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BackgroundAllogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders. However, it carries significant risk of morbidity and mortality. To improve patient outcomes, better graft selection strategies are needed, incorporating HLA matching with clinically important graft characteristics. Studies have shown that the cellular content of HCT grafts, specifically higher ratios of T regulatory (Tregs)/T cells, are important factors influencing outcomes when using adult peripheral blood mobilised grafts. So far, no equivalent study exists in umbilical cord blood (CB) transplantation due to the limitations of cryopreserved CB samples.Study design and methodsTo establish the most robust and efficient way to measure the Treg content of previously cryopreserved CB units, we compared the enumeration of Treg and CD3+ cells using flow cytometry and an epigenetic, DNA-based methodology. The two methods were assessed for their agreement, consistency and susceptibility to error when enumerating Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples.ResultsEpigenetic enumeration gave consistent and comparable results in both fresh and frozen CB samples. By contrast, assessment of Tregs and CD3+ cells by flow cytometry was only possible in fresh samples due to significant cell death following cryopreservation and thawing.ConclusionEpigenetic assessment offers significant advantages over flow cytometry for analysing cryopreserved CB; similar cell numbers were observed both in fresh and frozen samples. Furthermore, multiple epigenetic assessments can be performed from DNA extracted from small cryopreserved CB segments; often the only CB sample available for clinical studies.

Regulatory T cells (Tregs) are CD4+ T cells that are key players of immune tolerance. They are powerful suppressor cells, able to impact the function of numerous immune cells, including key effectors of inflammation such as effector T cells. For this reason, Tregs are an ideal candidate for the development of cell therapy approaches to modulate immune responses. Regulatory T cell (Treg) therapy has shown promising results so far, providing key knowledge on the conditions in which these cells can provide protection and demonstrating that they could be an alternative to current pharmacological immunosuppressive therapies. However, a more comprehensive understanding of their characteristics, isolation, activation, and expansion is needed to be able design cost effective therapies. Here we review the practicalities of making Tregs a viable cell therapy, in particular, discussing the challenges faced in isolating and manufacturing Tregs and defining what are the most appropriate applications for this new therapy.

It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25- cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25(high) and CD25(intermediate) CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T-cell populations such as those found in joints.

Despite substantial advances in our understanding of CD4+ CD25+ regulatory T cells, a possible equivalent regulatory subset within the CD8+ T cell population has received less attention. We now describe novel human CD8+/TCR alphabeta+ T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS-activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I-restricted fashion, by proliferation and production of IL-4, IL-5, IL-13 and TGFbeta1, but not IFN-gamma. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+ CD25+ regulatory T cells, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and Foxp3. They suppressed IFN-gamma production and proliferation by CD4+ T cells in vitro in a cell contact-dependent manner, which could be blocked using a CTLA-4-specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up-regulated in response to inflammation. We suggest that they are the CD8+ counterparts of CD4+ CD25+ regulatory T cells. They resemble recently described CD8+ regulatory cells in the rat that were able to abrogate graft-versus-host disease. Likewise, human HLA-restricted CD8+ regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.

Cross-reactive T cell recognition of self-heat shock proteins (hsp) has been ascribed a regulatory role in inflammatory arthritis in both animal models and human disease. The previous work implies that a repertoire for epitopes in self-hsp60 should exist in normal subjects. Accordingly, we sought to generate self-hsp60-reactive T cell clones from a healthy individual using a highly purified preparation of recombinant human (Hu) hsp60. Epitope mapping using synthetic peptides and truncated constructs indicated that the T cell clones obtained actually recognized hsp60 derived from Escherichia coli. Using a series of alanine-substituted peptides and additional appropriate synthetic peptides, it was demonstrated that the clones maintain self-tolerance because of their sensitivity to an asparagine to aspartic acid sequence difference between E. coli and HuHsp60 in the epitope-containing peptide. In addition, despite substantial conservation of sequence, the homologous peptide from HuHsp60 did not compete with the E. coli-derived peptide for recognition or antagonize responses by acting as an altered peptide ligand. The results suggest that, even when the immune system targets a highly conserved epitope in bacterial hsp60, self-tolerance is maintained. Furthermore, the finding that T cell clones specific for minor contaminant proteins in HuHsp60 preparations can readily be isolated raises the possibility that the HuHsp60 facilitates presentation of antigenic proteins to the immune system.

Transport of Ca2+ across the membrane by the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum involves the transfer of two Ca2+ ions from a pair of cytoplasmic sites to a pair of luminal sites, driven by phosphorylation of the ATPase. The ATPase is inhibited by Mg2+ at alkaline pH values. Inhibition follows from a decrease in the rate of release of Ca2+ from the phosphorylated ATPase. Phosphorylation-induced release of Ca2+ from the ATPase is biphasic at alkaline pH, which is consistent with sequential release of Ca2+ from the phosphorylated ATPase; the rates of both components decrease with increasing Mg concentration. The effect of Mg2+ on the slow phase of release follows from the binding of Mg2+ at the empty outer luminal site, vacated by the release of the first Ca2+ ion. The effect of Mg2+ on the rate of release of the first Ca2+ ion could follow from binding to a gating site also affecting the binding of Ca2+ to the cytoplasmic sites.

Effects of lipid structure on the function of the Ca2+-ATPase of skeletal muscle of sarcoplasmic reticulum are reviewed. Binding of phospholipids to the ATPase shows little specificity. Phosphatidylcholines with short (C14) or long (C24) fatty acyl chains have marked effects on the activity of the ATPase, including a change in the stoichiometry of Ca binding. Low ATPase activity in gel phase lipid follows from low rate of phosphorylation. Phosphatidylinositol 4-phosphate increases ATPase activity by increasing the rate of dephosphorylation of the phosphorylated ATPase. Stimulation is not seen with other anionic phospholipids; phosphatidic acid decreases ATPase activity in a Mg2+-dependent manner.

Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7-aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions.To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony-forming units (CFU) to determine if graft potency can be predicted using a "functional flow cytometry" approach.Significant numbers of CD34+ AnnV+ events were found within the 7AAD-gated population. Nonapoptotic cell dose (CD34+ AnnV-) correlated well with CFUs in both a small-scale (n = 10) and a large-scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration.Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.

It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25- cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25(high) and CD25(intermediate) CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T-cell populations such as those found in joints.

Because antigen-specific cells are the central coordinators of the immune response to infectious organisms, and the principal effector cells in autoimmune disease, there are many circumstances in which investigators may wish to examine the T-cell responses to particular antigens. This chapter outlines techniques for assessing the responses of polyclonal populations of T-lymphocytes by measuring a variety of outputs each of which gives different kinds of information about the response. The outputs discussed are proliferation and cytokine production, with methods for measuring cytokine secretion by the whole population together with techniques for making an estimate of the numbers of cells producing a cytokine in response to antigen, and examining the phenotype of the responsive cells. In many cases detailed information about responses to particular antigens requires the isolation and characterization of antigen-responsive T-cell clones, and this is also described together with methods of identifying unknown antigens by screening recombinant expression libraries. Lastly, because the techniques differ in many respects, methods for isolating antigen-specific CD8+ T-cells, particularly those which recognize bacteria, are also included.

Reactive arthritis (ReA) is unusual amongst inflammatory joint diseases because the cause of arthritis has been identified, namely preceding infection with certain bacteria, whereas the aetiology of rheumatoid arthritis or ankylosing spondylitis, or at least the triggering event, remains unknown. Most subjects infected by Chlamydia,Salmonella, Campylobacter,Yersinia or Shigella develop symptoms due to the effects of the infection in the genito-urinary (Chlamydia) or gastro-intestinal tracts, but resolve the infection without any long-term sequelae. A minority develop inflamed joints, and also inflammation at the sites of insertion of tendons and ligaments - a process termed enthesitis [1] -but the factors which confer susceptibility to arthritis have not been completely identified. HLA alleles play a part with HLAB27+ individuals being more likely to develop reactive arthritis, and to have more severe and long-lasting disease - indeed a proportion develop chronic arthritis, and these are nearly all HLA-B27+ [2,3]. Other HLA alleles including HLADRB1 *0103 have also been implicated [4]. The associations between disease inci-dence and/or severity with HLA imply that T-cell mediated immune responses are likely to be central to the pathogenesis of ReA, and that immune responses in subjects who develop arthritis will differ in some way from those who have uncomplicated infection. It is in this context that immune responses to heat shock proteins (HSP) have been studied in ReA.