Analysis Of Cell Subsets Research Articles

Abstract 2546: Analysis of immune cell subsets in a multidrug therapeutic regimen for patients with metastatic castration-resistant prostate cancer

Abstract Purpose: To investigate the effects of docetaxel-based combination therapy with bevacizumab and dexamethasone premedication on the immune response in patients with metastatic castration-resistant prostate cancer (mCRPC). Experimental Design: We studied immune responses in 13 patients enrolled in a phase II trial at the National Cancer Institute (NCI). The study was designed as a 13 patient expansion of a previously reported phase II study to evaluate the immunologic response after 2 cycles of treatment with a docetaxel-based chemotherapy regimen including docetaxel (75 mg/m2 every 3 weeks) and bevacizumab (15 mg/kg every 3 weeks). Dexamethasone pre-medication (4 mg) was given 12 h and 1 h prior to chemotherapy, and again after 12 h. Patients were evaluated before treatment and on day 40, 3 weeks after the second cycle. We compared PBMC and serum samples collected at baseline and after 40 days of treatment. We investigated CD4+ and CD8+ T-cells and regulatory T-cells (CD4+ CD25hi CD127- FoxP3+) by flow cytometry. T-cell proliferation, as well as NK-cell functional activity, was evaluated. Serum samples were analyzed for levels of cytokines, chemokines, sCD27, sCD40L and vascular endothelial growth factor (VEGF). Results: The baseline characteristics were: median age 64 years, Gleason score 9, PSA 100 ng/ml, and Halabi Predicted Survival 10.6 months. Patients had a median PSA decline of 66% after 2 cycles. The median TTP was 14.1 months, and OS 18.7 months. At 3 weeks after the second cycle we found no significant changes in absolute lymphocyte count, CD4+ and CD8+ T-cell proliferation and NK-cell function. The number of CD4+ T-cells decreased. CTLA4+ regulatory T-cells did not change. There was no change in the serum levels of IL-6, IL-8, IL-10 and TNFα. As expected, the serum levels of VEGF decreased substantially after therapy. The serum levels of sCD40L did not change after treatment. Interestingly, an increase in the serum level of sCD27 correlated with longer OS (P= 0.037, R= 0.58). Conclusions: Treatment of mCRPC patients with docetaxel-based combination therapy with bevacizumab and dexamethasone premedication for 40 days did not alter the immune response in a way that would decrease the likelihood of successful immunotherapy, either before or after this treatment. Citation Format: Caroline Jochems, Benjamin Boyerinas, Ravi A. Madan, Diane J. Poole, Yang-Min Ning, William D. Figg, David J. Liewehr, Seth M. Steinberg, James L. Gulley, Kwong-Yok Tsang, Jeffrey Schlom. Analysis of immune cell subsets in a multidrug therapeutic regimen for patients with metastatic castration-resistant prostate cancer. [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 2546. doi:10.1158/1538-7445.AM2014-2546

What makes a blood cell based miRNA expression pattern disease specific?--a miRNome analysis of blood cell subsets in lung cancer patients and healthy controls.

There is evidence of blood-borne miRNA signatures for various human diseases. To dissect the origin of disease-specific miRNA expression in human blood, we separately analyzed the miRNome of different immune cell subtypes, each in lung cancer patients and healthy individuals. Each immune cell type revealed a specific miRNA expression pattern also dependinging on the cell origin, line of defense, and function. The overall expression pattern of each leukocyte subtype showed great similarities between patients and controls. However, for each cell subtype we identified miRNAs that were deregulated in lung cancer patients including hsa-miR-21, a well-known oncomiR associated with poor lung cancer prognosis that was up-regulated in all leukocyte subtype comparisons of cancer versus controls. While the miRNome of cells of the adaptive immune system allowed only a weak separation between patients and controls, cells of the innate immune system allowed perfect or nearly perfect classification. Leukocytes of lung cancer patients show a cancer-specific miRNA expression profile. Our data also show that cancer specific miRNA expression pattern of whole blood samples are not determined by a single cell type. The data indicate that additional blood components, like erythrocytes, platelets, or exosomes might contribute to the disease specificity of a miRNA signature.

Spontaneous and Allo-Antibody (Ab)-Stimulated IFNγ Production in NK Cells as Detected By Cytokine Flow Cytometry (Allo-CFC) Decreased in HLA-Sensitized Kidney Transplant Patients (HS Tx Pts) Desensitized With Tocilizumab (TCZ, Anti-IL-6R)+Intravenous Immunoglobulin (IVIG).

Background: TCZ has anti-B cell and anti-inflammatory properties. Analysis of allo-Ab and Ab producing cell modification by desensitization (DES) with TCZ+IVIG in HS pts is underway. We have shown elevated IFNγ+ NK cellsvia ADCC-like mechanism after exposure to allo-PBMCsas analyzed by Allo-CFC. This suggests an important role of NK cells in antibody-mediated rejection. The effect of TCZ on ADCC is not known. Here, we examine the effects of “TCZ+IVIG” DES on IFNg production by NK cells stimulated by anti-HLA ADCC in HS pts with the DES. Methods: Blood from 6 pts desensitized with TCZ+IVIG (IVIG 2g/kg, x2, 1M apart; TCZ [8mg/kg], up to 7x, monthly) were submitted for NK cell subset analysis and Allo-CFC. In the Allo-CFC, IFNγ+ cell% in CD56+ NK cells were enumerated. Those obtained from 17 pts desensitized with rituximab+IVIG were used as control. PRA class I & class II (PRAI, PRAII) were performed by Luminex. Results: IFNγ+ NK cell% in the Allo-CFC significantly decreased by 4M post-TCZ in all 6 pts (100%, 3.8±5.1 vs. 5.6±6.1 pre-TCZ, p=0.03), and 3/4 pts (75%) with the available results at 0.5M post-TCZ already showed the reduction (3.9±3.3 vs. 5.9±2.6 pre-TCZ, p=0.19). Reduction of IFNγ+ cell% in NK cells without allo-PBMC stimulation (background) was also observed at 4M and 0.5M post-TCZ in all 6 and 4pts, respectively (1.4±0.8 vs. 3.0±1.0 pre-TCZ, p<0.02; 1.6±0.4 vs. 2.9±1.2, p=0.1). These trends were not observed in pts treated with “rituximab+IVIG” DES; Only 9/17 (53 vs. 75% in TCZ-DES, p<0.01) and 8/17 (47 vs. 100%, p<0.01) showed Allo-CFC and IFNγ background reduction at 0.5M post-rituximab, respectively. CD56+ (63±38/μl vs.49±35/μl pre-TCZ), CD16+ NK cell number (55±36/μl vs. 46±32 /μl), PRAI (86±26 vs. 88±23 %) & PRAII (93±7 vs. 91±9%), factors affecting the Allo-CFC (in vitro ADCC), did not significantly change by 4M post-TCZ. Conclusions: Spontaneous and allo-Ab-mediated IFNγ production in NK cells decreased after the “TCZ+IVIG” DES. This observation suggests that TCZ could suppress NK cell IFNg production in HS kidney Tx pts, which may regulate ADCC-mediated allograft injury post-Tx. DISCLOSURES:Jordan, S.: Grant/Research Support, CSL-Behring, Genentech Roche.

Clinical, molecular, and T cell subset analyses in a small cohort of Chinese patients with hyper-IgM syndrome type 1

Type 1 hyper-IgM syndrome (HIGM1) is a rare primary immunodeficiency disease caused by mutations in the CD40L gene. Patients often present with recurrent infections and autoimmune manifestations. We investigated the clinical and molecular characteristics of HIGM1 in thirteen patients from the Chinese mainland and examined the proportion of CD4+CD25+FoxP3+Treg, Th17, and Th1 cells in the peripheral blood. We identified ten distinct CD40L mutations in eleven patients: one missense mutation, one nonsense mutation, one insertion mutation (in frame), and seven deletions. Six of these mutations were novel. We observed the percentage of Tregs in the peripheral blood of HIGM1 patients decreased markedly compared with that in healthy controls, but no statistically significant differences was found in the percentages of Th17 and Th1. The identified mutations reflect the heterogeneity of the CD40L gene in HIGM1. Precise genetic diagnosis of HIGM1 will enable appropriate therapeutic interventions, reliable detection of carriers, and genetic counseling. Skewed Treg, Th17/Treg, and Th1/Treg profiles may be associated with immune responses to autoimmunity or infection, which requires replication in larger studies.

The mucosal adjuvant cyclic di-AMP exerts immune stimulatory effects on dendritic cells and macrophages.

The cyclic di-nucleotide bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) is a candidate mucosal adjuvant with proven efficacy in preclinical models. It was shown to promote specific humoral and cellular immune responses following mucosal administration. To date, there is only fragmentary knowledge on the cellular and molecular mode of action of c-di-AMP. Here, we report on the identification of dendritic cells and macrophages as target cells of c-di-AMP. We show that c-di-AMP induces the cell surface up-regulation of T cell co-stimulatory molecules as well as the production of interferon-β. Those responses were characterized by in vitro experiments with murine and human immune cells and in vivo studies in mice. Analyses of dendritic cell subsets revealed conventional dendritic cells as principal responders to stimulation by c-di-AMP. We discuss the impact of the reported antigen presenting cell activation on the previously observed adjuvant effects of c-di-AMP in mouse immunization studies.

T-cell regulation in lepromatous leprosy.

Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.

Differential activation of CD57-defined natural killer cell subsets during recall responses to vaccine antigens.

Natural killer (NK) cells contribute to the effector phase of vaccine-induced adaptive immune responses, secreting cytokines and releasing cytotoxic granules. The proportion of responding NK cells varies between individuals and by vaccine, suggesting that functionally discrete subsets of NK cells with different activation requirements may be involved. Here, we have used responses to individual components of the DTP vaccine [tetanus toxoid (TT), diphtheria toxoid (DT), whole cell inactivated pertussis] to characterize the NK cell subsets involved in interleukin-2-dependent recall responses. Culture with TT, DT or pertussis induced NK cell CD25 expression and interferon-γ production in previously vaccinated individuals. Responses were the most robust against whole cell pertussis, with responses to TT being particularly low. Functional analysis of discrete NK cell subsets revealed that transition from CD56bright to CD56dim correlated with increased responsiveness to CD16 cross-linking, whereas increasing CD57 expression correlated with a loss of responsiveness to cytokines. A higher frequency of CD56dim CD57− NK cells expressed CD25 and interferon-γ following stimulation with vaccine antigen compared with CD56dim CD57+ NK cells and made the largest overall contribution to this response. CD56dim CD57int NK cells represent an intermediate functional phenotype in response to vaccine-induced and receptor-mediated stimuli. These findings have implications for the ability of NK cells to contribute to the effector response after vaccination and for vaccine-induced immunity in older individuals.

Application of adenosine-triphosphate value of CD4+ T cells and T cell subsets analysis in monitoring immune function in post-liver transplantation recipients

Objective To investigate the significance of adenosine-triphosphate (ATP) value of CD4+ T cells and T cell subset analysis in monitoring immune function after liver transplantation.Methods Immuknow immune cell function kit was applied to test the ATP value of CD4 + T cells in peripheral blood from 48 patients undergoing liver transplantation and 30 healthy volunteers.Additionally,the frequency of CD3+,CD4+ and CD8+ cell subsets was analyzed by flow cytometry.Results For healthy volunteers,stable recipients,infectious recipients and recipients with acute rejection,the mean ATP levels were (331 ± 103),(364 ± 125),(182 ± 104) and (479 ± 116) μg/L,respectively.The ATP levels in acute rejection patients were higher than stable ones (P ＜ 0.01) and patients with infections (P ＜ 0.01).However,there was no statistically significant difference between stable recipients and recipients with acute rejection (P ＞ 0.05).T cell subsets analysis showed that the CD4+ cell counts were (25.53 ± 6.89) ％ in the infectious patients and (33.23 ± 11.34) ％ in stable group,there was a significantly different in two groups (P ＜ 0.05).Conclusion The ATP levels in CD4+ T cells and T cell subsets analysis can indicate the immune status after liver transplantation to a certain extent. Key words: Liver transplantation; CD4+ T cell; Lymphocyte subsets ; Immune

Phenotypic and Functional Analysis of Immune Cell Subsets in Pediatric-onset Multiple Sclerosis (MS): Towards Definition of Earliest Disease Mechanisms (P2.236)

Phenotypic and Functional Analysis of Immune Cell Subsets in Pediatric-onset Multiple Sclerosis (MS): Towards Definition of Earliest Disease Mechanisms (P2.236)

Comparative analysis of NK cell subsets in menstrual and peripheral blood of patients with unexplained recurrent spontaneous abortion and fertile subjects

Natural killer (NK) cells play a fundamental role in maintaining pregnancy. Based on the availability and non-invasive method of collection of menstrual blood (MB), here we investigated for the first time a comparative analysis of NK cell subsets in MB and peripheral blood (PB) of women with recurrent spontaneous abortion (RSA) and fertile women. PB and MB of healthy fertile (n=15) and RSA women (n=15) were sampled simultaneously on the second day of the menstrual cycle. Proportions of CD56+CD3−CD16+/−, CD56+CD3−CCR7+/−, and CD56+CD3−CD45RO+/− cells were analyzed using flow cytometry. In the MB of both groups, proportions of CD16+ and CD45RO− NK cells were significantly lower than in the PB. In parallel, CD56+CD16+CCR7− and CCR7+ cells were present in significantly smaller amounts in MB than in PB. However, the amounts of CD56+CD16−CCR7− and CCR7+ cells were greater in MB. In comparison to the fertile group, the percentage of MB CD45RO+ NK cells was significantly lower and frequencies of PB CD16−, CD45RO− and CD56+CD16+CCR7+ subsets were significantly higher in RSA patients. Different subsets of NK cells are differentially distributed in MB in comparison with PB in women with RSA and fertile subjects. Population differences of NK cell subsets in RSA patients and normal controls were more clearly reflected at the systemic level.

Natural killer (NK) cell deficit in coronary artery disease: no aberrations in phenotype but sustained reduction of NK cells is associated with low-grade inflammation

Although reduced natural killer (NK) cell levels have been reported consistently in patients with coronary artery disease (CAD), the clinical significance and persistence of this immune perturbation is not clarified. In this study we characterized the NK cell deficit further by determining (i) differentiation surface markers and cytokine profile of NK cell subsets and (ii) ability to reconstitute NK cell levels over time. Flow cytometry was used to analyse NK cell subsets and the intracellular cytokine profile in 31 patients with non-ST elevation myocardial infarction (non-STEMI), 34 patients with stable angina (SA) and 37 healthy controls. In blood collected prior to coronary angiography, the proportions of NK cells were reduced significantly in non-STEMI and SA patients compared with controls, whereas NK cell subset analyses or cytokine profile measurements did not reveal any differences across groups. During a 12-month follow-up, the proportions of NK cells increased, although not in all patients. Failure to reconstitute NK cell levels was associated with several components of metabolic syndrome. Moreover, interleukin (IL)-6 levels remained high in patients with sustained NK cell deficit, whereas a decline in IL-6 (P < 0·001) was seen in patients with a pronounced increase in NK cells. In conclusion, we found no evidence that reduction of NK cells in CAD patients was associated with aberrations in NK cell phenotype at any clinical stage of the disease. Conversely, failure to reconstitute NK cell levels was associated with a persistent low-grade inflammation, suggesting a protective role of NK cells in CAD.

Alterations In Specific Immune Cell Subsets In Children With Sickle Cell Disease

IntroductionPatients with sickle cell disease (SCD) are known to have altered immune systems. In the past, immunology studies in SCD patients have focused largely on splenic dysfunction and the increased risk of infection with encapsulated organisms. Increasingly, however, it is now being recognized that SCD patients also have evidence of abnormal immune activation with baseline chronic inflammation as well as high rates of red blood cell (RBC) alloimmunization and hematopoietic stem cell transplant (HSCT) graft rejection. We have previously demonstrated an increased risk of bone marrow transplant rejection with non-myeloablative conditioning in a SCD mouse model, with both increased T cells and natural killer (NK) cells implicated in this rejection. We have also previously shown that at baseline children with SCD have altered immune cell subsets. We now have expanded on this investigation of quantitative immune deviation in pediatric SCD by exploring how chronic transfusion (CT) or hydroxyurea (HU) therapy affects immunophenotype in a larger cohort of patients. MethodsOne hundred and eleven children (68 patients on CT only, 26 patients on HU only, 17 patients on neither CT nor HU) with SCD (SS or Sβ0) and 29 healthy, age-matched African American controls were recruited for this study. Exclusion criteria included recent illness or another disease associated with known abnormalities of the immune system. Flow cytometry was performed on samples from all participants to quantify the following immune cell populations: total white blood cells (WBC), total monocytes, total lymphocytes, T cells (total, CD4+, CD8+, naïve, central memory, effector memory, effector memory RA, and CD4+ T regulatory), B cells (total, naïve, and memory) and NK cells. ResultsSCD patients on CT and patients not receiving CT or HU (no CT/HU) had significantly increased total WBCs, granulocytes, monocytes, lymphocytes, T cells and B cells compared to healthy controls. Conversely, while SCD patients on HU also had mean counts that were elevated, they were not significantly greater than controls for any of these cell populations (Figure 1). Similarly, CD4+ T cells were significantly increased only in the CT and no CT/HU patients compared to controls (Figure 2). CD8+ T cells, however, were increased only in the CT group. CD4+ T cell subset analysis showed the most striking elevations in the CD4+ central memory and CD4+ effector memory (TEM) populations for both CT and no CT/HU patients compared to controls (Figure 3). CD4+ T regulatory cells were also increased in both of these groups. B cell analysis showed increased numbers of naïve but not memory B-cells in CT and no CT/HU patients. In distinct contrast to most populations that were elevated in CT patients, cytotoxic NK cells were uniquely increased only in no CT/HU patients. [Display omitted] [Display omitted] [Display omitted] ConclusionsChildren with SCD have quantitative differences in many different immune cell populations compared to age and race matched healthy controls. HU treatment appears to bring the absolute number of these cell subsets closer to those of healthy controls, while CT does not. It is unclear at this point to what extent CT therapy itself or baseline patient characteristics leading to the need for CT contribute to these findings. Future work is investigating whether certain cell subset abnormalities are associated with particular therapeutic complications such as RBC alloimmunization or HSCT graft rejection. Better understanding the immunology of SCD may allow for targeted therapies to improve SCD transfusion support and HSCT outcomes. Disclosures:Off Label Use: Abstract includes information related to the use of hydroxyurea in children with sickle cell disease.

Immune cell subtyping in the cerebrospinal fluid of patients with neurological diseases

The analysis of cerebrospinal fluid (CSF) with the assessment of CSF cell counts and proteins is an important method in the diagnostic workup of neurological diseases. As an addition to this standard approach, we here present data on the distribution of CSF immune cell subsets in common neurological diseases, and provide reference values along with cases of rare neurological diseases. CD4+ and CD8+ T cells, the CD4/CD8 ratio, B cells, plasmablasts, monocytes and NK cells in the CSF of 319 patients with inflammatory or non-inflammatory neurological diseases were analysed by seven-color flow cytometry. Diagnoses included headache, idiopathic intracranial hypertension, Guillain-Barré syndrome, multiple sclerosis, Lyme neuroborreliosis, bacterial and viral meningitis, human immunodeficiency virus (HIV) infection, stroke, and CNS malignancies, among others. T cells were the predominant population in the CSF with CD4+ T cells being more prevalent than CD8+ T cells. Mostly in HIV patients, and under other conditions of immunosuppression, CD4+ and CD8+ T cells were significantly altered and the CD4/CD8 ratio reduced. B cells and plasmablasts could hardly be detected in non-inflammatory diseases but were consistently elevated in inflammatory diseases. Monocytes were reduced in neuroinflammation and showed a negative correlation with B cells. NK cells were slightly elevated in neuroinflammation. Both monocytes and NK cells were slightly elevated in CNS malignancies. The analysis of immune cell subsets in the CSF adds valuable information to clinicians and is a promising tool for the differential diagnosis of neurological diseases.

Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung

The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.

Multicolor Flow Cytometric Analysis of Immune Cell Subsets in Tumor-Bearing Mice

This protocol describes a procedure for obtaining a single-cell suspension from mouse spleen or solid tissue. Once a single-cell suspension is prepared, multicolor flow cytometry is used to identify subsets of immune cells in the samples of interest. This protocol can be used to evaluate the following immune cell populations, defined by the expression of certain cell surface molecules: total leukocytes, T lymphocytes and CD4(+) and CD8(+) T-cell subsets, B lymphocytes, natural killer cells, dendritic cells, and immature myeloid cells.

Role of NK Cell Subsets in Organ-Specific Murine Melanoma Metastasis

Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b–, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche.

OP0139 A distinct subset of CD19-negative plasma cells resides in the human bone marrow

BackgroundPlasma cells (PC) are the unique source of protective and autoreactive immunoglobulin. Mature PC are resting and have downregulated CD20, rendering them refractory to almost all conventional and modern antirheumatic...

Variability of Memory B cell Markers in a Cohort of Common Variable Immune Deficiency Patients over 6 months

Common Variable Immunodeficiency Disorder (CVID) is a complex disorder that predisposes patients to recurrent and severe infections. Immunophenotypic classification schemes were developed to categorize patients with CVID into phenotypic and prognostic groups based on different memory B cell subsets. Whether the B cell subset analysis is stable over time has not been investigated. B cell phenotyping in patients with CVID (n = 15) and sex- and age-matched controls (n = 26) were carried out according to the three B cell classifications. Patients with CVID were evaluated monthly over 6 months. Controls were assessed once during the study. We scored how often each patient was assigned to the same group within each classification. The Freiburg classification assigned patients to the same group at a rate of 73% and the Paris classification at 88%. The EUROclass classification of smB- versus smB+ was at 90%. The two subclassifications [(smB-21low or smB-21norm) and transitional B] were at 87% and 97%, respectively. The level of naïve B cells measured in all patients with CVID during the 6-month evaluation was the most stable B cell subset. We conclude that all classifications systems show considerable variability, but the EUROclass classification was the most reliable scheme for our 15 CVID and 26 healthy cohorts. Our results indicate that phenotypic classifications within CVID will be difficult while there is variability of commonly used assays.

Higher cytotoxic capacity in peripheral blood of sarcoidosis patients (P3135)

Abstract BACKGROUND. Sarcoidosis is a granulomatous disease of unknown etiology, mainly affecting the lungs, where greatly increased numbers of CD4+ T cells typically are seen. However, CD8+ T cells can sometimes make up the majority of cells recovered by bronchoalveolar lavage (BAL). Previous studies have extensively studied Th1 cytokine-producing T cells and established their role in the pathological process. Little is however known about cytotoxic cells in sarcoidosis. METHODS: Peripheral blood (from 17 sarcoidosis patients and 11 healthy controls) and BAL fluid (from 19 patients and 4 controls) was obtained. Flow cytometric analysis of cell subsets was performed after staining with antibodies against the surface molecules CD3, CD4 and CD8 as well as antibodies against the cytotoxic intracellular mediators perforin, granzyme B and granulysine. The PBMC cytotoxic activity was quantified with the 51Cr release assay. RESULTS: Sarcoidosis patients had a significantly higher percentage of both perforin+ (p &amp;lt; 0.001) and granzyme B+ (p &amp;lt; 0.05) CD8+ T cells in blood. In BAL, however, a tendency in the opposite direction was seen. Higher cytotoxic activity was observed in PBMC from patients with sarcoidosis compared to controls. CONCLUSION: Cytotoxic cells, including CD8+ T cells, may be involved in the propagation or regulation of the inflammatory process in sarcoidosis, but further studies are needed to delineate their role in this disease.

Natural Killer Cell Dysfunction in Uremia: The Role of Oxidative Stress and the Effects of Dialysis

Immune deficiency is one of the numerous consequences of chronic renal failure. We can hypothesize that an abnormal natural killer (NK) cell response is present in patients with end-stage renal disease (ESRD). To characterize the immune defect in ESRD patients, we performed a NK cell subset analysis in 66 patients with ESRD treated by hemodialysis or peritoneal dialysis. Compared with healthy blood donors, patients undergoing chronic dialysis showed a profound decrease in NKG2D(+) cells within both CD8(+) T cell (58 vs. 67%, p = 0.03) and NK cell (39 vs. 56%, p = 0.002) populations. We further studied NK cell subsets in 55 hemodialysis patients and 17 patients treated by peritoneal dialysis. As compared to healthy donors, all these patients had significantly decreased NKG2D-positive NK cells. Patients using PMMA membranes (BK-F) or Helixone-FX membranes had a smaller decrease in NKG2D-positive NK cells when compared to patients treated with Nephral membranes. The expression of MICA on the cell surface of monocytes, which is a marker of inflammation induced by cellular stress, was also lower in patients using BK-F membranes. In conclusion, these data suggest that a subpopulation of NK cells is decreased in patients with ESRD. This decrease is associated with high circulating levels of the HLA-related molecule MICA. The dialysis membrane can influence the modulation of both the phenotype of NK cells and MICA overexpression.