Presence Of CD11c Research Articles

Impact of aquaporin-4 and CD11c + microglia in the development of ependymal cells in the aqueduct: inferences to hydrocephalus

AQP4 is expressed in the endfeet membranes of subpial and perivascular astrocytes and in the ependymal cells that line the ventricular system. The sporadic appearance of obstructive congenital hydrocephalus (OCHC) has been observed in the offspring of AQP4−/− mice (KO) due to stenosis of Silvio’s aqueduct. Here, we explore whether the lack of AQP4 expression leads to abnormal development of ependymal cells in the aqueduct of mice. We compared periaqueductal samples from wild-type and KO mice. The microarray-based transcriptome analysis reflected a large number of genes with differential expression (809). Gene sets (GS) associated with ependymal development, ciliary function and the immune system were specially modified qPCR confirmed reduced expression in the KO mice genes: (i) coding for transcription factors for ependymal differentiation (Rfx4 and FoxJ1), (ii) involved in the constitution of the central apparatus of the axoneme (Spag16 and Hydin), (iii) associated with ciliary assembly (Cfap43, Cfap69 and Ccdc170), and (iv) involved in intercellular junction complexes of the ependyma (Cdhr4). By contrast, genes such as Spp1, Gpnmb, Itgax, and Cd68, associated with a Cd11c-positive microglial population, were overexpressed in the KO mice. Electron microscopy and Immunofluorescence of vimentin and γ-tubulin revealed a disorganized ependyma in the KO mice, with changes in the intercellular complex union, unevenly orientated cilia, and variations in the planar cell polarity of the apical membrane. These structural alterations translate into reduced cilia beat frequency, which might alter cerebrospinal fluid movement. The presence of CD11c + microglia cells in the periaqueductal zone of mice during the first postnatal week is a novel finding. In AQP4−/− mice, these cells remain present around the aqueduct for an extended period, showing peak expression at P11. We propose that these cells play an important role in the normal development of the ependyma and that their overexpression in KO mice is crucial to reduce ependyma abnormalities that could otherwise contribute to the development of obstructive hydrocephalus.

CD11c regulates late-stage T cell development in the thymus.

CD11c, also named integrin αX, has been deemed solely as a dendritic cell marker for decades while the delineation of its biological function was limited. In the current study, we observed in mice that CD11c deficiency led to a defect in T cell development, demonstrated by the loss of CD4+CD8+ double positive (DP) T cells, CD4+CD8-, and CD4-CD8+ single positive (SP) T cells in the thymus and less mature T cells in the periphery. By using bone marrow chimera, we confirmed that CD11c regulated T cell development in the thymus. We further showed that CD11c deficiency led to an accelerated apoptosis of CD3 positive thymocytes, but not CD4-CD8- double negative (DN) T cells. Overall, this study added one more layer of knowledge on the regulatory mechanism of late-stage T cell development that the presence of CD11c in the thymus is critical for maintaining T cell survival.

CD11c+ B Cells Participate in the Pathogenesis of Graves' Disease by Secreting Thyroid Autoantibodies and Cytokines.

Graves’ disease (GD) is a common autoimmune disorder with an elevation in pathogenic autoantibodies, specifically anti-thyrotropin receptor antibodies (TRAbs), which are secreted by autoreactive B cells. To date, there has been little research on self-reactive B cells in GD. In the current study, we reported that a unique B-cell subset, CD11c+ B cells, was expanded in the peripheral blood (PB) of GD patients, as detected by flow cytometry. The frequency of CD11c+ B cells was positively correlated with serum TRAb levels. The flow cytometry data showed that CD11c expression was higher in a variety of B-cell subsets and that CD11c+ B cells presented a distinct immunophenotype compared to paired CD11c- B cells. Immunohistochemical and immunofluorescence staining indicated the presence of CD11c+CD19+ B cells in lymphocyte infiltration areas of the GD thyroid. Flow cytometric analysis of PB and fine-needle aspiration (FNA) samples showed that compared to PB CD11c+ B cells, CD11c+ B cells in the thyroid accumulated and further differentiated. We found that CD11c+ B cells from the PB of GD patients were induced to differentiate into autoreactive antibody-secreting cells (ASCs) capable of secreting TRAbs in vitro. Luminex liquid suspension chip detection data showed that CD11c+ B cells also secreted a variety of cytokines, including proinflammatory cytokines, anti-inflammatory cytokines, and chemokines, which might play roles in regulating the local inflammatory response and infiltration of lymphocytes in the thyroid. In addition, we performed a chemotaxis assay in a Transwell chamber to verify that CD11c+ B cells were recruited by thyroid follicular cells (TFCs) via the CXCR3-CXCL10 axis. In conclusion, our study determined that CD11c+ B cells were involved in the pathogenesis of GD in multiple ways and might represent a promising immunotherapeutic target in the future.

BCR activated CLL B cells use both CR3 (CD11b/CD18) and CR4 (CD11c/CD18) for adhesion while CR4 has a dominant role in migration towards SDF-1

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western world. In previous studies, various proportion of patients was found to carry CD11b+ or CD11c+ B cells whose presence was an unfavourable prognostic factor. The exact mechanism however, how these receptors contribute to the pathogenesis of CLL has not been revealed so far. Here we analysed the role of CD11b and CD11c on B cells of CLL patients in the adhesion to fibrinogen and in the migration towards stromal cell derived factor-1 (SDF-1) and studied the role of CR4 in the adherence of the CD11c+ B cell line BJAB. We observed that both CR3 and CR4 mediate adhesion of the malignant B cells. Moreover, we found, that CR4 was strongly involved in the migration of the leukemic cells towards the chemoattractant SDF-1. Our data suggest that CR3 and CR4 are not only passive markers on CLL B cells, but they might contribute to the progression of the disease. Since the role of SDF-1 is prominent in the migration of CLL cells into the bone marrow where their survival is supported, our findings help to understand how the presence of CD11c on leukemic B cells can worsen the prognosis of chronic lymphocytic leukaemia.

Evaluation of the host immune response and functional recovery in peripheral nerve autografts and allografts

Allogeneic peripheral nerve (PN) transplants are an effective bridge for stimulating regeneration of segmental PN defects, but there are currently no detailed studies about the timeline and scope of the immunological response for PN allografting. In this study, the cellular immune response in PN allografts and autograft was studied during the acute and chronic phases of a 1.0 cm critical size defect in the rat sciatic nerve at 3, 7, 14, 28 and 98 days after grafting autologous or allogeneic nerves without any immunosuppressive treatment. The assessment was based on functional, histomorphometrical and immunohistochemical criteria. Results showed modestly better functional outcomes for autografts with coordinate and adaptive immune response represented by the presence of CD11c, CD3, CD4, NKp46 and CD8 cells at 3 days, CD45R positive cells and CD25 positive cells at seven and CD45R positive cells at 14 days which seems an adaptive immune response. In contrast, allograft in the early time points showed innate immune response instead of adaptive immune response until day 14, when there was some increase in cell-mediated immunity. In conclusion, in PN autografts the immune system is synchronic initiating with a more robust early innate response that more rapidly transitions to adaptive while for PN allografts the infiltration of immune cells is slower and more gradually progresses to a moderate adaptive response.

CC chemokine receptor 2 promotes recruitment of myeloid cells associated with insulin resistance in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a common disease, closely associated with obesity and insulin resistance. We investigated the presence of a subset of myeloid cells associated with metabolic disturbance in the liver of patients with NAFLD and a murine model of obesity-induced liver disease. Gene and protein expression in liver and serum was investigated with RT-PCR or ELISA and correlated to clinical disease. Liver-infiltrating immune cells were isolated from normal or diseased human liver for flow cytometric analysis. In animal experiments, mice were fed a high-fat diet (60% of calories from fat) for 16 wk, or high-fat diet with 30% fructose for 32 wk to induce steatohepatitis and fibrosis. A small molecule inhibitor of CC chemokine receptor 2 (CCR2), CCX872, was administered to some mice. A subset of CD11c+CD206+ immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11c+CD206+ myeloid cells correlated with systemic insulin resistance. CD11c+CD206+ cells expressed high levels of CCR2, and liver CC chemokine ligand 2 (CCL2) expression was increased in nonalcoholic steatohepatitis and correlated with disease activity. In mice, CCR2 inhibition reduced infiltration of liver CD11b+CD11c+F4/80+ monocytes, which are functional homologs of human CD11c+CD206+ cells, and improved liver injury and glycemic control. A role for CCR2/CCL2 in human NAFLD has long been postulated. These data confirm a role for this chemokine/receptor axis, through mediating adipose and hepatic infiltration of myeloid cells. Inhibition of CCR2 improved hepatic inflammation and fibrosis in murine models of NAFLD. These data confirm the rationale for targeting CCR2 to treat NAFLD. NEW & NOTEWORTHY These data show for the first time that CD11c+CD206+ myeloid cells, previously associated with human adipose tissue inflammation, infiltrate into liver tissue in nonalcoholic fatty liver disease. These cells express CCR2. Inhibition of CCR2 in mice inhibits hepatic inflammation caused by a murine homolog of these myeloid cells and improves experimental liver disease.

CD11c+ cells are required for lymphocyte trafficking into previously infiltrated pancreatic islets during type 1 diabetes.

Abstract Type 1 diabetes (T1D) is a largely T cell mediated autoimmune disease that destroys the beta cells of the pancreatic islets. Inhibition of cell trafficking to an active disease site has been an effective therapy for multiple autoimmune diseases, but the requirements of lymphocyte trafficking to the islets are not fully understood. We show that lymphocyte entry into previously infiltrated islets is dependent on the presence of CD11c+ cells in the islets. T cells and B cells transferred prior to CD11c+ cell depletion were able to enter the islets; whereas, short-term CD11c+ cell depletion rapidly prevented further entry. CD11c+ cells include cells that are highly efficient antigen presenters; however, we show entry of both activated or naïve T cells into previously infiltrated islets was not reliant on antigen. An alternative role for CD11c+ cells in lymphocyte entry into the islets is the production of chemokines and cytokines. These molecules can then either directly recruit lymphocytes through chemoattraction or increased adhesion to the vasculature. Initial experiments show that there are no changes in adhesion to the vasculature after CD11c depletion, suggesting that CD11c+ cells act through recruitment by chemokines following lymphocyte arrest on the vascular endothelium. CD11c+ cells in the islets express high levels of CXCL9, a chemoattractant for T and B lymphocytes. CXCR3, the receptor for CXCL9, is present on both T and B cells in the islets. We hypothesize that CD11c+ cells facilitate the process of extravasation into the islets through chemokine production. Targeting CD11c+ produced chemokine production may be useful therapeutically for the treatment of T1D by preventing T cell entry into remaining or transplanted islets.

Role of heterogeneous cell population on modulation of dendritic cell phenotype and activation of CD8 T cells for use in cell-based immunotherapies

Dendritic cell (DC)-based immunotherapies have much utility in their ability to prime antigen-specific adaptive immune responses. However, there does not yet exist a consensus standard to how DCs should be primed. In this study, we aimed to determine the role of heterogeneous co-cultures, composed of both CD11c+ (DCs) and CD11c− cells, in combination with monophosphoryl lipid A (MPLA) stimulation on DC phenotype and function. Upon DC priming in different co-culture ratios, we observed reduced expression of MHCII and CD86 and increased antigen uptake among CD11c+ cells in a CD11c− dependent manner. DCs from all culture conditions were induced to mature by MPLA treatment, as determined by secretion of pro-inflammatory cytokines IL-12 and TNF-α. Antigen-specific stimulation of CD4+ T cells was not modulated by co-culture composition, in terms of proliferation nor levels of IFN-γ. However, the presence of CD11c− cells enhanced cross-presentation to CD8+ T cells compared to purified CD11c+ cells, resulting in increased cell proliferation along with higher IFN-γ production. These findings demonstrate the impact of cell populations present during DC priming, and point to the use of heterogeneous cultures of DCs and innate immune cells to enhance cell-mediated immunity.

Analysis of dendritic cell subpopulations in follicular lymphoma with respect to the tumor immune microenvironment

The immune cell composition of the follicular lymphoma (FL) tumor microenvironment is increasingly recognized as an important determinant for clinical outcome. Here, we explored frequency and distribution of dendritic cell (DC) subtypes in relation to regulatory T cells (Treg) by immunohistochemistry in lymph node biopsies from patients with de novo FL. We found that neoplastic follicles contained lower DC and higher Treg frequencies than hyperplastic follicles in control lymph nodes. Treg numbers particularly correlated with the subset of conventional CD11c+ DCs. Additionally, both a high intra- to interfollicular ratio of CD11c+ DCs and increased intrafollicular Treg frequencies were associated with decreased overall survival. This suggests that functional interactions between these cells may be relevant for FL progression/recurrence. The presence of CD11c+ DCs in the tumor microenvironment may assist tumor infiltration by Tregs, thus contributing to the suppression of an otherwise beneficial T-cell-dominated FL microenvironment.

Abstract 2554: DDX3X-specific effector T cells in small cell lung cancer patients reflect disease stage

Abstract [Background] Small cell lung cancer (SCLC) possesses high tendency to disseminate. However, SCLC patients with paraneoplastic syndrome mediated by immunity against onconeural antigens remain in limited-stage disease (LD) without distant metastases. We previously demonstrated that effector-dominant CD4+ T cell balance was observed only in LD-SCLC but not in extended-stage disease (ED)-SCLC. CD4+ T cell balance in peripheral blood of SCLC patients reflected disease stage and recurrence. Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, is responsible for distant metastases and recurrence. We previously reported that DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133+ murine melanoma cells exhibiting biological CSC features and that DDX3X-primed CD4+ T cells mediated potent antitumor therapeutic efficacy. We found that DDX3X is strongly expressed in small cell lung cancer (SCLC). [Objectives] In the current study, we examine that DDX3X-specific effector T cells exist in SCLC patients. [Patients and Methods] The present study comprised 20 consecutive SCLC patients, 5 long-term survivors, and 10 healthy volunteers from a single institution (Niigata University Medical and Dental Hospital). Specimens were collected after obtaining written informed consent approved by the Niigata University Ethical Committee. [Results] Six of 12 LD-SCLC patients possessed DDX3X-responsive effector T cells. CD62Llow CD4+ effector T cells obtained from peripheral blood of 5 LD-SCLC secreted significantly more IFNγ upon DDX3X antigen stimulation in the presence of CD11c+ autologous dendritic cells. CD62Llow CD8+ effector T cells from one LD-SCLC patient responded to DDX3X. In contrast, effector T cells obtained from ED-SCLC patients or healthy volunteers never responded to DDX3X. [Conclusion] DDX3X is likely one of major antigens that is expressed in SCLC and is recognized by T cell immune system. Antitumor immunotherapy to induce effector-dominant CD4+ T-cell immunity on DDX3X antigen may be a promising therapy to eliminate SCLC cells that mediate distant metastases. Citation Format: Natsue Igarashi, Hiroshi Kagamu, Koichiro Nozaki, Satoshi Shoji, Masaaki Okajima, Satoru Miura, Satoshi Watanabe, Hirohisa Yoshizawa, Ichiei Narita. DDX3X-specific effector T cells in small cell lung cancer patients reflect disease stage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2554. doi:10.1158/1538-7445.AM2014-2554

Identification of Adipose Tissue Dendritic Cells Correlated With Obesity-Associated Insulin-Resistance and Inducing Th17 Responses in Mice and Patients

T-cell regulation in adipose tissue provides a link between inflammation and insulin resistance. Because of alterations in adipose tissue T-cell composition in obesity, we aimed to identify the antigen-presenting cells in adipose tissue of obese mice and patients with insulin resistance. Dendritic cells (DCs) and T cells were studied in mice and in two cohorts of obese patients. In lean mice, only CD11c+ DCs were detected in adipose tissue. Adoptive transfer of naive CD4+ T cells in Rag1−/− mice led to a predominant Th1 response in adipose tissue. In contrast, during obesity DCs (human CD11c+CD1c+ and mouse CD11chighF4/80low) accumulated in adipose tissue. CD11chighF4/80low DCs from obese mice induced Th17 differentiation. In patients, the presence of CD11c+CD1c+ DCs correlated with the BMI and with an elevation in Th17 cells. In addition, these DCs led to ex vivo Th17 differentiation. CD1c gene expression further correlated with homeostatic model assessment-insulin resistance in the subcutaneous adipose tissue of obese patients. We show for the first time the presence and accumulation of specific DCs in adipose tissue in mouse and human obesity. These DCs were functional and could be important regulators of adipose tissue inflammation by regulating the switch toward Th17 cell responses in obesity-associated insulin resistance.

HIV-1 Infection of Human Intestinal Lamina Propria CD4+ T Cells In Vitro Is Enhanced by Exposure to Commensal Escherichia coli

Microbial translocation has been linked to systemic immune activation in HIV-1 disease, yet mechanisms by which microbes may contribute to HIV-associated intestinal pathogenesis are poorly understood. Importantly, our understanding of the impact of translocating commensal intestinal bacteria on mucosal-associated T cell responses in the context of ongoing viral replication that occurs early in HIV-1 infection is limited. We previously identified commensal Escherichia coli-reactive Th1 and Th17 cells in normal human intestinal lamina propria (LP). In this article, we established an ex vivo assay to investigate the interactions between Th cell subsets in primary human LP mononuclear cells (LPMCs), commensal E. coli, and CCR5-tropic HIV-1(Bal). Addition of heat-killed E. coli to HIV-1-exposed LPMCs resulted in increases in HIV-1 replication, CD4 T cell activation and infection, and IL-17 and IFN-γ production. Conversely, purified LPS derived from commensal E. coli did not enhance CD4 T cell infection. E. coli exposure induced greater proliferation of LPMC Th17 than Th1 cells. Th17 cells were more permissive to infection than Th1 cells in HIV-1-exposed LPMC cultures, and Th17 cell infection frequencies significantly increased in the presence of E. coli. The E. coli-associated enhancement of infection was dependent on the presence of CD11c(+) LP dendritic cells and, in part, on MHC class II-restricted Ag presentation. These results highlight a potential role for translocating microbes in impacting mucosal HIV-1 pathogenesis during early infection by increasing HIV-1 replication and infection of intestinal Th1 and Th17 cells.

The intranasal delivery of non-specific virus-like particles elicits heightened and accelerated immune responses to influenza virus (49.27)

Abstract We have previously shown that the intranasal delivery of a small heat-shock protein cage nanoparticle, a virus-like particle (VLP), elicits the formation of inducible bronchus-associated lymphoid tissue (iBALT), which provides protection against many diverse high-dose pathogen challenges. We demonstrate here that the presence of non-specific, VLP-induced iBALT facilitated accelerated influenza virus clearance and enhanced immunity through accelerated airway (CD103+) and parenchymal (CD11b+) dendritic cell antigen processing and trafficking. These responses were dependent upon the presence of CD11c+ cells, which had been directly exposed to VLPs, and migrated into the lung in a CCR2, VCAM-1, and ICAM-1-dependent manner. The initial innate response to influenza challenge was then followed by an accelerated expansion of influenza-specific CD4+ T cells. The combination of these events resulted in accelerated viral clearance and significantly ameliorated host-derived collateral damage. Furthermore, we show that the presence of iBALT, which is not specific to influenza virus, was efficient at clearing high-dose virus, independently of the TBLN’s help. Thus, we herein demonstrate the mechanism by which the presence of iBALT, derived from non-specific, non-pathogenic VLPs, and lacking any memory cells to agents of subsequent challenge, elicited alternative cellular trafficking and expansion patterns, and thus cleared virus at an accelerated rate, with less harmful consequence.

Contribution of immature muscle precursors to myositis pathogenesis: a source of type I interferon and a possible target of the immune attack

ObjectivesTo investigate the contribution of immature muscle precursors to the adaptive and innate immune responses in inflammatory myopathy.MethodMuscle biopsies from eight patients with polymyositis and dermatomyositis were investigated for the...

Prolonged Antigen Presentation Is Required for Optimal CD8+ T Cell Responses against Malaria Liver Stage Parasites

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization—a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites (137.16)

Abstract Immunization with irradiated sporozoites is the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization - a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate into liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite antigen.

Transport of Bacillus anthracis from the lungs to the draining lymph nodes is a rapid process facilitated by CD11c+ cells

Inhalational anthrax is established after inhaled Bacillus anthracis spores are transported to the lung associated lymph nodes. Dendritic cells (CD11c+ cells) located in the lungs are phagocytes that maintain many capabilities consistent with transport. This study investigates the role of dendritic cells as conduits of spores from the lung to the draining lymph nodes. The intratracheally spore-challenged mouse model of inhalational anthrax was utilized to investigate in vivo activities of CD11c+ cells. FITC labeled spores were delivered to the lungs of mice. Subsequently lung associated lymph nodes were isolated after infection and CD11c+ cells were found in association with the labeled spores. Further investigation of CD11c+ cells in early anthrax events was facilitated by use of the CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice in which CD11c+ cells can be transiently depleted by treatment with DT. We found that the presence of CD11c+ cells was necessary for efficient traffic of the spore to lung associated lymph nodes at early times after infection. Cultured dendritic cells were used to determine that these cells are capable of B. anthracis spore phagocytosis, and support germination and outgrowth. This data demonstrates that CD11c+ cells are likely carriers of B. anthracis spores from the point of inhalation in the lung to the lung associated lymph nodes. The cultured dendritic cell allows for spore germination and outgrowth supporting the concept that the CD11c+ cell responsible for this function can be a dendritic cell.

The Relationship between the Immunophenotypic Profile and Cytogenetic Abnormalities in Acute Myeloid Leukemia.

We studied the immunophenotypic characteristics of 459 consecutive adult AML patients and their correlation with karyotype. Immunophenotype was performed using a panel of 26 directly conjugated monoclonal antibodies. Cytogenetic analysis was performed using a standard G-banding technique. Karyotype was available in 394 patients (not done in 15, failed in 50): 1) 45 (11.4%) were t(15;17) APL patients with a mature myeloid phenotype (HLA-DR-/CD13+ and/or CD33+). CD2 and CD56 were expressed in 20% and 13.3% of cases, respectively. CD11b-positivity was less frequent than in the other cytogenetic groups. The markers significantly associated with t(15;17) were: presence of CD2 (two tailed Fisher exact test: p=.003) and CD117 (p=.01), absence of CD4dim (p<.001), CD7 (p<.001), CD11b (p<.001), CD11c (p<.001), CD14 (p<.001), CD34 (p<.001), TdT (p=.03) and HLA-DR (p<.001). 2) 12 (3%) showed t(8;21) and were characterized by CD34+/CD19+/CD13+<CD33+ in more than 80% of blasts. CD56 were expressed in 87.5%. CD11b was positive only in 8.3% and CD14 was constantly negative. In univariate analysis, t(8;21) was associated with CD11b- (p=.03), CD19+ (p<.001) and CD56+(p<.001). 3) 23 (5.8%) had inv(16) or t(16;16) with CD13+/CD33+ in >90% of blasts, CD34+ in 70%, MPO+ in 95.8% and HLA-DR+ in 89.3%. The association with CD14 and TdT was of borderline statistical significance. 4) 24 (6.1%) had −5/5q- with more than 80% of blasts CD117+/CD13+>CD33+. CD34 was positive in 62.9% of cases, CD7 in 33%, CD11b in 35.7%, CD11c in 67.8% and CD14 in 7.1%. CD15-positivity was less frequent than in other AML subtypes. Univariate analysis showed a trend of positive association with CD7 and CD117. 5) 40 (10.2%) showed −7/7q-, with CD13+>CD33+ detected in more than 80%, CD7 in 33.3%, CD11b in 72.7%, CD15 in 55%, CD34 in 73.3%. Abnormal CD16/CD33 and/or CD11b/CD66b myeloid cell pattern was detected in 40% of cases (p=.001). −7/7q- were significantly associated with CD34 (p=.02) and CD7 (p=.04). 6) 41 (10.4%), had complex karyotype (≥3 abn) with a similar antigenic profile than group 4) and 5) but with a more frequent expression of CD11b, CD14, CD15 and less frequent of MPO. In univariate analysis CD19 (p=.04), CD34 (p=.02) and MPO (p<.001) retained statistical significance. 7) 20 (5.1%) with +8 were CD13+ in 95.6%, while the other markers were present in less than 80% of cases, in particular lower CD33+ blasts (p=.01). 8) 17 (4.3%) had 11q23abn, with CD13+<CD33+, frequent TdT+ (61.5%) and rare CD34+ (31.8%, p=.02). CD11c and CD14 were more frequently expressed than in other subtypes (86.4% and 36.4%, respectively). T-lymphoid markers were present in 36.4%. Univariate analysis showed a positive association between 11q23abn and presence of CD11c (p=.04) and CD14 (p=.05). 9) 115 (29.2%) with a normal karyotype had a dishomogeneous antigenic profile. CD2 (p=.002), CD11c (p=.04) and HLA-DR (p<.001) were the most discriminant markers. Using a multivariate discriminant analysis, we identified a discriminant function only for group 1) and group 2), based on CD11c/CD13/CD34/MPO/HLA-DR (sensitivity >99%, specificity 94.7%) and CD7/CD11c/CD19/CD56/MPO/HLA-DR (sensitivity 83.3%, specificity 94.5%), respectively. We conclude that in AML patients some cytogenetic abnormalities are associated with peculiar antigenic profiles. In patients with normal karyotype the heterogeneity of antigenic pattern may reflect underlined distinct molecular abnormalities.

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood.

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.

Hematologic and Immunophenotypic Characterization of Human Umbilical Cord Blood

In this work, cord blood cells from 30 healthy term newborns were analyzed for complete blood counts with an automated cytometer and, in part of the sample, for surface molecules in cord blood monocytes, lymphocytes and CD34+ cells by two-color flow cytometry. Hematological parameters were as follows: WBC = 12.85 (5.24–15.10) ×10⁹/l; platelets = 304.33 (156.00–469.00) × 10⁹/l; Hb = 14.45 (11.90–17.82) g/dl; RBC = 3.99 (3.14–5.12) × 10¹²/l; MCV 107.25 (99.60–115.00) fl; reticulocytes = 157.80 (101.00–124.00) × 10⁹/l or 3.99 (2.45–6.01)%; erythroblasts = 0.88 (0.15–2.58) per 100 WBC or 6.63 (2.86–16.80) × 10⁹/l. The mononuclear population, as evaluated by flow cytometry, was composed of 22.9 ± 7.2% monocytes and 77.05 ± 7.24% lymphocytes, among which 46.59 ± 15.62% were T lymphocytes (43.94 ± 16.94% CD3+/CD4+ and 13.45 ± 7.46% CD3+/CD8+). CD34+ cells were on average 0.54 ± 0.24% of the mononuclear fraction. CD11c, CD49e and HLA-DR were found mainly on monocytes, and CD31 and CD62L occurred in similar levels on monocytes and lymphocytes. CD117+ cells were less than 5% of these populations. Among CD34+ cells, CD31 and HLA-DR were the molecules with higher frequencies (79.7 ± 19.9 and 65.7 ± 23.0%, respectively), followed by CD62L (41.8 ± 31.9%) and CD117 (20.1 ± 15.8%). The presence of CD11c and CD49e on CD34+ cells was low (below 10%). The results stress the phenotypic heterogeneity of cord blood CD34+ cells, and the different behavior of the cells when manipulated in vitro in different degrees of isolation.