Karin Backteman

B Cell Abnormalities in Wegener’s Granulomatosis and Microscopic Polyangiitis: Role of CD25+-expressing B Cells

Objective. The use of rituximab in vasculitis has increased interest in B cell biology. A subpopulation of B cells expressing CD25 shows antigen-presenting properties and may have regulatory functions. We assessed subpopulations of B cell maturation (Bm) and markers related to activity and antigen presentation, and related the findings to disease activity. Methods. Multiparameter flow cytometry was used to assess numbers and proportions of circulating lymphocytes from 34 patients with vasculitis (16 remission, 18 active) and 20 controls. Results. Active vasculitis samples showed decreased proportions of Bm1 (7.8% vs 11%; p = 0.041), Bm2’ (0.2% vs 0.7%; p = 0.002), and Bm3/Bm4 (0.1% vs 0.3%; p = 0.006), compared with controls; Bm2 cells were the most frequently occurring B cells but they were not significantly different in active vasculitis (74% vs 62%; p = 0.083). In patients with remission the proportion of CD25+ B cells was increased compared to controls (48% vs 29%, respectively; p = 0.006) and also compared to active vasculitis (23%; p = 0.006). The proportion of CD86+ B cells was also increased (31%) compared to active vasculitis (8%; p = 0.001), and to controls (6%; p = 0.0003). In multivariate analysis, Bm2’ cells and CD25+27– B cells were independently influencing the patient group. Conclusion. In active vasculitis, a lower proportion of Bm1 cells may indicate activated B cells. Patients in remission had higher proportions of CD25+ (α-chain of interleukin 2 receptor) and CD86+ (costimulatory molecule) B cells. We suggest that these B cells may have a regulatory role, or alternatively may result from previous treatment.

Persistent accumulation of interferon-γ-producing CD8+CD56+ T cells in blood from patients with coronary artery disease

OBJECTIVE There is emerging evidence for CD8(+) T cell alterations in blood from patients with coronary artery disease (CAD). We examined whether the distribution and phenotype of CD8(+)CD56(+) T cells differed according to the clinical manifestation of CAD. METHODS Patients with acute coronary syndrome (ACS, n = 30), stable angina (SA, n = 34) and controls (n = 36) were included. Blood was collected before and up to 12 months after referral for coronary investigation. CD8(+)CD56(+) T cells were assessed by flow cytometry for expression of surface markers, apoptosis, and intracellular expression of cytokines. RESULTS The proportions of CD8(+)CD56(+) T cells were significantly higher in both ACS and SA patients compared with controls, and remained so after 3 and 12 months. This was independent of age, sex, systemic inflammation and cytomegalovirus seropositivity. CD8(+)CD56(+) T cells differed from CD8(+)CD56(-) T cells in terms of lower CD28 expression and fewer apoptotic cells. Both CD8(+) T cell subsets were positive for interferon (IFN)-γ and tumor necrosis factor, although IFN-γ was significantly more confined to the CD8(+)CD56(+) T cells. CONCLUSION The persistent accumulation of CD8(+)CD56(+) T cells in ACS and SA patients share several features with immunological aging. It also contributes to a larger IFN-γ(+) pool in blood, and may thereby hypothetically drive the atherosclerotic process in a less favorable direction.

Lymphocyte Subpopulations in Lymph Nodes and Peripheral Blood: A Comparison between Patients with Stable Angina and Acute Coronary Syndrome

Objective Atherosclerosis is characterized by a chronic inflammatory response involving activated T cells and impairment of natural killer (NK) cells. An increased T cell activity has been associated with plaque instability and risk of acute cardiac events. Lymphocyte analyses in blood are widely used to evaluate the immune status. However, peripheral blood contains only a minor proportion of lymphocytes. In this study, we hypothesized that thoracic lymph nodes from patients with stable angina (SA) and acute coronary syndrome (ACS) might add information to peripheral blood analyses. Methods Peripheral blood and lymph nodes were collected during coronary by-pass surgery in 13 patients with SA and 13 patients with ACS. Lymphocyte subpopulations were assessed by flow cytometry using antibodies against CD3, CD4, CD8, CD19, CD16/56, CD25, Foxp3, CD69, HLA-DR, IL-18 receptor (R) and CCR4. Results Lymph nodes revealed a lymphocyte subpopulation profile substantially differing from that in blood including a higher proportion of B cells, lower proportions of CD8+ T cells and NK cells and a 2-fold higher CD4/CD8 ratio. CD4+CD69+ cells as well as Foxp3+ regulatory T cells were markedly enriched in lymph nodes (p<0.001) while T helper 1-like (CD4+IL-18R+) cells were more frequent in blood (p<0.001). The only significant differences between ACS and SA patients involved NK cells that were reduced in the ACS group. However, despite being reduced, the NK cell fraction in ACS patients contained a significantly higher proportion of IL-18R+ cells compared with SA patients (p<0.05). Conclusion There were several differences in lymphocyte subpopulations between blood and lymph nodes. However, the lymphocyte perturbations in peripheral blood of ACS patients compared with SA patients were not mirrored in lymph nodes. The findings indicate that lymph node analyses in multivessel coronary artery disease may not reveal any major changes in the immune response that are not detectable in blood.

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.

Flow-Cytometric Quantitation of Anti-D Antibodies

Objectives: Quantitation of Rh antibodies is important clinically in predicting the risk of hemolytic disease of the newborn. We describe a flow cytometry method for the quantitation of anti‐D antibodies that we developed in parallel to a recently described method. Methods: As a secondary antibody we used whole IgG instead of Fab molecules. The advantages, besides lower cost, include a stronge fluorescence signal with no need for amplification, and the possibility of diluting samples to minimize the risk of agglutination by IgM antibodies. We did extensive studies on reproducibility. Results: Reproducibility was superior to the autoanalyzer method. The two methods were roughly in agreement in estimating low, medium, or high levels of anti‐D with a correlation coefficient of 0.89. The autoanalyzer measures the in vitro agglutination of all anti‐D antibodies whereas flow cytometry measures the amount of IgG anti‐D bound to red cells, which is more like the in vivo situation. Conclusion: Further studies in a clinical setting will show whether flow‐cytometric quantitation may improve the diagnostic value of anti‐D concentration measurement.

Expansions of CD4+CD28- and CD8+CD28- T cells in granulomatosis with polyangiitis and microscopic polyangiitis are associated with cytomegalovirus infection but not with disease activity.

Objective. T helper cells lacking CD28 (CD4+CD28–) have been implicated in the pathogenesis of granulomatosis with polyangiitis (Wegener; GPA) and microscopic polyangiitis (MPA). Expansions of CD4+CD28– and CD8+CD28– T cells have also been associated with latent cytomegalovirus (CMV) infection. We assessed these T cells with and without coexpression of CD56 and CD57 in relation to vasculitis as well as CMV status. Methods. Blood from 16 patients in remission (12 GPA, 4 MPA), 18 patients with active vasculitis (12 GPA, 6 MPA), and 20 healthy controls was examined by flow cytometry for expression of CD4, CD8, CD56, CD57, and CD28 on T cells. The influence of age, CMV status, presence of disease, and disease activity on T cell subpopulations was tested with multiple regression analyses. Results. In active vasculitis, the total numbers and proportion of lymphocytes were decreased. Total numbers of CD4+, CD8+, CD4+CD28–, CD8+CD28–, CD4+CD57+, and CD8+CD57+ T subpopulations were decreased to the same extent, implying unchanged proportions. Multivariate analyses showed no associations between vasculitis and CD28– or CD57+ T subpopulations, whereas immunoglobulin G antibodies to CMV were associated with expanded proportions of CD28– and CD57+ T cells, in both the CD4+ and the CD8+ compartments. Conclusion. CD28– and CD57+ T cells were associated with latent CMV infection and not with a diagnosis of GPA or MPA. Vasculitis assessment should include CMV status.

A rapid and reliable flow cytometric routine method for counting leucocytes in leucocyte-depleted platelet concentrates

Background and Objectives To ensure a proper quality control it is important to use a reliable method to count low numbers of leucocytes in leucocyte‐reduced platelet concentrates (PCs).

Soluble Fas ligand is associated with natural killer cell dynamics in coronary artery disease

OBJECTIVE Apoptosis of natural killer (NK) cells is increased in patients with coronary artery disease (CAD) and may explain why NK cell levels are altered in these patients. Soluble forms of Fas and Fas ligand (L) are considered as markers of apoptosis. Here, we investigated whether plasma levels of Fas and FasL were associated with NK cell apoptosis and NK cell levels in CAD patients. METHODS Fas and FasL in plasma were determined by ELISA in 2 cohorts of CAD patients; one longitudinal study measuring circulating NK cells and apoptotic NK cells by flow cytometry 1 day, 3 months and 12 months after a coronary event and one cross-sectional study measuring NK cell apoptosis ex vivo. Both studies included matched healthy controls. Fas and FasL were also determined in supernatants from NK cells undergoing cytokine-induced apoptosis in cell culture. RESULTS In the 12-month longitudinal study, plasma FasL increased by 15% (p<0.001) and NK cell levels by 31% (p<0.05) while plasma Fas did not change. Plasma FasL and NK cell levels were significantly related at 3 months and 12 months, r=0.40, p<0.01. Furthermore, plasma FasL, but not plasma Fas, correlated with NK cell apoptosis ex vivo in CAD patients, r=0.54, p<0.05. In vitro, cytokine-induced apoptosis of NK cells resulted in abundant release of FasL. CONCLUSION In CAD patients, FasL in plasma is associated with both apoptotic susceptibility of NK cells and dynamic changes in circulating NK cells. NK cells are also themselves a potential source of soluble FasL. Our findings link NK cell status to a soluble marker with possible atheroprotective effects thereby supporting a beneficial role of NK cells in CAD.