Low expression of CD39+/CD45RA+ on regulatory T cells (Treg) cells in type 1 diabetic children in contrast to high expression of CD101+/CD129+ on Treg cells in children with coeliac disease

Type 1 diabetes (T1D) and coeliac disease are both characterized by an autoimmune feature. As T1D and coeliac disease share the same risk genes, patients risk subsequently developing the other disease. This study aimed to investigate the expression of T helper (Th), T cytotoxic (Tc) and regulatory T cells (T_reg) in T1D and/or coeliac disease children in comparison to healthy children. Subgroups of T cells (Th : CD4⁺ or Tc : CD8⁺); naive (CD27⁺CD28⁺CD45RA⁺CCR7⁺), central memory (CD27⁺CD28⁺CD45RA⁻CCR7⁺), effector memory (early differentiated; CD27⁺CD28⁺CD45RA⁻CCR7⁻ and late differentiated; CD27⁻CD28⁻CD45RA⁻CCR7⁻), terminally differentiated effector cells (TEMRA; CD27⁻CD28⁻CD45RA⁺CCR7⁻) and T_reg (CD4⁺CD25⁺FOXP3⁺CD127⁻) cells, and their expression of CD39, CD45RA, CD101 and CD129, were studied by flow cytometry in T1D and/or coeliac disease children or without any of these diseases (reference group). Children diagnosed with both T1D and coeliac disease showed a higher percentage of TEMRA CD4⁺ cells (P < 0·05), but lower percentages of both early and late effector memory CD8⁺ cells (P < 0·05) compared to references. Children with exclusively T1D had lower median fluorescence intensity (MFI) of forkhead box protein 3 (FoxP3) (P < 0·05) and also a lower percentage of CD39⁺ and CD45RA⁺ within the T_reg population (CD4⁺CD25⁺FOXP3⁺CD127⁻) (P < 0·05). Children with exclusively coeliac disease had a higher MFI of CD101 (P < 0·01), as well as a higher percentage of CD129⁺ (P < 0·05), in the CD4⁺CD25^hi lymphocyte population, compared to references. In conclusion, children with combined T1D and coeliac disease have a higher percentage of differentiated CD4⁺ cells compared to CD8⁺ cells. T1D children show signs of low CD39⁺/CD45RA⁺ T_reg cells that may indicate loss of suppressive function. Conversely, children with coeliac disease show signs of CD101⁺/CD129⁺ T_reg cells that may indicate suppressor activity.     

The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins

Rabbit anti-thymocyte globulins (rATG) induce CD4⁺CD25⁺forkhead box P3 (FoxP3⁺) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4⁺CD25⁺FoxP3⁺CD127^−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25^neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25⁺ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4⁺CD25⁺FoxP3⁺CD127^−/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3⁺T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3⁺ T cells. The rATG tacrolimus-induced CD25⁺ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4⁺CD25⁺FoxP3⁺CD127^−/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25⁺ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-γ, perforin and granzyme B in contrast to natural CD25⁺ T cells (all P = 0·03), while FoxP3 was expressed at a lower level (P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4⁺CD25⁺ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.     

Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4+CD45RA+CD27+ T cells: the potential involvement of interleukin-7 in this process

The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4⁺ T-cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA⁺ CD27⁺ CD4⁺ T cells over time. In contrast, the increase in CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells in CMV-seropositive donors are specific for this virus. CD45RA⁺ CD27⁻ CD4⁺ T cells have significantly reduced CD28, interleukin-7 receptor α (IL-7Rα) and Bcl-2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T-cell receptor activation compared with the other T-cell subsets in the same donors. Despite this, the CD45RA⁺ CD27⁻ subset is as multifunctional as the CD45RA⁻ CD27⁺ and CD45RA⁻ CD27⁻ CD4⁺ T-cell subsets, indicating that they are not an exhausted population. In addition, CD45RA⁺ CD27⁻ CD4⁺ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4⁺ memory T cells re-expressing CD45RA can be generated from the CD45RA⁻ CD27⁺ population by the addition of IL-7 and during this process these cells down-regulated expression of IL-7R and Bcl-2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA⁺ CD27⁻ CD4⁺ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated.     

The imbalance of T helper 17/regulatory T cells and memory B cells during the early post‐transplantation period in peripheral blood of living donor liver transplantation recipients under calcineurin inhibitor‐based immunosuppression

There is limited clinical research regarding the changes in peripheral lymphocyte subsets during the early post-operative period of liver transplantation. Serial changes of T cells and B cells in living donor liver transplantation (LDLT) recipients during the early post-transplantion period were prospectively investigated. From June 2010 to February 2011, 27 consecutive LDLT recipients were enrolled. Percentages of T helper type 1 (Th1; interferon-γ-producing), Th2 (interleukin-4-producing), Th17 (interleukin-17-producing), regulatory T (Treg; CD4⁺ CD25⁺ FoxP3⁺), memory B (CD19⁺ CD24^hi CD38⁻) and mature B (CD19⁺ CD24^int CD38^int) cells were measured using fluorescence-activated cell sorting. Patients were followed up for a median of 9·9 months (range 6·8−15·5 months) after transplantation. Serial monitoring of immunological profiles showed no significant suppression of Th1, Th2, Th17, mature B or memory B cells, whereas frequencies of Treg cells significantly decreased. Interleukin-17 production by central and effector memory cells was not suppressed during the early post-operative period. The continuous production of interleukin-17 by the memory T cells may contribute to the persistence of Th17 cells. This prospective study demonstrated that current immunosuppression maintained the effector T or memory B cells during the early post-transplantation period but significantly suppressed Treg cells. Serial immune monitoring may suggest clues for optimal or individualized immunosuppression during the early post-operative period in clinical practice.     

CD4(+) CD25(-) FoxP3(+) regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs

The distribution and dynamic changes of CD4⁺ CD25⁺ FoxP3⁺ and CD4⁺ CD25⁻ FoxP3⁺ regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post-inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue-residing Treg cells, and decreased the frequencies of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) producing CD4⁺ CD25⁻ Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL-10 and TGF-β producing CD4⁺ CD25⁻ Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV-caused diarrhoea (r = −1, P < 0·0001). Hence, higher frequencies of functional CD4⁺ CD25⁻ Treg cells can be an indicator for poorer protective immunity against rotavirus. Our results highlighted the importance of CD4⁺ CD25⁻ Treg cells over CD4⁺ CD25⁺ Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4⁺ CD25⁻ Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF-β producing Treg cells post-challenge. The reduced suppressive effect of Treg cells in AttHRV-vaccinated pigs would unleash effector/memory T-cell activation upon challenge. Preserving TGF-β producing CD4⁺ CD25⁻ Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T-cell responses after primary rotavirus infection/vaccination and challenge.     

Co‐inhibitory profile and cytotoxicity of CD57+PD‐1– T cells in end‐stage renal disease patients

Blockade of the CD80/86‐CD28 pathway by belatacept after kidney transplantation is associated with an increased risk of rejection compared with standard, calcineurin inhibitor (CNI)‐based therapy. CD28^– T cells, which express CD57, are not susceptible to belatacept treatment. High numbers of CD4⁺CD57⁺programmed death 1 (PD‐1)^– T cells pretransplantation have been associated with a higher chance of rejection, although conflicting data have been reported. To investigate the working mechanism behind this possible higher chance of rejection, we studied the expression of co‐inhibitory molecules (CD223, CD244 and PD‐1), proliferative capacity and cytotoxic potential of fluorescence activated cell sorted (FACS) CD4⁺CD57⁺PD‐1^– and CD8⁺CD57⁺PD‐1^– T cells, and their CD57^– control populations, after alloantigen stimulation. The effect of belatacept on the cytotoxic capacity of pretransplantation peripheral blood mononuclear cells from 20 patients who received belatacept post‐transplantation was also tested. Expression of co‐inhibitory molecule CD223 increased by approximately 10‐fold after allogeneic stimulation in all four T cell subsets. Proliferation and up‐regulation of CD244 and PD‐1 was observed for CD4⁺CD57^‐PD‐1^– T cells after allogeneic stimulation, but no up‐regulation of these markers occurred on CD8⁺ T cells or CD4⁺CD57⁺PD‐1^– T cells. However, CD4⁺CD57⁺PD‐1^– T cells and, to a lesser extent, CD8⁺CD57⁺PD‐1^– T cells displayed higher cytotoxicity as indicated by granzyme B expression. Belatacept inhibited the cytotoxic potential of CD4⁺CD57⁺PD‐1^– T cells (median of inhibition 31%, P < 0·01) and CD8⁺CD57⁺PD‐1^– T cells (median of inhibition 10%, P < 0·05). In conclusion, alloantigen‐activated CD4⁺CD57⁺PD‐1^– T cells exhibited a less proliferative but more cytotoxic profile than their CD57^– counterparts. Their cytotoxic capacity can be inhibited partly by belatacept and was not associated with development of rejection after kidney transplantation.     

Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)-γ-producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.     

Increased frequency and suppressive activity of CD127low/− regulatory T cells in the peripheral circulation of patients with head and neck squamous cell carcinoma are associated with advanced stage and nodal involvement

The presence of regulatory T (Treg) cells is thought to be an important mechanism by which head and neck squamous cell carcinoma (HNSCC) successfully evades the immune system. Using multicolour flow cytometry, the frequency and functional capacity of two CD4⁺ CD127^low/− Treg cell populations, separated on the basis of different levels of CD25 expression (CD25^inter and CD25^high), from the peripheral circulation of newly presenting HNSCC patients were assessed with regard to clinicopathological features and healthy controls. The frequency of circulating Treg cells was similar between HNSCC patients and healthy controls, and for patients with HNSCC developing from different subsites (laryngeal compared with oropharyngeal). However, patients with advanced stage tumours and those with nodal involvement had significantly elevated levels of CD4⁺ CD25^high CD127^low/− Treg cells compared with patients who had early stage tumours (P = 0·03) and those without nodal involvement (P = 0·03), respectively. CD4⁺ CD25^high CD127^low/− Treg cells from the entire HNSCC patient cohort and from patients whose tumours had metastasized to the lymph nodes were also shown to suppress the proliferation of effector T cells significantly more, compared with those from healthy controls (P = 0·04) or patients with no nodal involvement (P = 0·04). Additionally, CD4⁺ CD25^inter CD127^low/− Treg cells consistently induced greater suppressive activity than CD4⁺ CD25^high CD127^low/− Treg cells on the proliferation of the effector T-cell populations (CD4⁺ CD25⁻ CD127^−/+ and CD4⁺ CD25⁺ CD127⁺). Peripheral Treg cells, identified by the CD127^low/− phenotype, have been shown to be influenced by a patient''s tumour stage and/or nodal status in HNSCC; suggesting a role in tumour progression that could be manipulated by future immunotherapy.     

Human cytomegalovirus-specific CD8+ T-cell expansions contain long-lived cells that retain functional capacity in both young and elderly subjects

The immune response to human cytomegalovirus (HCMV) infection is characterized by the accumulation of HCMV-specific CD8⁺ T cells, particularly in the elderly; such expansions may impair immune responses to other pathogens. We investigated mechanisms underlying HCMV-specific expansions in 12 young and 21 old healthy subjects (although not all analyses were performed on all subjects). Phenotypically, HCMV-pentamer⁺ CD8⁺ T cells were characterized by marked Vβ restriction, advanced differentiation (being predominantly CD27⁻ CD28⁻), and variable CD45RO/RA expression. Although more common and larger in older subjects, expansions had similar phenotypic characteristics in the young. In one old subject, repeated studies demonstrated stability in size and Vβ distribution of pentamer⁺ populations over 6 years. We tested whether HCMV-specific CD8⁺ T-cell expansions arose from accelerated proliferation or extended lifespan by in vivo labelling with deuterated glucose and ex vivo Ki-67 expression. Uptake of deuterated glucose was lower in pentamer⁺ cells than in pentamer^– CD8⁺ CD45RO⁺ or CD8⁺ CD45RA⁺ cells in three old subjects, consistent with reduced proliferation and extended lifespan. Similarly Ki-67 labelling showed no evidence for increased proliferation in HCMV-specific CD8⁺ expansions in older subjects, although pentamer^– CD45RA⁺ cells from young donors expressed very little Ki-67. We investigated Bcl-2 and CD95 as possible anti-apoptotic mediators, but neither was associated with pentamer-positivity. To investigate whether expansion represents a compensatory response to impaired functionality, we performed two tests of functionality, peptide-stimulated proliferation and CD107 expression; both were intact in pentamer⁺ cells. Our data suggest that HCMV-specific CD8⁺ expansions in older subjects accumulate by extended lifespan, rather than accelerated proliferation.     

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (T_reg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (−64%) but TLR-9-induced IFN-α secretion only slightly (−20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4⁺forkhead box protein 3 (FoxP3)⁺ regulatory T cells, but did not affect the generation of suppressive CD8⁺CD38⁺lymphocyte activation gene (LAG)-3⁺ T_reg. In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4⁺ T cell proliferation and induce CD4⁺FoxP3⁺ regulatory T cell generation.     

Regulatory T cells and chronic immune activation in human immunodeficiency virus 1 (HIV-1)-infected children

The function of CD4⁺ T cells with regulatory activity (T_regs) is the down-regulation of immune responses. This suppressive activity may limit the magnitude of effector responses, resulting in failure to control human immunodeficiency virus 1 (HIV-1) infection, but may also suppress chronic immune activation, a characteristic feature of HIV-1 disease. We evaluated the correlation between viral load, immune activation and T_regs in HIV-1-infected children. Eighty-nine HIV-1-infected children (aged 6–14 years) were included in the study and analysed for HIV-1 plasmaviraemia, HIV-1 DNA load, CD4 and CD8 cell subsets. T_reg cells [CD4⁺ CD25^highCD127^lowforkhead box P3 (FoxP3^high)] and CD8-activated T cells (CD8⁺CD38⁺) were determined by flow cytometry. Results showed that the number of activated CD8⁺CD38⁺ T cells increased in relation to HIV-1 RNA plasmaviraemia (r = 0·403, P < 0·0001). The proportion of T_regs also correlated positively with HIV-1 plasmaviraemia (r = 0·323, P = 0·002), but correlated inversely with CD4⁺ cells (r = −0·312, P = 0·004), thus suggesting a selective expansion along with increased viraemia and CD4⁺ depletion. Interestingly, a positive correlation was found between the levels of T_regs and CD8⁺CD38⁺ T cells (r = 0·305, P = 0·005), and the percentage of T_regs tended to correlate with HIV-1 DNA load (r = 0·224, P = 0·062). Overall, these findings suggest that immune activation contributes to the expansion of T_reg cells. In turn, the suppressive activity of T_regs may impair effector responses against HIV-1, but appears to be ineffective in limiting immune activation.     

Combined Env- and Gag-specific T cell responses in relation to programmed death-1 receptor and CD4+ T cell loss rates in human immunodeficiency virus-1 infection

Additional progression markers for human immunodeficiency virus (HIV) infection are warranted. In this study we related antigen-specific responses in CD4⁺ and CD8⁺ T cells to CD38, reflecting chronic immune activation, and to CD4⁺ T cell loss rates. Clones transiently expressing CD107a (CD8⁺) or CD154 (CD4⁺) in response to Gag, Env and Nef overlapping peptide pools were identified, along with their expression of the inhibitory programmed death-1 receptor (PD-1) in fresh peripheral blood mononuclear cells (PBMC) from 31 patients off antiretroviral treatment (ART). HIV-specific CD8⁺ T cell responses dominated over CD4⁺ T cell responses, and among CD8⁺ responses, Gag and Nef responses were higher than Env-responses (P < 0·01). PD-1 on CD8⁺ HIV-specific subsets was higher than CMV-specific CD8⁺ cells (P < 0·01), whereas PD-1 on HIV-specific CD4⁺ cells was similar to PD-1 on CMV-specific CD4⁺ cells. Gag and Env CD8⁺ responses correlated oppositely to the CD4 loss rate. Env/Gag CD8⁺ response ratios, independently of PD-1 levels, correlated more strongly to CD4 change rates (r = −0·50 to −0·77, P < 0·01) than the total number of Gag-specific CD8⁺ cells (r = 0·44–0·85, P ≤ 0·02). The Env/Gag ratio performed better than CD38 and HIV-RNA in logistic regression analysis predicting CD4 change rate as a measure of progression. In conclusion, HIV-specific CD8⁺CD107a⁺ Env/Gag response ratio was a stronger predictor for progression than CD38 and HIV-RNA. The Env/Gag ratio may reflect the balance between possibly beneficial (Gag) and detrimental (Env) CD8⁺ T cell responses and should be explored further as a progression marker.     

Generation and characterization of chicken bone marrow‐derived dendritic cells

Dendritic cells (DCs) are bone marrow-derived professional antigen-presenting cells. The in vitro generation of DCs from either bone marrow or blood is routine in mammals. Their distinct morphology and phenotype and their unique ability to stimulate naïve T cells are used to define DCs. In this study, chicken bone marrow cells were cultured in the presence of recombinant chicken granulocyte–macrophage colony-stimulating factor (GM-CSF) and recombinant chicken interleukin-4 (IL-4) for 7 days. The cultured population showed the typical morphology of DCs, with the surface phenotype of major histocompatibility complex (MHC) class II⁺ (high), CD11c⁺ (high), CD40⁺ (moderate), CD1·1⁺ (moderate), CD86⁺ (low), CD83⁻ and DEC-205⁻. Upon maturation with lipopolysaccharide (LPS) or CD40L, surface expression of CD40, CD1·1, CD86, CD83 and DEC-205 was greatly increased. Endocytosis and phagocytosis were assessed by fluorescein isothiocyanate (FITC)-dextran uptake and fluorescent bead uptake, respectively, and both decreased after stimulation. Non-stimulated chicken bone marrow-derived DCs (chBM-DCs) stimulated both allogeneic and syngeneic peripheral blood lymphocytes (PBLs) to proliferate in a mixed lymphocyte reaction (MLR). LPS- or CD40L-stimulated chBM-DCs were more effective T-cell stimulators in MLR than non-stimulated chBM-DCs. Cultured chBM-DCs could be matured to a T helper type 1 (Th1)-promoting phenotype by LPS or CD40L stimulation, as determined by mRNA expression levels of Th1 and Th2 cytokines. We have therefore cultured functional chBM-DCs in a non-mammalian species for the first time.     

Increase in tumour‐infiltrating lymphocytes with regulatory T cell immunophenotypes and reduced ζ‐chain expression in nasopharyngeal carcinoma patients

The pathological significance of the mechanisms of tumour immune-evasion and/or immunosuppression, such as loss of T cell signalling and increase in regulatory T cells (T_regs), has not been well established in the nasopharyngeal carcinoma (NPC) microenvironment. To evaluate the T_reg immunophenotypes in tumour-infiltrating lymphocytes (TILs), we performed a double-enzymatic immunostaining for detection of forkhead box P3 (FoxP3) and other markers including CD4, CD8, and CD25 on 64 NPC and 36 non-malignant nasopharyngeal (NP) paraffin-embedded tissues. Expression of CD3ζ and CD3ε was also determined. The prevalence of CD4⁺FoxP3⁺ cells in CD4⁺ T cells and the ratio of FoxP3⁺/CD8⁺ were increased significantly in NPC compared with those in NP tissues (P < 0·001 and P = 0·025 respectively). Moreover, the ratio of FoxP3⁺/CD25⁺FoxP3⁻ in NPC was significantly lower than that in NP tissues (P = 0·005), suggesting an imbalance favouring activated phenotype of T cells in NPC. A significant negative correlation between the abundance of FoxP3⁺ and CD25⁺FoxP3⁻ cells (P < 0·001) was also identified. When histological types of NPC were considered, a lower ratio of FoxP3⁺/CD25⁺FoxP3⁻ was found in non-keratinizing and undifferentiated carcinomas. Increased CD4⁺FoxP3⁺/CD4⁺ proportion and FoxP3⁺/CD8⁺ ratio were associated with keratinizing squamous cell carcinoma. A reduced expression of CD3ζ in TILs was found in 20·6% of the NPC tissues but none of the NP tissues. These data provide evidence for the imbalances of T_reg and effector T cell phenotypes and down-regulation of signal-transducing molecules in TILs, supporting their role in suppression of immune response and immune evasion of NPC.     

Specific overexpression of tumour necrosis factor‐α‐induced protein (TNFAIP)9 in CD14+CD16− monocytes in patients with rheumatoid arthritis: comparative analysis with TNFAIP3

The tumour necrosis factor (TNF)-α-induced proteins (TNFAIP)9 and TNFAIP3 play an important pathogenic role in murine arthritis. To clarify their pathophysiological roles in patients with rheumatoid arthritis (RA), we examined their expression and localization in peripheral blood mononuclear cells (PBMC). TNFAIP9 and TNFAIP3 mRNA expression was determined in PBMC of RA patients and healthy subjects (control). Flow cytometry was used to analyse the main TNFAIP9- and TNFAIP3-expressing cell populations. TNFAIP9 and TNFAIP3 mRNA expression levels were examined in vitro on CD14⁺ cells stimulated with TNF-α and lipopolysaccharide (LPS). The expression levels of TNFAIP9 and TNFAIP3 mRNA were also measured before and 12 weeks after treatment with tocilizumab and abatacept. TNFAIP9 expression was significantly higher, while TNFAIP3 expression was lower in PBMC of RA (n = 36) than the control (n = 24) (each P < 0·05). TNFAIP9 was expressed on CD14⁺ cells, especially in human leucocyte antigen D-related (HLA-DR)⁺CD14^brightCD16⁻cells, while TNFAIP3 was expressed mainly on CD3⁺ T cells. TNF-α and LPS induced TNFAIP9 and TNFAIP3 in human CD14⁺monocytes in vitro. Treatment with tocilizumab (n = 13), but not abatacept (n = 11), significantly reduced TNFAIP9 mRNA expression in PBMC, which was associated with reduction in the number of circulating CD14^bright monocytes. The expression of TNFAIP9 in CD14⁺ cells was specifically elevated in patients with RA, regulated by TNF-α and LPS, and suppressed by tocilizumab, while TNFAIP3 in PBMC showed different localization and induction patterns.     

Phenotypic characterization of T cells in bronchoalveolar lavage fluid (BALF) and peripheral blood of patients with diffuse panbronchiolitis; the importance of cytotoxic T cells

We investigated the contribution of T cells in diffuse panbronchiolitis (DPB) by identifying T cell subsets in BALF of 36 patients with DPB, before and after long-term treatment with macrolide antibiotics, and 16 healthy control subjects. The percentages of lymphocytes and CD3⁺γδ⁺ cells in BALF of DPB patients and control subjects were similar, but the absolute number of these cells was higher in DPB patients. Treatment resulted in a significant reduction in the absolute number of these cells. A further two-colour analysis of T cell subsets in BALF showed a significantly higher ratio and number of CD8⁺HLA-DR⁺ cells in DPB patients. Treatment resulted in a significant reduction of activated T cells. Most BALF CD8⁺ cells were CD8⁺CD11b⁻ cytotoxic T cells. The number of these cells in BALF of DPB patients (26.69 ± 5.86 × 10³/ml) was higher than the control (2.02 ± 0.38 × 10³/ml; P< 0.001), and a significant reduction was observed after treatment (7.69 ± 2.59 × 10³/ml; P< 0.01). The number of CD4⁺ cells was also higher in DPB patients than in controls, and most were CD4⁺CD29⁺ memory T cells. However, treatment did not influence the number of these cells. The number of lymphocytes, CD3⁺γδ⁺, CD8⁺CD11b⁻, CD8⁺HLA-DR⁺, and CD4⁺CD29⁺ cells was higher in patients with bacterial infection than in those without bacterial infection, and interestingly, macrolide therapy reduced the number of lymphocytes, CD3⁺γδ⁺, CD8⁺CD11b⁻ and CD8⁺HLA-DR⁺ cells, irrespective of bacterial infection. In peripheral blood, the percentage of CD8⁺HLA-DR⁺ cells was also higher in DPB patients than in healthy subjects, and significantly decreased after treatment. The percentage of CD8⁺CD11b⁻ cells in peripheral blood was similar in DPB patients and normal subjects, and treatment significantly reduced the percentage of these cells. Finally, the expression of the adhesion molecules CD11a/CD18 (α/β-chains of LFA-1) on lung CD3⁺ cells and CD49d (α-chain of VLA) on lung CD4⁺ cells was enhanced compared with that on peripheral blood in DPB patients. Our results suggest that elevation of memory T cells and activation of CD8⁺ cells, mainly cytotoxic T cells, in the airway lumen of DPB patients may contribute to chronic bronchial inflammation, possibly through up-regulation of adhesion molecules. Our findings also indicate that macrolide antibiotics may have a direct or indirect suppressive effect on cytotoxic T cells, and as such, reduce inflammation and improve clinical condition.     

How immunosuppressive therapy affects T cells from kidney transplanted patients of different age: the role of latent cytomegalovirus infection

The average age of patients receiving renal transplantation is increasing as programmes have been established which support the donation of organs from elderly donors to older recipients. Little is known about the effect of immunosuppressive therapy on the immune system of older patients. In this study, T cell function and the composition of the T cell repertoire were analysed in immunosuppressed renal transplant recipients of different age and cytomegalovirus (CMV) status in comparison to age-and CMV-matched controls. Independent of age and CMV status, the production of interleukin (IL)-2 and interferon (IFN)-γ by T cells was decreased in the patient groups and autologous serum from patients was capable of inhibiting the proliferation of CD3⁺ T cells. CXCR5 expression on T cells was increased in patients versus controls reflecting reduced endogenous IL-2 signalling under immunosuppressive therapy. In CMV-seronegative patients kidney transplantation and immunosuppressive therapy did not induce changes in the CD8⁺ T cell pool, but there was a moderate increase in CD4⁺CD28⁻ effector T cells when compared to age-matched controls. In contrast, latent CMV infection triggered a shift from early to late differentiated CD4⁺ and CD8⁺ T cells in patients and controls. This shift was most pronounced in elderly transplant patients under immunosuppressive therapy. In conclusion, our results demonstrate that immunosuppressive therapy following kidney transplantation is effective in patients older than 65 years. Latent CMV infection, however, accelerates age-related changes in the T cell repertoire in elderly people under immunosuppressive therapy. These patients should therefore be monitored with special care.