Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment

Background

Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4⁺, murine CD4^−/−, HLA-DR3⁺ mice.

Methodology/Principal Findings

Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45⁺ showed depletion of CD4⁺, CD8⁺, CD19⁺, and CD117⁺ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4⁺ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death.

Conclusions/Significance

NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Functional Contribution of Elevated Circulating and Hepatic Non-Classical CD14+CD16+ Monocytes to Inflammation and Human Liver Fibrosis

Background

Monocyte-derived macrophages critically perpetuate inflammatory responses after liver injury as a prerequisite for organ fibrosis. Experimental murine models identified an essential role for the CCR2-dependent infiltration of classical Gr1/Ly6C⁺ monocytes in hepatic fibrosis. Moreover, the monocyte-related chemokine receptors CCR1 and CCR5 were recently recognized as important fibrosis modulators in mice. In humans, monocytes consist of classical CD14⁺CD16⁻ and non-classical CD14⁺CD16⁺ cells. We aimed at investigating the relevance of monocyte subpopulations for human liver fibrosis, and hypothesized that ‘non-classical’ monocytes critically exert inflammatory as well as profibrogenic functions in patients during liver disease progression.

Methodology/Principal Findings

We analyzed circulating monocyte subsets from freshly drawn blood samples of 226 patients with chronic liver disease (CLD) and 184 healthy controls by FACS analysis. Circulating monocytes were significantly expanded in CLD-patients compared to controls with a marked increase of the non-classical CD14⁺CD16⁺ subset that showed an activated phenotype in patients and correlated with proinflammatory cytokines and clinical progression. Correspondingly, CD14⁺CD16⁺ macrophages massively accumulated in fibrotic/cirrhotic livers, as evidenced by immunofluorescence and FACS. Ligands of monocyte-related chemokine receptors CCR2, CCR1 and CCR5 were expressed at higher levels in fibrotic and cirrhotic livers, while CCL3 and CCL4 were also systemically elevated in CLD-patients. Isolated monocyte/macrophage subpopulations were functionally characterized regarding cytokine/chemokine expression and interactions with primary human hepatic stellate cells (HSC) in vitro. CD14⁺CD16⁺ monocytes released abundant proinflammatory cytokines. Furthermore, CD14⁺CD16⁺, but not CD14⁺CD16⁻ monocytes could directly activate collagen-producing HSC.

Conclusions/Significance

Our data demonstrate the expansion of CD14⁺CD16⁺ monocytes in the circulation and liver of CLD-patients upon disease progression and suggest their functional contribution to the perpetuation of intrahepatic inflammation and profibrogenic HSC activation in liver cirrhosis. The modulation of monocyte-subset recruitment into the liver via chemokines/chemokine receptors and their subsequent differentiation may represent promising approaches for therapeutic interventions in human liver fibrosis.     

Low-Level Vibrations Retain Bone Marrow's Osteogenic Potential and Augment Recovery of Trabecular Bone during Reambulation

Mechanical disuse will bias bone marrow stromal cells towards adipogenesis, ultimately compromising the regenerative capacity of the stem cell pool and impeding the rapid and full recovery of bone morphology. Here, it was tested whether brief daily exposure to high-frequency, low-magnitude vibrations can preserve the marrow environment during disuse and enhance the initiation of tissue recovery upon reambulation. Male C57BL/6J mice were subjected to hindlimb unloading (HU, n = 24), HU interrupted by weight-bearing for 15 min/d (HU+SHAM, n = 24), HU interrupted by low-level whole body vibrations (0.2 g, 90 Hz) for 15 min/d (HU+VIB, n = 24), or served as age-matched controls (AC, n = 24). Following 3 w of disuse, half of the mice in each group were released for 3 w of reambulation (RA), while the others were sacrificed. RA+VIB mice continued to receive vibrations for 15 min/d while RA+SHAM continued to receive sham loading. After disuse, HU+VIB mice had a 30% greater osteogenic marrow stromal cell population, 30% smaller osteoclast surface, 76% greater osteoblast surface but similar trabecular bone volume fraction compared to HU. After 3 w of reambulation, trabecular bone of RA+VIB mice had a 30% greater bone volume fraction, 51% greater marrow osteoprogenitor population, 83% greater osteoblast surfaces, 59% greater bone formation rates, and a 235% greater ratio of bone lining osteoblasts to marrow adipocytes than RA mice. A subsequent experiment indicated that receiving the mechanical intervention only during disuse, rather than only during reambulation, was more effective in altering trabecular morphology. These data indicate that the osteogenic potential of bone marrow cells is retained by low-magnitude vibrations during disuse, an attribute which may have contributed to an enhanced recovery of bone morphology during reambulation.     

Tip-DC Development during Parasitic Infection Is Regulated by IL-10 and Requires CCL2/CCR2, IFN-γ and MyD88 Signaling

The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b⁺Ly6C⁺CD11c⁺ TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b⁺Ly6C⁺ monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b⁺Ly6C⁺ monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b⁺Ly6C⁺ monocytic cells to immature inflammatory DCs (CD11c⁺ but CD80/CD86/MHC-II^low) which is IFN-γ and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-II^high) TNF and NO producing Tip-DCs which is IFN-γ and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b⁺Ly6C⁺ monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular.     

Erythroid Progenitor Cells Expanded from Peripheral Blood without Mobilization or Preselection: Molecular Characteristics and Functional Competence

Background

Continued development of in-vitro procedures for expansion and differentiation of erythroid progenitor cells (EPC) is essential not only in hematology and stem cell research but also virology, in light of the strict erythrotropism of the clinically important human parvovirus B19.

Methodology/Principal Findings

We cultured EPC directly from ordinary blood samples, without ex vivo stem cell mobilization or CD34+ cell in vitro preselection. Profound increase in the absolute cell number and clustering activity were observed during culture. The cells obtained expressed the EPC marker combination CD36, CD71 and glycophorin, but none of the lymphocyte, monocyte or NK markers. The functionality of the generated EPC was examined by an in vitro infection assay with human parvovirus B19, tropic for BFU-E and CFU-E cells. Following infection (i) viral DNA replication and mRNA production were confirmed by quantitative PCR, and (ii) structural and nonstructural proteins were expressed in >50% of the cells. As the overall cell number increased 100–200 fold, and the proportion of competent EPC (CD34+ to CD36+) rose from <0.5% to >50%, the in vitro culture procedure generated the EPC at an efficiency of >10 000-fold. Comparative culturing of unselected PBMC and ex vivo-preselected CD34+ cells produced qualitatively and quantitatively similar yields of EPC.

Conclusions/Significance

This approach yielding EPC directly from unmanipulated peripheral blood is gratifyingly robust and will facilitate the study of myeloid infectious agents such as the B19 virus, as well as the examination of erythropoiesis and its cellular and molecular mechanisms.     

Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival

It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.     

Peripheral NKT Cells in Simian Immunodeficiency Virus-Infected Macaques

NKT cells are a specialized population of T lymphocytes that have an increasingly recognized role in immunoregulation, including controlling the response to viral infections. The characteristics of NKT cells in the peripheral blood of macaques during simian immunodeficiency virus (SIV) or chimeric simian/human immunodeficiency virus (HIV) (SHIV) infection were assessed. NKT cells comprised a mean of 0.19% of peripheral blood lymphocytes across the 64 uninfected macaques studied. Although the range in the percentages of NKT cells was large (0 to 2.2%), levels were stable over time within individual macaques without SIV/SHIV infection. The majority of NKT cells in macaques were CD4⁺ (on average 67%) with smaller populations being CD8⁺ (21%) and CD4/CD8 double positive (13%). A precipitous decline in CD4⁺ NKT cells occurred in all six macaques infected with CXCR4-tropic SHIV_mn229 early after infection, with a concomitant rise in CD8⁺ NKT cells in some animals. The depletion of CD4⁺ NKT cells was tightly correlated with the depletion of total CD4⁺ T cells. R5-tropic SIV_mac251 infection of macaques resulted in a slower and more variable decline in CD4⁺ NKT cells, with animals that were able to control SIV virus levels maintaining higher levels of CD4⁺ NKT cells. An inverse correlation between the depletion of total and CD4⁺ NKT cells and SIV viral load during chronic infection was observed. Our results demonstrate the infection-driven depletion of peripheral CD4⁺ NKT cells during both SHIV and SIV infection of macaques. Further studies of the implications of the loss of NKT cell subsets in the pathogenesis of HIV disease are needed.     

Inactivated Simian Immunodeficiency Virus-Pulsed Autologous Fresh Blood Cells as an Immunotherapy Strategy

Practical immunotherapies for human immunodeficiency virus infection are needed. We evaluated inactivated simian immunodeficiency virus (SIV) pulsed onto fresh peripheral blood mononuclear cells in 12 pigtail macaques with chronic SIV_mac251 infection for T-cell immunogenicity in a randomized cross-over design study. The immunotherapy was safe and convincingly induced high levels of SIV-specific CD4⁺ T-cell responses (mean, 5.9% ± 1.3% of all CD4⁺ T cells) and to a lesser extent SIV-specific CD8⁺ T-cell responses (mean, 0.7% ± 0.4%). Responses were primarily directed toward Gag and less frequently toward Env but not Pol or regulatory/accessory SIV proteins. T-cell responses against Gag were generally broad and polyfunctional, with a mean of 2.7 CD4⁺ T-cell epitopes mapped per animal and more than half of the SIV Gag-specific CD4⁺ T cells expressing three or more effector molecules. The immunogenicity was comparable to that found in previous studies of peptide-pulsed blood cells. Despite the high-level immunogenicity, no reduction in viral load was observed in the chronically viremic macaques. This contrasts with our studies of immunization with peptide-pulsed blood cells during early SIV infection in macaques. Future studies of inactivated virus-pulsed blood cell immunotherapy during early infection of patients receiving antiretroviral therapy are warranted.     

FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Background

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.

Methodology

FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.

Principal Findings

HIV infected individuals had significantly higher frequencies of CD4⁺FOXP3⁺ T cells (median of 8.11%; range 1.33%–26.27%) than healthy controls (median 3.72%; range 1.3–7.5%; P = 0.002), despite having lower absolute counts of CD4⁺FOXP3⁺ T cells. There was a significant positive correlation between the frequency of CD4⁺FOXP3⁺ T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = −0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/µl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%–26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4⁺ T cells following antigenic or other stimulation.

Conclusions/Significance

FOXP3 expression in the CD4⁺ T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Detection of Gene Expression in an Individual Cell Type within a Cell Mixture Using Microarray Analysis

Background

A central issue in the design of microarray-based analysis of global gene expression is the choice between using cells of single type and a mixture of cells. This study quantified the proportion of lipopolysaccharide (LPS) induced differentially expressed monocyte genes that could be measured in peripheral blood mononuclear cells (PBMC), and determined the extent to which gene expression in the non-monocyte cell fraction diluted or obscured fold changes that could be detected in the cell mixture.

Methodology/Principal Findings

Human PBMC were stimulated with LPS, and monocytes were then isolated by positive (Mono+) or negative (Mono−) selection. The non-monocyte cell fraction (MonoD) remaining after positive selection of monocytes was used to determine the effect of non-monocyte cells on overall expression. RNA from LPS-stimulated PBMC, Mono+, Mono− and MonoD samples was co-hybridised with unstimulated RNA for each cell type on oligonucleotide microarrays. There was a positive correlation in gene expression between PBMC and both Mono+ (0.77) and Mono− (0.61–0.67) samples. Analysis of individual genes that were differentially expressed in Mono+ and Mono− samples showed that the ability to detect expression of some genes was similar when analysing PBMC, but for others, differential expression was either not detected or changed in the opposite direction. As a result of the dilutional or obscuring effect of gene expression in non-monocyte cells, overall about half of the statistically significant LPS-induced changes in gene expression in monocytes were not detected in PBMC. However, 97% of genes with a four fold or greater change in expression in monocytes after LPS stimulation, and almost all (96–100%) of the top 100 most differentially expressed monocyte genes were detected in PBMC.

Conclusions/Significance

The effect of non-responding cells in a mixture dilutes or obscures the detection of subtle changes in gene expression in an individual cell type. However, for studies in which only the most highly differentially expressed genes are of interest, separating and analysing individual cell types may be unnecessary.     

Decreased NK Cell FcRγ in HIV-1 Infected Individuals Receiving Combination Antiretroviral Therapy: a Cross Sectional Study

Background

FcRγ is an immunoreceptor tyrosine-based activation motif (ITAM)-signalling protein essential for immunoreceptor signaling and monocyte, macrophage and NK cell function. Previous study from our laboratory showed that FcRγ is down-regulated in HIV-infected macrophages in vitro. FcRγ expression in immune cells present in HIV-infected individuals is unknown.

Methodology/Principal Findings

We compared FcRγ expression in peripheral blood mononuclear cells isolated from HIV-1-infected individuals receiving combination antiretroviral therapy and healthy, HIV-1-uninfected individuals. FcRγ mRNA and protein levels were measured using quantitative real-time PCR and immunoblotting, respectively. CD56⁺ CD94⁺ lymphocytes isolated from blood of HIV-1 infected individuals had reduced FcRγ protein expression compared to HIV-uninfected individuals (decrease = 76.8%, n = 18 and n = 12 respectively, p = 0.0036). In a second group of patients, highly purified NK cells had reduced FcRγ protein expression compared to uninfected controls (decrease = 50.2%, n = 9 and n = 8 respectively, p = 0.021). Decreased FcRγ expression in CD56+CD94+ lymphocytes was associated with reduced mRNA (51.7%, p = 0.021) but this was not observed for the smaller group of patients analysed for NK cell expression (p = 0.36).

Conclusion/Significance

These data suggest biochemical defects in ITAM-dependent signalling within NK cells in HIV-infected individuals which is present in the context of treatment with combination antiretroviral therapy.     

Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Background

Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34⁺ cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion.

Methodology/Principal Findings

CB CD34⁺ cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34⁺ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone.

Conclusion/Significance

Our data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34⁺ cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.     

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56⁺CD3⁻ NK cell products could be routinely generated from freshly selected CD34⁺ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34⁺ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56⁺ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34⁺ cells for cancer immunotherapy.     

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4⁺CD25⁺FoxP3⁺CD127^low regulatory T cells, contraction of CD4⁺ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.     

Yersinia enterocolitica Targets Cells of the Innate and Adaptive Immune System by Injection of Yops in a Mouse Infection Model

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80⁺, 11% of CD11c⁺, 7% of CD49b⁺, 5% of Gr1⁺ cells, 2.3% of CD19⁺, and 2.6% of CD3⁺ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19⁺CD21⁺CD23⁺ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.     

Differential expression of CD163 on monocyte subsets in healthy and HIV-1 infected individuals

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16− monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16− monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16− subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16− monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals.     

Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome

Innate immune cells like monocytes patrol the vasculature and mucosal surfaces, recognize pathogens, rapidly redistribute to affected tissues and cause inflammation by secretion of cytokines. We previously showed that monocytes are reduced in blood but accumulate in the airways of patients with Puumala virus (PUUV) caused hemorrhagic fever with renal syndrome (HFRS). However, the dynamics of monocyte infiltration to the kidneys during HFRS, and its impact on disease severity are currently unknown. Here, we examined longitudinal peripheral blood samples and renal biopsies from HFRS patients and performed in vitro experiments to investigate the fate of monocytes during HFRS. During the early stages of HFRS, circulating CD14–CD16+ nonclassical monocytes (NCMs) that patrol the vasculature were reduced in most patients. Instead, CD14+CD16– classical (CMs) and CD14+CD16+ intermediate monocytes (IMs) were increased in blood, in particular in HFRS patients with more severe disease. Blood monocytes from patients with acute HFRS expressed higher levels of HLA-DR, the endothelial adhesion marker CD62L and the chemokine receptors CCR7 and CCR2, as compared to convalescence, suggesting monocyte activation and migration to peripheral tissues during acute HFRS. Supporting this hypothesis, increased numbers of HLA-DR+, CD14+, CD16+ and CD68+ cells were observed in the renal tissues of acute HFRS patients compared to controls. In vitro, blood CD16+ monocytes upregulated CD62L after direct exposure to PUUV whereas CD16– monocytes upregulated CCR7 after contact with PUUV-infected endothelial cells, suggesting differential mechanisms of activation and response between monocyte subsets. Together, our findings suggest that NCMs are reduced in blood, potentially via CD62L-mediated attachment to endothelial cells and monocytes are recruited to the kidneys during HFRS. Monocyte mobilization, activation and functional impairment together may influence the severity of disease in acute PUUV-HFRS.     

Very low oxygen concentration (0.1%) reveals two FDCP-Mix cell subpopulations that differ by their cell cycling,differentiation and p27KIP1 expression

Oxygen (O₂) concentrations in bone marrow vary from 4% in capillaries to <0.1% in subendosteum, in which hematopoietic stem cells reside in specific niches. Culture at low O₂ concentrations (3, 1 and 0.1%) influences hematopoietic stem and progenitor cells survival, proliferation and differentiation, depending on their level of differentiation. Culture of human CD34⁺ cells at low O₂ concentrations (O₂ ⩽3%) maintains stem cell engraftment potential better than at 20% O₂ (NOD/Scid xenograft model). In contrast, progenitors disappear from cultures at/or <1% O₂ concentrations. A very low O₂ concentration (0.1%) induces CD34⁺ quiescence in G₀. The exploration of molecules and mechanisms involved in hematopoietic stem and progenitor cells'' quiescence and differentiation related to low O₂ concentrations is unfeasible with primary CD34⁺ cells. Therefore, we performed it using murine hematopoietic nonleukemic factor-dependent cell Paterson (FDCP)-Mix progenitor cell line. The culture of the FDCP-Mix line at 0.1% O₂ induced in parallel G₀ quiescence and granulo-monocytic differentiation of most cells, whereas a minority of undifferentiated self-renewing cells remained in active cell cycle. Hypoxia also induced hypophosphorylation of pRb and increased the expression of p27^KIP1, the two proteins that have a major role in the control of G₀ and G₁ to S-phase transition.