[36]

Cultured cells can be encouraged to assemble primary

[36]

Cultured cells can be encouraged to assemble primary cilia by removing serum from their growing medium to induce exit from the cell cycle.[3] Madin Darby Canine Kidney (MDCK) and Inner Medullary Collecting Duct 3 (IMCD3) are commonly used renal epithelial cells lines that assemble primary cilia and have proved invaluable for investigating components involved in cilium-based signalling pathways. Techniques have also been developed to study the primary cilia produced by cultured metanephric mesenchyme.[37] Similarly, cultured mouse embryonic fibroblasts derived from knockout and transgenic strains are widely used to this website study the genetic basis of primary cilium function. As a general rule, immunolocalization of ciliary components is easier in cultured cells than kidney sections. Most of the reagents used for electron microscopy are hazardous and provision needs to be made for their safe handling and disposal. A fume cupboard and appropriate protection are essential. For best preservation mouse kidneys are perfusion fixed. The mouse is deeply anaesthetized with ketamine anaesthetic and perfused via the left ventricle

of the heart with nicking of the inferior ABT-263 research buy vena cava to allow blood and perfusate to escape. Perfusion takes place on an absorbent pad, or on a tray with a hole draining to a beaker in the fume hood sink. This allows escaping perfusate to be collected so that it can be disposed of appropriately. see more Perfusion should not exceed normal mouse blood pressure (100–130 mmHg) to avoid damaging the kidney. Gravity fed perfusion systems are frequently used and will give a pressure equivalent to approximately 75 mmHg if perfusion fluid is at an elevation of 1 m above the animal. Some custom made and commercial perfusion apparatus (e.g. Leica Perfusion One) use

a chamber with controlled air pressure to regulate perfusion pressure. Perfusion begins with phosphate buffered saline (PBS) at 37°C until blood is flushed and is followed by fixative composed of 2.5% glutaraldehdye and 2% formaldehyde in phosphate buffer or cacodylate buffer. Phosphate buffer is the easier non-toxic option; however, toxic cacodylate buffer may offer better preservation and less chance of precipitate forming in the specimen. The kidneys are removed and cut into several smaller pieces, immersed in fixative for 2–5 h, washed three times in buffer, post-fixed in 1% osmium tetroxide in buffer for 1 h, washed in buffer then three changes of water. A perfusion fixation approach is also applicable to rat kidneys.[38] Kidneys from embryonic mice are dissected out at the desired developmental stage and can be immersion fixed intact because of their small size. Human kidney samples are cut into small pieces and immersion fixed using the same sequence of fixatives.

3B and C) Among subjects with detectable virus-specific IL-10+ C

3B and C). Among subjects with detectable virus-specific IL-10+ CD8+ T cells, co-production of IFN-γ was observed in the majority of CMV-specific cells, while only a minority of HCV-specific IL-10+ CD8+ T cells co-produced IFN-γ (Fig. 3D). In contrast

to HIV-1, the expression of FoxP3 and CD25 in these CMV- and HCV-specific populations was heterogeneous (Fig. 3E). HIV-1-specific IL-10+ T cells have been defined as immunosuppressive on the basis of the effects of their depletion on other HIV-specific T-cell populations, such as enhancement of cytolytic, proliferative and IL-2-producing capacities in vitro [6, 21]. However, interpretation of these data could be confounded by the method of depletion used, which would have led to removal of spontaneous IL-10-producing cells

(monocytes and B cells) in addition click here to virus-specific T cells (Supporting Information Fig. 1). Obeticholic Acid price To address this, we examined the effects of selectively depleting HIV-specific IL-10+ CD8+ T cells on the responses of other T cells and of peripheral blood monocytes following stimulation with HIV-1 gag peptides (see Materials and methods and schema in Fig. 4A). We confirmed that removal of the CD8+ IL-10-producing T-cell population resulted in a decrease in IL-10 accumulating in the supernatant during subsequent culture (Fig. 4B). The depletion of IL-10+ CD8+ cells led to a small but statistically significant increase in the frequency of activated (CD38+ HLA-DR+) CD8+

T cells after Digestive enzyme subsequent culture (Fig. 4C) but had no effect on the activation of CD4+ T cells, as indicated by expression of CD38 and HLA-DR (Supporting Information Fig. 3), or on the T-cell effector functions, indicated by production of IL-2, IL-4, IFN-γ and TNF-α during an 18-h culture (data not shown). However, levels of IL-6, which is predominantly secreted by innate cells including peripheral blood monocytes in both HIV-infected and -uninfected individuals [22-24], were upregulated by a median 1.4-fold (range 0.6- to 3.4-fold, p = 0.013) (Fig. 4D). Using intracellular cytokine staining, we confirmed that CD14+ monocytes were the predominant source of IL-6 in gag-stimulated PBMCs in ART-naïve individuals; this population accounted for more than 85% of IL-6+ cells in the majority of subjects tested (Fig. 4E). In addition to augmenting IL-6 production, depletion of HIV-1 gag-specific IL-10+ CD8+ T cells led to a modest yet significant upregulation of CD38 in CD14+ monocytes (p = 0.001), and the magnitude of the change in CD38 expression was directly correlated with the magnitude of IL-10+ CD8+ T-cell population that was depleted (r = 0.91, p = 0.0005, Fig. 4C). In contrast to CD8+ T cells, increased CD38 expression in monocytes was not accompanied by a significant change in cell surface HLA-DR expression (data not shown).

This result contrasts with the effects of simvastatin on SOCS3 in

This result contrasts with the effects of simvastatin on SOCS3 induction that were maximal after 24 hr of stimulation. When we examined the effects of simvastatin on the early events in the TGF-β signal transduction cascade, we did not observe any augmentation of Smad3 phosphorylation. In contrast, the major effects of simvastatin were associated with a decreased induction of Smad6/7, inhibitory Smads that inhibit TGF-β signalling by blocking the phosphorylation of Smad2/3.

We favour the view that simvastatin can directly block the induction of Smad6/7 expression, as the drug also inhibited the induction of Smad6/7 at 72 hr in the presence of a TCR signal alone in the absence of TGF-β. Alternatively, it is possible that the effects of simvastatin on Smad6/7 expression are mediated indirectly via a direct effect on Foxp3 expression as Fantini et al.22 have click here demonstrated that transfection of Foxp3 is capable of blocking TGF-β-induced Smad7 expression by acting directly on the Smad7 promoter. This mechanism is consistent with our findings that Smad6/7 cannot be induced in Foxp3+ nTregs following TGF-β signalling. Although it is difficult to extrapolate from our in vitro model systems to the in vivo situation, our results that simvastatin can markedly

enhance the induction of Foxp3 expression Talazoparib purchase in the presence of Neratinib cell line low concentrations of TGF-β strongly suggest that some of the beneficial effects of simvastatin include the generation of Tregs in the inflammatory milieu of the atherosclerotic

plaque. Further analysis of the mechanism of action of simvastatin will require identification of the targets of geranylgeranylation at different time-points after T-cell activation. Ras, Rho, CDC42 and many different GTPases are important for early signal transduction after engagement of the TCR and may play a role in induction of SOCS3. However, our findings suggest that the effects of simvastatin are on proteins synthesized 24 hr after TCR stimulation. At the very least, our study strongly implies that an analysis of TCR-specific protein prenylation is a potential pathway for pharmacological manipulation of Tregs in vivo. This study was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD). The authors have no conflict of interest. Figure S1. Simvastatin does not induce cell death or alter the cell cycle of Foxp3− cells. “
“The composition of the peripheral blood lymphocyte compartment underlies developmental changes during ontogeny. Recently, several new B cell populations have been characterized which were suggested to develop in an age-dependent manner. However, age-dependent reference values for distinct B cell populations have rarely been reported.

At 8 weeks after surgery, the CD-augmented tissues contained laye

At 8 weeks after surgery, the CD-augmented tissues contained layered SMA-positive cells, urothelium uroplakin beta-catenin cancer III -positive urothelium, and S100 fibers, similar to normal bladder tissue. The SIS-augmented bladders showed similar results. At 8 weeks after augmentation, the bladder volume of CD-augmented bladders was larger than that at 4 weeks, while the SIS-augmented bladders were the same as those at 4 weeks. The bladder volume of the non-augmented group did not increase. The bladder compliance of the

CD-augmented bladders at 8 weeks was significantly higher than at earlier times. The bladder compliance of neither the non-augmented nor the SIS-augmented groups increased during the study period. Conclusion: Acellular bovine pericardium-derived material could be a suitable biomaterial for bladder augmentations “
“Many theories attempt to explain the complex etiology of overactive bladder syndrome (OAB), but the exact mechanisms of the pathophysiology JNK inhibitor have yet to be fully

understood. Recent findings have suggested that hypercholesterolemia is related with detrusor overactivity (DO), which, in turn, is usually associated with OAB. The present report examines published studies that have associated hypercholesterolemia with DO to determine the grounds on which such studies were based. According to our analysis, OAB and DO are closely related with hypercholesterolemia. Furthermore, DO and OAB may be affected not just by a single factor like hypercholesterolemia, but rather by all components of metabolic syndrome. Several mechanisms, including

autonomic overactivity, artherosclerosis, ischemic change, alteration of nitric oxide synthase (NOS)/NO system and increased Rho-kinase activity may have a role in the relationship between OAB and hypercholesterolemia. Further studies are warranted, however, to evaluate more about the pathophysiology of OAB. Overactive bladder syndrome (OAB) is characterized by an “urgency, with or without urge incontinence, usually with frequency and nocturia”, and detrusor overactivity (DO) is a urodynamic observation characterized by involuntary detrusor contractions during the filling phase and may of be spontaneous or provoked according to the International Continence Society.1 The diagnosis of OAB does not require urodynamic confirmation of DO, although it often is stated that the patient-reported sensation of urinary urgency is the result of a concomitant involuntary detrusor contraction.2 Hence, DO is often, but not invariably, associated with OAB.3 The pathophysiology of OAB is difficult to explain with simply one etiology. Neurologic dysfunction, as well as obstruction-related, congenital, behavioral, age-related, myogenic, ischemic, inflammatory, and many other factors are considered to be causes of OAB.4–6 Likewise, the pathophysiological basis of DO remains incompletely understood.

[27] For the toxin-neutralization

[27]. For the toxin-neutralization Dabrafenib cost assay, 20 pg/mL of EHEC-derived Stx2 was preincubated with an equal volume of 100-fold diluted sera from mice immunized with mStx2-His or PBS for 1 hr at 37°C. For the in vivo assays, each Stx2-His was serially diluted with PBS and 0.5 mL of each dilution injected

intraperitoneally into at least five female ICR mice (6 weeks of age, Japan SLC, Hamamatsu, Japan). The animals were observed for 1 week and their deaths were recorded. The MLD was calculated from the dilution that killed all animals. Ten micrograms of mStx2-His containing 0.05% (w/v) of aluminum hydroxide (which has been clinically used as an adjuvant) in 0.2 mL of PBS was injected s.c. twice at a 2-week interval into 25 female ICR mice (6 weeks of age). For a control group, PBS containing 0.05% (w/v) of aluminum hydroxide was injected into five mice instead of

mStx2-His. Two weeks after the secondary immunization, the animals were tail bled to determine the specific serum antibody titer by ELISA. The mice immunized with mStx2-His were then divided into three groups that were intraperitoneally challenged with a 10-, 100-, or 1000-fold lethal doses of Stx2-His and their survivability was monitored for 1 week. All animal experiments were conducted according to the Guidelines for the Management of Laboratory Animals at Fujita Health University. Flat-bottom, 96-well plates were coated with 1 μg/100 μL of Stx2-His overnight at 4°C. After washing the plates three times with T-PBS, each well was blocked see more using 200 μL of S-PBS for 1.5 hr at 37°C. After washing the plates three times, 100 μL of immunized or untreated (normal) mice sera serially diluted with S-PBS was added to the plates and incubated for 1 hr at 37°C. The plates were washed three times and incubated with 100 μL of HRP-conjugated anti-mouse IgG goat Immunoglobulin (Jackson ImmunoResearch, West Grove, PA, USA) for 1 hr at 37°C. After washing the plates,

the wells were reacted with 100 μL of citrate buffer (pH 5.0) containing 0.04% (w/v) o-phenylenediamine and 0.02% (v/v) hydrogen peroxide for 30 min at 37°C. The reaction was stopped by the addition of 100 μL of 1 M H2SO4 and the absorbance measured Tolmetin at 492 nm using a microplate reader (Tecan, Mannedorf, Switzerland). The absorbance value for each sample was compared with that of normal serum at the same dilution, and the antibody titer was determined as a reciprocal of the highest dilution with the lowest positive difference of the 1.5 × absorbance value of normal serum subtracted from the 1 × absorbance value of each sample. Cell lysates from transformants were prepared using previously described methods [25]. The sample proteins were resolved on a 15% polyacrylamide gel. The gel was stained with CBB-R250 or electroblotted onto a PVDF membrane using the iBlot gel transfer system (Invitrogen).

Cells were washed once with Hanks’s balanced salt solution and cu

Cells were washed once with Hanks’s balanced salt solution and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 5% fetal

calf serum (Gibco, Paisley, UK), 1% L-glutamine (Sigma, St Louis, MO, USA), 1% non-essential amino acids (Sigma), 2 × 10−5 M 2-mercaptoethanol (Amresco, Solon, OH, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). All cells were adjusted to 2 × 106 cells/ml. MNC suspensions (4 × 105) obtained above were seeded in triplicate in 96-well, round-bottomed microtitre plates at different lymphocyte : astrocyte ratios (10:1, 1:1 and 1:5). Cells were stimulated with 25 μg/ml MOG35–55 peptide for Ibrutinib ic50 72 h. For anti-CD3/CD28-induced

cell proliferation, 96-well culture plates were coated with purified anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) (5 μg/ml each; eBioscience, Ltd, Ireland, UK). ConA (Sigma, St Louis, MO, NVP-AUY922 nmr USA) was used at 5 μg/ml. Proliferation was measured by [3H]-thymidine (specific activity, 60 μCi/mmol; Institute of Atomic Energy, China; 0·5 μCi/well) incorporation after 72 h in complete DMEM medium. Astrocytes were cultured at a concentration of 1 × 106 cells/well in 12-well plates, then incubated with 2 μg/ml goat anti-mouse-IL-27 antibody (R&D Systems, Minneapolis, MN, USA) [37] or isotype control immunoglobulin (Ig)G2a in 2 ml medium for 12 h to neutralize IL-27. ifoxetine Astrocytes were co-cultured with MNCs (1 × 107) harvested from the lymph nodes of EAE mice in 2 ml lymphocyte culture medium. The cells were incubated at 37°C, 5% CO2 for 72 h. Supernatants were collected for measurement of the levels of soluble cytokines. Astrocytes (1 × 106) were co-cultured with lymph node lymphocytes (1 × 107) harvested from 7 dpi mice in 2 ml lymphocyte culture medium. Where indicated, lymphocytes were also seeded in Transwell™ insert (24-well plates, 3 μm pore size;

Corning, NY, USA). Twenty-five μg/ml MOG35–55 peptide was incubated as antigen and the supernatants were collected 72 h later. Measurement of cytokine levels in cell culture supernatants was performed by enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits, in accordance with the manufacturer’s instructions. IFN-γ, IL-17 and IL-4 ELISA kits were purchased from Peprotech (Rocky Hill, NJ, USA). The TGF-β ELISA kit was obtained from Boster, China. Results are expressed as pg/ml. Total RNA was prepared from spinal cords or lymph node MNCs using TRIzol reagent (Invitrogen). cDNA was synthesized using a reverse transcription–polymerase chain reaction (RT–PCR) kit from TaKaRa (Kyoto, Japan). RT–PCR was used to detect MHC-II genes using the following forward 5′-GATCGGATCCAACCCTGCTGAGGATTCA-3′ and reverse 5′-GATCGGATCCTGTCCTCGGCTGGGAAGA-3′ primers.

Substantial differences in the analyte profiles were notable, wit

Substantial differences in the analyte profiles were notable, with the group demonstrating the highest level of periodontitis showing elevated levels of IL-6, IL-8 and LBP and significantly decreased levels of PGE2 and BPI. By the time of delivery, and following ligation of teeth in four quadrants, all animals had a CIPD >20 (not periodontally healthy). Again, the most diseased animals provided a profile of serum analytes that was distinctive from animals expressing primarily gingival inflammation,

with a lower level of destructive disease. These data suggested that the variation in naturally occurring periodontal Hydroxychloroquine nmr inflammation and disease in the female baboons was reflected by patterns of systemic inflammation. Moreover, those animals that responded more robustly to the infection burden accompanying ligation generally

demonstrated a unique profile of mediator levels. As we have observed previously, these findings are consistent with a subset of these non-human primates that show an increased susceptibility to dysregulated local responses eliciting greater disease and allowing a more substantial challenge to the systemic inflammatory apparatus. These outcomes would also suggest that animals with a more effective adaptive immune response to the microbial challenge would demonstrate less disease, as we have reported previously [46,55], and less systemic challenge with lower serum inflammatory responses. Examination of the relationships between the inflammatory mediators and antibody in serum showed that elevated or decreased antibody specificities were coincident selleck screening library with levels of selected mediators. However, identification of a particular pattern of antibodies that best described the systemic inflammatory response profiles was somewhat complex. Generally, the acute phase reactants were delineated by

unique patterns of antibody responses that were observed at specific time-points during the study. The chemokines IL-8 and MCP-1 demonstrated some similarity in the patterns of antibody correlations, particularly at baseline only and mid-pregnancy. IL-6 levels were best described by distinctive antibody specificities during the protocol. However, of the 20 antibody specificities that were evaluated, levels of F. nucleatum, P. gingivalis, A. actinomycetemcomitans and C. rectus showed some consistency in contributing to relationships with the range of inflammatory mediators analysed. However, within the model system, a pattern of the serum analytes provided some insight into describing the expression of disease. We observed a clear association of IL-6, IL-8 and LBP levels across disease expression and throughout pregnancy. When broken down further, we observed that these relationships were related primarily to the characteristics of the disease expression in the individual animal, and generally related less to the stage of pregnancy at which the sample was obtained.

Any therapeutic manipulation aimed at improving viral control by

Any therapeutic manipulation aimed at improving viral control by reducing Blimp-1 expression has to avoid the point at which further reduction of Blimp-1 becomes harmful. D.T.F. & J.E.D.T. are funded by the

Wellcome Trust. The authors declare no financial or commercial conflict of interest. “
“Dipeptidyl peptidase 2 (DPP2) is an N-terminal dipeptidase, required for maintaining lymphocytes in a resting state. Mutant mice with T-cell-specific KPT-330 price knock-down (kd) of DPP2 (lck-DPP2 kd) were generated and analyzed for their phenotype. Normal thymocyte development and a modest increase in the proportions of peripheral T cells were observed in these mice compared with littermate controls. Interestingly, the peripheral T cells were hyperactive upon TCR stimulation in vitro, although they did not express any activation markers. Furthermore, CD3-crosslinking in the naïve CD4+ and CD8+ T cells of lck-DPP2 kd mice resulted mainly in IL-17 production. Similarly, the mutant T cells secreted primarily

IL-17 after in vivo priming and in vitro antigen-specific restimulation. These data suggest that IL-17 production is the default program for T-cell differentiation in the buy Cobimetinib absence of DPP2. Thus, DPP2 seems to impose a threshold for quiescent T cells, preventing them from drifting into cell cycle. Dipeptidyl peptidase 2 (DPP2), a member of the serine dipeptidyl peptidase family, is an N-terminal protease that is ubiquitously transcribed in most tissues 1. It is localized in intracellular vesicles and is also secreted upon cellular activation 2. The DPP2 expression level is particularly high in quiescent T cells and fibroblasts, but is significantly downregulated upon activation of these cells 3. We previously demonstrated that DPP2 inhibition in vitro causes apoptosis in quiescent, but not activated, T cells 4 and fibroblasts due to a deregulated entry into the cell cycle 5. In order to analyze the role of DPP2 in quiescent T lymphocytes in vivo, we generated mutant mice where DPP2 is specifically downregulated

in the T-cell lineage. The majority of T cells in the body are in a resting state until encounter with a pathogen. In the presence of exogenous cytokines, TCR-stimulation of naïve CD4+ and CD8+ T cells Tau-protein kinase leads to their maturation into various TH cell subsets and CTL effector cells. CD4+ cells can differentiate into the classical Th1 or Th2 subsets 6 or one of the more recently discovered lineages, Th17 7 and inducible Tregs 8. Differentiation into Th1 and Th2 cells depends on exogenous IL-12 and IL-4, respectively. In contrast, Th17 differentiation can be achieved with TGF-β and IL-6, two cytokines with opposing effects, while TGF-β alone induces iTregs 8. Ghoreschi et al. recently demonstrated that IL-1 and/or TGF-β in conjuction with IL-6, IL-21 and IL-23 promote Th17 development 9. Thus, the cytokine environment determines TH effector differentiation, a mechanism mediated through selective STAT proteins 10, 11.

e HLA-B*57, etc ), we interpret that NK

cells can contri

e. HLA-B*57, etc.), we interpret that NK

cells can contribute to both resistance against infection and to viral control once infected (Table 3). Together with data illustrating increased activation [10,20,91] and function [6,19] of NK cells in HESNs, these results suggest that NK cells fit the model of a candidate cell type whose retained function and heightened activation status may contribute to both control over HIV-1 replication and resistance to HIV-1 NVP-LDE225 in HESN subjects. The identification of highly exposed but persistently uninfected individuals that maintain resistance to HIV-1 infection despite high-risk exposure has generated hope that mechanisms of natural resistance to HIV-1 may some day be translated into a sterilizing vaccine to prevent infection. The failure of T cell vaccine strategies [34,35] and pre-existing CTL responses in HESN subjects to HIV-1 to protect against HIV-1 infection [38–40] has dampened interest

in the potential role of T cells in sterilizing immunity. Similarly, a recent study from Africa documenting an absence of consistent HIV-specific IgA responses in plasma or cervicovaginal lavage from HESN sex workers [59] is in agreement with previous findings indicating a lack of a direct correlation between HIV-resistance and IgA responses [60]. Collectively, the presence of HIV-specific selleck inhibitor humoral or cellular responses has not been a unifying functional attribute among HESN subjects, thereby highlighting the potential role of non-adaptive mechanisms of immunity in protection from HIV-1. Genotypic and functional association between increased NK activity and resistance to HIV-1 infection in multiple cohorts of HESN subjects suggests that the innate immune response may play a greater role than proposed to date in maintaining natural ZD1839 molecular weight resistance to infection in high-risk subjects. Alternatively, synergistic responses involving both the innate and adaptive immune compartments against HIV-1 may act in concert to resist infection with HIV-1. Examples of the co-operative response

between the adaptive and innate immune system include the targeting of MHC class I highly expressing cells by CD8 T cells and the targeting of MHC-class I down-regulated cells by NK cells. Similarly, HIV-specific IgA antibodies may act alone in neutralizing HIV-1 (dimeric IgA), or may increase HIV-1 clearance by binding to macrophages or neutrophils via the monomeric IgA Fc receptor, CD89 [56,57]. During chronic infection, HIV-specific IgGs are known to mediate neutralization of viral particles while also complementing well with NK cells to trigger antibody-mediated antibody-dependent cytotoxicity of infected target cells. Moving forward, non-human primate studies modelling HESN resistance to infection will be critical in investigating the complementary role of innate and adaptive immunity in resistance to HIV-1 infection. As shown in Fig.

5–7 A small number of phase I/II clinical studies have been compl

5–7 A small number of phase I/II clinical studies have been completed and have confirmed that sufficient numbers of genetically

modified T cells can be generated ex vivo, that TCR-transduced autologous T cells can persist after adoptive transfer and that anti-tumour activity in melanoma patients was feasible.8 However, further improvements are required to optimize the efficacy of TCR gene transfer in the clinical setting. Idelalisib chemical structure The efficiency of TCR gene transfer, and the subsequent function of the TCR-transduced T cell, is influenced by the vector delivery system, the TCR transgenes and the transduction conditions. To date, most TCR gene-transfer protocols have utilized gamma-retroviral vectors. Stable genomic integration of retroviral vectors requires full T-cell activation and proliferation during the transduction process. This process requires stimulation through the TCR complex using antibodies against CD3, with or without anti-CD28, in order to stimulate progression through the cell cycle, followed by RAD001 mw a period of in vitro expansion in the presence of interleukin (IL)-2. During this in vitro activation process, T-cell differentiation occurs and cell-surface molecules important for homing to secondary lymphoid organs (i.e. CD62L) or costimulation (i.e. CD28) are down-regulated. There are theoretical advantages to redirecting the antigen

specificity of less-differentiated cells and this can be achieved using lentiviral vectors, which permit gene transfer into non-dividing T cells.9,10 These approaches are currently being explored by a number of research teams, together with TCR transfer into selected central memory or naïve T cells and co-transfer of specific homing molecules. A number of challenges remain, including: (i) to maximize the cell-surface expression of the introduced TCR; (ii) to minimize or eliminate the mispairing of introduced

TCR-α and TCR-β chains with endogenous TCR chains; (iii) to improve the association of the introduced TCR with molecules of the CD3 complex; and (iv) to enhance the functional avidity of the TCR-transduced T cells. The relevant steps in the generation of antigen-specific T cells by TCR gene transfer are Adenosine indicated in a schematic representation (Fig. 1). TCR assembly and expression is a complex process.11 Before cell-surface expression, the TCR-α and TCR-β chains have to form a heterodimer. This process is influenced by the secondary and tertiary structures of both the variable and constant domains. The TCR-αβ then associates with the CD3 complex within the endoplasmic reticulum (ER), which involves interactions between the TCR constant domain (both intracellular and intramembrane portions) and the CD3 molecules. Finally, the TCR–CD3 complex is released from the ER and translocates to the cell membrane.