Major Cell Subsets Research Articles

Complement receptor C5aR1/CD88 and dipeptidyl peptidase-4/CD26 define distinct hematopoietic lineages of dendritic cells.

Differential display of the integrins CD103 and CD11b are widely used to distinguish two major dendritic cell (DC) subsets in nonlymphoid tissues. CD103(+) DCs arise from FLT3-dependent DC precursors (preDCs), whereas CD11b(hi) DCs can arise either from preDCs or FLT3-independent monocytes. Functional characterization of these two lineages of CD11b(hi) DCs has been hindered by the lack of a widely applicable method to distinguish between them. We performed gene expression analysis of fractionated lung DCs from C57BL/6 mice and found that monocyte-derived DCs (moDCs), including CD11b(hi)Ly-6C(lo) tissue-resident and CD11b(hi)Ly-6C(hi) inflammatory moDCs, express the complement 5a receptor 1/CD88, whereas preDC-derived conventional DCs (cDCs), including CD103(+) and CD11b(hi) cDCs, express dipeptidyl peptidase-4/CD26. Flow cytometric analysis of multiple organs, including the kidney, liver, lung, lymph nodes, small intestine, and spleen, confirmed that reciprocal display of CD88 and CD26 can reliably distinguish FLT3-independent moDCs from FLT3-dependent cDCs in C57BL/6 mice. Similar results were obtained when DCs from BALB/c mice were analyzed. Using this novel approach to study DCs in mediastinal lymph nodes, we observed that most blood-derived lymph node-resident DCs, as well as tissue-derived migratory DCs, are cDCs. Furthermore, cDCs, but not moDCs, stimulated naive T cell proliferation. We anticipate that the use of Abs against CD88 and CD26 to distinguish moDCs and cDCs in multiple organs and mouse strains will facilitate studies aimed at assigning specific functions to distinct DC lineages in immune responses.

Fetomaternal Microchimerism Is Associated with Better Outcome in Haploidentical Hematopoietic Stem Cell Transplantation

▪Haploidentical hematopoietic stem cell transplantation (hHSCT) from a parent to a child has become well established for pediatric patients with high risk leukemia in the absence of an HLA-matched donor. Donor choice in hHSCT has been an issue addressed by several groups. Some showed a beneficial impact of certain Killer immunoglobulin-like receptor (KIR)/KIR ligand constellations in different transplantation settings. Others demonstrated an improved outcome after mother-to-child transplantations compared to father-to-child transplantations in terms of lower transplant-related mortality and lower incidence of graft-versus-host disease (GvHD). Obviously, pregnancy leaves an imprint on the mother’s immune system, which is beneficial in hHSCT from mother to child. During pregnancy, a fetal microchimerism (FM) is established in the mother by fetal cells crossing the placental barrier dividing maternal and fetal circulation. In some mothers, fetal cells can be detected in peripheral blood for more than three decades after birth. Yet, not all mothers develop a persisting FM. CD56brightNK cells make up approximately 70% of the lymphocytes in the decidua during the first trimester of pregnancy and represent the major cell subset interacting with cells of the developing fetus. Not surprisingly, certain maternal KIR/fetal KIR ligand combinations are associated with recurrent spontaneous abortions or preeclampsia. We hypothesized that (i) FM plays a role in hHSCT, and that (ii) FM may be influenced by the KIR pattern on maternal NK cells and KIR ligands on the child’s cells.In a multicenter retrospective study, we analyzed 46 pediatric patients from 6 German transplantation centers (Tübingen, Frankfurt, Düsseldorf, Heidelberg, Jena, and Hamburg) who underwent first T-cell depleted hHSCT from a parent following conditioning with fludarabine, thiotepa, melphalan and ATG for standard indications: acute lymphoblastic leukemia (n=10), acute myeloid leukemia (n=10), juvenile myelomonocytic leukemia (n=1), lymphoproliferative disease (n=2), myelodysplastic syndrome (n=4), hemophagocytic lymphohistiocytosis (n=3), immune deficiencies (n=9), and others (n=7). Excess DNA of the donors extracted from peripheral blood prior to transplantation was assayed for fetal DNA (in mother-to-child transplantations), and for KIR genotype by real-time PCR. Mothers who had at least one fetal cell within 250,000 maternal cells could be detected were considered as FM+. Although we confirmed that, overall mothers as a group are better donors than fathers, in closer analysis this held only true for FM+ mother. Overall survival was significantly higher in the group of patients receiving stem cells from their FM+ mother compared to the patients grafted from their FM¯ mother (72% vs. 29), or father (72% vs. 50%). Most interestingly, looking at the minimal residual disease (MRD) negative acute leukemia patients undergoing first allogeneic HSCT for standard indications, overall survival was also significantly higher after hHSCT from FM+ mothers than from FM¯ mothers (66% vs. 26%) and there was a trend towards lower risk of relapse in the FM+ group (25% vs. 52%). There was no case of grade IV acute GvHD and only one or no case of acute GvHD grade III in all groups. Percentages of grade I + II acute GvHD did not differ significantly among FM+and FM¯ groups.Reasons for persistence of fetal cells in some, but not all mothers are controversial. While fetal microchimerism is detected during pregnancy and early after birth in almost all mothers, it is reported to decrease below the detection limit in many mothers over time. In our cohort, we found no correlation of FM with age or gender of the respective child. Since a poor outcome after hHSCT has been reported for patients that are homozygous for HLA-C1, we also grouped our cohort into a HLA-C1 heterozygous and a homozygous group. Interestingly, not only an improved outcome, but also a higher average FM could be seen in the HLA-C1 heterozygous group compared to the C1 homozygous group. Furthermore, mothers expressing KIR2DS1 showed a significantly higher level of FM compared to those negative for this activating KIR. These findings suggest a certain role for KIR and HLA-C on the persistence of fetal microchimerism in mothers.In conclusion, detection of fetal microchimerism may contribute to identification of the most promising parental donor in haploidentical stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

The gene expression profile of CD11c+ CD8α- dendritic cells in the pre-diabetic pancreas of the NOD mouse.

Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c+CD8α− DCs (strong CD4+ T cell proliferation inducers) and the CD8α+CD103+ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c+CD8α− DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c+CD8α− DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c+CD8α− DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c+CD8α− DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c+CD8α− DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.

Adult Thymus Contains FoxN1− Epithelial Stem Cells that Are Bipotent for Medullary and Cortical Thymic Epithelial Lineages

SummaryWithin the thymus, two major thymic epithelial cell (TEC) subsets—cortical and medullary TECs—provide unique structural and functional niches for T cell development and establishment of central tolerance. Both lineages are believed to originate from a common progenitor cell, yet the cellular and molecular identity of these bipotent TEC progenitors/stem cells remains ill defined. Here we identify rare stromal cells in the murine adult thymus, which under low-attachment conditions formed spheres (termed “thymospheres”). These thymosphere-forming cells (TSFCs) displayed the stemness features of being slow cycling, self-renewing, and bipotent. TSFCs could be significantly enriched based on their distinct surface antigen phenotype. The FoxN1 transcription factor was dispensable for TSFCs maintenance in situ and for commitment to the medullary and cortical TEC lineages. In summary, this study presents the characterization of the adult thymic epithelial stem cells and demonstrates the dispensability of FoxN1 function for their stemness.

Evaluation of PBMC processing in the Precision Bioservices laboratory network (HUM7P.325)

Abstract Cryopreservation of peripheral blood mononuclear cells (PBMC) is a good alternative to testing fresh PBMC in clinical trials as functional immunologic assays may be batched to increase efficiency. Functional assays are essential for accurately evaluating the performance of vaccines, antiviral and anticancer agents. Optimal retention of PBMC functionality during cryopreservation depends on optimized and standardized cell isolation, freezing and storage conditions. Collection and analysis of PBMC is a significant technical challenge in multi-center clinical trials due to pre-analytical variability in processing, freezing and storage. It is vital that all centers participating in a multi-center collaboration operate with consistent protocols. To this end, Precision has developed a Laboratory Network (PLN) to ensure quality PBMC processing across multi-center trials. The current study was performed within the PLN to ensure samples processed at each laboratory achieved similar results. We report the recovery, viability and functionality of PBMC obtained from participating laboratories. Phenotypic characterization of the major cell subset was also analyzed and the percent obtained was comparable across laboratories. This data provides confidence that PBMC processing in a multi-center clinical trial operating with the same standard operating procedures (SOPs) produces comparable data, thus accelerating drug and vaccine development.

In Vivo Administration of a JAK3 Inhibitor during Acute SIV Infection Leads to Significant Increases in Viral Load during Chronic Infection

The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8+ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4+ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a+ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses.

HIV Type 1 Infection of Plasmacytoid and Myeloid Dendritic Cells Is Restricted by High Levels of SAMHD1 and Cannot be Counteracted by Vpx

Dendritic cells are professional antigen-presenting cells of the immune system and are major producers of type-I interferon. Their role in HIV-1 infection is not well understood. They express CD4 and CCR5 yet appear to be resistant to infection. In culture, infection of the cells with HIV-1 is inhibited by the host cell restriction factor SAMHD1. Lentiviruses such as HIV-2/SIVmac counteract the restriction by encoding Vpx, a virion-packaged accessory protein that induces the proteasomal degradation of SAMHD1. In this study we investigated SAMHD1-mediated restriction in the two major dendritic cell subsets: plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC). The cells were highly resistant to HIV-1 and expressed high levels of SAMHD1. SAMHD1 amino acid residue T592, a target of CDK1 phosphorylation, was unphosphorylated, corresponding to the antiviral form of the enzyme. The resistance to infection was not counteracted by Vpx and SAMHD1 was not degraded in these cells. Treatment of pDCs with a cocktail of antibodies that blocked type-I interferon signaling partially restored the ability of Vpx to induce SAMHD1 degradation and caused the cells to become partially permissive to infection. pDCs and mDCs responded to HIV-1 virions by inducing an innate immune response but did not appear to sense newly produced Gag protein. The findings suggest that in vivo, dendritic cells serve as sentinels to alert the immune system to the virus but do not themselves become infected by virtue of high levels of SAMHD1.

NK cells dysfunction in systemic lupus erythematosus: relation to disease activity

Through their cytotoxic capacities and cytokine production, natural killer (NK) cells modulate autoimmune diseases. However, their role in the pathogenesis of systemic lupus erythematosus (SLE) has not been extensively studied. The aim of this study was to analyse the immunophenotypic and functional characteristics of the two major NK cell subsets in SLE and relate them with disease activity. Peripheral blood samples from 44 patients with active (n = 18) and inactive SLE (n = 26) and 30 controls were analysed by flow cytometry to evaluate NK cell subsets, according to: the differential expression of CXCR3 and CD57; expression of granzyme B and perforin; and production of interferon gamma (IFN-γ) and tumor necrosis alpha (TNF-α), after PMA/ionomycin activation. A clear decrease in absolute and relative numbers of circulating NK cells was found in SLE, particularly in active disease, while the proportions of the major NK cell subsets were unaffected. Active SLE was associated with a reduced CXCR3 expression on both NK cell subsets and a lower frequency of CD56(dim) NK cells expressing CXCR3. Furthermore, granzyme B expression was decreased in both SLE groups, but the percentage of NK cells expressing granzyme B and perforin was higher, particularly in active disease. We found a significant decrease in the percentage of CD56(bright) and CD56(dim) NK cells producing TNF-α and of its expression on CD56(dim) NK cells in active disease, while IFN-γ expression on CD56(bright) NK cells was increased in both SLE groups. Our findings suggest that NK cell subsets exhibit unique phenotypic and functional changes that are particularly evident in active SLE, and they may have the potential to affect the disease outcome.

Functional diversity of human vaginal APC subsets in directing T-cell responses.

Human vaginal mucosa is the major entry site of sexually transmitted pathogens and thus has long been attractive as a site for mounting mucosal immunity. It is also known as a tolerogenic microenvironment. Here, we demonstrate that immune responses in the vagina are orchestrated by the functional diversity of four major antigen-presenting cell (APC) subsets. Langerhans cells (LCs) and CD14− lamina propria (LP)-DCs polarize CD4+ and CD8+ T cells toward Th2, whereas CD14+ LP-DCs and macrophages polarize CD4+ T cells toward Th1. Both LCs and CD14− LP-DCs are potent inducers of Th22. Due to their functional specialties and the different expression levels of pattern-recognition receptors on the APC subsets, microbial products do not bias them to elicit common types of immune responses (Th1 or Th2). To evoke desired types of adaptive immune responses in the human vagina, antigens may need to be targeted to proper APC subsets with right adjuvants.

Th17 cells: critical mediators of host responses to burn injury and sepsis.

Th cells have long been recognized as vital components of the adaptive immune system. Until recently, CD3(+)CD4(+) Th cells were divided into cell-mediated Th1 or humoral Th2 responses. However, the Th1-Th2 hypothesis failed to accommodate the more recently described Th17 cells. Today, the major Th cell subsets include Th1, Th2, Th9, Th17, Th22, and Tregs, each of which produce specific effector cytokines under unique transcriptional regulation. Specifically, Th17 cells produce effector cytokines IL-17, IL-21, and IL-22 under the regulation of ROR-γt. Th17 lymphocytes were first described as orchestrators of neutrophil recruitment and activation and as key players in chronic inflammation and autoimmunity. More recent evidence suggest that Th17 lymphocytes and their effector cytokines play a crucial role in maintaining mucosal immunity and barrier integrity, including the skin, lung, and gut. Burn injury induces global changes to the systemic immune response, including suppressed immune function and increased susceptibility to infection. Moreover, burn trauma is associated with remote organ injury. This relationship between burn and remote organ injury supports the hypothesis that immune suppression may facilitate the development of sepsis, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome in critically ill burn patients. Herein, we discuss this emerging adaptive cell subset in critical care settings, including burn injury and clinical sepsis, and highlight the potential therapeutic role of IL-22.

Early Induction of CCL7 Downstream of TLR9 Signaling Promotes the Development of Robust Immunity to Cryptococcal Infection

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.

Natural killer cell subsets in man and rodents

NK cells are important contributors to the early immune defence against infected or transformed cells. They are rapidly activated in response to cytokines, whereby they exert their effector functions. NK cell responses are controlled by a multitude of receptors, which are expressed by subpopulations of NK cells with distinct phenotypes and functionalities. Direct comparisons between species are often difficult because of differences in the expression of NK cell receptors and other markers. In addition, NK cells change their phenotype and effector functions during differentiation, by tissue-specific factors, or upon activation, complicating interpretations. We will here review the similarities and differences between the major NK cell subsets in man and two well-characterized rodent models.

Pulmonary mucosal dendritic cells in T-cell activation: implications for TB therapy

Mycobacterium tuberculosis, the causative agent of pulmonary TB, causes chronic intracellular infection of lung-resident antigen-presenting cells, including macrophages and dendritic cells (DCs). Life-long bacterial control requires robust T-cell immune responses. Lung DCs are critical for initiating and co-ordinating adaptive immune responses against TB. The recent description of previously uncharacterized DC subsets has prompted the re-examination of lung DCs and their role in priming antimycobacterial T-cell responses. While there is some data on these new DCs in their naive state, very little is known about how these DCs respond to pulmonary mycobacterial infection. In this article, we attempt to identify the major antigen-presenting cell subsets that may be critical to lymph node homing, T-cell priming and controlling mycobacterial infection. We further examine the areas of DC heterogeneity that may relate to differential susceptibility between mouse strains. Furthermore, we discuss how DCs may be manipulated and exploited as a cell-based prophylactic TB vaccine and the prospect of using this strategy for post-M. tuberculosis exposure settings.

Abstract 5328: Expansion of CD11b+CD33lowCD15high immunosuppressive myeloid cell population in patients with bladder carcinoma

Abstract Recent studies highlighted the immunoregulatory role of CD11b myeloid cells in cancer-associated immune non-responsiveness. Increased presence of myeloid-derived suppressor cells (MDSC) is one of the major factors responsible for immune suppression in tumor hosts. Here we describe the expanded CD11b myeloid cell subset in peripheral blood obtained from patients with bladder cancer. Based on expression of CD33 and CD15 myeloid cells markers, the circulating CD11b-positive cells in patients with bladder cancer could be subdivided into two major subpopulations: CD33lowCD15high and CD33highCD15low. First subpopulation (CD11b+CD33lowCD15high cells) has granulocyte-type morphology, express G-CSF receptor (CD114) and secrete elevated amounts of pro-inflammatory cytokines and chemokines such as G-CSF, MCP-1 MIP-1alpha, MIP-1beta. Second major myeloid cell subset from cancer patients (CD11b+CD33high CD15lowcells) displays a monocyte-type cell morphology and co-express CD14, M-CSF receptor (CD115), CCR2 and HLA-DR. We show here that proportion of granulocytic CD33lowCD15high cells is highly elevated in cancer patients as compared to healthy individuals. Patients with bladder cancer but not healthy volunteers show weak T cell proliferative response after in vitro stimulation. We demonstrate that granulocyte-type CD33lowCD15high cell population is involved in T cell hyporesponsiveness, since specific depletion of this cell subset restored the T cell proliferation. Obtained results suggest that fraction of PBMC in patients with bladder cancer patients contains markedly elevated proportion of CD11b+CD33lowCD15high granulocyte-type myeloid cell population which contributes to cancer-associated inflammation and tumor-induced immune suppression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5328.

Thymic Stromal Lymphopoietin-Activated Plasmacytoid Dendritic Cells Induce the Generation of FOXP3+ Regulatory T Cells in Human Thymus

Human thymus contains major dendritic cell (DC) subsets, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs). We previously showed that mDCs, educated by thymic stromal lymphopoietin (TSLP) produced by the epithelial cells of the Hassall's corpuscles, induced differentiation of CD4(+)CD25(-) thymocytes into Forkhead Box P3(+) (FOXP3(+)) regulatory T cells (T(R)) within the medulla of human thymus. In this study, we show that pDCs expressed the TSLP receptor and IL-7 receptor alpha complexes upon activation and became responsive to TSLP. TSLP-activated human pDCs secrete macrophage-derived chemokine CCL-22 and thymus- and activation-regulated chemokine CCL-17 but not Th1- or Th2-polarizing cytokines. TSLP-activated pDCs induced the generation of FOXP3(+) T(R) from CD4(+)CD8(-)CD25(-) thymocytes, which could be strongly inhibited by Th1-polarizing cytokine IL-12 or Th2-polarizing cytokine IL-4. Interestingly, the FOXP3(+) T(R) induced by the TSLP-pDCs expressed more IL-10 but less TGF-beta than that induced by the TSLP-mDCs. These data suggest that TSLP expressed by thymic epithelial cells can activate mDCs and pDCs to positively select the FOXP3(+) T(R) with different cytokine production potential in human thymus. The inability of TSLP to induce DC maturation without producing Th1- or Th2-polarizing cytokines may provide a thymic niche for T(R) development.

Statistical mixture modeling for cell subtype identification in flow cytometry

Statistical mixture modeling provides an opportunity for automated identification and resolution of cell subtypes in flow cytometric data. The configuration of cells as represented by multiple markers simultaneously can be modeled arbitrarily well as a mixture of Gaussian distributions in the dimension of the number of markers. Cellular subtypes may be related to one or multiple components of such mixtures, and fitted mixture models can be evaluated in the full set of markers as an alternative, or adjunct, to traditional subjective gating methods that rely on choosing one or two dimensions. Four color flow data from human blood cells labeled with FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PE-Cy5-conjugated anti-CD4, and APC-conjugated anti-CD19 Abs was acquired on a FACSCalibur. Cells from four murine cell lines, JAWS II, RAW 264.7, CTLL-2, and A20, were also stained with FITC-conjugated anti-CD11c, PE-conjugated anti-CD11b, PE-Cy5-conjugated anti-CD8a, and PE-Cy7-conjugated-CD45R/B220 Abs, respectively, and single color flow data were collected on an LSRII. The data were fitted with a mixture of multivariate Gaussians using standard Bayesian statistical approaches and Markov chain Monte Carlo computations. Statistical mixture models were able to identify and purify major cell subsets in human peripheral blood, using an automated process that can be generalized to an arbitrary number of markers. Validation against both traditional expert gating and synthetic mixtures of murine cell lines with known mixing proportions was also performed. This article describes the studies of statistical mixture modeling of flow cytometric data, and demonstrates their utility in examples with four-color flow data from human peripheral blood samples and synthetic mixtures of murine cell lines.

The Ontogeny and Fate of NK Cells Marked by Permanent DNA Rearrangements

A subset of NK cells bears incomplete V(D)J rearrangements, but neither the consequence to cell activities nor the precise developmental stages in which recombination occurs is known. These are important issues, as recombination errors cause cancers of the B and T lineages. Using transgenic recombination reporter mice to examine NK cell dynamics in vivo, we show that recombination(+) NK cells have distinct developmental patterns in the BM, including reduced homeostatic proliferation and diminished Stat5 phosphorylation. In the periphery, both recombination(+) and recombination(-) NK cells mediate robust functional responses including IFN-gamma production, cytolysis, and tumor homing, suggesting that NK cells with distinct developmental histories can be found together in the periphery. We also show that V(D)J rearrangement marks both human cytolytic (CD56(dim)) and immunoregulatory (CD56(bright)) populations, demonstrating the distribution of permanent DNA rearrangements across major NK cell subsets in man. Finally, direct quantification of rag transcripts throughout NK cell differentiation in both mouse and man establishes the specific developmental stages that are susceptible to V(D)J rearrangement. Together, these data demonstrate that multipotent progenitors rather than lineage-specified NK progenitors are targets of V(D)J recombination and that NK cells bearing the relics of earlier V(D)J rearrangements have different developmental dynamics but robust biological capabilities in vivo.

Elevated CD56dimCD16neg NK Cells on Day 28 after HCT: Role in Immunity Against Viral Infections or Residual Tumor Cells?.

Natural killer (NK) cells represent a lymphocyte subset important in the immune surveillance against virus infected and malignant cells. In humans, two major NK cell subsets have been defined by CD56 and CD16 staining:1.Regulatory NK cells (CD56 high CD16 neg), which are potent cytokine (IFNg) producers; and2.Cytolytic NK cells (CD56 dim CD16 pos) that are involved in target cell lysis.Functions of other NK cell subset are unknown. One of such subset, CD56 dimCD16 neg NK cells have recently been reported to represent cytolytic cells, degranulated after contact with target cells. NK cells are the first lymphocyte subset recovering to normal quantity and function after an allogeneic or autologous HCT, typically by 4 weeks post HCT. In the present investigation, we attempted to determine percentages of following NK cell subsets (Regulatory NK cells, Cytolytic NK cells, CD56dimCD16 neg, CD56 highCD16 pos and CD56neg CD16 high NK cells) at multiple time points (day 28, 56, 84, 180 and 365) after allogeneic (n= 32) and autologous (n= 25) HCT and compared with that of healthy individuals (n=14). Whole blood specimen was incubated with fluorochrome-conjugated monoclonal antibodies (CD3-ECD, CD14-PE, CD56-FITC and CD16-PC5) and analyzed by four color flowcytometry. NK cells were defined as mononuclear cells expressing CD16 or CD56 and not expressing CD3 or CD14. The most striking finding was that of significant increase in percentage of CD56dimCD16−ve cells (among NK cells) on day 28. This was true for both autologous HCT recipient (n=24; median = 1.02% of total NK cells) and allogeneic HCT recipient (n=26; median = 1.1% of total NK cells) in comparison to the healthy individuals (n=14; median = 0.56% of total NK cells). We speculate the rise in CD56dimCD16−ve NK cells at 1 month post HCT may be due to increased degranulation of cytolytic NK cells in response to viral infections or residual malignancy.

Plasmacytoid dendritic cells represent a major dendritic cell subset in sentinel lymph nodes of melanoma patients and accumulate in metastatic nodes

Plasmacytoid dendritic cells (pDC) represent the main source of interferon-α, a cytokine with antitumor activity. However, in vitro studies point to pDC as a key subset for induction of tolerance. Herein, we investigated pDC in sentinel lymph nodes (SLN) of melanoma patients. We report that pDC were constantly found in SLN and represented, with Langerhans cells, the most frequent dendritic cell subset. Their frequency in positive (with metastasis) SLN was significantly higher than in negative (without metastasis) SLN. PDC were observed in the T cell-rich areas of lymph nodes, particularly around high endothelial venules and, in metastatic nodes, they accumulated in close vicinity with melanoma nests. Finally, pDC capability to produce interferon-α in situ was impaired. Consistently, pDC expressed CD86, but neither CD80 nor CD83, suggesting a not complete activation in melanoma-draining lymph nodes. These results are consistent with the hypothesis of a tolerogenic role played by pDC in tumor immunology.

Relevance of DC-SIGN in DC-induced T cell proliferation

The role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in DC-T cell communication was assessed by analyzing the effect of DC-SIGN-blocking mAb in MLR. The results show that the degree of inhibition by DC-SIGN and LFA-1 mAb depends on the magnitude of the MLR and the maturation status of the DC. Addition of DC-SIGN mAb at several time-points during MLR showed that DC-SIGN is involved early on in DC-T cell contacts. This initial role is masked by strong adhesive and costimulatory mechanisms, indicating a short-lived effect of DC-SIGN in DC-T cell interactions. To examine this concept in more detail, the percentage of PBL capable of binding DC-SIGN was determined. Analysis of several donors revealed that 1-20% PBL bind to beads coated with recombinant DC-SIGN, and the DC-SIGN-binding cells comprised all major cell subsets found in blood. PBL isolated from a donor with high DC-SIGN-binding capacity were more prone to blocking by DC-SIGN mAb in MLR than PBL from a donor with low DC-SIGN-binding capacity. This study indicates an initial and transient role for DC-SIGN in T cell proliferation, which becomes apparent when T cell proliferation is low and when the percentage of DC-SIGN binding PBL is high.