The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories’ own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNγ-secretion.

Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans, three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However, the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements, making it difficult to draw conclusions as to their functional profile on the cellular level. In this study, we have investigated lipoteichoic acid (LTA)- and lipopolysaccharide (LPS)-induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine-secreting cells on the single-cell level and uses fluorescent detection, allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach, human monocytes from healthy volunteers could be divided into several subgroups as IL-1β, IL-6, TNF-α and MIP-1β were secreted by larger populations of responding cells (25.9–39.2%) compared with the smaller populations of GM-CSF (9.1%), IL-10 (1.3%) and IL-12p40 (1.2%). Furthermore, when studying co-secretion in FluoroSpot, an intricate relationship between the monocytes secreting IL-1β and/or IL-6 and those secreting TNF-α, MIP-1β, GM-CSF, IL-10 and IL-12p40 was revealed. In this way, dissecting the secretion pattern of the monocytes in response to TLR-2 or TLR-4 stimulation, several subpopulations with distinct cytokine-secreting profiles could be identified.

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In vivo electroporation (EP) has proven to significantly increase plasmid transfection efficiency and to augment immune responses after immunization with plasmids. In this study, we attempted to establish an immunization protocol using intradermal (i. Vaccination with genes was first described in the early 1990s and is becoming an alternative to traditional vaccine strategies. DNA vaccines possess several advantages, such as the capacity to induce a balanced immune response including humoral as well as cellular immune responses similar to those induced during natural infection with intracellular pathogens. The potential of DNA vaccines has been shown in numerous preclinical studies and by the licensure of veterinary DNA vaccines against infectious diseases and cancer (3,9,20). However, immunogenicity has been limited in humans, and ways to enhance the potency of these vaccines are being investigated. Besides gene optimization and the use of adjuvants (17), the most promising approach for plasmid vaccines administered as a single modality is by the use of in vivo electroporation (EP). EP has been shown to considerably increase the transfection efficacy of plasmid vaccines, ultimately leading to enhanced and long-lasting expression (10, 24) and improved immunogenicity (13, 21, 27, 28) of the encoded antigen. Furthermore, the electric pulses cause mild inflammation, with resulting recruitment of antigen-presenting cells (APCs) to the site of injection (19,24), which further enhances the immunogenicity.dAlthough intramuscular (i.m.) delivery of DNA vaccines, with or without the addition of EP, has been studied most extensively, DNA vaccine delivery to skin is becoming increasingly popular. Unlike muscle tissue, the dermal tissue has a large population of resident APCs, including Langerhans cells and dermal dendritic cells, that can facilitate the induction of vaccine-specific immune responses (2, 16). There is also a more rapid turnover of cells in the skin than in muscle, which together with the large number of APCs can lead to a rather fast removal of plasmids from the site of injection (24). This feature is positive for vaccination, as transient expression of the encoded antigen is sufficient to induce strong immune responses. The rapid removal of vaccine plasmids might also explain why more DNA is usually required to induce the same level of expression as that induced by i.m. delivery (10, 15). The skin is also an assessable tissue, making both monitoring and evaluation of immune responses easy to perform. More importantly, the addition of EP after intradermal (i.d.) delivery appears safe, as it does not affect the persistence or integration of vaccine plasmids (5, 24).Laddy et al. conducted a head-to-head comparison of EP-augmented i.m. and i.d. delivery of equal amounts of influenza virus-encoding vaccine plasmids in rhesus macaques. Immune reactivity assessed after three immunizations revealed that i.m. EP induced the highest levels of cellular immune responses, whereas i.d. EP was superior for induction of c...

In sepsis, large quantities of inflammatory cytokines are released into the bloodstream. The cellular source of these cytokines is unclear, and we have here investigated to what extent circulating cells in blood contributed to this production. We used the enzyme-linked immunospot technique to study the spontaneous as well as the lipopolysaccharide (LPS)-induced secretion of the proinflammatory cytokines interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor, IL-1β, IL-12p40, and the anti-inflammatory cytokine IL-10 from whole-blood cells. The study comprised 32 septic patients (24 with septic shock) and 30 healthy controls. Despite significantly increased plasma cytokine levels in the septic patients, the number of spontaneous cytokine-secreting cells was small or nonexistent and did not differ between the two groups. Lipopolysaccharide stimulation of cells from the same samples triggered substantially increased numbers of cytokine-producing cells in both patients and controls. However, although the numbers of IL-6- and tumor necrosis factor α-secreting monocytes were very similar in both groups, significantly fewer IL-1β-, IL-10-, IL-12p40-, and granulocyte-macrophage colony-stimulating factor-secreting monocytes were seen in samples from septic patients as compared with healthy controls. The reduced number of cytokine-secreting cells in response to LPS stimulation correlated with disease severity, as expressed by Sequential Organ Failure Assessment score and the stage of sepsis. In summary, circulating leukocytes did not appear to be responsible for the increased plasma levels of cytokines observed in sepsis. A selective sepsis-induced downregulation of cytokine secretion in response to LPS underscores the complexity of cytokine regulation in sepsis.