Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation

Cytomegalovirus (CMV) is a latent virus which causes chronic activation of the immune system. Here, we demonstrate that cytotoxic and pro-inflammatory CD4+CD28null T cells are only present in CMV seropositive donors and that CMV-specific Immunoglobulin (Ig) G titers correlate with the percentage of these cells. In vitro stimulation of peripheral blood mononuclear cells with CMVpp65 peptide resulted in the expansion of pre-existing CD4+CD28null T cells. In vivo, we observed de novo formation, as well as expansion of CD4+CD28null T cells in two different chronic inflammation models, namely the murine CMV (MCMV) model and the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis (MS). In EAE, the percentage of peripheral CD4+CD28null T cells correlated with disease severity. Pre-exposure to MCMV further aggravated EAE symptoms, which was paralleled by peripheral expansion of CD4+CD28null T cells, increased splenocyte MOG reactivity and higher levels of spinal cord demyelination. Cytotoxic CD4+ T cells were identified in demyelinated spinal cord regions, suggesting that peripherally expanded CD4+CD28null T cells migrate towards the central nervous system to inflict damage. Taken together, we demonstrate that CMV drives the expansion of CD4+CD28null T cells, thereby boosting the activation of disease-specific CD4+ T cells and aggravating autoimmune mediated inflammation and demyelination.

Moreover, due to the persistent nature of CMV, substantial accumulation of CMV-specific memory T cells (on average 10% of the total memory T cell compartment) can occur 18,[22][23][24] , albeit with varying degrees, which may be caused by differences in infectious dose 25 . As a consequence of this large percentage of CMV-specific T cells, immune surveillance could become less effective over time, thereby compromising normal immunity 18,26 . Indeed, CMV seropositivity has been correlated with a worse MS disease course, although disease limiting effects have also been stated (Reviewed in ref. 16). The most important finding indicating a disease promoting role is the enrichment of CMV-specific antibodies in MS 27 . When these antibodies were present in MS patients, this was correlated to a decreased time to relapse, an increase in the number of relapses and enhanced brain atrophy [28][29][30] . In contrast, another study concluded that the presence of CMV-specific antibodies was associated with a better clinical outcome, an increased age of disease onset and decreased brain atrophy 31 . A recent meta-analysis on 1341 MS patients and 2042 healthy controls did not yield a conclusive result on the relationship between CMV infection and the occurrence of MS 32 .
In this study we investigated whether CMV by itself is able to trigger the expansion of CD4 + CD28 null T cells and aggravate MS disease, using a combination of human data and in vivo animal model systems.

CMV expands CD4 + CD28 null T cells via repeated antigenic stimulation. To determine whether
CMV infection is linked to expansion of CD4 + CD28 null T cells (>2% of CD4 + T cells), an association study between CMV serology and the percentage of CD4 + CD28 null T cells was performed. In our cohort, the percentage of CD4 + CD28 null T cells is significantly higher in CMV seropositive (CMV+) donors compared to CMV seronegative (CMV−) donors (p < 0.0001, Fig. 1a and b), with no differences between MS and HC, which is in line with other studies 9 . Furthermore, CMV-specific IgG titers positively correlate with the percentage of CD4 + CD28 null T cells (ρ s = 0.6, p < 0.0001, Fig. 1c). To test whether this correlation is CMV specific, we examined the serology of EBV, another chronic and latent virus which has been implicated in MS 33 . No significant correlation was found between the percentage of CD4 + CD28 null T cells and EBNA IgG titers (Fig. 1d). Furthermore, EBV IgG levels did not differ between donors with versus without CD4 + CD28 null T cell expansion (respectively: 9 ± 4 vs 8 ± 4, p > 0.05). In contrast, donors with CD4 + CD28 null T cell expansion have significantly higher CMV IgG titers compared to donors without expansion (respectively: 219 ± 92.8 vs 5 ± 0, p < 0.0001).
Since CD4 + CD28 null T cell expansion only occurred in CMV infected individuals and correlated with the level of CMV-specific antibody titers, we investigated whether CMV infection can drive expansion of CD4 + CD28 null T cells, using in vitro and in vivo models. Since there is no significant difference in the percentage of CD4+CD28 null T cells between HC and MS patients, we did not discriminate between both populations in the following experiment. To mimic chronic TCR triggering by CMV, PBMCs from MS patients and HC, who were either CMV+ or CMV− and exhibited CD4 + CD28 null T cell expansion (exp+) or not (exp−), were repeatedly stimulated with a CMV peptide (CMVpp65) in vitro. The percentage of CD4 + CD28 null T cells significantly increased over time in CMV+ exp+ donors, as opposed to CMV+ exp− and CMV−exp− donors (Fig. 1e). IL-2 by itself did not induce expansion of CD4+CD28 null T cells (Fig. 1f). Repetitive CMV peptide stimulation in vitro did not induce the generation of CD4 + CD28 null T cells in exp− donors over the duration of the experiment (20 days). To investigate the long term effect of CMV infection on formation and expansion of CD4 + CD28 null T cells, we used the in vivo MCMV mouse model, the most widely used and relevant model for human CMV infection 25 . MCMV infected mice showed a significant increase of CD4 + CD28 null T cells in the spleen over time, with a 2-fold increase at day 8 (p < 0.05) and 20-fold increase at day 250 post-infection compared to non-infected mice (d0, p < 0.0001, Fig. 1g). In non-infected mice, the CD4 + CD28 null T cell levels were below the threshold for expansion (1 ± 0.2%), indicating that CMV infection induces loss of CD28 in CD4 + T cells in vivo. In summary, repeated in vitro stimulation with CMV peptide expands pre-existing CD4 + CD28 null T cells, whereas in vivo CMV infection induces CD28 loss in CD4 + T cells and drives expansion of CD4 + CD28 null T cells.
To determine whether CMV induces the loss of CD28 on CD4 + T cells via repeated antigenic triggering or via interaction with its ligands CD80 and CD86, we infected CD80/86 −/− mice with MCMV. MCMV infection induced the expansion of CD4 + CD28 null T cells to a similar extent in CD80/86 −/− mice and WT mice (Fig. 1h), indicating that the loss of CD28 is not caused by binding with their ligands CD80 and CD86. These findings further strengthen our notion that CD28 loss is caused by repeated antigenic triggering via the TCR.
CD4 + CD28 null T cells are increased in EAE mice and correlate with disease severity. CD4 + CD28 null T cells are cytotoxic, accumulate in MS lesions and at least a subpopulation is autoreactive in nature 14 . To test the hypothesis that CD4 + CD28 null T cells are associated with the severity of neuroinflammation, an EAE experiment was performed. Follow-up time (Fig. 2a) was extended compared to the standard protocol (30 day p.i.), to test whether CD4 + CD28 null T cells expand during acute and chronic stages of EAE (Fig. 2b). While limited numbers of CD4 + CD28 null T cells were found in CFA control mice, a significant increase above the 2% threshold for expansion was only found in the EAE mice (EAE: 3 ± 0.7%, p = 0.004 and control: 1.8 ± 0.3%, p > 0.05, Fig. 2b).
From previous studies, it is known that human CD4 + CD28 null T cells produce IFNγ and granzyme B, and that they show low expression of CD62L, CD127 and CD27 9,14,[34][35][36] . To determine whether mouse CD4 + CD28 null T cells have a similar phenotype, we analysed these cells, which were present in the peripheral blood of EAE mice. We found that they indeed phenotypically resemble their human counterparts as evidenced by a low expression of CD62L, CD127 and CD27, and production of IFNγ and granzyme B (Fig. 2c), identifying them as proinflammatory and cytotoxic effector memory T cells.
Furthermore, the percentage of CD4 + CD28 null T cells positively correlated with the EAE disease score (ρ s = 0.6, p = 0.0002, Fig. 2d). Long-term follow-up indicated that there was no further expansion of CD4 + CD28 null T cells in the chronic phase of EAE (after d30).
The increase in CD4 + CD28 null T cells in EAE mice could result from repeated auto-antigenic stimulation. To test this hypothesis, human MBP-specific T cell clones, generated and sustained in vitro by stimulation rounds with MBP or PHA, were analysed for the presence of CD4 + CD28 null T cells (Fig. 2e). The number of CD4 + CD28 null T cells increased after each successive round of stimulation. Thus, repeated MBP stimulation leads to the expansion of CD4 + CD28 null T cells in vitro, indicating that the expansion of CD4 + CD28 null T cells in MS patients may result from chronic auto-antigenic stimulation in vivo. Of note, in vitro stimulation with tetanus toxoid also induced expansion of CD4 + CD28 null T cells (Supplementary Figure S2), indicating that the expansion is not antigen specific, but rather due to the chronicity of the antigen exposure.

CMV infection exacerbates clinical symptoms of EAE.
Our results indicate that CD4 + CD28 null T cells expand after repeated immune activation, either as a result of CMV infection or after the induction of autoimmunity. Here, we investigated whether CMV infection and subsequent expansion of CD4 + CD28 null T cells correlate with a worse EAE outcome. The interplay between these different factors was investigated by infecting mice with MCMV and subsequently inducing EAE 8 days later. The EAE disease score of mice that were pre-exposed to MCMV was significantly higher compared to the EAE control group (mean cumulative score: 56 ± 4 vs 47 ± 3, p < 0.01; mean maximal score: 3.8 ± 0.26 vs 3.5 ± 0, p < 0.02; mean end score: 3.1 ± 0.35 vs 2.2 ± 0.27 p = 0.002). Furthermore, the MCMV group experienced a relapse between day 26 and day 30 after immunization, whereas EAE control mice did not (Fig. 3a). The percentage of CD4 + CD28 null T cells in the spleen increased at least eight-fold in each group (CMV: 8 ± 2%, p < 0.001, EAE: 12 ± 3%, p < 0.0001 and CMV+ EAE: 14 ± 2%, p < 0.0001) compared to baseline (1 ± 0.2%) (Fig. 3b). These results provide further evidence that both CMV infection and EAE induction lead to the expansion of CD4 + CD28 null T cells and that prior CMV infection aggravates EAE symptoms.
Since we showed that CMV exacerbates EAE disease, we asked whether this is due to increased autoimmune reactivity. To answer this question, CD4 + T cell reactivity to MOG peptide was measured in the spleen. The MCMV infected EAE group displayed enhanced MOG-specific CD4 + T cell reactivity compared to the control groups (EAE: p < 0.004, CMV: p < 0.002). Furthermore, this MOG response correlated to the percentage of CD4 + CD28 null T cells in the spleen of these mice (Fig. 3c). Also, we detected splenic CMV-specific CD4 + T cell reactivity in the MCMV infected groups, however they were not increased by EAE induction (data not shown). Viral load measured in the salivary glands at the end of the experiment indicate that the virus was still present in high amounts in both the MCMV and the MCMV infected EAE groups (data not shown). These data indicate that CMV infection increases the percentage of MOG-specific CD4 + T cells, thereby increasing autoimmune mediated neuroinflammation, and that CD4 + CD28 null T cells take part in this overall MOG response.

CMV infection increases demyelination in EAE.
In MS patients, CD4 + CD28 null T cells accumulate in brain lesions and are in close contact with neural cells 14 . Since CMV infection leads to a worse EAE disease course, we next questioned whether demyelination of the spinal cord, the predominant location of lesions in this model, is also increased in these animals. No demyelination was found in the spinal cord of MCMV infected mice ( Fig. 4a and b). MCMV infected EAE animals exhibited enhanced demyelination compared to the EAE control group (Fig. 4b), indicating that CMV infection accelerates autoimmune-mediated CNS damage. Furthermore, the extent of demyelination is strongly correlated with the percentage of spleen-derived CD4 + CD28 null T cells (R = 0.71, p < 0.05, Fig. 4c). We further identified the presence of CD4 + GranzymeB + T cells in the spinal cord

Discussion
Here, we demonstrate that CD4 + CD28 null T cells expand during EAE and positively correlate with disease severity. In addition, we show that CMV by itself is able to enhance activation of disease-specific CD4 + T cells, trigger the expansion of CD4 + CD28 null T cells and worsen EAE. Overall, our findings support a detrimental role for CMV in autoimmune neuroinflammation.
Our group, together with others have shown that CD4 + CD28 null T cells are associated with the pathogenesis of chronic inflammatory disorders 15,37,38 . In MS, a direct link with disease severity has not been demonstrated so far. However, indirect evidence, such as their target tissue infiltrating capacity and cytotoxic activity towards oligodendrocytes, certainly alludes to this hypothesis 14,35 . In this study, we made use of the widely documented mouse model for MS, EAE. Although this model is certainly not fully equivalent to the human situation, it does recapitulate the inflammatory response that arises in patients with MS, which is the focal point of our study 39 . Here, we demonstrate that peripheral CD4 + CD28 null T cells are increased in EAE animals and that the percentage of CD4 + CD28 null T cells is strongly correlated with the amount of demyelination and disease severity. Mouse-derived CD4 + CD28 null T cells displayed an effector memory (CD62L low CD127 low CD27 low IFNγ + ) and cytotoxic (granzyme B + ) phenotype, indicating that they are similar to their human counterparts 9, 14, 34-36 . Our findings are in line with evidence found in collagen-induced arthritis (CIA), the animal model for RA, where an increase in the number of CD4 + CD28 − NKG2D + T cells was observed after immunization 40 . The increase in peripheral CD4 + CD28 null T cells in EAE mice could be attributed to repeated auto-antigenic stimulation caused by chronic autoimmune inflammation. Indeed, as evidenced by our in vitro data, repeated MBP stimulation of MBP-specific T cell clones leads to CD4 + CD28 null T cell expansion. In vivo, we found a direct correlation between the percentage of CD4 + CD28 null T cells and the anti-MOG response level in the spleen of EAE mice. Together, these findings confirm the autoreactive nature of CD4 + CD28 null T cells 11 . After 30 days p.i., there was no further expansion of CD4 + CD28 null T cells in the blood of EAE mice. Instead, starting from day 60, the memory pool maintained a steady state. This is as expected with regards to the homeostasis of the memory pool: expansion is followed by contraction and ultimately maintenance of the remaining memory T cell pool 41 .
In contrast to EAE mice, not all MS patients have CD4 + CD28 null T cell expansion. Therefore, in humans additional components could be important in the generation of CD4 + CD28 null T cells. Potential triggers include: 1) chronic inflammation 42 ; and 2) viral infections 7 , of which CMV, as a persistent virus, is a promising candidate. Our data demonstrate that repetitive in vitro CMV peptide stimulation of human PBMCs expands pre-existing CD4 + CD28 null T cells. IL-2, which enhances T-cell proliferation and differentiation, does not lead to the expansion of CD4 + CD28 null T cells. EBV, another chronic and latent virus implicated in MS, is not associated with CD4 + CD28 null T cell expansion. These findings further support the hypothesis that CD4 + CD28 null T cells arise after CMV infection, which corresponds with previous reports by other groups 9,17 . Of note, we did not measure proliferation; therefore the increase in CD4 + CD28 null T cells after CMV stimulation could be due to survival rather than proliferation. However, van Leeuwen et al. indicated that CD4 + CD28 null T cells proliferate after addition of CMV antigens, suggesting the latter is true 17 . In vivo, CMV infection leads to continuous activation, enabling us to study chronic repeated antigenic challenge. Although human CMV and MCMV are different viruses, the MCMV mouse model is widely used and is the most relevant mouse model which mimics human CMV infection 25 . MCMV virus in the salivary gland is thought to be important for spreading the virus from mouse to mouse. Whereas in all organs the virus is latent in less than a few weeks, in the salivary glands the virus replicates for months 43 . Thus the amount of virus in the salivary gland is not influencing the titers in other organs, such as spleen and lymph nodes, but is instead set by the initial infection dose, and the local and pre-existing immunity conditions. Using this model, we clearly show formation and expansion of CD4 + CD28 null T cells in all MCMV infected animals over time. These findings are in line with those of other groups 7 . Since CMV is unable to infect T cells, CMV cannot directly reduce CD28 expression on T cells, but rather exerts its effects due to its persistent nature. In this study, we show that the loss of CD28 is caused by continued antigenic triggering and not by binding with their ligands CD80 and CD86, since the number of CD4 + CD28 null T cells did not differ between MCMV-infected CD80/86 −/− mice and WT. Furthermore, studies in mice and humans have indicated that the number and phenotypes of CMV-specific T cells correlate with viral load 25,44,45 ; higher viral loads drive higher expansions, establishing the antigen-driven aspect of the response. In this study, we used a relatively high dose of MCMV leading to a higher amount of antigen-specific T cells, including CD4 + CD28 null T cells. In the human population, the dose of CMV is not evenly distributed, leading to variability in the number of antigen specific T cells between individuals. This heterogeneity explains the difference in the percentage of CD4 + CD28 null T cells among CMV seropositive donors. In this respect, it is of interest to note that the CMV Ig titers correlate with a higher percentage of CD4 + CD28 null T cells. This implies that individuals with a higher CMV exposure may develop more CD4 + CD28 null T cells and associated disease. Also, the proportion of CMV-seropositive individuals increases with age 46 , as does the percentage of CD4 + CD28 null T cells 47 .
In MS patients, CMV seropositivity and high IgG titers are correlated with increased percentages of CD4 + CD28 null T cells. This link between CMV and CD4 + CD28 null T cell expansion was previously also reported for RA, ankylosing spondylitis and cardiovascular diseases 17,38,[48][49][50][51][52] , indicating that CMV infection and CD28 null T cell expansion form a common pathogenic background in these diseases 9 . The logical next step is to confirm the possible link between CMV, CD4 + CD28 null T cells and autoimmunity. Here, we demonstrate that CD4 + CD28 null T cells are increased in MCMV, EAE and MCMV+ EAE mice after 30 days p.i. MCMV infected EAE animals had a higher disability score and experienced a relapse, compared to the EAE control mice. Furthermore, MCMV infection increased demyelination in EAE mice, which correlated with higher CD4 + CD28 null T cell percentages in the periphery. Since we found CD4 + GranzymeB + T cells in the spinal cord of EAE and MCMV infected EAE mice, this suggests that CD4 + CD28 null T cells accumulate in the CNS to inflict damage in line with our previous observations in post-mortem MS brain material 14 . Thus, CMV infection exacerbates EAE disease course and does this by boosting the autoimmune response, as indicated by an increased MOG response. Indeed, T cell expansion preferentially occurred in MOG specific T cells, since the overall T cell responsiveness (no peptide control) in the spleen was comparable between all groups (data not shown). This is in accordance with others, where EAE induction combined with viral infection (γ-herpes virus, Semlike Forest virus or Sindbis virus) accelerated or exacerbated disease as a result of enhanced immune cell infiltration and polarization of the adaptive immune response [53][54][55] . Furthermore, MCMV infection rendered EAE-resistant BALB/c mice susceptible for EAE induction 56 . In another murine model of MS, namely Theiler's murine encephalitis virus (TMEV) model, opposite findings were demonstrated; CMV infection attenuated TMEV disease course 57 . However, the immune response in TMEV is largely CD8 mediated, whereas in EAE and MS CD4 + T cells are the main players 58 . We believe that the EAE model better represents what is going on in MS, namely a primary autoimmune mediated attack of the CNS, in contrast to the TMEV model, where primary viral induced neurotoxicity induces secondary autoimmunity.
An important question still remains to be answered: is the disease exacerbating effect and enhanced demyelination directly caused by CMV infection itself or attributable to the increased expansion of CD4 + CD28 null T cells? While technically challenging, an adoptive transfer study is needed to indisputably prove a direct cause-and-effect relationship of CD4 + CD28 null T cells and disease severity. CMV was previously reported to be present in the CNS, where it could damage local cells and tissues directly 33 . The ensuing cell death could then enhance autoimmunity as a result of the release and spreading of self-epitopes from degenerating tissue 59 . However, since demyelination was not present in animals only infected with CMV, it is unlikely that CMV by itself leads to CNS damage as proposed by the epitope spreading hypothesis. On the other hand, reactivation of CMV during ongoing MS could trigger the activation of autoreactive T cells (molecular mimicry) thereby enhancing subsequent demyelination. Of note, CMV-specific T cells were previously identified in MS lesions 60 . Evidence for cross reactivity between a CMV antigen (UL86 981-1003 ) and the myelin oligodendrocyte glycoprotein epitope (MOG  ) has been found in rats and non-human primates 61,62 . However, our data show that CMV infection alone did not mount a significant MOG response in the spleen, which would have been the case if molecular mimicry was involved. Another possible way by which CMV could directly contribute to autoimmunity is through bystander activation, where the immune response against CMV leads to robust inflammation, triggering the non-specific activation of autoreactive T cells 63 . We postulate that these bystander activated autoreactive T cells are mainly responsible for exacerbating EAE disease severity.
In summary, CMV infection and EAE induction lead to the expansion of CD4 + CD28 null T cells. Both CMV infection and CD4 + CD28 null T cells aggravate autoimmune mediated CNS inflammation, since EAE disease severity, measured by EAE score and the extent of neuroinflammation and demyelination, correlated with increasing amounts of CD4 + CD28 null T cells and the presence of a CMV infection. Overall, CMV infection drives the expansion of CD4 + CD28 null T cells, thereby amplifying the activation of disease-specific CD4 + T cells, and exacerbating EAE disease. Future studies will address whether this is also the case in MS patients. However, CMV vaccination to prevent the formation of CD4 + CD28 null T cells and the adverse effects of the infection itself, could be beneficial for people at risk of developing MS.

Methods
Study subjects. Human. Peripheral blood samples (Li-Heparin coated tubes) were collected from 63 healthy controls (HC) and 227 MS patients in collaboration with the University Biobank Limburg (UBiLim). CMV and Epstein-Barr virus (EBV) status and titers (CMV IgG and EBV EBNA IgG) were determined in serum samples via Vidas ELFA (bioMérieux, Marcy l'Etoile, France) and Architect immunoassay (Abbott, Illinois, USA). Clinical data are presented in Table 1; there were no significant differences between CMV positive or negative donors, neither in MS patients nor in healthy controls.
Mice. Female C57BL/6 mice were purchased from Harlan (Horst, the Netherlands). CD80/86 −/− mice 64 were bred in LUMC to the C57BL/6 background. EAE induction. 10 week old C57BL/6J mice were immunized subcutaneously with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG   Flow cytometry. Human. All donors included in this study were analysed for the percentage of CD4 + CD28 null T cells. This was done by isolating peripheral blood mononuclear cells (PBMCs) from whole blood by density gradient centrifugation (Cedarlane lympholyte, Sheffield, UK). Cells were double stained with anti-human CD4 FITC and CD28 PE (both BD Biosciences, Franklin Lakes, NJ). The gating strategy consists of a lymphocyte gate using the forward and side scatter signal, after which CD4 + cells were gated and subsequently CD28 expression was monitored within this gate (Supplementary Figure S1a). Cells were acquired using a FACSAria II cytometer, and data were analysed using BD FACSDiva software. Significant expansion of CD4 + CD28 null T cells was arbitrary defined as a percentage ≥2% of the total CD4 + T cell population, as this was the minimal percentage of cells that allowed discrimination of a distinctive population 14 .
Mice. Single cell suspensions were prepared from spleens by mincing the tissue through a 70-μm cell strainer (BD Bioscience). Erythrocytes were lysed in a hypotonic ammonium chloride buffer. The gating strategy consists of a lymphocyte gate using the forward and side scatter signal, after which CD3 + CD4 + cells were gated and subsequently CD28 expression was monitored within this gate (Supplementary Figure S1b). Surface and intracellular cytokine staining were used to identify and characterize CD4 + CD28 null T cells. MOG-specific CD4 + T cell responses were determined after in vitro stimulation with MOG 35  In vitro CMV stimulation assay. PBMCs from 12 HC and 8 MS patients were isolated from whole blood via density gradient centrifugation. These donors differed according to their CMV status and CD4 + CD28 null T cell expansions (Table 2). PBMCs were cultured in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% foetal bovine serum (FBS; Hyclone Europe, Erembodegem, Belgium), 1% nonessential amino acids, 1% sodium pyruvate, 50 U/ml penicillin and 50 mg/ml streptomycin (all Life technologies).

Generation of MBP reactive T cell clones.
MBP-specific T cell clones were generated as described previously 65 . Briefly, MBP-reactive T-cell lines were generated from the blood of MS patients via limiting dilution analysis (LDA), cloned with phytohemagglutinin (PHA) in the presence of allogeneic accessory cells and further expanded by successive rounds of restimulation with MBP or PHA and autologous antigen presenting cells (APCs). Statistical analysis. Statistical analyses were performed using GraphPad Prism version 6 and SAS 9.3. Parametric analyses include t-tests (2 groups), 1-way ANOVA and 2-way ANOVA (multiple groups). Nonparametric tests encompass Mann-Whitney tests (2 groups) and Kruskal-Wallis tests (multiple groups). Parametric data are shown as mean ± SD, nonparametric data as median ± interquartile range. A p-value < 0.05 was considered significant.
Ethics approval and consent to participate. Experiments involving human samples and data were approved by the Medical Ethics Committee UZ KU Leuven and experiments were performed in accordance with its guidelines and regulations. Informed consents were obtained from all donors.
All animal studies were in accordance with the EU directive 2010/63/EU for animal experiments and were approved by the Ethical Committee Animal Experiments UHasselt.