Stephan Fuhrmann

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.

Tuberculin-Specific T Cells Are Reduced in Active Pulmonary Tuberculosis Compared to LTBI or Status Post BCG Vaccination

Functional characteristics of tuberculosis (TB)-specific CD4 T cells were studied in clinically active pulmonary TB (n = 21) and high TB exposure including LTBI (n = 17). Following tuberculin stimulation, activated CD4 T cells were identified by flow-cytometry (CD154 up-regulation, degranulation, interferon γ [IFN-γ], tumor necrosis factor α [TNF-α], and interleukin 2 [IL-2\ production). Interestingly, CD154 up-regulation accounted for ∼80% of activated CD4 T cells in the active TB group but just 40% in the controls, whereas IFN-γ accounted for only ∼50% of activated cells in each group. The frequencies of CD4 T cells displaying at least 1 activation marker discriminated better between the groups than those displaying degranulation or IFN-γ production alone.

The phenotypic distribution and functional profile of tuberculin-specific CD4 T-cells characterizes different stages of TB infection

Recent publications have suggested that altered proportions of functional CD4 T‐cell subsets correlate with active pulmonary TB. Also, CD27‐expression on tuberculin‐activated IFN‐γ+ CD4 T‐cells is known to differ significantly between patients with active pulmonary TB and healthy TB‐unexposed BCG vaccinees. Here, we explore links between CD4 T‐cell phenotype, multiple functional subsets, and control of TB.

Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets

Multi-colour flow cytometry is the only technological platform that can analyse the highly complex cellular composition of the immune system in parallel and at single cell resolution. Analysis of the T cell compartment, in particular, requires the simultaneous measurement of multiple markers in order to account for lineage, phenotype and function. Flow cytometry also enables the analysis of intracellular signalling events. By combining the expression of surface markers, intracellular cytokines, phosphorylated versus unphosphorylated kinases, cell proliferation and DNA profile, mechanistic and kinetic information of subset-specific signalling may be obtained: this has not previously been achieved. Here we present a protocol which permits all of these aspects to be explored simultaneously. By comparing basic procedures previously described we were able to optimise different variables, including the choice of antibody/fluorochrome pairs, permeabilisation, fixation and labelling time, to obtain the best DNA staining of different cell types. We applied this method to study subset-specific signalling related to cytokine production and DNA synthesis in T cells responding to specific antigens.

T Cell Response to the Cytomegalovirus Major Capsid Protein (UL86) Is Dominated by Helper Cells with a Large Polyfunctional Component and Diverse Epitope Recognition

T cells are crucial in controlling cytomegalovirus (CMV) infection. The CMV major capsid protein (UL86) is frequently recognized by these cells, but the nature of this response has not been explored in detail. In this study, healthy CMV-exposed individuals were examined, and ex vivo peptide stimulation of peripheral blood mononuclear cells and flow-cytometry were used to obtain data, including response prevalence, magnitude, functional profiles, and recognized epitopes. Of 24 subjects, 19 (79%) had a UL86-specific CD4 T cell response rate between 0.03% and 1.4%. This group of individuals exhibited a similar percentage of polyfunctional T cells in their UL86-specific and pp65-specific responses. A total of 8 CD4 T cell epitopes were identified. In contrast, CD8 T cell responses to UL86 were rare and small. UL86 is of interest for monitoring the response to CMV.

Dissection of the CMV specific T-cell response is required for optimized cardiac transplant monitoring.

Despite the success of antivirals in preventing clinically overt CMV disease in cardiac allograft recipients, sub‐clinical active CMV infection remains a major concern because of its association with allograft rejection and vasculopathy. The measurement of CMV specific T‐cell responses is a promising approach to assessing this situation. For simplicity, class‐I MHC/peptide‐multimers staining CD8 T‐cells directly are often used but this ignores a much wider range of responses including the whole CD4 T‐cell compartment. CD4 T‐cells, however, were recently shown to be critical to reducing CMV load early after transplantation. To determine how extensive T‐cell responses to CMV are, the responses to two dominant CMV proteins, IE‐1 and pp65, were dissected in detail accounting for T‐cell lineage, frequencies, epitope recognition and changes over time in more than 25 heart transplant recipients. Cross‐sectional results from over 30 healthy CMV‐carriers were analyzed for comparison. Responses were unexpectedly complex, with considerable inter‐individual variation in terms of dominance, breadth, and recognized epitopes. Whereas the use of MHC/peptide‐multimers for clinical CD8 T‐cell response monitoring alone can be justified in some situations, short term T‐cell activation combined with intracellular cytokine staining was clearly found to be of more general usefulness. The performance of IFN‐gamma, TNF‐alpha, or IL‐2 as single read‐outs in identifying activated T‐cells was examined and confirmed that the frequently used IFN‐gamma was best suited. These results should be used to inform the design of clinically applicable and diagnostically useful approaches to monitoring CMV specific responses in heart transplant recipients. J. Med. Virol. 80:1604–1614, 2008.

Cyclosporin A and tacrolimus reduce T‐cell polyfunctionality but not interferon‐γ responses directed at cytomegalovirus

Cytomegalovirus (CMV) ‐specific immunity is often estimated by the number of in vitro CMV antigen‐inducible interferon‐γ‐positive (IFN‐γ+) T cells. However, recent work indicates that simultaneous production of IFN‐γ, tumour necrosis factor‐α (TNF‐α) and interleukin‐2 (IL‐2) (referred to as ‘polyfunctionality’) is more relevant for anti‐viral protection. Here, we compared polyfunctionality of CMV‐specific T cells (pp65 and IE‐1 proteins) in 23 solid‐organ transplant patients and seven healthy controls by flow cytometry. The proportions of TNF‐α+/IFN‐γ+/IL‐2 cells among the activated cells were significantly reduced in transplant patients but not the frequencies of IFN‐γ+ CD8+ T cells. Immunosuppression reduces polyfunctionality, which reflects the increased infection risk in this patient group.

Using hydroxymethylphenoxy derivates with the SPOT technology to generate peptides with authentic C-termini

The SPOT technology can fulfill most requirements for highly parallel, multiple peptide synthesis of soluble peptides within the upper microgram range. Here, we report on an improved method using hydroxymethylphenoxyacetic acid (HMPA) for 19 amino acids and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) for proline as acidic labile linkers in SPOT synthesis. Using this approach we could reduce side-chain reactions normally occurring during conventional alkaline peptide cleavage from cellulose membranes. All synthesis steps were adapted to fully-automated SPOT synthesis and therefore represent a time- and cost-saving procedure. Furthermore, the improved cleavage and washing steps resulted in peptides with authentic C-termini in a purity range of 60-95%. Our improved method is ideal for synthesizing many thousand different peptides subsequently used directly for different biological assays requiring authentic C-termini, such as CD8 T-cell epitope screening, vaccine immunization, or tumor imaging.

Expression of CD56 defines a distinct subgroup in childhood T-ALL with inferior outcome. Results of the ALL-BFM 2000 trial

This study reports the prognostic impact of the expression of the natural killer cell marker CD56 in a large series of risk‐adapted paediatric patients with T cell acute lymphoblastic leukaemia (T‐ALL; n = 493) treated within the ALL‐Berlin‐Frankfurt‐Münster (BFM) 2000 protocol. The immunophenotype was analysed centrally at diagnosis using flow cytometry and correlated with clinical parameters and outcome. CD56 expression was detected in 7·1% and early T‐cell precursor (ETP) phenotype in 6·7% of all T‐ALL patients. The percentage of ETP in the CD56+ T‐ALL cohort was 4‐fold higher than in the whole cohort. CD56+ T‐ALL frequently expressed the progenitor marker CD34 and myeloid antigens CD13 and CD33. The 5‐year event‐free survival (EFS) rates for the European Group for the Immunological classification of Leukaemias/World Health Organization subgroups and the ETP phenotype were not statistically different. By contrast, patients with CD56 expression had a significantly reduced EFS (60 ± 8%) and overall survival (60 ± 8%) at 5 years, with a hazard ratio of 2·46 (P = 0·002) and 2·99 (P < 0·001), respectively. Moreover, CD56 expression in combination with the minimal residual disease (MRD)‐based high risk assignment defined a population with a ‘very‐high’ risk probability of relapse in the ALL‐BFM 2000 trial. The CD56 marker has the potential to augment MRD‐based risk stratification and may serve as a molecular target for antibody‐based treatment strategies in childhood T‐ALL.