Therapeutic drug monitoring is routinely performed to maintain optimal tacrolimus concentrations in kidney transplant recipients. Nonetheless, toxicity and rejection still occur within an acceptable concentration-range. To have a better understanding of the relationship between tacrolimus dose, tacrolimus concentration, and its effect on the target cell, we developed functional immune tests for the quantification of the tacrolimus effect. Twelve healthy volunteers received a single dose of tacrolimus, after which intracellular and whole blood tacrolimus concentrations were measured and were related to T cell functionality. A significant correlation was found between tacrolimus concentrations in T cells and whole blood concentrations (r = 0.71, p = 0.009), while no correlation was found between tacrolimus concentrations in peripheral blood mononuclear cells (PBMCs) and whole blood (r = 0.35, p = 0.27). Phytohemagglutinin (PHA) induced the production of IL-2 and IFNγ, as well as the inhibition of CD71 and CD154 expression on T cells at 1.5 h post-dose, when maximum tacrolimus levels were observed. Moreover, the in vitro tacrolimus effect of the mentioned markers corresponded with the ex vivo effect after dosing. In conclusion, our results showed that intracellular tacrolimus concentrations mimic whole blood concentrations, and that PHA-induced cytokine production (IL-2 and IFNγ) and activation marker expression (CD71 and CD154) are suitable readout measures to measure the immunosuppressive effect of tacrolimus on the T cell.

In mice vaccination with Streptococcus pneumoniae results in an increase in anti-oxLDL IgM antibodies due to mimicry of anti-phosphorylcholine (present in the cell wall of S. pneumoniae) and anti-oxLDL IgM. In this study we investigated the human translation of this molecular mimicry by vaccination against S. pneumoniae using the Prevenar-13 vaccine. Twenty-four healthy male volunteers were vaccinated with Prevenar-13, either three times, twice or once in a double-blind, placebo-controlled, randomized single center clinical study. Anti-pneumococcal wall, oxLDL and phosphorycholine antibody levels were measured at a fixed serum dilution, as well as circulating lipid levels over the course of 68 weeks. A significant increase in anti-oxLDL IgG and IgM was seen in the group receiving two doses six months apart compared to the placebo. However, these differences were not observed in the groups receiving a single dose, two doses one month apart, or three doses. This study shows that vaccination with Prevenar-13 does not result in robust anti-oxLDL IgM levels in humans. Further research would be required to test alternative pneumococcal-based vaccines, vaccination regimens or study populations, such as cardiovascular disease patients.

Based on its wide range of immunosuppressive properties, hydroxychloroquine (HCQ) is used for the treatment of several autoimmune diseases. Limited literature is available on the relationship between HCQ concentration and its immunosuppressive effect. To gain insight in this relationship, we performed in vitro experiments in human PBMCs and explored the effect of HCQ on T and B cell proliferation and Toll-like receptor (TLR)3/TLR7/TLR9/RIG-I-induced cytokine production. In a placebo-controlled clinical study, these same endpoints were evaluated in healthy volunteers that were treated with a cumulative dose of 2400 mg HCQ over 5 days. In vitro, HCQ inhibited TLR responses with IC₅₀s > 100 ng/mL and reaching 100% inhibition. In the clinical study, maximal HCQ plasma concentrations ranged from 75 to 200 ng/mL. No ex vivo HCQ effects were found on RIG-I-mediated cytokine release, but there was significant suppression of TLR7 responses and mild suppression of TLR3 and TLR9 responses. Moreover, HCQ treatment did not affect B cell and T cell proliferation. These investigations show that HCQ has clear immunosuppressive effects on human PBMCs, but the effective concentrations exceed the circulating HCQ concentrations under conventional clinical use. Of note, based on HCQ’s physicochemical properties, tissue drug concentrations may be higher, potentially resulting in significant local immunosuppression. This trial is registered in the International Clinical Trials Registry Platform (ICTRP) under study number NL8726.

Human whole blood cultures are widely used for the investigation of physiological pathways and drug effectsin vitro. Detailed information on the effect of “sample aging” (the time span between blood collection and experimental start) on the experimental outcome is not readily available in the public domain. We studied the effect of sample aging on the ability of immune cells to respond to cell-specific immune triggers (LPS, PMA/ionomycin, and SEB). Sample aging at room temperature profoundly inhibited the LPS-induced monocytic cytokine release in minimally diluted whole blood cultures. The reduction ranged from 20–50% after 30 minutes to 80–100% after 10 hours and differed between cytokines (IL-1β, IL-2, IL-6, IFNγ, and TNFα). Sample storage at 4°C or 37°C even worsened this. PMA/ionomycin- and SEB-induced cytokine release, both mainly T-cell-driven, were also reduced by sample aging but to a lesser extent (20–50% after 24 hours). Intracellular cytokine staining revealed that the number of LPS-responding cells was not impacted by sample aging and reduced LPS responsivity could also not be explained by apoptosis or downregulated TLR4 expression. Thus, we speculate that sample aging induces an inhibitory pathway downstream from TLR4 in monocytes. These results underline the importance of quick sample handling when investigating innate immune responses in whole blood, especially for monocyte responses.

Novel compounds targeting the adaptive immune system are commonly initially investigated in healthy volunteers (HV). HV frequently lack constitutively expressed drug‐target engagement biomarkers, complicating the evaluation of pharmacological activity. The keyhole limpet hemocyanin (KLH) neo‐antigen challenge elicits a controlled immune response in HV and is a potential model for investigating compounds targeting the adaptive immune system. A randomized, double‐blind, placebo‐controlled study in HV was conducted, investigating the effects of repeated immunizations with KLH on Days 1, 15, and 29, followed by intradermal KLH administration on Day 50. Systemically, KLH‐specific antibodies were evaluated. Leukocytes were stimulated ex vivo to determine KLH‐specific responses using ELISpot. Locally, KLH skin response was evaluated 0–48 hours post intradermal administration, using imaging, suction blister fluid analysis, and biopsy analysis. Twelve male HV aged 18–42 years completed the study. KLH antibody levels increased incrementally. ELISpot analyses demonstrated KLH‐specific IFN‐γ and IL‐13 responses. Intradermal KLH injection drove a peak response 24 h after administration, observed as erythema and increased perfusion. Blister fluid analysis showed cell influx (T cells; B cells; monocytes; dendritic cells) at 24 h, largely decreasing at 48 h, as confirmed by immunofluorescent staining of skin biopsies. These findings demonstrate a T‐cell‐mediated response to intradermal KLH that is more rapid than the classical delayed‐type hypersensitivity response. This study highlights the importance of the timing of measurements and combining non‐invasive and invasive biomarkers. The KLH neo‐antigen challenge model presented in this study offers a framework to study the effect of novel immunomodulatory compounds on the adaptive immune system early in clinical development.