CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Clinical significance of detection of soluble interleukin 2 receptor alpha chain in the assessment of rheumatoid arthritis disease activity / 北京大学学报(医学版)

OBJECTIVE@#To evaluate soluble interleukin-2 receptor alpha chain (sIL-2Rα, sCD25) in serum for the determination of rheumatoid arthritis (RA) activity.@*METHODS@#Peripheral blood was collected from 108 patients with RA, 39 patients with osteoarthritis (OA) and 50 healthy control subjects, and synovial fluids were from 40 patients with RA. The sera from the patients with RA, the disease control group (osteoarthritis), the healthy control group, and the synovial fluids of the RA patients were detected by enzyme-linked immunosorbent assay (ELISA).The clinical manifestations and laboratory parameters of the patients with RA were recorded and the correlation with the serum sCD25 level was analyzed.@*RESULTS@#The serum sCD25 concentration in RA group was (2 886±1 333) ng/L, the serum sCD25 concentration in OA group was (2 090±718) ng/L, and the serum sCD25 concentration in healthy group was (1 768±753) ng/L. The serum sCD25 level in the patients with RA was significantly higher than that in the disease controls and healthy controls (P<0.001). Sensitivity of serum sCD25 in the diagnosis of RA was 66.1% and specificity was 83.0%;serum sCD25 levels and erythrocyte sedimentation rate (r=0.321, P=0.001), C-reactive protein (r=0.446, P<0.001), DAS28 score (r=0.324, P<0.001), joint tenderness count (r=0.203, P=0.024), D-dimer levels (r=0.383, P<0.001), age (r=0.24, P=0.007), IgG (r=0.207, P=0.028), HRF-IgG (r=0.345, P=0.034) showed a significant positive correlation, and disease duration (r=-0.206, P=0.021) showed a negative correlation with sCD25;In patients with rheumatoid arthritis, the positive rates of serum ESR, CRP, and sCD25 were 14.3% (2 cases), 14.3% (2 cases), and 71.4% (10 cases) in the low disease activity group. The positive rates of serum ESR, CRP and sCD25 in the moderate disease activity group were 94.2% (49 cases), 82.7% (43 cases), and 86.5% (45 cases). The positive rates of serum ESR, CRP, and sCD25 in the high disease activity group were 100% (42 cases), 95.2% (40 cases), and 90.5% (38 cases);36 cases of ESR and/or CRP were negative (about 33.3%) in 108 patients, serum sCD5 levels of 17 cases in these 36 cases (about 47.2%)increased, of which 14 cases (about 82.4%) had a DAS28 score higher than 3.2.@*CONCLUSION@#The serum sCD25 has a high specificity for diagnosis of RA and a poor sensitivity. The serum level is closely related to the activity of RA, indicating that sCD25 may be involved in the inflammatory process of RA and may become a new inflammatory marker of RA. It is more meaningful for detection of serum sCD25 when RA is active, but ESR and/or CRP is negative.

Systemic mastocytosis with associated acute myeloid leukemia with t (8; 21) (q22; q22).

Systemic mastocytosis with associated clonal hematological nonmast cell lineage disease (SM-AHNMD) is a subtype of mastocytosis associated commonly with myeloid neoplasms, Non-Hodgkin's lymphoma, or other hematological neoplasms. In these conditions, mastocytosis needs to be differentiated from mast cell hyperplasia or mast cell activation states. Neoplastic nature of mastocytosis is proved either by morphology, aberrant immunophenotype, or detection of point mutation at codon-816 of c-kit gene. This is a rare entity, even more so in pediatric population. Herein, we report a case of 14-year-old girl with SM associated with acute myeloid leukemia with maturation with t(8;21). Multifocal dense infiltrate of spindle-shaped mast cells on bone marrow aspirate and biopsy with coexpression of CD2 and CD25 by flow cytometric analysis proved the SM component at the time of diagnosis and persistence at post induction status also.

Leukemic phase of anaplastic lymphoma kinase positive, anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a distinct type of CD30+ T/null-cell non-Hodgkin's lymphoma that frequently involves nodal and extranodal sites. The presence of leukemic phase in ALCL is extremely rare and occurs exclusively with ALK1-positive ALCL. We describe two patients with ALK1-positive ALCL who developed a leukemic phase with rapid progression of the disease. Immunophenotypic pattern assessed on peripheral blood by flow cytometry revealed CD45, CD30, and CD25 positivity in both cases but NPM-ALK1 was expressed in only one case. Both patients developed leukemic phase as a terminal event of the disease and we share the immunophenotypic features of both cases.

Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study.

Background: Hepatocellular carcinoma (HCC) is notorious for poor prognosis with limited therapeutic options. A better understanding of the role of regulatory T-cells (Tregs) in HCC is important for design of immunotherapy based clinical protocol. The objective of the present study was to evaluate the presence of Tregs in tumor microenvironment in patients with HCC compared to chronic hepatitis (CH). Materials and Methods: The frequency of CD4 + CD25 + Treg cells was evaluated from peripheral blood (PB) of 28 patients of HCC and 30 controls including CH cases and healthy donors using flowcytometry. Intratumoral Treg were also analyzed in tissue samples from 17 HCC cases and 15 CH cases. In addition the expression of FOXP3 and CTLA-4 was also studied by RT-PCR. Results: Frequency of CD4 + CD25 + cells in the PBMCs of HCC cases was significantly higher than in HC (10.8 ± 7.64 vs 3.05 ± 1.30, P < 0.005) and CH patients (2.88 ± 1.92, P < 0.005). Also Treg population was significantly higher in HCC tumor microenvironment compared to CH biopsies (15.8 ± 5.32 vs 5.51 ± 3.40, P < 0.05). Expression of FOXP3 and CTLA-4 was also significantly higher in HCC patients ( P < 0.05) compared to CH group. Conclusions: We provide evidence of an increased population of Treg not only in the PB but also in tumor microenvironment of HCC patients, suggesting association of enhanced Treg activity with poor immune responses to tumor antigens. These findings may in future play a significant role in designing immunotherapeutic approaches in HCC.