Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines.

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.

Key role of effector memory CD4+ T lymphocytes in a short-incubation heparin-binding hemagglutinin gamma interferon release assay for the detection of latent tuberculosis.

The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing the Mycobacterium tuberculosis reservoir. Therefore, powerful LTBI screening tools are indispensable. A gamma interferon release assay (IGRA) in response to the stimulation of peripheral blood mononuclear cells by the latency antigen native heparin-binding hemagglutinin (nHBHA-IGRA) has proven its potential for this purpose. We have evaluated its possible optimization through a reduction of incubation time from 96 to 24 h, while compensating for this by adding interleukin 7 (IL-7) to the medium. We have also investigated the phenotypes of the gamma interferon (IFN-γ)-producing cells after both short and long incubation times. One hundred thirty-one nonimmunocompromised patients were recruited from 3 Brussels-based university hospitals. They were divided into 1 of 4 subgroups according to their M. tuberculosis infection status (LTBI, TB infection, undetermined M. tuberculosis infection status, and noninfected controls). The novel 24-h nHBHA-IGRA was performed for all subjects, and a simultaneous 96-h classical HBHA-IGRA was performed for 79 individuals. The results showed a good correlation between the two tests, and the novel 24-h nHBHA-IGRA maintained the principal advantages of the classical test, namely, a high specificity for LTBI diagnosis, an absence of interference of Mycobacterium bovis BCG vaccination during infancy, and a relative discrimination between LTBI and TB infection. Whereas the commercialized IGRAs show a greater sensitivity for recent than for remote M. tuberculosis infections, the 24-h nHBHA-IGRA appears to have comparable diagnostic powers for recent and remote LTBI. The IFN-γ detected by the 24-h nHBHA-IGRA was mainly secreted by effector memory CD4(+) T lymphocytes, a finding suggestive of continuous HBHA presentation during latency.

Different T cell memory in preadolescents after whole-cell or acellular pertussis vaccination.

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.

Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.

Rho GTPase Cdc42 is essential for human T-cell development.

BACKGROUND: Rho GTPases are involved in the regulation of many cell functions, including some related to the actin cytoskeleton. Different Rho GTPases have been shown to be important for T-cell development in mice. However, their role in human T-cell development has not yet been explored. DESIGN AND METHODS: We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration. RESULTS: We found that the expression of several Rho GTPases is differently regulated during successive stages of T-cell development in man, suggesting a specific role in human thymopoiesis. In chimeric fetal thymus organ culture, T-cell development was not or only mildly affected by expression of dominant negative Rac1 and Rac2, but was severely impaired in the presence of dominant negative Cdc42, associated with enhanced apoptosis and reduced proliferation. Kinetic analysis revealed that Cdc42 is necessary in human T-cell development both before and after expression of the pre-T-cell receptor. Using inhibitors and retrovirally transferred mutants of the aforementioned Rho GTPases, we showed that only Rac1 is necessary for migration of different thymocyte subsets, including the early CD34(+) fraction, towards stromal cell-derived factor-1 alpha. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1 alpha and T-cell development to different degrees. CONCLUSIONS: This is the first report on Rho GTPases in human T-cell development, showing the essential role of Cdc42. Our data suggest that enhanced apoptotic death and reduced proliferation rather than disturbed migration explains the decreased thymopoiesis induced by dominant negative Cdc42.

Surface-expressed viral proteins in feline infectious peritonitis virus-infected monocytes are internalized through a clathrin- and caveolae-independent pathway.

Infection with feline infectious peritonitis virus (FIPV), a feline coronavirus, frequently leads to death in spite of a strong humoral immune response. In previous work, we reported that infected monocytes, the in vivo target cells of FIPV, express viral proteins in their plasma membranes. These proteins are quickly internalized upon binding of antibodies. As the cell surface is cleared from viral proteins, internalization might offer protection against antibody-dependent cell lysis. Here, the internalization and subsequent trafficking of the antigen-antibody complexes were characterized using biochemical, cell biological and genetic approaches. Internalization occurred through a clathrin- and caveolae-independent pathway that did not require dynamin, rafts, actin or rho-GTPases. These findings indicate that the viral antigen-antibody complexes were not internalized through any of the previously described pathways. Further characterization showed that this internalization process was independent from phosphatases and tyrosine kinases but did depend on serine/threonine kinases. After internalization, the viral antigen-antibody complexes passed through the early endosomes, where they resided only briefly, and accumulated in the late endosomes. Between 30 and 60 min after antibody addition, the complexes left the late endosomes but were not degraded in the lysosomes. This study reveals what is probably a new internalization pathway into primary monocytes, confirming once more the complexity of endocytic processes.

Tumor necrosis factor promotes T-cell at the expense of B-cell lymphoid development from cultured human CD34+ cord blood cells.

OBJECTIVE: Human CD34+ cord blood (CB) cells are hematopoietic progenitors useful for stem cell transplantation, even after ex vivo expansion. We investigated the effect of tumor necrosis factor (TNF) on lymphoid development from cultured CD34+ CB cells. MATERIALS AND METHODS: Human CD34+ CB cells were cultured in cytokine mixes with or without TNF. Preculture during 60 hours was followed by in vitro differentiation assays, including fetal thymus organ culture and coculture on murine stromal MS-5 cells. In a next step, experiments were extended to CD34+CD38- and CD34+CD38+ CB cells and prolonged preculture. RESULTS: Preculture in the presence of TNF improved differentiation into T cells and diminished the ability to generate B cells, while NK potential and myeloid development were unaffected. Sorted CD34+CD38- CB cells were more potent T-cell precursors after preculture in TNF, compared to CD34+CD38+ CB cells. In precultured CD34+CD38- CB cells, TNF increased GATA3 but decreased EBF1 expression, in line with the skewed lymphoid differentiation induced by TNF. However, when preculture in the presence of TNF was extended to 1 week, T-cell precursors were lost. CONCLUSION: After short-term culture of CD34+ CB cells in the presence of TNF, T-cell generation is stimulated at the expense of B-cell generation. T-cell progenitors are enriched in the CD34+CD38- fraction. These results have implications on the culture conditions to be used for CB CD34+ cells prior to transplantation.

Multiple gene knock-down by a single lentiviral vector expressing an array of short hairpin RNAs

RNA interference (RNAi), mediated by short double-stranded RNAs, is a powerful mechanism for posttranscriptional gene silencing. Sustained expression of short hairpin RNA (shRNA) can be accomplished in mammalian cells by viral delivery systems. Using lentiviral constructs, stable gene silencing is established both in dividing and non-dividing cells. Targeting one single gene can lead to the development of escape mutants or may be insufficient to silence redundant pathways. Therefore, simultaneous targeting of multiple genes may be necessary. We have generated a lentiviral vector-based system for expression of multiple shRNAs from a single viral vector, which also encodes an EGFP reporter protein. We show that knock-down of each single gene from multiple target vectors is achieved at an efficiency comparable to that obtained after transduction using single target viral vectors. In this way, we were able to knock-down several members of the human Rho-family GTPases in T cells. Double and triple knock-down persisted after multiple passages of the cells. The ability to inhibit two or more genes simultaneously from one single expression vector further widens the application spectrum of RNAi, both in functional studies and therapeutic strategies.