Characterization of BAT activity in rats using invasive and non-invasive techniques.

INTRODUCTION: Brown adipose tissue (BAT) is considered as a potential target for combating obesity in humans where active BAT metabolizes glucose and fatty acids as fuel resulting in heat production. Prospective studies in humans have been set up to further study the presence and metabolic activity of BAT mostly using Positron Emission Tomography (PET) imaging in cold-stimulated conditions with the radiolabeled glucose derivative [18F]FDG. However, radiotracers beyond [18F]FDG have been proposed to investigate BAT activity, targeting various aspects of BAT metabolism. It remains questionable which tracer is best suited to detect metabolic BAT activity and to what extent those results correlate with ex vivo metabolic BAT activity. METHODS: PET and Single Photon Emission Computed Tomography (SPECT) imaging, targeting different aspects of BAT activation such as glucose metabolism, fatty acid metabolism, noradrenergic stimulation, blood perfusion and amino acid transport system, was performed immediately after injection of the tracer in rats under different temperatures: room temperature, acute cold (4 °C for 4 h) or acclimated to cold (4 °C for 6 h per day during 28 days). Furthermore, Magnetic Resonance Spectroscopy (MRS)-derived BAT temperature was measured in control and cold-acclimated rats. RESULTS: At room temperature, only [18F]FDG visualized BAT. Glucose metabolism, fatty acid metabolism, noradrenergic stimulation and blood perfusion showed a clear tracer-dependent twofold increase in BAT uptake upon cold exposure. Only the tracer for the amino acid transport system did not show BAT specific uptake under any of the experimental conditions. MRS demonstrated that cold-acclimated animals had BAT with a stronger heat-production compared to control animals. CONCLUSION: BAT activity following cold exposure in rats was visualized by several tracers, while only [18F]FDG was also able to show BAT activity under non-stimulated conditions (room temperature). The variances in uptake of the different tracers should be taken into account when developing future clinical applications in humans.

The prevalence of antibodies against the HLA-DRB3 protein in kidney transplantation and the correlation with HLA expression.

Human leukocyte antigen (HLA)-DRB3 is a functional HLA class II gene, which has a limited allele diversity in the human population. Furthermore, the HLA-DRB3 gene is only present in a subset of individuals. Therefore, in organ transplantation, this HLA molecule is frequently mismatched between patient and graft donor and thus antibodies against this mismatched HLA molecule can develop. In this study, we aimed to evaluate the prevalence and reactivity of these antibodies and aimed to identify factors that underlie antibody formation against HLA-DRB3. We showed in our patient cohort that HLA-DRB3 antibodies are identified in about 7% of all patients that were screened with solid phase assays. In these assays, we observed multiple antibody reactivity patterns indicating that HLA-DRB3 harbours multiple epitopes. In those cases, where we succeeded at tracing back the induction of these antibodies to the molecular HLA typing of the immunogenic event, we noticed a different frequency of HLA-DRB1 allele groups in the donors as compared to a control group. To a certain extent this distribution (e.g. HLA-DRB1*11 individuals) could be linked to an altered expression level. However, it also appears that different HLA-DRB3 alleles (e.g. HLA-DRB3*01 group) vary in their immunogenicity without having an expression difference. In conclusion, our study provides information on the immunogenicity and reactivity patterns of antibodies against HLA-DRB3 in kidney transplantation, and it points towards the possibility of HLA expression as a factor underlying antibody formation.

Fractionated Radiotherapy with 3 x 8 Gy Induces Systemic Anti-Tumour Responses and Abscopal Tumour Inhibition without Modulating the Humoral Anti-Tumour Response.

Accumulating evidence indicates that fractionated radiotherapy (RT) can result in distant non-irradiated (abscopal) tumour regression. Although preclinical studies indicate the importance of T cells in this infrequent phenomenon, these studies do not preclude that other immune mechanisms exhibit an addition role in the abscopal effect. We therefore addressed the question whether in addition to T cell mediated responses also humoral anti-tumour responses are modulated after fractionated RT and whether systemic dendritic cell (DC) stimulation can enhance tumour-specific antibody production. We selected the 67NR mammary carcinoma model since this tumour showed spontaneous antibody production in all tumour-bearing mice. Fractionated RT to the primary tumour was associated with a survival benefit and a delayed growth of a non-irradiated (contralateral) secondary tumour. Notably, fractionated RT did not affect anti-tumour antibody titers and the composition of the immunoglobulin (Ig) isotypes. Likewise, we demonstrated that treatment of tumour-bearing Balb/C mice with DC stimulating growth factor Flt3-L did neither modulate the magnitude nor the composition of the humoral immune response. Finally, we evaluated the immune infiltrate and Ig isotype content of the tumour tissue using flow cytometry and found no differences between treatment groups that were indicative for local antibody production. In conclusion, we demonstrate that the 67NR mammary carcinoma in Balb/C mice is associated with a pre-existing antibody response. And, we show that in tumour-bearing Balb/C mice with abscopal tumour regression such pre-existing antibody responses are not altered upon fractionated RT and/or DC stimulation with Flt3-L. Our research indicates that evaluating the humoral immune response in the setting of abscopal tumour regression is not invariably associated with therapeutic effects.

Potency of Both Human Th1 and NK Helper Cell Activation is Determined by IL-12p70-Producing PAMP-Matured DCs.

Besides T helper (Th) cells, natural killer (NK) cells have also been described to participate in the shaping of dendritic cell (DC)-mediated adaptive immune responses. At present, it remains unclear to what extent the induction of these NK helper cell immune mechanisms is coupled with Th responses and whether both helper immune responses are induced by the same DC upon specific pathogen recognition receptor (PRR) stimulation. In this study, we demonstrate that maturation of DCs with a cocktail containing FMKp (membrane fragments of Klebsiella pneumoniae) mounts both Th cell and NK cell helper responses in a PRR-triggered dose-dependent manner as determined by the capacity of the helper cells to produce IFN-γ. Furthermore, by triggering an additional PRR pathway [FMKp in combination with poly(I:C) lyovec], we reveal that both approaches modulate the amount of DC-derived IL-12p70 and that this cytokine is the key determinant of the DC-induced Th1 and NK cell helper responses. Moreover, all PRR triggers able to induce IL-12-producing mature DCs are sufficient to induce these helper responses. We propose the existence of a single program used by DCs to induce potent cellular immune responses by stimulating both T helper and NK cell helper processes. This knowledge can help to select the proper PRR triggers in preventive and therapeutic vaccine design.

Monitoring the initiation and kinetics of human dendritic cell-induced polarization of autologous naive CD4+ T cells.

A crucial step in generating de novo immune responses is the polarization of naive cognate CD4+ T cells by pathogen-triggered dendritic cells (DC). In the human setting, standardized DC-dependent systems are lacking to study molecular events during the initiation of a naive CD4+ T cell response. We developed a TCR-restricted assay to compare different pathogen-triggered human DC for their capacities to instruct functional differentiation of autologous, naive CD4+ T cells. We demonstrated that this methodology can be applied to compare differently matured DC in terms of kinetics, direction, and magnitude of the naive CD4+ T cell response. Furthermore, we showed the applicability of this assay to study the T cell polarizing capacity of low-frequency blood-derived DC populations directly isolated ex vivo. This methodology for addressing APC-dependent instruction of naive CD4+ T cells in a human autologous setting will provide researchers with a valuable tool to gain more insight into molecular mechanisms occurring in the early phase of T cell polarization. In addition, it may also allow the study of pharmacological agents on DC-dependent T cell polarization in the human system.

Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells.

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

Klebsiella pneumoniae-triggered DC recruit human NK cells in a CCR5-dependent manner leading to increased CCL19-responsiveness and activation of NK cells.

Besides their role in destruction of altered self-cells, NK cells have been shown to potentiate T-cell responses by interacting with DC. To take advantage of NK-DC crosstalk in therapeutic DC-based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK-cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5-dependent manner. An additional mechanism of DC-induced NK-cell recruitment is characterized by the induction of CCR7 expression on CD56(dim) CD16(+) NK cells after physical contact with membrane fraction of K. pneumoniae-matured DC, resulting in an enhanced migratory responsiveness to the lymph node-associated chemokine CCL19. Bacterial fragment-matured DC do not only mediate NK-cell migration but also meet the prerequisites needed for augmentation of NK-cell cytotoxicity and IFN-γ production, the latter of which contributes to Th1 polarization.

In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+ cells mature in the thymus.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(c)(-/-) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alphabeta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT.