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Background: It is now well known that the outcome after allogeneic transplantation, such as incidence of acute rejections, very much depends on the individual's immune reactivity status. There is also increasing evidence that the presence of preexisting memory T cells can affect antigraft immune responses., Methods: In a prospective study, we monitored peripheral CD4 and CD8 central memory, effector memory, and terminal differentiated effector memory (TEMRA) T cells in 55 patients who underwent deceased liver transplantation and received conventional immunosuppressive treatment with or without basiliximab induction. The primary endpoint of the study was acute allograft rejection during a 1-year follow-up period., Results: We observed significantly increased proportions of CD4 and CD8 TEMRA cells in patients before transplantation compared with healthy controls (P=0.006 and 0.009, respectively). This characteristic was independent of the underlying disease. In patients with no signs of acute rejection, we observed an immediate reduction of CD4 TEMRA cells. In contrast, patients who experienced acute cellular rejection, and especially antibody-mediated rejection, displayed persistent elevated TEMRA cells (P=0.017 and 0.027, respectively). Basiliximab induction therapy did not influence CD4 and CD8 TEMRA numbers., Conclusions: Conventional immunosuppressive or basiliximab treatment cannot control the persistence of TEMRA T cells, which may contribute to acute cellular rejection and antibody-mediated rejection after liver transplantation. In the future, specific targeting of TEMRA cells in selected patients may prevent the occurrence of difficult to treat steroid-resistant rejections, thereby leading to improved patient outcome.

Background: 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., Methods: We examined ex-vivo overnight tuberculin activated CD4 T-cells in regards to CD27-expression and the activation markers, CD154 upregulation, IFN-γ, TNF-α, IL-2, and degranulation in 44 individuals, including cases of clinically active pulmonary TB, and hospital staff with prolonged TB exposure, some of whom had latent TB., Results: Active pulmonary TB generally showed an excess of TNF-α(+) subsets over IFN-γ(+) subsets, paralleled by decreased CD27 expression on activated IFN-γ(+) or CD154(+) CD4 T-cells. The single subset distinguishing best between active pulmonary TB and high TB exposure was CD154(+) /TNF-α(+) / IFN-γ(-) /IL-2(-) /degranulation(-) (AUROC 0.90). The ratio between the frequencies of TNF-α(+) /IFN-γ(+) CD4 T-cells was an effective alternative parameter (AUROC 0.87)., Conclusions: Functional subsets and phenotype of tuberculin induced CD4 T-cells differ between stages of TB infection. Predominance of TNF-α(+) CD4 T-cells in active infection suggests an increased effort of the immune system to contain disease., (Copyright © 2012 International Clinical Cytometry Society.)

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., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

Dengue is a major cause of illness among travelers and a threat to military troops operating in areas to which it is endemic. Before and during World War II, dengue frequently occurred in US military personnel in Asia and the South Pacific. From the 1960s into the 1990s, dengue often occurred in US troops in Vietnam, the Philippines, Somalia, and Haiti. We found attack rates as high as 80% and periods of convalescence up to 3-1/2 weeks beyond the acute illness. The increase in dengue throughout the world suggests that it will remain a problem for military personnel until an effective vaccine is licensed.

Purpose of Review: Transfer of human regulatory T cells (Tregs) has become an attractive therapeutic alternative to improve the long-term outcome in transplantation and thus reduce the side-effects of conventional immunosuppressive drugs. Here, we summarize the recent findings on human Treg subsets, their phenotype and in-vivo function., Recent Findings: In the last 2 years, it has become apparent that several Treg subsets exist that specifically regulate Th1-driven, Th2-driven, or Th17-driven immune responses; these subsets are very unstable and rapidly change their phenotype, for example, there is loss of Foxp3 expression upon extensive ex-vivo expansion and only the administration of rapamycin has been shown to be able to interfere reproducibly. New humanized mouse models incorporating human solid-organ grafts have been developed, which have been used to test the human Treg in-vivo function, and the first human Treg-cell products have been tested for safety and efficacy in stem cell transplantation., Summary: With the recent findings, we have gained a better understanding of Treg heterogeneity, plasticity and function. Using the outcomes of clinical trials in stem cell transplantation, we have learned that adoptive therapy of Tregs is well tolerated and we are now awaiting the first result in solid-organ transplantation from the 'ONE Study'.

The application of flow cytometry has hugely advanced the field of tuberculosis (TB) across all areas of research ranging from diagnostic tests to understanding the underlying immunology. As cellular responses are understood to be the mainstay of the immune response in the control of TB it is very likely that polychromatic flow will become the tool of choice for assessing the effects of vaccination. Results are particularly encouraging in this area. The development of a new type of diagnostic test, a prototype of which has been reported, may be the spin-off of a broad and systematic approach to understanding and profiling the T-cell response to TB. It is obvious that flow cytometry will not be able to address all research questions in the field of TB. However, its enormous flexibility as a technology will make it the tool of choice in many situations. An ever increasing availability of flow cytometers, even in resource-poor countries, will rapidly change the face of TB research and management in the years ahead.

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.

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.

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity., Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mug nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amino-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFNgamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFNgamma, IL2, IL27 and T-bet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low., Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes.

Background: Tuberculin-specific T-cell responses have low diagnostic specificity in BCG vaccinated populations. While subunit-antigen (e.g. ESAT-6, CFP-10) based tests are useful for diagnosing latent tuberculosis infection, there is no reliable immunological test for active pulmonary tuberculosis. Notably, all existing immunological tuberculosis-tests are based on T-cell response size, whereas the diagnostic potential of T-cell response quality has never been explored. This includes surface marker expression and functionality of mycobacterial antigen specific T-cells., Methodology/principal Findings: Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively M. tuberculosis specific ESAT-6 protein were used as stimulants. A set of classic surface markers of T-cell naïve/memory differentiation was selected and IFN-gamma production was used to identify T-cells recognizing these antigens. The percentage of tuberculin-specific T-helper-cells lacking the surface receptor CD27, a state associated with advanced differentiation, varied considerably between individuals (from less than 5% to more than 95%). Healthy BCG vaccinated individuals had significantly fewer CD27-negative tuberculin-reactive CD4 T-cells than patients with smear and/or culture positive pulmonary tuberculosis, discriminating these groups with high sensitivity and specificity, whereas individuals with latent tuberculosis infection exhibited levels in between., Conclusions/significance: Smear and/or culture positive pulmonary tuberculosis can be diagnosed by a rapid and reliable immunological test based on the distribution of CD27 expression on peripheral blood tuberculin specific T-cells. This test works very well even in a BCG vaccinated population. It is simple and will be of great utility in situations where sputum specimens are difficult to obtain or sputum-smear is negative. It will also help avoid unnecessary hospitalization and patient isolation.

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.

The analysis of T-cell responses to peptides has recently become a busy area of immunologic research. Peptides may be used as single stimulants, pools or libraries, or as part of peptide/major histocompatibility complexes (MHC) for direct T-cell receptor staining. For stimulating T cells, peptides must be bound to MHC molecules. In this study we have used 9- or 10-amino acid peptides, 15-amino acid peptides containing stimulating shorter sequences, and peptides with modified C-terminal function. On average 67% of the T cells from healthy cytomegalovirus-positive donors that bound a frequently used cytomegalovirus pp65/HLA-A*0201 tetramer were able to produce interferon-gamma on stimulation with the respective 9-amino acid peptide. Peptides of 15 amino acids length used at the same concentration (in microg/ml) stimulated CD8 T cells somewhat less efficiently (on average 77% of the frequencies induced with the respective shorter peptides). Modifications of 9-amino acid peptides such as addition of amino acids or functional groups often resulted in a decreased ability to stimulate. However, based on our own results, published data, and theoretic considerations, we conclude that sets of peptides of 15 amino acids length with 11 amino acids overlap represent a good compromise for stimulating both CD8 and CD4 T cells in a number of applications. These parameters may be modified subject to the purpose of a study.