Mathias Streitz

Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study

BackgroundImmune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry.MethodsSix leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons.ResultsOptimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies.ConclusionsLocal performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.

Loss of receptor on tuberculin-reactive T-cells marks active pulmonary tuberculosis.

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-γ 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.

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.

NS1 specific CD8+ T-cells with effector function and TRBV11 dominance in a patient with parvovirus B19 associated inflammatory cardiomyopathy

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/μg 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-amico-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 IFNγ 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 (IFNγ, 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.

Elevation of CD4+ differentiated memory T cells is associated with acute cellular and antibody-mediated rejection after liver transplantation.

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.

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.

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.

Age and gender leucocytes variances and references values generated using the standardized ONE-Study protocol

Flow cytometry is now accepted as an ideal technology to reveal changes in immune cell composition and function. However, it is also an error‐prone and variable technology, which makes it difficult to reproduce findings across laboratories. We have recently developed a strategy to standardize whole blood flow cytometry. The performance of our protocols was challenged here by profiling samples from healthy volunteers to reveal age‐ and gender‐dependent differences and to establish a standardized reference cohort for use in clinical trials. Whole blood samples from two different cohorts were analyzed (first cohort: n = 52, second cohort: n = 46, both 20–84 years with equal gender distribution). The second cohort was run as a validation cohort by a different operator. The “ONE Study” panels were applied to analyze expression of >30 different surface markers to enumerate proportional and absolute numbers of >50 leucocyte subsets. Indeed, analysis of the first cohort revealed significant age‐dependent changes in subsets e.g. increased activated and differentiated CD4+ and CD8+ T cell subsets, acquisition of a memory phenotype for Tregs as well as decreased MDC2 and Marginal Zone B cells. Males and females showed different dynamics in age‐dependent T cell activation and differentiation, indicating faster immunosenescence in males. Importantly, although both cohorts consisted of a small sample size, our standardized approach enabled validation of age‐dependent changes with the second cohort. Thus, we have proven the utility of our strategy and generated reproducible reference ranges accounting for age‐ and gender‐dependent differences, which are crucial for a better patient monitoring and individualized therapy.