Cell Subset Distribution Research Articles

Impact of Acute Exercise on Natural Killer Cell Subset Distributions in Selected Mouse Tissues

PURPOSE: Natural killer (NK) cells can be divided into several subsets, based on the expression intensity of cell surface markers. In mice, CD11b (Mac-1) expression is a marker of maturation and the tumor necrosis factor receptor superfamily member CD27 is a functional marker. Mouse Mac-1hi and CD27lo NK cells have similarities to human CD56dim NK cells and CD27hi NK cells are similar to human CD56bright NK cells (Hayakawa et al., Immunol Rev, 2006). However, little is known about distribution of these NK cell subsets between various tissues under stressful conditions. We thus examined the impact of acute exercise on mouse NK cell subset distributions in the blood, lungs, liver and spleen. METHODS: Male C57B/6 mice (7-8wk of age) were exercised on a treadmill for 30 min at 0% grade and a speed of 35 m/min. Animals were sacrificed without exercise (PRE, n=4), just after exercise (END, n=4), 30 min after exercise (POST 30, n=3) and 120 min after exercise (Post 120, n=3). Peripheral blood mononuclear cells (PBMCs) from the aforementioned tissues were isolated and were incubated with monoclonal antibodies. Flow cytometric analysis was performed to determine the cell subsets. Data were analyzed for statistical significance by using one-factor ANOVA. RESULTS: In blood, the collected number of PBMCs increased at END (p=0.0360) but returned to baseline at POST 30. The opposite pattern was observed in the spleen. There were no significant time-related changes in the absolute number of PBMCs in the lungs and liver. There were no changes in subset proportions (T, NK, NKT) in any tissues. However, altered proportions of NK cell subsets were observed in the liver. Thus, proportions of Mac-1lo CD27lo NK cells were significantly increased (p=0.0012) at POST 30 (p=0.0005) and POST 120 (p=0.0255), and those of Mac-1lo CD27hi NK cells were significantly elevated (p=0.0234) at POST 120 (p=0.0096). CONCLUSION: These results suggested that mouse lymphocyte were mobilized in both the blood and the spleen during acute exercise. In addition, exercise had delayed impact on NK cell subsets distribution in the liver. This study was supported by the Grant-in-Aid for Scientific Research (B), Japan Society for the Promotion of Science, No. 21300257.

γδT cells in Juvenile Idiopathic Arthritis: Higher Percentages of Synovial Vδ1+ and Vγ9+ T Cell Subsets Are Associated with Milder Disease

To analyze γδT cell subsets in peripheral blood (PB) and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA), and to correlate γδT cell subsets with clinical characteristics. γδT cell subsets as percentages of CD3+ T cells in samples of PB (n = 25) and SF (n = 93) were analyzed by flow cytometry in 93 JIA patients. The percentage of Vγ9+ γδT cells after 10 days of in vitro expansion with either interleukin 2 (IL-2) or isopentenyl pyrophosphate (IPP) plus IL-2 was determined. Both Vδ1+ and Vγ9+ γδT cell subsets were detected in SF of all patients, but only the percentage of Vδ1+ cells was higher in SF compared to PB (p < 0.01). The distribution of γδT cell subsets was similar in different JIA subgroups, whereas antinuclear antibody (ANA)-positive patients had a higher percentage of SF Vδ1+ T cells than ANA-negative patients (p < 0.01). The percentage of SF Vδ1+ T cells was inversely associated with age at onset, recurrence of synovitis, and erythrocyte sedimentation rate; and that of SF Vγ9+ T cells was inversely correlated with age at onset and was higher in patients who recovered from disease (n = 15). IPP-induced expansion of SF Vγ9+ T cells correlated with disease remission, whereas the expansion of SF Vγ9+ T cells in media with IL-2 alone was significantly greater in patients with uveitis. The percentage of Vδ1+ and Vγ9+ γδT cells among the SF T cells and their ability to respond to IPP or IL-2 correlated with specific outcomes of JIA, suggesting their role in the immunopathogenesis of this disease.

Critical Role of cRel Subunit of NF-κB in Sepsis Survival

NF-κB is a critical regulator of gene expression during severe infections. NF-κB comprises homo- and heterodimers of proteins from the Rel family. Among them, p50 and p65 have been clearly implicated in the pathophysiology of sepsis. In contrast, the role of cRel in sepsis is still controversial and has been poorly studied in single-pathogen infections. We aimed to investigate the consequences of cRel deficiency in a cecal ligation and puncture (CLP) model of sepsis. We have approached the underlying mechanisms of host defense by analyzing bacterial clearance, systemic inflammation, and the distribution of spleen dendritic cell subsets. Moreover, by using a genome-wide technology, we have also analyzed the CLP-induced modifications in gene expression profiles both in wild-type (wt) and in rel(-/-) mice. The absence of cRel enhances mortality due to polymicrobial sepsis. Despite normal pathogen clearance, cRel deficiency leads to an altered systemic inflammatory response associated with a sustained loss of the spleen lymphoid dendritic cells. Furthermore, a whole-blood microarray study reveals that the differential outcome between wt and rel(-/-) mice during sepsis is preceded by remarkable changes in the expression of hundreds of genes involved in aspects of host-pathogen interaction, such as host survival and lipid metabolism. In conclusion, cRel is a key NF-κB member required for host antimicrobial defenses and a regulatory transcription subunit that controls the inflammatory and immune responses in severe infection.

Loss of CD127 &amp; increased immunosenescence of T cell subsets in HIV infected individuals

Background & objectives:HIV infection is characterized by a perturbation in T cell homeostasis, leading to alteration in T cell subsets. In addition to alteration in differentiation, HIV infection also leads to change in T cell survival and regenerative capacity, as suggested by differential expression of CD127 and CD57. We evaluated the expression patterns of CD127 and CD57 on CD4 and CD8 effector, memory and naïve T cell subsets in HIV-infected and uninfected individuals.Methods:We characterized T cell subsets based on expression of these markers, and compared their expression pattern in HIV infected subjects and uninfected controls. We further assessed therapy generated changes in these subsets and expression of CD127 and CD57 on them.Results:There was a generalized decrease in naïve CD4 and CD8 T cells in HIV infected subjects. These changes in T cell subset distribution were related to antigen load. CD127 expression was significantly reduced in T cells from HIV infected subject. In association to this, HIV infected subjects had higher percentage of T cell subsets expressing CD57. Increased CD57 and reduced CD127 expression correlated with plasma viraemia and CD8 T cell activation state. Incomplete restoration of T cell subset proportions was observed, despite suppression of viral replication and increase in CD4 T cell counts. Further, the improvement was more pronounced in CD127 expression.Interpretation & conclusions:HIV infected subjects have reduced T cell regenerative capacity along with increased senescence, highlighting decreased proliferation and effector activities.

Phase I Study of Intravenous Plerixafor Added to a Mobilization Regimen of G-CSF In Lymphoma Patients Undergoing Autologous Stem Cell Collection

AbstractAbstract 823 Background:Plerixafor, a reversible inhibitor of the interaction between stromal cell-derived factor 1 (SDF-1) and CXCR4, improves autologous stem cell collection in patients with non-Hodgkin lymphoma (NHL), multiple myeloma, and Hodgkin lymphoma (HL). In Phase II and III trials, plerixafor has been combined with the standard G-CSF regimen and administered as a subcutaneous (sc) injection of 0.24 mg/kg, given 9–11 hours before pheresis. Intravenous (IV) administration of plerixafor may result in a faster rise and higher peak in the peripheral CD34+ cell count, allowing administration of plerixafor the same day as pheresis and improving stem cell collection. Methods:The primary objectives of this Phase I/II study were to determine the maximum tolerated dose (MTD) of IV plerixafor, up to 0.40 mg/kg combined with G-CSF, and to determine the efficacy of IV plerixafor + G-CSF to mobilize ≥ 2 × 106 CD34+ cells/kg from patients with lymphoma. Patients were given an initial dose of plerixafor 0.24 mg/kg sc on day -5, to assess the mobilization of CD34+ cell subsets, and then started mobilization with G-CSF (10 ug/kg SC daily) on days -4 thru -1 and on each day of pheresis. IV plerixafor was given over 30 min. 4 hrs. before each pheresis, beginning day 0. Samples for CD34+ cell subsets were collected after treatment with plerixafor only on day -5, after 5 days of G-CSF on day 0 (G-CSF only), and 4 hrs after plerixafor administration on day 0 (G-CSF + plerixafor). Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection CD34 isolation kit. Results:In the Phase I component of the study, 25 adult pts. (median age 49) with NHL (n=15) or HL (n=10) were treated with IV plerixafor at escalating doses (10 pts. at 0.16 mg/kg, 3 at 0.24 mg/kg, 6 at 0.32 mg/kg, and 6 at 0.40 mg/kg). One dose-limiting toxicity (grade 2 chest pain) was observed at 0.32 mg/kg, and no grade 3/4 toxicities occurred at 0.40 mg/kg. 24 of 25 pts. (96%) met the goal collection of ≥ 2.0 × 106 CD34+ cells/kg. 21 of 25 pts. (84%) collected ≥ 5.0 × 106 CD34+ cells/kg in a median 1 day of pheresis, including 6 of 6 pts. in the 400 ug/kg cohort. Peripheral blood CD34+ cell count increased median 27-fold (range, 4–174) after G-CSF + plerixafor IV and 2.1-fold (range, 0.8–7.0) 4 hours after the first dose of IV plerixafor. There was a positive correlation between peripheral blood CD34+ cell count 6 hr. after plerixafor SC on day -5 and both peripheral blood CD34+ cell count after G-CSF mobilization and first day stem cell collection. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) were divided into four distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Rα): (1) CD34+CD45RA-CD123+/− primitive HSPCs, (2) CD34+CD45RA+CD123+/− committed progenitors, (3) CD34dimCD45RA+CD123hi plasmacytoid dendritic cell (pDC) progenitors and (4) CD34+CD45RA-CD123hi cells of unknown function. Table 1 shows the CD34+ cell subset distribution following sequential mobilization with plerixafor, G-CSF, and plerixafor + G-CSF (PL+G). G-CSF mobilized grafts were enriched with CD45RA-CD123+/− primitive HSPCs while plerixafor preferentially mobilized the CD34dimCD45RA+CD123hi pDC precursors and CD34+CD45RA-CD123hi cells. We found no significant differences in the CD34+ subset distribution between HL and NHL pts. Flow cytometric analyses showed that the CD34+ subsets preferentially mobilized by plerixafor expressed high levels of cell surface CXCR4. Conclusion:Plerixafor IV, at doses up to 0.40 mg/kg, is well-tolerated and effective when added to G-CSF for the mobilization of stem cells from patients with lymphoma, with mobilization kinetics and stem cell collections that compare favorably with sc dosing. The Phase II study is proceeding at the 0.40 mg/kg dose. In addition, our data suggest that G-CSF and plerixafor mobilize distinct subsets of human CD34+ HSPCs. The impact of these cells on the engraftment and function of plerixafor mobilized grafts requires further study.Table 1Phenotype of human CD34+ stem cells.PhenotypePlerixafor (n = 18)G-CSF (n = 16)PL + G (n = 22)34+RA-123+/−, %47.8 ± 1478.6 ± 5.957.9 ± 1334+RA+123+/−, %29.2 ± 8.019.9 ± 5.834.8 ± 1134dimRA+123hi, %18.2 ± 9.20.8 ± 0.64.4 ± 2.134+RA-123hi, %3.8 ± 4.40.7 ± 0.52.2 ± 2.0 Disclosures:Rettig:Genzyme Corp.: Consultancy, Honoraria. Vij:Genzyme: Honoraria, Speakers Bureau. Uy:Genzyme: Consultancy, Honoraria, Research Funding.

Chemokine-mediated distribution of dendritic cell subsets in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) represents one of the most immunoresponsive cancers. Antigen-specific vaccination with dendritic cells (DCs) in patients with metastatic RCC has been shown to induce cytotoxic T-cell responses associated with objective clinical responses. Thus, clinical trials utilizing DCs for immunotherapy of advanced RCCs appear to be promising; however, detailed analyses concerning the distribution and function of DC subsets in RCCs are lacking.MethodsWe characterized the distribution of the different immature and mature myeloid DC subsets in RCC tumour tissue and the corresponding normal kidney tissues. In further analyses, the expression of various chemokines and chemokine receptors controlling the migration of DC subsets was investigated.ResultsThe highest numbers of immature CD1a+ DCs were found within RCC tumour tissue. In contrast, the accumulation of mature CD83+/DC-LAMP+ DCs were restricted to the invasive margin of the RCCs. The mature DCs formed clusters with proliferating T-cells. Furthermore, a close association was observed between MIP-3α-producing tumour cells and immature CCR6+ DC recruitment to the tumour bed. Conversely, MIP-3β and SLC expression was only detected at the tumour border, where CCR7-expressing T-cells and mature DCs formed clusters.ConclusionIncreased numbers of immature DCs were observed within the tumour tissue of RCCs, whereas mature DCs were found in increased numbers at the tumour margin. Our results strongly implicate that the distribution of DC subsets is controlled by local lymphoid chemokine expression. Thus, increased expression of MIP-3α favours recruitment of immature DCs to the tumour bed, whereas de novo local expression of SLC and MIP-3β induces accumulation of mature DCs at the tumour margin forming clusters with proliferating T-cells reflecting a local anti-tumour immune response.

Immunosenescence and Cytomegalovirus: where do we stand after a decade?

Since Looney at al. published their seminal paper a decade ago it has become clear that many of the differences in T cell immunological parameters observed between young and old people are related to the age-associated increasing prevalence of infection with the persistent beta-herpesvirus HHV-5 (Cytomegalovirus). Ten years later, studies suggest that hallmark age-associated changes in peripheral blood T cell subset distribution may not occur at all in people who are not infected with this virus. Whether the observed changes are actually caused by CMV is an open question, but very similar, rapid changes observed in uninfected patients receiving CMV-infected kidney grafts are consistent with a causative role. This meeting intensively discussed these and other questions related to the impact of CMV on human immune status and its relevance for immune function in later life.

Viral load reduction improves activation and function of natural killer cells in patients with chronic hepatitis B

Natural killer (NK) cells play a major role in anti-viral immunity as first line defense and regulation of virus-specific T cell responses. This study aimed to investigate phenotype and function of NK cells in patients with chronic hepatitis B virus (HBV) infection and to study the effect of anti-viral therapy. Peripheral blood NK cells from 40 chronic HBV patients were compared to NK cells of 25 healthy controls. The effect of entecavir-induced viral load reduction on NK cell phenotype and function was investigated in 15 chronic HBV patients. NK cell numbers and subset distribution did not differ between HBV patients and normal subjects. In chronic HBV patients, the cytotoxic capacity was retained, but NK cell activation and subsequent IFNγ and TNFα production, especially of the CD56(dim) subset, were strongly hampered. This functional dichotomy was paralleled by an altered activation state, elevated expression of NKG2A, and downregulated expression of CD16 and NKp30, which correlated with serum HBV-DNA load. Anti-viral therapy partially restored NK cell phenotype, as shown by NKG2A downregulation. Moreover, viral replication inhibition improved IFNγ production as a result of an increased ability of CD56(dim) NK cells to become activated de novo. This improved NK cell activation and function which correlated with therapy-induced reduction in serum ALT levels, but not HBV-DNA load. The specific defect in CD56(dim) NK cell activation and the reduced capacity to produce anti-viral and Th1-skewing cytokines may play a role in HBV persistence. Restoration of this NK cell cytokine-producing capacity, as achieved by viral load reduction, could therefore contribute to definite clearance of the virus.

Preferential Mobilization of CD34+ Plasmacytoid Dendritic Cell Precursors by Plerixafor.

Abstract 32 Background:Plerixafor (Mozobil®) is a CXCR4 antagonist that was recently approved by the Food and Drug Administration for use in combination with granulocyte-colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSCs) in patients with non-Hodgkin's lymphoma and multiple myeloma undergoing autologous transplantation. As part of our ongoing clinical evaluation of plerixafor, we observed that the drug efficiently mobilized a CD34dim population of HSCs. Here, we characterize the CD34dim population and assess its relative frequency following mobilization with G-CSF and/or plerixafor in apheresis samples and in non-mobilized cord blood samples. Methods: Aliquots of apheresis products were obtained from 16 healthy donors following treatment with a single injection of plerixafor or 5 days of G-CSF (10 μg/kg/day). In a separate study, five patients with lymphoma were mobilized sequentially with plerixafor (240 μg/kg), G-CSF (10 μg/kg/day), and plerixafor + G-CSF. These patients received an injection of plerixafor on day −5. Twenty-four hours later, patients were treated with G-CSF for 4 days. On the fifth day (day 0), patients received a dose of G-CSF and a dose of plerixafor followed four hours later by apheresis. Samples were collected after treatment with plerixafor on day −5 as well as immediately before and 4 hrs after plerixafor administration on day 0. Cord blood units were obtained from the St. Louis Cord Blood Bank. Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection CD34 isolation kit. We used Affymetrix Human Genome U133+2 arrays to generate gene expression profiles for 24 samples of CD34+ cells collected following mobilization of healthy donors with either Plerixafor (n=12) or G-CSF (n=12). Results: Human CD34+ cells can be divided into three distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Rα): (i.) CD34+CD45RA−CD123+/− primitive HSCs, (ii.) CD34+CD45RA+CD123+/− committed progenitors, and (iii.) CD34dimCD45RA+CD123hi cells. Table 1 shows the relative frequencies of each of these CD34+ cell subsets in apheresis products obtained from healthy donors mobilized with G-CSF or plerixafor as well as in cord blood units. Strikingly, we observed that each graft type was significantly enriched for one of the CD34+ cell subsets compared to the other two grafts. G-CSF mobilized grafts contained more CD45RA−CD123+/− primitive HSCs, cord blood units were enriched with CD45RA+CD123+/− committed progenitors and plerixafor mobilized products had significantly more CD34dimCD45RA+CD123hi cells. Table 1 also shows the CD34 cell subset distribution following sequential mobilization of lymphoma donors with plerixafor, G-CSF, and plerixafor + G-CSF (PL+G). This data agrees with the results we obtained from healthy donors and confirms that G-CSF grafts are enriched with CD34+CD45RA−CD123+/− cells while plerixafor preferentially mobilizes the CD34dimCD45RA+CD123hi subset. Extensive FACS and functional analyses determined that the CD34dim population represents a plasmacytoid pro-DC2 (for progenitor of pre-dendritic cell type 2) progenitor compartment as indicated by their CD45RA+CD123hiBDCA−2+BDCA−4+CD36+CXCR4hiCD4dimCD25−CD13− phenotype and inability to form CFU-GM colonies in vitro. Finally, we found that the gene signature of plerixafor-mobilized CD34+ cells is distinct from that of G-CSF-mobilized CD34+ cells. Plerixafor-mobilized CD34+ cells expressed significantly more of the specific transcriptional regulator of plasmacytoid DC (pDC) development, E2-2, as well as additional transcriptional factors (SpiB, IRF7, IRF8) and cell surface markers (BDCA-2, ILT7) that are specific to the pDC lineage. Conclusions: This data suggest that the CD34dimCD45RA+CD123hi cells preferentially mobilized by plerixafor are precursor pDCs. Further study is required to determine the impact these cells have on the engraftment and function of plerixafor mobilized grafts after hematopoietic stem cell transplantation.Table 1Phenotype of human CD34+ stem cellsPhenotypeHealthy DonorsLymphoma PatientsPlerixafor (n=7)G-CSF (n=9)Cord (n=4)Plerixafor (n=5)G-CSF (n=5)PL + G (n=5)34+RA−123+/−, %67.1 ± 7.381.6 ± 9.566.5 ± 3.639.4 ± 8.877.5 ± 2.659.6 ± 1334+RA+123+/−, %18.7 ± 4.116.7 ± 9.230.5 ± 4.537.5 ± 7.920.6 ± 2.234.2 ± 1234dimRA+123hi, %12.2 ± 5.31.0 ± 0.82.2 ± 0.819.6 ± 4.01.4 ± 0.74.4 ± 0.9Values represent Mean ± SD Disclosures:DiPersio:Genzyme Corp.: Honoraria.

The Natural Cytotoxicity Receptor NKp46 Is Dispensable for IL-22-Mediated Innate Intestinal Immune Defense against Citrobacter rodentium

Natural cytotoxicity receptors (including NKp30, NKp44, and NKp46 in humans and NKp46 in mice) are type I transmembrane proteins that signal NK cell activation via ITAM-containing adapter proteins in response to stress- and pathogen-induced ligands. Although murine NKp46 expression (encoded by Ncr1) was thought to be predominantly restricted to NK cells, the identification of distinct intestinal NKp46(+) cell subsets that express the transcription factor Rorc and produce IL-22 suggests a broader function for NKp46 that could involve intestinal homeostasis and immune defense. Using mice carrying a GFP-modified Ncr1 allele, we found normal numbers of gut CD3(-)GFP(+) cells with a similar cell surface phenotype and subset distribution in the absence of Ncr1. Splenic and intestinal CD3(-)NKp46(+) cell subsets showed distinct patterns of cytokine secretion (IFN-gamma, IL-22) following activation via NK1.1, NKp46, IL-12 plus IL-18, or IL-23. However, IL-22 production was sharply restricted to intestinal CD3(-)GFP(+) cells with the CD127(+)NK1.1(-) phenotype and could be induced in an Ncr1-independent fashion. Because NKp46 ligands can trigger immune activation in the context of infectious pathogens, we assessed the response of wild-type and Ncr-1-deficient Rag2(-/-) mice to the enteric pathogen Citrobacter rodentium. No differences in the survival or clinical score were observed in C. rodentium-infected Rag2(-/-) mice lacking Ncr1, indicating that NKp46 plays a redundant role in the differentiation of intestinal IL-22(+) cells that mediate innate defense against this pathogen. Our results provide further evidence for functional heterogeneity in intestinal NKp46(+) cells that contrast with splenic NK cells.

A high proportion of bone marrow T cells with regulatory phenotype (CD4+CD25hiFoxP3+) in Ewing sarcoma patients is associated with metastatic disease

Immunosuppressive CD4+CD25(hi)FoxP3+ T cells (T(reg) cells) have been found at increased densities within the tumor microenvironment in many malignancies and interfere with protective antitumor immune responses. Osseous Ewing sarcomas (ESs) are thought to derive from a bone marrow (BM) mesenchymal cell of origin, and microscopic marrow involvement defines a subpopulation of patients at a high risk of relapse. We hypothesized that BM-resident T cells may contribute to a permissive milieu for immune escape of ESs. Using 6-color-flow cytometry, we investigated the pattern of immune cell subset distribution including NK cells, gammadelta T cells, central and effector memory CD8+ and CD4+ T cells as well as T cells with regulatory phenotype (T(reg) cells) in BM obtained at diagnosis from 45 primary or relapsed ES patients treated within standardized protocols. Although patients at relapse had an inverted CD4:CD8 T-cell ratio, neither CD8+ effector/memory T-cell subsets nor T(reg) cells significantly differed from patients at diagnosis. No significant associations of innate and effector/memory T-cell subpopulations with known risk factors were found, including age, gender, tumor site, primary metastases and histological tumor response. By contrast, T(reg) cells were found at significantly higher frequencies in patients with primary metastatic disease compared with localized ESs (5.0 vs. 3.3%, p = 0.01). Thus, increased BM T(reg) cells in patients with metastasized ES may reflect an immune escape mechanism that contributes to the development of metastatic disease. Immunotherapeutic strategies will have to adequately consider the regulatory milieu within areas of Ewing tumor-immune interactions.

Dendritic Cells and Monocyte/Macrophages That Create the IL-6/APRIL-Rich Lymph Node Microenvironments Where Plasmablasts Mature

IL-6 and APRIL influence the growth, differentiation, and survival of normal and neoplastic Ab-forming cells (AFC). In this study, we identify two subsets of myeloid cells that associate with the AFC and are the main producers of these factors during a T-dependent Ab response to alum-precipitated protein in mouse lymph nodes. First CD11c(+)CD8alpha(-) dendritic cells located in the perivascular area of the T zone provide about half of the IL-6 mRNA produced in the node together with significant amounts of APRIL mRNA. The number of these cells increases during the response, at least in part due to local proliferation. The second subset comprises Gr1(+)CD11b(+)F4/80(+) monocyte/macrophages. These colonize the medullary cords during the response and are the other main IL-6 mRNA producers and the greatest source of APRIL mRNA. This medullary cord monocyte/macrophage subset results in local increase of APRIL mRNA that mirrors the polarity of CXCL12 expression in the node. The distribution of these myeloid cell subsets correlates with a gradient of AFC maturation assessed by progressive loss of Ki67 as AFC pass from the B cell follicle along the perivascular areas to the medullary cords.

Intracytoplasmic Cytokine Expression and T Cell Subset Distribution in the Peripheral Blood of Patients with Ankylosing Spondylitis

To determine the role of inflammatory mediators in the pathogenesis of ankylosing spondylitis (AS), we investigated peripheral blood lymphocyte subsets and their intracellular cytokine production. The percentages of T and B lymphocytes, natural killer (NK) cells, activated T lymphocytes, CD4+ T helper (Th), and CD8+ T cytotoxic (Tc) cells were determined by flow cytometry in 42 patients with AS compared to 52 healthy controls. In order to assess circulating Th1/Th2 and Tc1/Tc2 subsets, we used a whole-blood cytometric assay based on the intracellular interferon-gamma, interleukin 4 (IL-4), and IL-10 expression of the cells. In the peripheral blood, the frequencies of CD4+ T helper and CD56+ NK cells were higher in AS (54.8% and 16.2%, respectively) compared to controls (45.3% and 10.8%) (p < 0.05). The frequencies of Th0 (1.9% vs 0.8%) and Tc0 (2.1% vs 0.8%) cells were higher, while that of Tc1 cells was lower (26.6% vs 40.1%) in patients with AS versus controls (p < 0.05). The percentage of IL-10-producing Tc cells was significantly higher in AS (18.4%) versus controls (8.5%) (p < 0.05). Finally, the active phase of AS was associated with significantly lower percentage of IL-10-producing Tc cells in the peripheral blood (6.6%) compared to patients with inactive AS (23.1%). Our results provide further evidence for an altered T cell subset distribution and intracytoplasmic cytokine balance in AS.

Plasmacytoid Dendritic Cells Are Proportionally Expanded at Diagnosis of Type 1 Diabetes and Enhance Islet Autoantigen Presentation to T-Cells Through Immune Complex Capture

OBJECTIVE—Immune-mediated destruction of β-cells resulting in type 1 diabetes involves activation of proinflammatory, islet autoreactive T-cells, a process under the control of dendritic cells of the innate immune system. We tested the hypothesis that type 1 diabetes development is associated with disturbance of blood dendritic cell subsets that could enhance islet-specific autoimmunity.RESEARCH DESIGN AND METHODS—We examined blood dendritic cells (plasmacytoid and myeloid) in 40 patients with recent-onset diabetes (median duration 28 days) and matched control subjects. We also examined the relative ability of different dendritic cell subsets to process and present soluble or immune complexed islet cell autoantigen (the islet tyrosine phosphatase IA-2) to responder CD4 T-cells.RESULTS—The balance of blood dendritic cells was profoundly disturbed at diabetes diagnosis, with a significantly elevated proportion of plasmacytoid and reduction of myeloid cells compared with control subjects. Dendritic cell subset distribution was normal in long-standing disease and in patients with type 2 diabetes. Both dendritic cell subsets processed and presented soluble IA-2 to CD4 T-cells after short-term culture, but only plasmacytoid dendritic cells enhanced (by as much as 100%) autoantigen presentation in the presence of IA-2+ autoantibody patient serum.CONCLUSIONS—The plasmacytoid subset of dendritic cells is overrepresented in the blood close to diabetes onset and shows a distinctive ability to capture islet autoantigenic immune complexes and enhance autoantigen-driven CD4 T-cell activation. This suggests a synergistic proinflammatory role for plasmacytoid dendritic cells and islet cell autoantibodies in type 1 diabetes.

Costimulation molecule expression and subset distribution of blood dendritic cells in normal children and newly diagnosed pediatric leukemia and lymphoma patients

To characterize dendritic cell (DC) numbers, subset distribution, phenotype, and costimulation molecule expression in a normal pediatric population and in a lymphoblastic malignant pretherapy pediatric population. DC are potent antigen-presenting cells and are crucial for initiating specific immune responses. We first analyzed peripheral blood samples of healthy pediatric controls (n=72). Once a range of normal parameters was established, we compared these to newly diagnosed pediatric leukemia and lymphoma patients prior to receiving therapy (n=69). Using flow cytometry, we examined blood DC cell-surface expression of CD80, CD86, CD40, CD18, CD50, CD83, CD123, CD58, CD54, and CD11c. Expression of each of these molecules was significantly altered except for CD80, CD83, and CD58. When compared to healthy children, absolute blood DC were reduced in children with leukemia or lymphoblastic lymphoma (p<0.0001) and children with Hodgkin's disease (p=0.0028). Additionally, lymphocyte function in vitro, was impaired (p=0.0489) for children with lymphoblastic malignancies, while patients with Hodgkin's disease had normal proliferative function. Our results show that peripheral blood DC from children with newly diagnosed leukemia or lymphoma are significantly altered in number, subset distribution, and costimulation molecule expression, and that lymphocyte function is impaired compared to healthy pediatric controls.

Effects of interferon-gamma and tumour necrosis factor-alpha on the development of cytotoxic T lymphocytes in autologous mixed lymphocyte tumour cultures with human melanoma

We have studied the influence of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the development of cytotoxic T lymphocytes (CTL) against melanoma in mixed lymphocyte tumour cultures (MLTC). In these MLTC, TNF-alpha at 10(4) U/ml increased the expansion of the CTL up to 10(4)-fold over recombinant IL-2 (rIL-2) alone. IFN-gamma at 10(4) U/ml and combinations of TNF-alpha plus IFN-gamma at 10(2)-10(3) U/ml promoted the proliferation more variably. MLTC generated with rIL-2 showed a predominance of CD8+ cells, while 2 weeks of culture in the presence of IFN-gamma at 10(4) U/ml, or with IFN-gamma and TNF alpha at 1 x 10(2)-10(3) U/ml, favoured the emergence of CD4+ cell populations. The cytotoxic activity of the lymphocytes generated in these MLTC showed a consistent decline of K562 cytotoxic activity following exposure to the combination of IFN-gamma and TNF-alpha. Despite the altered T cell subset distribution with different combinations of cytokines, no consistent alteration in the specific anti-tumour cytotoxicity against melanoma was detected. These results suggest that TNF-alpha and IFN-gamma influence the activation, phenotypic, and functional outcome of MLTC-generated CTL, and may account for the phenotypic variations observed in T cell populations generated in vitro.

Heterogeneous effects of exogenous IL-2 on HIV-specific cell-mediated immunity (CMI).

A characteristic feature associated with HIV-1 infection of the human host is a chronic decline in circulating CD4+ T helper/inducer cell numbers. Impaired cell-mediated immune functions usually occur in parallel with the decline in CD4+ T cells. Activated CD4+ T helper cells are a major source of endogenous IL-2 which is required for the immunoregulation of both antigen-specific B cells and CD8+ T cells. HIV-specific T cell proliferative responses are said to be weak and inconsistent, even during the asymptomatic phase of disease. We thus wished to determine how exogenous IL-2 affected HIV-specific T cell proliferation at different stages of the disease. Our cohort of 81 included both asymptomatic and symptomatic HIV-infected patients as well as uninfected normal donors. Proliferative responses of peripheral blood mononuclear cells (PBMC) that were elicited during culture with an immunodominant gp41-derived synthetic peptide, gp41[8], and which were known to be CD8+ cell-associated in asymptomatics only, were used to analyse the effects of exogenous IL-2. IL-2 had three main effects on HIV-specific proliferation, namely (i) an additive effect, (ii) a synergistic effect, and (iii) an induced effect. More specifically, low dose exogenous IL-2 frequently augmented lymphoproliferation in both asymptomatic and symptomatic gp41[8] responders. In most symptomatics, however, who were predominantly gp41[8] non-responders, exogenous IL-2 induced lymphoproliferation. Flow cytometric analyses using dual immunofluorescence were used to analyse the T cell subset distribution of proliferating PBMC cultures. During culture with gp41[8], both CD4+ and CD8+ T cell numbers increased. However, after the addition of exogenous IL-2 to gp41[8]-containing cultures, CD8+ cell-associated lymphoproliferative responses were preferentially augmented. These results suggest that in symptomatics there is an inadequate supply of endogenous IL-2 to help maintain the strong and effective CD8+ cell-associated anti-viral immunity, and an exogenous supply of IL-2 may be required.

Altered natural killer cell subset distributions in resolved and persistent hepatitis C virus infection following single source exposure

Background:Natural killer (NK) cells may be impaired in patients with persistent hepatitis C virus (HCV) infection, but studies to date have yielded inconsistent findings due to patient and virus heterogeneity...

B Lymphocyte Depletion by CD20 Monoclonal Antibody Prevents Diabetes in Nonobese Diabetic Mice despite Isotype-Specific Differences in FcγR Effector Functions

NOD mice deficient for B lymphocytes from birth fail to develop autoimmune or type 1 diabetes. To assess whether B cell depletion influences type 1 diabetes in mice with an intact immune system, NOD female mice representing early and late preclinical stages of disease were treated with mouse anti-mouse CD20 mAbs. Short-term CD20 mAb treatment in 5-wk-old NOD female mice reduced B cell numbers by approximately 95%, decreased subsequent insulitis, and prevented diabetes in >60% of littermates. In addition, CD20 mAb treatment of 15-wk-old NOD female mice significantly delayed, but did not prevent, diabetes onset. Protection from diabetes did not result from altered T cell numbers or subset distributions, or regulatory/suppressor T cell generation. Rather, impaired CD4+ and CD8+ T cell activation in the lymph nodes of B cell-depleted NOD mice may delay diabetes onset. B cell depletion was achieved despite reduced sensitivity of NOD mice to CD20 mAbs compared with C57BL/6 mice. Decreased B cell depletion resulted from deficient FcgammaRI binding of IgG2a/c CD20 mAbs and 60% reduced spleen monocyte numbers, which in combination reduced Ab-dependent cellular cytotoxicity. With high-dose CD20 mAb treatment (250 microg) in NOD mice, FcgammaRIII and FcgammaRIV compensated for inadequate FcgammaRI function and mediated B cell depletion. Thereby, NOD mice provide a model for human FcgammaR polymorphisms that reduce therapeutic mAb efficacy in vivo. Moreover, this study defines a new, clinically relevant approach whereby B cell depletion early in the course of disease development may prevent diabetes or delay progression of disease.

Phenotypic Studies of Natural Killer Cell Subsets in Human Transporter Associated with Antigen Processing Deficiency

Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56bright cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, 51Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56bright NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56dimCD16− NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56bright NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.