Robust Functional Responses Research Articles

Characterization and clinical enrichment of HLA-C*07:02-restricted Cytomegalovirus-specific CD8+ T cells.

Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV’s immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07:02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer+ T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFNγ+; median = 5.02%) in healthy individuals. However, MHC-multimer+ and IFNγ-secreting T cell frequencies showed a relatively weak correlation (r2 = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C*07:02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer+ T cells were still high (median = 6.86%) and correlated now strongly (r2 = 0.96) with IFNγ-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C*07:02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8+ T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HLA-C*07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HLA-C*07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT.

Safety, tolerability, and immunogenicity of a 4-antigen Staphylococcus aureus vaccine (SA4Ag): Results from a first-in-human randomised, placebo-controlled phase 1/2 study

BackgroundA prophylactic Staphylococcus aureus four-antigen vaccine (SA4Ag) is under development for prevention of invasive S. aureus disease. A preliminary S. aureus three-antigen vaccine (SA3Ag) was reformulated to include a novel manganese transporter protein (MntC or rP305A). This study describes the first-in-human dose-finding, safety, and immunogenicity results for SA4Ag. MethodsIn this double-blind, sponsor-unblind, placebo-controlled, phase 1/2 study, 454 healthy adults aged 18–64years were randomised to receive a single dose of one of three formulations of SA4Ag with escalating dose levels of rP305A or placebo. Functional immune responses were measured using opsonophagocytic activity (OPA) killing and fibrinogen-binding inhibition (FBI) assays; antigen-specific immunogenicity was assessed using a four-plex competitive Luminex® immunoassay (cLIA). ResultsA high proportion of SA4Ag recipients met the pre-defined antibody thresholds for each antigen at Day 29. A substantial and dose-level dependent immune response was observed for rP305A, with up to 18-fold rises in cLIA titres at Day 29. Robust functional responses were demonstrated, with >80-fold and >20-fold rises in OPA assay titres at Day 29 using S. aureus strains expressing capsular polysaccharide serotypes 5 and 8, respectively. Durable antibody responses were observed through month 12, gradually waning from peak levels achieved by days 11–15. SA4Ag was well tolerated, and no vaccine-related serious adverse events were reported. ConclusionsSingle-dose vaccination of SA4Ag in healthy adults aged 18–64years safely induced rapid and robust functional immune responses that were durable through month 12, supporting further development of this vaccine. Trial registration number: NCT01364571

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe(223) residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs.

Humanised mice have functional human neutrophils

The differences between murine and human neutrophils mean that findings in mice may not translate to humans, and therefore an in vivo model with human neutrophils would be an important methodological advance. We generated humanised mice by injecting human cord blood derived CD34+ stem cells into irradiated NOD-scid-γc−/− mice. At least 3months after engraftment, treatment of mice with GCSF mobilised circulating human neutrophils, which comprised 2.6% of human leukocytes, and led to L-selectin shedding and upregulation of CD66b, CD11b and CD63. Subsequent in vivo LPS treatment led to further downregulation of L-selectin with upregulation of CD66b and CD63, and also resulted in human neutrophil sequestration in the lungs. Furthermore, human neutrophils from these mice were capable of robust functional responses. They were shown to undergo a respiratory burst, and to degranulate with upregulation of CD63 and CD66b, in response to fMLP and Escherichia coli. These data show that functional human neutrophils develop from CD34+ cord blood stem cells in NOD-scid-γc−/− mice. They suggest that this approach may facilitate the in vivo study of human neutrophils in clinically relevant models of infection and autoimmunity.

The Ontogeny and Fate of NK Cells Marked by Permanent DNA Rearrangements

A subset of NK cells bears incomplete V(D)J rearrangements, but neither the consequence to cell activities nor the precise developmental stages in which recombination occurs is known. These are important issues, as recombination errors cause cancers of the B and T lineages. Using transgenic recombination reporter mice to examine NK cell dynamics in vivo, we show that recombination(+) NK cells have distinct developmental patterns in the BM, including reduced homeostatic proliferation and diminished Stat5 phosphorylation. In the periphery, both recombination(+) and recombination(-) NK cells mediate robust functional responses including IFN-gamma production, cytolysis, and tumor homing, suggesting that NK cells with distinct developmental histories can be found together in the periphery. We also show that V(D)J rearrangement marks both human cytolytic (CD56(dim)) and immunoregulatory (CD56(bright)) populations, demonstrating the distribution of permanent DNA rearrangements across major NK cell subsets in man. Finally, direct quantification of rag transcripts throughout NK cell differentiation in both mouse and man establishes the specific developmental stages that are susceptible to V(D)J rearrangement. Together, these data demonstrate that multipotent progenitors rather than lineage-specified NK progenitors are targets of V(D)J recombination and that NK cells bearing the relics of earlier V(D)J rearrangements have different developmental dynamics but robust biological capabilities in vivo.

Acid–base regulation of bone metabolism

It has been known for almost a century that systemic acidosis causes depletion of the skeleton—an effect assumed to result from physicochemical dissolution of bone mineral. However, our work has shown that resorption pit formation by cultured osteoclasts is dependent on extracellular acidification. Osteoclasts are almost inactive above pH 7.4 and show maximum acid- activation at about pH 6.9. Within this pH range, small shifts in H + concentration can cause large changes in resorption. Bone mineralisation by cultured osteoblasts is inhibited in a reciprocal manner by acidosis. In vivo, acidosis can occur systemically as a result of renal, bronchial or gastrointestinal disease, diabetes, severe (anaerobic) exercise, excessive protein intake, ageing, or the menopause. Acidosis can also occur locally as a result of inflammation, infection, wounds, tumours or ischaemia (due to increased anaerobic metabolism and reduced perfusion). The robust functional responses of bone cells to extracellular pH changes probably represent a primitive 'failsafe' to correct systemic acidosis by releasing alkaline bone mineral when the lungs and kidneys are unable to remove sufficient H + equivalent. The association of acidosis with hypoxia led us to investigate the effects of oxygen tension on bone cell function. We found that hypoxia causes impressive stimulation of osteoclast formation, independent of pH changes, whereas osteoblast growth and differentiation are blocked. Our results provide strong evidence for the critical role of the vasculature in the main- tenance of bone health. © 2006 Elsevier B.V. All rights reserved.

Analytical Design for Six Sigma for multiple response products

Here, we developed a step-by-step procedure of analytical Design for Six Sigma (DFSS) for conflicting multiple responses. Through a case study, the paper shows how analytical DFSS is able to provide engineers and managers with a scientific basis to make accountable decisions. The procedure basically starts with a development of analytical models of the system we deal with. Having developed the analytical models, the influence of design variables to the functional responses are then derived via graphical approach and Sobol's global sensitivity analysis. Multi-objective optimisation is finally applied to search for design variable settings that give Pareto-optimal and robust functional responses.

Reshaping the past: Strategies for modulating T-cell memory immune responses

Memory T cells are generated following an initial encounter with antigen, persist over the lifetime of an individual, and mediate rapid and robust functional responses upon antigenic recall. While immune memory is generally associated with protective immune response to pathogens, memory T cells can be generated to diverse types of antigens including autoantigens and alloantigens through homologous or crossreactive priming and comprise the majority of circulating T cells during adulthood. Memory T cells can therefore play critical roles in propagating and perpetuating autoimmune disease and in mediating allograft rejection, although the precise pathways for regulation of memory immune responses remain largely undefined. Moreover, evaluating and designing strategies to modulate memory T-cell responses are challenging given the remarkable heterogeneity of memory T cells, with different subsets predominating in lymphoid versus non-lymphoid tissue sites. In this review, we discuss what is presently known regarding the effect of current immunomodulation strategies on the memory T-cell compartment and potential strategies for controlling immunological recall.