Effect of the in vivo application of granulocyte colony‐stimulating factor on NK cells in bone marrow and peripheral blood

Granulocyte colony‐stimulating factor (G‐CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor‐derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post‐allo‐HSCT. The aim of this study was to evaluate the effect of G‐CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post‐G‐CSF in vivo application. Relative expansion of CD56^bri NK cells led to a decreased ratio of CD56^dim and CD56^bri NK subsets in BM and PB post‐G‐CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G‐CSF treatment. G‐CSF treatment decreased the IFN‐γ‐secreting NK population (NK1) dramatically in BM and PB, but increased the IL‐13‐secreting NK (NK2), TGF‐β‐secreting NK (NK3) and IL‐10‐secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft‐vs‐host disease post‐transplantation. Taken together, our results show that the in vivo application of G‐CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56^dim and CD56^bri NK cells as well as decreased NK1 populations in both PB and BM.     

Leukocyte trafficking in free-flowing cerebrospinal fluid of normal rhesus macaques (Macaca mulatta)

Abstract: Cellular components in free-flowing cerebrospinal fluid (CSF) of normal rhesus macaques were characterized. Microscopic counting enumerated the total number of leukocytes, percentage of polymorphonuclear cells (PMN), leukocytes with nonspecific esterase (NSE), and those reducing nitro blue tetrazolium (NBT). Flow cytometric analysis further identified CD4, CD8, CD14, and CD20 positive leukocytes. These experiments established reliable techniques for evaluating cellular components in CSF from rhesus macaques and documented the difference in the CD4/CD8 ratio between peripheral blood (PB) and CSF compartments under normal physiological conditions.     

IL-2 stimulated but not unstimulated NK cells induce selective disappearance of peripheral blood cells: concomitant results to a phase I/II study

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9-14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/-) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(-) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/-)CD69(+)NCR(high)CD62L(-) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.     

Increased proportion of CD4+CDw29+CD45R-UCHL-1+ lymphocytes in the cerebrospinal fluid of both multiple sclerosis patients and healthy individuals

In this study we confirm earlier reports of an increase of the proportion of T and CD4+ lymphocytes and a decrease of B and CD8+ lymphocytes in cerebrospinal fluid (CSF) as compared to peripheral blood (PB) in MS patients. In addition we now demonstrate that this difference between CSF and PB lymphocyte populations is of the same magnitude in healthy individuals suggesting that it is physiological and not associated with disease. Functionally distinct subsets of the T human helper cell (CD4+) population have previously been defined by the monoclonal antibodies 4B4 (CDw29), Leu-18 (CD45R), and UCHL-1. In the present investigation we demonstrate a selective increase in the proportion of CD4+CDw29+CD45R-UCHL-1+ lymphocytes in CSF as compared to PB of both MS patients and healthy individuals, which strongly indicates that also this enrichment is physiological rather than associated with disease. A possible relationship between this subset of CD4+ lymphocytes and T memory cells is discussed.     

Lack of dendritic cell mobilization into the peripheral blood of cancer patients following standard- or high-dose chemotherapy plus granulocyte-colony stimulating factor

BACKGROUND: Dendritic cells (DC), the most specialized antigen-presenting cells, can be detected in the peripheral blood (PB) and divided into two subsets of populations, DC1 and DC2, endowed with different functions. The aim of this study was to evaluate the effect on DC release and on their subsets of three regimens utilized to mobilize CD34+ cells into the PB in cancer patients and in normal CD34+ cell donors. PATIENTS AND METHODS: The mobilizing sequences were: standard-dose epirubicin+taxol+granulocyte-colony-stimulating factor (G-CSF; 15 patients with advanced breast cancer), high-dose cyclophosphamide (CTX)+G-CSF (10 patients with breast cancer patients and 7 with non-Hodgkin's lymphoma, NHL), and G-CSF alone (5 normal donors of CD34+ cells for allogeneic transplantation). Comparative data were obtained from the steady-state PB of 20 healthy volunteers. For flow cytometric analysis, DC were gated as negative for specific lineage markers (CD3, CD11b, CD14, CD16, CD56, CD19, CD20, CD34) and positive for HLA-DR. The DC1 and DC2 subsets were defined as CD11c and CDw123 positive, respectively. RESULTS: The percentages of DC at baseline and the time of CD34+ cell peak were: 0.48 and 0.51 for standard-dose chemotherapy (CT); 0.55 and 0.63 for breast cancer after high-dose CTX+G-CSF; 0.53 and 0.71 for NHL after high-dose CTX+G-CSF; and 0.51 and 0.54 for normal donors of CD34+ cells after G-CSF alone (all p=n.s.).Mean DC1/DC2 ratios in each study group at the time of CD34+ cell peak were 0.10, 0.12, and 0.18, respectively. Finally, in the group of healthy volunteers, the percentage of circulating DC was 0.95 and the mean DC1/DC2 ratio was 1.28. CONCLUSION: To our knowledge, this is the first report that demonstrates that both standard-dose or high-dose CT, when utilized together with G-CSF, do not induce DC mobilization into the PB, whereas a reversed DC1/DC2 ratio is observed. Furthermore, a lack of significant DC mobilization after G-CSF alone was also seen, in contrast to what was previously observed by others. These data should be taken in account when evaluating clinical correlations between DC number and CPC engraftment in both the transplantation setting, when monitoring the effects on the immune system of combinations of new drugs and/or cytokines, and when high numbers of DC are required for both experimental and clinical applications.     

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO+ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA+ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5+ and CCR3+ cells within the CD4+CD45RO+ and CD8+CD45RO+ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA+ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO+ memory T cells, as well as on CD45RA+ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4+ and CD8+ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.     

Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells

There is some evidence that patients with chronic fatigue syndrome (CFS) suffer from immune abnormalities, such as immune activation and decreased immune cell responsivity upon polyclonal stimili. This study was designed to evaluate lymphocyte activation in CFS by using a CD69 expression assay. CD69 acts as a costimulatory molecule for T- and natural killer (NK) cell activation. We collected whole blood from CFS patients, who met CDC criteria, and healthy volunteers. The blood samples were stimulated with mitogens during 18 h and the levels of activated T and NK cells expressing CD69 were measured on a Coulter Epics flow cytometer using a three color immunofluorescence staining protocol. The expression of the CD69 activation marker on T cells (CD3+, CD3+CD4+, and CD3+CD8+) and on NK cells (CD45+CD56+) was significantly lower in CFS patients than in healthy subjects. These differences were significant to the extent that a significant diagnostic performance was obtained, i.e. the area under the ROC curve was around 89%. No differences either in the number of leukocytes or in the number or percentage of lymphocytes, i.e. CD3, CD4, CD8 and CD19, could be found between CFS patients and the controls. Patients with CFS show defects in T- and NK cell activation. Since induction of CD69 surface expression is dependent on the activation of the protein kinase C (PKC) activation pathway, it is suggested that in CFS there is a disorder in the early activation of the immune system involving PKC.     

Restoration of CD3+CD56+ cell level improves skin lesions in severe psoriasis: A pilot clinical study of adoptive immunotherapy for patients with psoriasis using autologous cytokine-induced killer cells

Psoriasis is a chronic inflammatory skin disorder mediated by the cells and molecules of both the innate and adaptive immune systems. Autologous cytokine-induced killer (CIK) cell infusion is considered an effective and safe cancer treatment and is licensed for this use in China. Accumulated evidence indicating that CD3⁺CD56⁺ cells are significantly decreased in psoriatic patients prompted us to investigate if the restoration of CD3⁺CD56⁺ cells may be beneficial for psoriatic patients. We designed a clinical trial for psoriasis treatment that involved CIK cell infusion because CIK cells include a large amount of CD3⁺CD56⁺ T cells (NCT01894373 at www.clinicaltrials.gov). Six patients with severe psoriasis were initially enrolled, and four of them exhibited markedly lower levels of CD3⁺CD56⁺ cells in their peripheral blood (PB) relative to healthy donors. CIK cell infusion-associated toxicity was not observed in any infusion. The percentage of CD3⁺CD56⁺ cells in the PB markedly increased and the psoriasis area and severity index (PASI) synchronously decreased in four patients with lower CD3⁺CD56⁺ cell contents, and two of them obtained a more than 4-month PASI75 after completing a four-cycle treatment. However, a decrease in the CD3⁺CD56⁺ cells was observed concomitantly with disease recurrence after short-term amelioration. In contrast, no obvious improvement was observed in the two patients with nearly normal CD3⁺CD56⁺ cells in the PB before treatment. These observations suggest that the normalization of the CD3⁺CD56⁺ cell level may improve the skin lesions of severe psoriasis and warrant further clinical trials for severe psoriasis using repeated CIK adoptive immunotherapy.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

NKT cells in the induced sputum of severe asthmatics

To determine whether there was a specific inflammatory process in severe asthmatics, the phenotypic characteristics of induced sputum immune cells were analysed among patients with severe asthma. Twenty-two induced sputa (10 severe asthmatics) were studied. Flow cytometric analysis was performed using immune cells of the sputum and monoclonal antibodies to CD3, CD4, CD8, CD56, CD25, and TCRgammadelta. The number of NKT (CD3(+) CD56(+)) cells was significantly higher in the sputum of severe asthmatics compared with mild asthmatic and healthy control groups (P < .05). CD8(+)CD56(+) cells were the predominant subtype of the increased NKT cells in severe asthmatics. CD3(+)CD56(+)Valpha24(+), TCRgammadelta(+) CD56(+), and CD4(+)CD25(+) T cells were significantly increased in severe asthmatic patients. These results suggest that the immunopathogenesis of severe asthmatics vary between severe and mild asthmatics, and that CD8(+)CD56(+) NKT cells may play an important role in the immunopathogenesis of severe asthma.     

Spontaneous apoptosis, oxidative status and immunophenotype markers in blood lymphocytes of AIDS patients.

Peripheral blood mononuclear cells (PBMC) from 251 HIV-positive drug abusers of known clinical stage and from 40 healthy donors were tested for conventional immunologic markers (CD3, CD4, CD8, CD19, CD14, CD16/CD56, CD45 and HLA-DR). Additional cell parameters and the occurrence of spontaneous apoptosis (programmed cell death) were investigated on freshly isolated PBMC by flow cytometric measurement of either annexin-V bound to plasma membrane phosphatidylserine or propidium iodide uptake. The activity of gamma-glutamyltransferase (gamma-GT), an ectoenzyme contributing to the synthesis of the intracellular antioxidant glutathione (GSH) and involved in early apoptosis, was also determined in these cells. Immunocompetent T-cell counts were lower in HIV+ patients, with the exception of CD8+ and HLA-DR+ lymphocytes. The external binding of annexin-V was significantly higher in HIV+ PBMC and occurred in both CD8+ and CD4+ T-lymphocyte subsets. The activity of gamma-GT, was significantly lower in the PBMC from HIV+ patients, indicating that the redox status of PBMC may be affected in HIV+ individuals. Finally, the most dominant features characterising patients receiving antiretroviral therapy were greater long-term stability in the distribution of various cell parameters excepted the level of apoptosis.     

Expression of Fas antigen and Fas ligand in bronchoalveolar lavage from silicosis patients

OBJECTIVE: To understand the role of apoptosis through Fas/Fas ligand (FasL) interaction in the pathogenesis of silicosis, we examined the expression of Fas antigen, FasL and apoptosis in bronchoalveolar lavage fluid lymphocytes obtained from patients with silicosis. MATERIALS AND METHODS: Ten patients with silicosis, and 10 healthy controls were studied. Non-adherent cells were separated and analysed by cytometry for the expression of Fas antigen, FasL, and the co-expression of Fas/FasL. By double staining, we studied the FasL expression on CD4, CD8, CD56 and CD45RO-positive cells. DNA fragmentation was investigated by the terminal deoxy(d) UTP nick end labelling (TUNEL) method. RESULTS: We have found Fas and FasL expression in silicosis patients to be significantly higher than those in healthy controls. Interestingly, 6-18% of lymphocytes from silicosis patients co-expressed Fas and FasL. In silicosis patients, FasL was highly expressed on CD4+, CD56+ and CD45RO+ bronchoalveolar lavage cells. Fas antigen expressing cells showed DNA fragmentation characteristic for apoptosis. CONCLUSION: FasL was significantly expressed on cytotoxic effector and memory cells. The Fas/FasL system is implicated in the inflammatory process observed in silicosis patients.     

The activation processes of the immune system during low intensity exercise without relaxation

The activation processes of T-, B- and NK-cells were studied during 8-week low intensity strength training without relaxation. After the long-term training, no changes occurred in the peripheral blood contents of the main subpopulations of immunocompetent cells (CD3+, CD4+, CD8+, CD19+ and CD16+/CD56+ lymphocytes) and serum levels of immunoglobulin A, M, and G. At the same time, training was accompanied by positive activation of the immunocompetent cells which was evident from increased percentage of CD3+, CD19+ and /CD56+ cells expressing CD69 after activation (PHA, PW and II.2, respectively). The final period of the training course was also associated with a decrease in the level of lymphocyte apoptosis and increase of proliferative responses of lymphocytes.     

Intracellular cytokine profiles and T cell activation in pulmonary sarcoidosis

In granulomatous inflammatory lung diseases such as sarcoidosis, the balance of cytokine production by activated T cells in the lungs may influence clinical disease outcome. To investigate the potential of T lymphocytes to produce cytokines and contribute to this process, T cells from bronchoalveolar lavage (BAL) and PB from 19 patients with active lung disease were stimulated, stained, and analysed by flow cytometry for intracellular production of cytokines and expression of the activation marker CD69. Higher proportions of BAL cells expressed CD69 compared with PB, in the absence of in vitro stimulation. The expression of IFN-γ was similar in unstimulated BAL and PB T cells, and there was no association between the expression of CD69 and IFN-γ. Following stimulation, there were increased numbers of IFN-γ⁺ T cells. A similar trend was found with IL-2⁺ T cells, but there were lower levels of IL-4⁺ T cells in BAL compared with PB, and similar levels of IL-10⁺ T cells. The presence of activated T lymphocytes in BAL samples from patients with sarcoidosis, with the potential to produce Th1 type 1 cytokines may contribute to the inflammatory processes in this granulomatous lung disease. The use of intracellular flow cytometry to investigate cytokine production by BAL T cells could help to indicate potential targets for future therapy.     

Comparative analysis of differential expression of sialic acids and adhesion molecules on mononuclear cells of bone marrow and peripheral blood in childhood acute lymphoblastic leukaemia at diagnosis and clinical remission

Childhood acute lymphoblastic leukaemia (ALL) is characterized by the neoplasm of immature haematopoietic precursor cells (HPCs). We report significant differences between the expression of sialoglycoproteins and adhesion molecules on mononuclear cells (MNCs) of bone marrow (BM) and peripheral blood (PB) from individual children at diagnosis of the disease. Lymphoblasts in PB predominantly expressed 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs), sialic acid, alpha2-3 linked sialic acid, L- and P-selectins and vascular cell adhesion molecule -1 (VCAM-1) on their surface compared to BM, as determined with selective lectins and monoclonal antibodies (mAbs) by flow cytometric analysis. CD34+CD38+ cells present either in diagnostic PB or BM always showed enhanced expression of both alpha2-3 and alpha2-6 linked sialic acids, Neu5,9Ac2-GPs, L- and P-selectins and VCAM-1, compared to CD34+CD38- population, as confirmed by higher mean fluorescence intensity (MFI). Expression of ICAM-1 was reverse. However, MFI of Neu5,9Ac2-GPs was always higher both in CD34+CD38+ and CD34+CD38- population in PB compared to BM. Diverse trend of these cell surface macromolecules was observed during clinical remission. This is the first comparative study between PB and BM, where significant differential distribution of sialylated macromolecules and adhesion molecules was observed. Hence, supervising these cell surface macromolecules at various stages of treatment might help in minimal residual disease detection, identifying mobilization factor(s) and in isolation of normal HPCs for autologous BM transplantation.     

Effects of 10-day confinement on the immune system and psychological aspects in humans.

We investigated the changes in percentages of leukocyte subpopulations, natural killer (NK) cells, CD69-expressing lymphocytes, and psychological aspects in 10 subjects who participated in a 10-day confinement study. Suppression of lymphocyte proliferative reaction and changes in leukocyte distribution are known to occur in space. These responses are similar to those induced by psychological stress. Ground-based confinement studies are suitable for validating the effects of stress arising only due to confinement. Two groups, consisting of five male subjects (ages 20-27 yr, mean 22.8 yr) each, participated in a 10-day confinement study. Blood samples were taken once before, three times during, and once after the confinement and activated with an anti-CD2 agonistic antibody cocktail. The percentages of leukocyte subpopulations, NK (CD45(+)CD56+) cells, and activated lymphocytes (CD45(+)CD69+) were measured by flow cytometric assay. The face scale test was used to measure psychological aspects. The percentage of CD69+ lymphocytes decreased during the period of confinement. This was mostly caused by changes in the ratio between NK and non-NK lymphocytes. The face scale showed that the subjects' moods improved toward the postconfinement period. Consistent with the face scale, the percentages of innate immune cells, such as NK cells and granulocytes, increased during the postconfinement period. We concluded that the changes in the distribution of immune cells caused by stress plays an important role in suppression of proliferative reactivity. The observed physiological reactions were specific to the confined environment, and the stress caused by confinement plays a role in the immune changes observed in space.     

Lymphocyte subsets and specific T-cell immune response in thalassemia

Infection is very common in thalassemia and is one of the major causes of death. To date, it is not quite clear why these patients are susceptible to infection. In this study, lymphocyte immunophenotyping for CD3(+) (T-cells), CD3(+)CD4(+) (T-helper/inducer cells), CD3(+)CD8(+) (T-suppressor/cytotoxic cells), CD3(-)CD19(+) (B-cells), and CD3(-)CD16/56(+) (natural killer cells) subsets and expression of the activation antigen CD69 on CD3(+)CD4(+) and CD3(+)CD8(+) T-cells were determined in the whole blood of thalassemia patients, using a three-color flow cytometric technique. Results showed that only splenectomized beta-thalassemia/hemoglobin (Hb) E patients displayed a marked increase in absolute number of all lymphocytes. In addition, splenectomized beta-thalassemia/Hb E showed a significantly lower percentage of CD3(+) cells, with a corresponding increase in CD19(+) cells. These differences, when compared with normal subjects and other thalassemia patients, may be attributed to splenectomy. alpha-thalassemia patients, on the other hand, showed no significant difference from the normal group. While lymphocyte subsets in splenectomized beta-thalassemia/Hb E patients showed an abnormal distribution, T-cell activation in these patients was not different from the activation seen in normal subjects. This implies that thalassemia patients, during the steady state of disease, appear to have normal T-lymphocyte function with only moderate abnormalities of T- and B-lymphocyte subsets.     

CD4+CD25+/highCD127low/- regulatory T cells are enriched in rheumatoid arthritis and osteoarthritis joints—analysis of frequency and phenotype in synovial membrane,synovial fluid and peripheral blood

Introduction

CD4⁺CD25^+/highCD127^low/- regulatory T cells (Tregs) play a crucial role in maintaining peripheral tolerance. Data about the frequency of Tregs in rheumatoid arthritis (RA) are contradictory and based on the analysis of peripheral blood (PB) and synovial fluid (SF). Because Tregs exert their anti-inflammatory activity in a contact-dependent manner, the analysis of synovial membrane (SM) is crucial. Published reports regarding this matter are lacking, so we investigated the distribution and phenotype of Tregs in concurrent samples of SM, SF and PB of RA patients in comparison to those of osteoarthritis (OA) patients.

Methods

Treg frequency in a total of 40 patients (18 RA and 22 OA) matched for age and sex was assessed by flow cytometry. Functional status was assessed by analysis of cell surface markers representative of activation, memory and regulation.

Results

CD4⁺ T cells infiltrate the SM to higher frequencies in RA joints than in OA joints (P = 0.0336). In both groups, Tregs accumulate more within the SF and SM than concurrently in PB (P < 0.0001). Relative Treg frequencies were comparable in all compartments of RA and OA, but Treg concentration was significantly higher in the SM of RA patients (P = 0.025). Both PB and SM Tregs displayed a memory phenotype (CD45RO⁺RA^-), but significantly differed in activation status (CD69 and CD62L) and markers associated with Treg function (CD152, CD154, CD274, CD279 and GITR) with only minor differences between RA and OA.

Conclusions

Treg enrichment into the joint compartment is not specific to inflammatory arthritis, as we found that it was similarly enriched in OA. RA pathophysiology might not be due to a Treg deficiency, because Treg concentration in SM was significantly higher in RA. Synovial Tregs represent a distinct phenotype and are activated effector memory cells (CD62L^-CD69⁺), whereas peripheral Tregs are resting central memory cells (CD62L⁺CD69^-).     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

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 CD56(bright) 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, (51)Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56(bright) NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56(dim)CD16(-) 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 CD56(bright) 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.