme by showing that mesenchymal cells migrate into the epithelial thymus to establish a network of fibroblasts and associated extracellular matrix. We propose that the latter might be important for T cell development through integrin and/or cytokine interactions with immature thymocytes.

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a longstanding puzzle in immunology. In the past few years it has become clear that production of mu heavy chain and subsequent assembly with surrogate light chain to form the pre-B cell receptor complex is critical to promote development of adult B cell precursors in mouse bone marrow. Recently we found that instead of promoting pre-B cell expansion as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver, providing a previously unrecognized mechanism for alteration of the B cell repertoire with age. The consequence is very distinct primary repertoires for development of fetal B1 cells and adult bone marrow B2 cells.

The acute graft-versus-host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell-mediated attack on host lymphohematopoietic populations, resulting in the reconstitution of the host with donor hematopoietic stem cells. We examined the effect of GVHD on the donor and host hematopoiesis in parental-induced acute GVHD. The bone marrow was hypoplastic and the number of hematopoietic progenitor cells significantly decreased at 4 weeks after GVHD induction. However, extramedullary splenic hematopoiesis was present and the number of hematopoietic progenitor cells in the spleen significantly increased at this time. Fas expression on the host spleen cells and bone marrow cells significantly increased during weeks 2 to 8 of GVHD. Host cell incubation with anti-Fas Ab induced apoptosis, and the number of hematopoietic progenitor cells decreased during these weeks. A significant correlation between the augmented Fas expression on host bone marrow cells and the decreased number of host bone marrow cells by acute GVHD was observed. Furthermore, the injection of Fas ligand (FasL)-deficient B6/gld spleen cells failed to affect host bone marrow cells. Although Fas expression on repopulating donor cells also increased, Fas-induced apoptosis by the repopulating donor cells was not remarkable until 12 weeks, when more than 90% of the cells were donor cells. The number of hematopoietic progenitor cells in the bone marrow and the spleen by the repopulating donor cells, however, decreased over an extended time during acute GVHD. This suggests that Fas-FasL interactions may regulate suppression of host hematopoietic cells but not of donor hematopoietic cells. Hematopoietic dysfunctions caused by the reconstituted donor cells are independent to Fas-FasL interactions and persisted for a long time during parental-induced acute GVHD., (Copyright 1999 Academic Press.)

Induction of experimental autoimmune diseases often relies on immunization with the organ-specific autoantigens in CFA, which contains heat-killed mycobacteria. In several of these models, including collagen-induced arthritis, endogenous IFN-gamma acts as a disease-limiting factor in the pathogenesis of the disease. Here we show that in collagen-induced arthritis the protective effect of IFN-gamma depends on the presence of mycobacteria in the adjuvant. Omission of mycobacteria inverts the role of endogenous IFN-gamma to a disease-promoting factor. Thus, the mycobacterial component of CFA opens a pathway by which endogenous IFN-gamma exerts a protective effect that supersedes its otherwise disease-promoting effect. Extramedullary hemopoiesis and expansion of the Mac-1+ cell population accompanied the accelerated and more severe disease course in the IFN-gamma receptor knockout mice immunized with CFA. Treatment of such mice with Abs against the myelopoietic cytokines IL-6 or IL-12 inhibited both disease development and the expansion of the Mac-1+ population. We postulate that mycobacteria in CFA stimulate the expansion of the Mac-1+ cell population by a hemopoietic process that is restrained by endogenous IFN-gamma. These results have important implications for the validity of animal models of autoimmunity to study the pathogenesis and to evaluate cytokine-based therapy of autoimmune diseases.

Inflammatory granulomatous reactions in liver elicited by schistosomal infection have been shown to function as active extramedullar myelopoietic sites, producing potentially all the myeloid lineages. We have now addressed the question of the extramedullar B lymphopoiesis in these sites. We have shown the presence of early B cell precursors (pro-B cells) in the granulomas by immunophenotyping. Their total number in the liver was equivalent to the pro-B cells in the bone marrow of one femur. In agreement with their phenotype, the RT-PCR analysis showed that these cells expressed RAG-1 and lambda5 genes. However, the conversion of the pro-B to pre-B cells was not observed and no clonogenic B cell precursors could be detected in semi-solid cultures stimulated by IL-7. The granulomatous stroma was shown to produce IL-7 and express c-kit, and was able to sustain the full B lymphopoiesis in vitro. Conversely, the granuloma supernatant was shown to inhibit actively the development of B lymphocytes. We conclude that the granuloma environment elicits homing and proliferation of totipotent hematopoietic precursors, and that it is permissive for early commitment to the B cell lineage, but the full extramedullar production of B cell is abrogated by soluble factors produced inside the granulomas.

Bacterial DNA and the synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the adaptive immune system (lymphocytes) and the innate immune system (APCs) in a sequence-dependent manner. Here, we addressed whether CpG-ODNs affect hemopoiesis. Challenging mice with immunostimulatory CpG-ODN sequences led to transient splenomegaly, with a maximum increase of spleen weight at day 6. The induction of splenomegaly by CpG-ODNs was sequence-specific, dose-dependent, and associated with an increase in splenic cell count, in numbers of granulocyte-macrophage CFUs (GM-CFUs), and early erythroid progenitors (burst-forming units-erythroid). The transfer of spleen cells from CpG-ODN-pretreated animals into lethally irradiated syngeneic mice yielded an increase of spleen CFUs. Furthermore, the challenge of sublethally irradiated mice with CpG-ODNs caused radioprotective effects, in that recovery of GM-CFUs and cytotoxic T cell function was enhanced. The increase in GM-CFU and CTL function correlated with an enhanced resistance to Listeria infection in irradiated mice. We conclude from these data that CpG-ODNs trigger extramedullary hemopoiesis, and that this finding could be of therapeutic relevance in myelosuppression.

Cytokines interact not only with membrane anchored receptors, but also with specific soluble receptors which circulate in the bloodstream. In general, soluble cytokine receptors such as soluble tumor necrosis factor receptor, soluble interleukin 1 receptor, and soluble interleukin 4 receptor compete with their membrane-bound counterparts for the ligands and therefore act as antagonists. In contrast, soluble receptors for cytokines of the interleukin-6 (IL-6) family complex with their ligands act agonistically. Interestingly, the complex of IL-6 and the soluble interleukin 6 receptor (sIL-6R) activates target cells that do not express the membrane-bound IL-6R and therefore cannot respond to IL-6. To identify cellular responses that are due to IL-6/sIL-6R but not to IL-6 alone, IL-6/sIL-6R double-transgenic mice were generated and compared with IL-6 single-transgenic mice. IL-6/sIL-6R transgenic mice develop a severe phenotype showing 1) marked hepatocellular hyperplasia frequently surrounded by peliosis and necrosis, 2) significant acceleration and aggravation of plasmacytoma formation, and 3) excessive activation of extramedullary hematopoiesis in spleen and liver followed by a subsequent increase of all cellular components in the peripheral blood. These in vivo data suggest that the sIL-6R recruits primarily unresponsive cell populations such as hematopoietic progenitor cells and hepatocytes to IL-6-induced proliferation, but also enhances the known mitogenic effect of IL-6 on plasma cells and thereby contributes to plasmacytoma formation.

FLT3 ligand (FL) is a recently described hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors. We have investigated the effect of FL on murine hematopoiesis and dendritic cell (DC) generation and accumulation in lymphoid tissues and liver in vivo and in vitro evaluating the morphologic, phenotypic, and functional characteristics of these DC. We have observed extramedullary hematopoiesis in the mouse spleen with all lineages of hematopoietic cells represented after the administration of FL. Injection of FL results in a time-dependent and reversible accumulation of DC in the spleen, bone marrow, lymph nodes, and liver. Both flow cytometry and immunohistochemistry revealed a significant accumulation of DC in these tissues. Results of mixed leukocyte reaction suggested that these cells, isolated from murine bone marrow or spleen, were active as antigen presenting cells. Furthermore, cultivation of splenic and marrow cells with GM-CSF and IL-4 gave rise to large numbers of functionally active mature DC. Thus, the results of this study suggest that FL is a promising growth factor that stimulates the generation of large number of DC and may be a useful cytokine for the immunotherapy of cancer.

Deficiency of the cytokine receptor common gamma chain (gamma c) results in abnormal lymphoid development and a severe immunodeficiency disease due to the combined loss of the receptors for interleukins (IL)-2, -4, -7, -9, and -15. We have observed the development of secondary hematopoiesis with circulating hematopoietic progenitor cells in adult mice harboring a null mutation in gamma c. These extramedullary changes were not secondary to bone marrow failure or to an inability to maintain circulating blood counts. These results suggested that gamma c-dependent cytokine signaling pathways modulate hematopoietic development. An intrinsic defect in gamma c- hematopoietic stem cell commitment appeared unlikely, as fetal liver hematopoiesis was unaltered in gamma c- embryos. Furthermore, the absence of natural killer cells in gamma c- mice was not responsible for the observed hematopoietic changes. Peripheral TCR alpha beta T cells from gamma c- mice were characterized by an activated phenotype (CD62Llo, CD44hi, CD69hi) and showed increased levels of transcripts for hematopoietic stimulating cytokines, including IL-3 and granulocyte/macrophage-colony-stimulating factor. A predominance of these cells was detected in the bone marrow, suggesting a role for residual T cells in the enhanced hematopoiesis. Strikingly, the elimination of residual T cells from gamma c- mice reduced splenic and circulating hematopoietic precursor frequencies to normal levels. These results clearly implicate a deregulated TCR alpha beta T cell population in the observed hematopoietic changes in gamma c- mice, and emphasize the importance of gamma c-dependent cytokine interactions in modulating mature T cell responses.

Amyloid deposition in varying amounts and with variable patterns of distribution (focal or diffuse) was demonstrated in eight canine and two feline extramedullary plasmacytomas expressing lambda light chains. Frequently, the neoplastic plasma cells had been displaced by the amyloid deposits. Foreign-body giant cells were regularly detected in the vicinity of the amyloid. In all 10 cases, Congo-red staining of the amyloid was resistant to potassium permanganate oxidation. Immunohistochemically, the amyloid reacted positively with cross-reacting antibodies against human and equine A lambda amyloids. Extramedullary plasmacytomas accompanied by localized AL amyloidosis have so far been described in human beings, dogs, cats and horses.

IL-13, a recently identified Th2 cytokine, shares some, but not all, IL-4 functions, including inhibition of monocyte and macrophage activation, stimulation of human B cells, and induction of growth and differentiation of mouse bone marrow cells in vitro. We have now tested the in vivo effects of recombinant mouse IL-13 (rIL-13) from stably transfected, high expressing BW5147 thymoma cells. After purification by anion exchange chromatography, rIL-13 was administered in the peritoneal cavity of BALB/c mice via osmotic pump for 7 days. Spleens from the rIL-13-treated mice were significantly enlarged compared with control spleens due to increased cellularity. In particular, increased numbers of immature erythroblasts and megakaryocytes were observed in splenic sections after rIL-13 treatment. Spleen cells from rIL-13-treated mice showed greatly increased responsiveness in vitro to recombinant forms of mouse IL-3, mouse granulocyte-macrophage CSF, or human CSF-1 and, to a lesser extent, to mouse IL-4 or IL-13. Moreover, the rIL-13-treated mice also showed significant increases in CFU-E, CFU-C, and erythroid burst colonies in the spleen, further indicating the presence of increased numbers of hemopoietic precursors. Hematologic analyses indicated that rIL-13 treatment induced slight anemia and striking monocytosis. Finally, spleen cells from rIL-13-treated mice produced significantly more IL-6 upon LPS stimulation. Interestingly, the strong Th2 response induced by Nippostrongylus brasiliensis infection was also accompanied by an increase in hemopoietic precursor frequencies in the spleen. Collectively, these data indicate that exogenous rIL-13 induces extramedullary hemopoiesis in mice and suggest that endogenous IL-13 may contribute to replenishment of effector cells during strong Th2 responses.

Problem: Decidual tissue is an interface between the host and fetus, which is regarded as a natural allograft. We have reported the possible presence of extrathymic T cells in human decidua through the expression of recombinase-activating genes in the decidual CD16-CD56bright large granular lymphocytes., Methods: In this study we analyzed expression of TCR V gamma delta gene segments in peripheral lymphocytes and decidual and nonpregnant endometrial samples using RT-PCR and oligonucleotide hybridization., Results: Interestingly, a limited repertoire of V gamma gene segments were discovered in decidual lymphocytes of normal pregnancy when compared with peripheral lymphocytes, whereas usage of V delta gene segments were ubiquitous in peripheral and decidual lymphocytes. Strong expression of V gamma I gene segment, often exceeding 50% of total V gamma, were noted in all (13/13) pregnant decidual tissues obtained at different gestational stages. Interestingly, this predominance of V gamma I gene is also noted in nonpregnant endometrial samples and the decidua of patients with ectopic pregnancies., Conclusions: Our results suggest the nature of decidual V gamma I T cells as possible suppressors of rejective maternal cytotoxic T cells during the maintenance of normal pregnancies.

The mouse mutant Ft displays thymic hyperplasia and fused toes (Ft) of the forelimbs. Both phenotypic abnormalities are caused by transgene insertion in the D region of chromosome 8. While the forelimb defect is probably caused by developmentally dysregulated programmed cell death, the mechanism underlying thymic hyperplasia has not been characterized. In this work, we show that expansion of the thymocyte compartment progresses with time, is polyclonal, and affects all major thymocyte subsets, including the earliest CD4-8- subset, i.e., CD44+ CD25- cells; hyperplasia is not an autonomous property of mutant T cells, but is caused indirectly by a primary defect in thymic stromal. The rate of cell division and the cell turnover of immature CD4-8- and CD4+8+ thymocytes under steady state conditions are not altered in hyperplastic Ft thymi. Immature CD4+8+ thymocytes of mutant mice, however, are less susceptible to induction in vitro of programmed cell death by different modes (TCR cross-linking, cortisone, or radiation). Increased production of thymocytes results in increased export of T cells, yet the size and composition of the peripheral T cell pool are normal. Overproduction of immature CD4+8+ thymocytes is offset partly by a reduced conversion rate of CD4+8+ double positive to single positive thymocyte growth control by epithelial cells, and may serve as a model to study the regulation of early thymopoiesis.

NK cell cytotoxicity may provide an important first line of defense against the implantation of circulating tumor emboli. Previously we reported that murine hind limb amputation induces a rapid and marked impairment of perioperative NK cell cytotoxicity that is associated with increased metastatic activity compared to unmanipulated tumor-bearing controls. Because tumor emboli are systemically discharged in patients undergoing solid tumor resection, we thought it pertinent to begin examining the mechanism underlying the perioperative impairment of murine NK cell cytotoxicity. Studies presented in this report suggest that the mechanism of impairment is multifactorial and includes the surgical stress-induced expansion of splenic erythroblasts that successfully compete with NK cells for YAC-1 target binding sites. In addition, although there is no surgically mediated decrease in splenic NK cell content (as defined morphologically, phenotypically, and functionally), there is an acute decrease in maximal NK cell recycling capacity. An accurate understanding of the mechanisms mediating perioperative impairment of NK cell cytotoxicity may be important in the future development of NK-specific perioperative immunomodulation strategies.