Shara B.A Cohen

Analysis of the cytokine production by cord and adult blood

To date, over 400 human umbilical cord blood cord blood (CB) transplants have been reported from different centres world-wide and it is generally agreed that CB represents an encouraging alternative to bone marrow (BM) transplantation. There are a variety of reasons for this which include the wider availability and easier access of CB compared to BM. In addition it has been suggested that there is a reduced graft-versus-host-disease (GvHD) with CB compared to BM transplantation. The explanations for this implied benefit are numerous, but research into this area is only just beginning. Nevertheless, it is clear that both T cells and natural killer (NK) cells have reduced function when isolated from CB compared to adult and both these cell types have been implicated in GvHD pathogenesis. How and why the function is reduced is yet to be determined. Many laboratories have tried to answer these questions and the majority have done this by comparing the function of lymphocytes obtained from adult blood with those compared with CB. Since cytokine production by a cell is an indication of the cells function it is important to determine the differences between adult and CB with respect to production of these soluble factors. Here, we have reviewed the current research regarding these CB and adult cell comparisons with an emphasise on cytokine production. Our aim is to obtain a clearer understanding of the mechanisms which may be involved in causing a reduced GvHD in CB compared to BM transplantation.

Hepatitis B third‐generation vaccines: improved response and conventional vaccine non‐response – evidence for genetic basis in humans

The lack of response to hepatitis B vaccination remains a problem for those individuals directly at risk of hepatitis B infection, particularly those who work in the health care industry. The factors associated with non‐response to hepatitis B vaccination have been investigated in 86 non‐responder health care workers who had received multiple ‘S’ vaccinations without sustained production of anti‐HBs. This group received a recently developed hepatitis B vaccine, HepageneTM, which included proteins derived from the envelope region of HBV, not present in currently licensed vaccines. The pre‐S1 and pre‐S2 proteins were included in HepageneTM in order to circumvent anti‐HBs non‐responsiveness which had previously been demonstrated in the inbred mouse model. The inclusion of these additional proteins in HepageneTM enabled some seroconverion, from non‐responder to responder; however, a proportion of the vaccinees remained non‐responders and the reasons for this have been investigated here, with reference to HLA alleles and the demographic predisposition. Here the mechanisms that underlie hepatitis B vaccine non‐response have considered the distribution of HLA alleles, age, sex, height and weight in addition to the T‐cell responses to HepageneTM derived antigens.

The influence of exogenous peptide on beta2-microglobulin exchange in the HLA complex: analysis in real-time.

Abstract We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor β2–microglobulin (β2m) exchange within the HLA-A2 molecule, whereby native β2m in the complex is replaced by β2m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (kass), dissociation rate (kdiss) and affinity constants (KA) for the β2m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of β2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1.55–1.88×109 M–1) of HLA-A2 for immobilized β2m compared with the control (KA =1.14±0.04×109 M–1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that β2m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines.

Can cord blood cells support the cytokine storm in GvHD

Cord blood has a high number of proliferating hematopoietic progenitors and is therefore used as an alternative source of hematopoietic cells for allogeneic transplantation. In addition there is a wider availability of cord blood and a lower cost of procurement compared to bone marrow. However one of the most interesting immunological benefits of a cord blood transplant that has been proposed is the low severity of Graft versus Host Disease (GvHD). This review aims to address some of the immunological reasons why this may be the case by assessing the role of cord blood cytokines in the cytokine storm of GvHD.

Cytokine profile data.

The Th1/Th2 paradigm is now well established. In the mouse, a CD4+ cell that makes interleukin 4 (IL-4) and not interferon γ (IFN-γ) is termed Th2 and a cell that makes IFN-γ and not IL-4 is termed Th1 (Ref. 1xTwo types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. Mosmann, T.R. et al. J. Immunol. 1986; 136: 2348–2357PubMedSee all ReferencesRef. 1). This nomenclature has been extended to other cell types such as CD8+ cytotoxic T cells (Tc1, Tc2)2xDiffering lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Salgame, P. et al. Science. 1991; 254: 279–282Crossref | PubMedSee all References2 and natural killer cells (NK1, NK2)3xDifferentiation of human NK cells into NK1 and NK2 subsets. Peritt, D. et al. J. Immunol. 1998; 161: 5821–5824PubMedSee all References3. These clear cut cell types, defined by their cytokine profile, define the function of the cell.In humans this distinction between cell types is less clear cut because, depending on the stimulus, cells can make both IFN-γ and IL-4. Therefore, many researchers use a ratio of IFN-γ:IL-4 to define the phenotype of the cell. Although using a ratio would define the function of the cell, it does not take into account the quantity of cytokine produced or the strength of the signal that was used to stimulate cytokine production.For example, stimulation of a CD4+ cell might produce 1 unit of IFN-γ and 2 units of IL-4. The IFN-γ:IL-4 ratio would be 0.5 and the cell would be defined as Th2. If the strength of the signal was increased so that the cell made 2 units of IFN-γ and 4 units of IL-4, the cell would still have a ratio of 0.5 and thus be defined as Th2. If the cell was stimulated with an antigen that drives a Th1-type response and made 4 units of IFN-γ and 2 units of IL-4, the ratio would be 2 and the cell would be defined as Th1. In this latter case, the Th1 cells (defined by ratio) make a lot of proinflammatory cytokine but produce the same level of IL-4 as the Th2 cell in the first example.In vivo cytokines have an effect locally (unless they become neutralized), therefore the quantity of cytokine made is important. I propose that a more physiologically relevant ratio to use when stimulating cells in vitro would be the cell phenotype defining cytokine (CPDC), in this example IFN-γ or IL-4, divided by a cytokine that is made by every T cell.IL-3 is an example of a cytokine that could be used. Using a ratio such as this to look at skewing of T-cell cytokine profiles would first, not introduce artefacts due to signal strength (by normalizing against a control cytokine) and second, provide information on the role of the CPDC. This type of analysis is similar to using actin as an internal control when looking at mRNA made by cells. Molecular biologists take actin mRNA (that is always made by live cells) and standardize their analysis on this. Perhaps cytokine biologists should use similar internal controls?

Cord blood serum does not increase lymphocyte responses in comparison to adult serum

To date, over 1000 cord blood (CB) transplants have been reported from different centers worldwide and it is generally agreed that CB represents an encouraging alternative to bone marrow (BM) transplantation. There are a variety of reasons for this, however, possibly the two most controversial aspects are (a) whether there is less graft versus host disease (GVHD) with CB compared to BM transplantation, and (b) whether we can use more HLA mismatches with CB transplantation. The major theory regarding the reduced immunological response of CB lymphocytes is that CB T and NK cells are naive and, therefore, not primed for activation. However, the naive phenomena that has been noted in vitro may be bypassed in vivo by unforeseen factors. We show evidence that there are differences in the soluble factors present in CB and adult serum and that these differences play a role in T cell function. Thus, adult serum will enhance both mitogen and IL-2 specific T cell growth whereas CB serum has no effect, suggesting that there is an activation/growth factor present in adult sera, which is absent in CB sera. This work could enable us to identify the molecular mechanisms which are associated with a lower GVHD in CB compared to BM transplanted individuals.

Real-time analysis of cell surface HLA class I interactions

Characterisation of the kinetics of assembly and dissociation of the HLA class I heterotrimeric complex provides valuable insights into the relative contributions of each element to complex stability. However, to date there has been no real-time binding analysis on whole cells. Here we have developed an optical biosensor model to investigate the binding of class I HLA complexes on whole cells to human beta 2-microglobulin (beta 2m) and the effects of different HLA-specific peptides on this binding. We immobilised beta 2m on an IAsys biosensor surface and established conditions to analyse the binding of this to HLA-A2 expressing cells (T2 cells). Using 721.221 cells as an HLA negative control we showed that HLA-A2 binding was optimal using a) a carboxymethylated dextran surface and b) no growth factors or supplements in the culture medium at the binding event. Using these conditions we verified specificity of binding by inhibition of the reaction with free beta 2m and determined the dissociation rate constant for T2 cell binding to beta 2m (0.03 s-1). In addition, we demonstrated the ability of different HLA specific peptides to modulate cellular HLA-A2 binding to beta 2m. This is the first time that interactions of cell surface HLA class I molecules has been investigated using real-time analysis. Furthermore, our peptide analysis has shown that this model can be used to characterise peptide specific HLA-binding responses on the whole cell surface in real-time.

Naive T cells from cord blood have the capacity to make Types 1 and 2 cytokines

We wanted to determine whether naive T cells could make the Types 1, 2 and 0 defining cytokines Interleukin (IL)-4 and Interferon (IFN)gamma. We show that stimulation of naive T cells (CD3+ CD45RA+) derived from cord blood by phorbol ester (phorbol-12-myristate-13-acetate: PMA) plus lonomycin induced detection of Types 1, 2 and 0 cells. Conversely, when we stimulated the naive T cells through the T cell receptor (with anti-CD3 monoclonal antibody alone) there was no detection of IFNgamma or IL-4 producing cells. Stimulation with PMA and CD3 induced detection of only Type 2 cells. This unexpected finding shows that there is a high frequency of Type 2 cells within the naive T cell population, contrary to previously published reports. The highest percentage of Type 2 naive cells (10.5%) was obtained with 50 ng/ml PMA plus 50 microg/ml anti-CD3. Thus, we have shown that naive T cells derived from cord blood have the capacity to make both Types 1 and 2 cytokines and the frequency of cells producing these cytokines can be greater than 20%, depending on the stimulus used.

Cord blood serum affects T cells ability to produce and respond to IL-2

The current literature suggests that cord blood (CB) cells are functionally immature. We previously reported that CB sera inhibit T cell proliferation and suggested that the microenvironment in which CB T cells reside may be, in part, responsible for their reduced function. In this study we have tried to explain some of the actions of the CB sera on peripheral blood mononuclear cells (PBMC). We showed that, as expected CB sera decreased the anti-CD3 and anti-CD28-induced proliferative response of PBMC (p < 0.01) but unexpectedly, increased the interleukin-2 (IL-2) specific proliferation of both a human T cell line (p < 0.005) and T cells within a mononuclear cell population (p < 0.05). These findings prompted us to analyse the effect of CB sera on the T cell ability to make and respond to IL-2. Stimulation of T cells in the presence of CB sera increased the frequency of IL-2 producing cells (p < 0.005) (but not the amount of IL-2 secreted) and resulted in a higher expression of CD25 (p < 0.05). Furthermore CB sera (in the presence and absence of IL-2) made the cells apoptose less (p < 0.005) than adult sera. Our results go some way to explaining the effect of the CB microenvironment on CB cellular function.

The effect of cord blood sera on CD69 expression

We have reported previously that cord blood (CB) sera inhibit T cell proliferation and cytokine production, and have suggested that the microenvironment that CB T cells come from may be responsible for the reduced function of CB T cells. In the present study we analysed the effect of CB sera on the expression of CD69, the earliest activation marker, on T and natural killer (NK) cells. CD69 expression on adult NK cells was higher than on CB NK cells (p<0.05). CB and adult blood T cells presented with a similar proportion of CD69 expressing cells. Incubation of peripheral blood mononuclear cells (PBMC) in the CB sera enhanced CD69 expression on alloactivated T cells (p<0.05). However there was no difference in the CD69 expression within the population of alloactivated adult NK cells. Stimulation of PBMC with phytohaemagglutinin (PHA) in the presence of different sera resulted in higher CD69 expression on T cells incubated with CB sera (p<0.05), but was similar on NK cells. Our former experiments documented similar effect of CB sera on CD25 expression on mitogen and allostimulated T cells. Thus CB sera seem to have similar influences on the expression of activation markers on stimulated T cells within PBMC, implying that there may be a factor(s) within CB sera affecting lymphocyte responses and activation status.