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The highly diversified variable regions of immunoglobulin (Ig) molecules contain immunogenic determinants denoted idiotopes. We have previously reported that T cells from multiple sclerosis (MS) patients recognize IgG from autologous cerebrospinal fluid (CSF), and mapped a T-cell epitope to an IgG idiotope. To test the ability of CSF IgG molecules to elicit a broad polyclonal T-cell response in MS, we have analysed T-cell responses in the blood and CSF against idiotope peptides spanning complementarity determining region (CDR) 3 and somatic mutations within the variable regions of monoclonal CSF IgG. Consistent with a diversified idiotope-specific T-cell repertoire, CD4+ T cells from both patients recognized several idiotope peptides presented by HLA-DR molecules. Mutations were critical for T-cell recognition, as T cells specific for a mutated CDR1 peptide did not recognize corresponding germline-encoded peptides. One T-cell clone recognized both an idiotope peptide and the B-cell clone expressing this idiotope, compatible with endogenous processing and presentation of this idiotope by B cells. These results suggest that mutated CSF IgG from MS patients carry several T-cell epitopes, which could mediate intrathecal IgG production and inflammation in MS through idiotope-driven T–B-cell collaboration.

Inherent properties of dendritic cell (DC) subsets are important in the regulation of naïve T-cell differentiation (e.g. Th1 versus Th2 cells), whereas effector memory T cells are believed to produce a fixed cytokine repertoire independent of the type of antigen presenting cell. Here we show that two distinct human DC subsets, plasmacytoid DC (PDC) and myeloid CD11c+ DC, induced autologous mumps virus-specific memory CD4(+) T cells to produce markedly different cytokine patterns upon antigen stimulation. PDC stimulated the T cells to produce gamma-interferon (IFN-gamma) and interleukin-(IL)-10, whereas CD11c+ DC induced lower levels of IFN-gamma, virtually no IL-10, but significant levels of IL-5. Analysis of intracellular cytokine production showed simultaneous production of IL-10 and IFN-gamma in mumps-specific T cells activated by PDC, a cytokine pattern similar to that described for Th1-like regulatory cells. Introduction of CpG oligodeoxynucleotides in PDC/T-cell co-cultures had synergistic effect on virus-dependent IFN-gamma production, whereas the other cytokines remained unchanged. Together, our results show that the type of DC involved in reactivation of previously primed T cells may have significant impact on the resulting cytokine response and suggest that targeting of viral antigens and adjuvant to specific DC subsets should be considered in the design of therapeutic antiviral vaccines.

It has been suggested that human cytomegalovirus (HCMV) evades the immune system by infecting and paralyzing antigen-presenting cells. This view is based mainly on studies of dendritic cells (DCs) obtained after culture of monocytes (moDCs). It is contradicted by the asymptomatic course of HCMV infection in healthy persons, indicating that other key antigen-presenting cells induce an efficient immune response. Here we show that HCMV activates CD11c+ DCs and plasmacytoid DCs (PDCs). In contrast to moDCs, CD11c+ DCs and PDCs produced interferon (IFN) type 1 when exposed to HCMV. Autocrine IFN type 1 partially protected CD11c+ DCs against infection, whereas PDCs were resistant to HCMV even when IFN type 1 activity was inhibited. HCMV exposure induced the maturation of CD11c+ DCs by IFN type 1-dependent and -independent mechanisms. Importantly, CD11c+ DCs infected by inhibiting IFN type 1 activity retained full capacity to stimulate T cells. Renal transplant recipients receiving immunosuppressive treatment had lower frequencies of CD11c+ DCs and PDCs in blood than did healthy controls. The results show that HCMV activates the immune system by interacting with CD11c+ DCs and PDCs and that recipients of renal transplants have low frequencies of these cell types in blood.

T -cells are present in the spinal cord from patients with amyotrophic lateral sclerosis (ALS), and could attack neurons or activate microglia through secretion of cytokines. We report that interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-4, IL-5 and IL-10 could not be detected in cerebrospinal fluid (CSF) samples from 15 ALS patients and 23 out of 25 controls with a multiplexed cytometric bead assay. In vivo activated T-cell clones were established from CSF (n = 26) and blood (n = 21) of one ALS patient. The proliferative capacity of CSF T-cell clones was lower than that of T-cell clones from blood (p = 0.0007). All CD4+ CSF T-cell clones produced IFN-gamma, compatible with a predominant T helper (h) 1 phenotype, but several T-cell clones also produced Th2 cytokines. These data suggest that in vivo activated intrathecal T-cells can be induced to secrete cytokines which may play a role in ALS.

Background Plasmacytoid dendritic cells (PDCs) accumulate in the nasal mucosa of allergic rhinitis patients, but their function in upper airway allergy has not been determined. CpG oligodeoxynucleotides, potent adjuvants in immunotherapeutic strategies in animal models, are especially effective at activating PDCs. These cells are therefore potential targets for immunomodulation in humans. Objective In this study, PDCs were compared with CD11c + dendritic cells (DCs), a very potent antigen-presenting cell type, for their capacity to induce allergen-dependent activation of T H 2 memory cells. Then, we investigated whether CpG-activated PDCs were able to modulate the allergen-specific T H 2 memory response. Methods DCs were isolated from patients with upper airway allergy and cocultured with autologous CD4 + T cells with or without grass pollen extract and CpG. In some experiments cells were restimulated with allergen-pulsed monocyte-derived DCs. T-cell activation was measured by their proliferative response and cytokine production. Results PDCs stimulated allergen-dependent T-cell proliferation and T H 2 cytokine production as efficiently as CD11c + DCs. CpG-activated PDCs inhibited allergen-dependent proliferation of T H 2 memory cells and markedly increased IFN-γ production in PDC/T-cell cocultures; the former effect depended on the CpG-induced IFN-α/β production by the PDCs. Conclusion Our results demonstrated that PDCs efficiently drive allergen-dependent T H 2 memory responses, suggesting that they play an active role in the allergic reaction. However, in the presence of CpG, PDCs were responsible for increased production of the T H 1-related cytokines IFN-α and IFN-γ, indicating that mucosal PDCs may be targets for CpG-based immunotherapeutic strategies against airway allergy.

BACKGROUND: It has been suggested that allergic diseases are caused by defective suppression of allergen-specific Th2 cells by CD4(+)CD25(+) regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25-expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127(lo), allowing discrimination between these distinct T cell subpopulations. OBJECTIVE: The aim of this study was to study whether the putative regulatory subset defined as CD4(+)CD25(+)CD127(lo) was involved in grass pollen-reactive T cell responses. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non-atopic controls out of season. Grass pollen-induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4(+)CD25(+), CD4(+)CD25(+)CD127(hi) or CD4(+)CD25(+)CD127(lo) T cells. RESULTS: Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non-atopic controls. Depletion of all CD25(+) T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4(+)CD25(+)CD127(lo) cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non-atopic group. CONCLUSION: Our study showed that T cells from grass pollen-allergic patients and non-atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.

GvHD is a serious and potentially life-threatening side-effect of allogeneic BMT, caused by alloreactive cells attacking normal host cells. A number of different approaches have been attempted to remove allo-activated cells from the graft prior to transplantation. When developing such assays, there is a need to control for unwanted removal of cells, as well as depletion efficiency related to activation kinetics.The specific activation induced by the superantigens SEB and TSST-1 of T cells with defined Vbeta chains was utilized to follow activation of bystander cells and the kinetics of specific cellular activation by flow cytometry.The activation marker CD69 was up-regulated on bystander T cells, and was only transiently highly expressed on the specific T cells, making this marker unreliable for removal of alloreactive cells. In contrast, CD25 was found only on specifically activated T cells and was stably expressed over several days. However, it was not detected on all specific cells until day 6. Likewise, proliferation occurred only in T cells expressing the expected Vbeta chains, with all activated cells having undergone at least one cell cycle by day 4.In conclusion, our assay demonstrates that only temporary bystander activation occurs when polyclonally activating T cells by SEB or TSST-1, and that CD25, but not CD69, can be used for removal of specifically activated cells. Furthermore, this assay is useful for monitoring methods aiming at specific removal of cycling cells.

Dendritic cells (DCs) are believed to regulate T cell-mediated immunity primarily by directing differentiation of naive T cells. Here, we show that a large fraction of CD4+ memory cells produce IL-10 within the first hours after interaction with plasmacytoid DCs (PDCs). In contrast, CD11c+ DCs induce IFN-γ and little IL-10. IL-10–secreting T cells isolated after 36 hours of culture with PDCs suppressed antigen-induced T-cell proliferation by an IL-10–dependent mechanism, but were distinct from natural and type 1 regulatory T cells. They proliferated strongly and continued to secrete IL-10 during expansion with PDCs, and after restimulation with immature monocyte-derived DCs or CD11c+ DCs. The IL-10–producing T cells acquired the ability to secrete high levels of IFN-γ after isolation and subsequent coculture with PDCs or CD11c+ DCs. Compared to CD11c+ DCs, PDCs were superior in their ability to selectively expand T cells that produced cytokines on repeated antigenic challenge. The DC-dependent differences in cytokine profiles were observed with viral recall antigen or staphylococcal enterotoxin B and were independent of extracellular type I interferon or IL-10. Our results show that DCs can regulate memory responses and that PDCs rapidly induce regulatory cytokines in effector T cells that can suppress bystander activity.

Epstein-Barr virus-specific CD4+ T cells could be involved in the pathogenesis of multiple sclerosis, provided they can gain entry to the intrathecal compartment. The authors have previously demonstrated that cerebrospinal fluid T cells from multiple sclerosis patients recognize autologous Epstein-Barr virus-transformed B cells. They now report that CD4+ T cells specific for the Epstein-Barr virus DNA polymerase peptide EBV 627-641 were present in the cerebrospinal fluid from one of two multiple sclerosis patients, and that a high proportion of these CD4+ T cells cross-recognized an immunodominant myelin basic protein peptide, MBP 85-99. In the observed patient, the proportion of EBV 627-641-specific CD4+ T cells seemed to exceed 1/10,000 in cerebrospinal fluid, compared to approximately 1/100,000 in blood. These findings prove that Epstein-Barr-virus specific CD4+ T cells can gain access to the intrathecal compartment, and suggest that Epstein-Barr virus-specific CD4+ T cells could target myelin basic protein in the central nervous system.

Production of nitric oxide (NO) subsequent to expression of inducible NO synthase (iNOS) contributes to the development of ischemic renal injury and inflammation. Here we investigate the effects of GW274150, a potent, long-acting and highly selective inhibitor of iNOS activity, on NO production by primary cultures of rat proximal tubular cells (PTC).Pure populations of PTC were isolated from the cortex of kidneys obtained from male Wistar rats using a combination of collagenase digestion, sieving and Percoll centrifugation. Confluent PTC cultures were incubated for 1-24 h with MEM, interferon-gamma (IFN-gamma, 100 microg/ml), bacterial lipopolysaccharide (LPS, 10 microg/ml) in combination after which NO production was determined. PTC were also incubated with IFN-gamma (100 microg/ml) and LPS (10 microg/ml) and increasing concentrations of GW274150 or L-N6-(1-iminoethyl)lysine (L-NIL) (10 nM-1 mM) for 24 h after which nitrite levels in the incubation medium were measured.IFN-g and LPS in combination produced a significant, time-dependent increase in NO production. Both GW274150 and L-NIL produced a significant and concentration-dependent inhibition of NO production. However, GW274150 was markedly more potent (EC50 approximately 100 nM) than L-NIL (EC50 approximately 10 microM).GW274150 inhibits NO production by primary cultures of PTCs and may therefore be useful in conditions associated with nitrosative stress of the kidney.

High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal models of shock via modulation of the expression of adhesion molecules and pro-inflammatory enzymes. As renal inflammation plays an important role in the development of ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the ability of HDL to alleviate renal dysfunction and injury in a rat model of renal I/R. HDL (80 mg/kg, intravenous) was administered to male Wistar rats 30 min before bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h. After 6-h reperfusion, HDL significantly reduced (1) renal and tubular dysfunction, (2) tubular and reperfusion-injury, and (3) histologic evidence of renal injury. HDL also improved renal function (after 24-h and 48-h reperfusion) and reduced histologic signs of renal injury (after 48-h reperfusion). Administration of HDL significantly reduced the numbers of polymorphonuclear leukocytes (PMN) infiltrating into renal tissues during reperfusion, which was reflected by an attenuation of the increase in renal myeloperoxidase activity caused by I/R. Furthermore, HDL markedly reduced expression of the adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), and P-selectin during reperfusion. The increase in renal malondialdehyde levels caused by renal I/R was also significantly reduced by HDL, suggesting attenuation of lipid peroxidation subsequent to oxidative stress. These results demonstrate that HDL significantly reduces renal I/R injury and severity of ischemic acute renal failure. It is proposed that the mechanism of protection involves reduction of the expression of adhesion molecules, resulting in reduction of PMN infiltration and oxidative stress.

The tyrosine kinase inhibitor tyrphostin AG126 reduces renal ischemia/reperfusion injury in the rat. Background We investigate the effects of tyrphostin AG126, an inhibitor of tyrosine kinase activity, on the renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the kidney. Methods Tyrphostin AG126 (5 mg/kg intraperitoneally) was administered to male Wistar rats 30 minutes prior to bilateral renal ischemia for 45 minutes followed by reperfusion for up to 48 hours. Biochemical markers of renal dysfunction and injury were measured and renal sections assessed for renal injury. Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and formation of nitrotyrosine and poly (ADP) ribose (PAR) were assessed using immunohistochemistry. Rat proximal tubular cells (PTCs) were incubated with interferon-γ (100 IU/mL), bacterial lipopolysaccharide (10 μg/mL), and with increasing concentrations of tyrphostin AG126 (0.0001–1 mmol/L) for 24 hours. Nitric oxide production was measured in both plasma from rats subjected to I/R and in incubation medium from PTCs. Results After 6 hours of reperfusion, tyrphostin AG126 significantly reduced the increase in serum and urinary indicators of renal dysfunction and injury caused by I/R and reduced histologic evidence of renal injury. Tyrphostin AG126 also improved renal function (after 24 and 48 hours of reperfusion) and reduced the histologic signs of renal injury (after 48 hours of reperfusion). Tyrphostin AG126 reduced the expression of iNOS and nitric oxide levels in both rat plasma and in PTC cultures, as well as expression of COX-2. Tyrphostin AG126 also reduced nitrotyrosine and PAR formation, suggesting reduction of nitrosative stress and poly (ADP-ribose) polymerase (PARP) activation, respectively. Conclusion Taken together, these results show that tyrphostin AG126 significantly reduces the renal dysfunction and injury caused by I/R of the kidney. We propose that inhibition of tyrosine kinase activity may be useful against renal I/R injury.

Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury. Background Nitric oxide (NO), produced via inducible nitric oxide synthase (iNOS), is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of the iNOS inhibitors L- N 6 -(1-iminoethyl)lysine (L-NIL) and aminoethyl-isothiourea (AE-ITU) on ( a ) renal dysfunction and injury mediated by bilateral I/R of rat kidneys in vivo and ( b ) cytokine-stimulated NO production by primary cultures of rat proximal tubule (PT) cells. Methods Male Wistar rats subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h). Rats were administered either L-NIL (3 mg/kg IV bolus 15min prior to I/R followed by 1 mg/kg/h throughout I/R) or AE-ITU (1 mg/kg IV bolus 15min prior to I/R followed by 1 mg/kg/h throughout I/R). Serum and urinary biochemical indicators of renal dysfunction and injury were measured; serum creatinine (S Cr , glomerular dysfunction), fractional excretion of Na + (FE Na , tubular dysfunction), serum aspartate aminotransferase (sAST, I/R injury) and urinary N -acetyl-˂-D-glucosaminidase (uNAG, tubular injury). Additionally, renal sections were used for histological grading of renal injury and for immunological evidence of nitrotyrosine formation. Nitrate/nitrate levels in plasma were measured using the Griess assay and used as an indicator of NO production. Primary cultures of rat PT cells were incubated with interferon-γ (IFN-γ, 100 IU/mL) and lipopolysaccharide (LPS, 10 μg/mL) for 24 h, either in the absence or presence of increasing concentrations of L-NIL or AE-ITU (0.001 to 1 mmol/L) after which nitrite/nitrate levels were measured using the Griess assay. Results L-NIL and AE-ITU significantly reduced the I/R-mediated increases in S Cr , FE Na , sAST and uNAG, indicating attenuation of I/R-mediated renal dysfunction and injury. Specifically, L-NIL and AE-ITU reduced the I/R-mediated glomerular and tubular dysfunction and biochemical and histological evidence of tubular injury. Both L-NIL and AE-ITU attenuated the plasma levels of nitrate (indicating reduced NO production) and the immunohistochemical evidence of the formation of nitrotyrosine. In vitro, L-NIL and AE-ITU both significantly reduced cytokine-stimulated NO production by primary cultures of rat PT cells in a dose-dependent manner. Conclusions These results suggest that L-NIL and AE-ITU reduce the renal dysfunction and injury associated with I/R of the kidney, via inhibition of iNOS activity and subsequent reduction of NO (and peroxynitrite) generation. We propose that selective and specific inhibitors of iNOS activity may be useful against the NO-mediated renal dysfunction and injury associated with I/R of the kidney.

GW274150, a potent and highly selective inhibitor of iNOS, reduces experimental renal ischemia/reperfusion injury. Background Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of GW274150, a novel, highly selective, potent and long-acting inhibitor of iNOS activity in rat and mouse models of renal I/R. Methods Rats were administered GW274150 (5 mg/kg intravenous bolus administered 30 minutes prior to I/R) and subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Serum and urinary indicators of renal dysfunction, tubular and reperfusion injury were measured, specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N -acetyl-β-D-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine formation and poly [adenosine diphosphate (ADP)-ribose] (PAR). Nitrate levels were measured in rat plasma using the Griess assay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOS-/-) were subjected to bilateral renal ischemia (30 minutes) followed by reperfusion (24 hours) after which renal dysfunction (serum urea, creatinine), renal myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured. Results GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicating reduction of renal dysfunction and injury caused by I/R. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. GW274150 abolished the rise in the plasma levels of nitrate (indicating reduced NO production). GW274150 also reduced the renal dysfunction in wild-type mice to levels similar to that observed in iNOS-/- mice subjected to I/R. Renal MPO activity and MDA levels were significantly reduced in wild-type mice administered GW274150 and iNOS-/- mice subjected to renal I/R, indicating reduced polymorphonuclear leukocyte (PMN) infiltration and lipid peroxidation. Conclusions These results suggest that ( 1 ) an enhanced formation of NO by iNOS contributes to the pathophysiology of renal I/R injury and ( 2 ) GW274150 reduces I/R injury of the kidney. We propose that selective inhibitors of iNOS activity may be useful against renal dysfunction and injury associated with I/R of the kidney.