Systemic Lupus Erythematosus (SLE) is an autoimmune disease affecting over 3 million people globally. SLE significantly impacts patients’ life through chronic pain, fatigue, disability and dysfunction of vital organs imposing substantial physical, mental and socio-economic challenges on patients. SLE predominantly affects women, with over 90% of cases occurring in females, particularly during the child-bearing years. Disease etiology is complex resulting from interplay between environmental factors and genetic predispositions that trigger an aberrant autoimmune response (1). The clinical manifestations and severity of SLE are highly heterogeneous, complicating both treatment choices and the conduct of clinical trials. Therefore, there is a need to identify biomarkers that can aid in stratifying and managing SLE patients more effectively.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphoinositides at the 3-OH position. PI3Ks are grouped into three classes (I, II, and III) based on the subunit structure, substrate specificity, and final phosphoinositide products. PI3Kδ, a class I enzyme predominantly found in leukocytes, is activated by upstream receptor tyrosine kinases (RTKs), T-cell receptor (TCR), B-cell receptor (BCR), and cytokine receptors. PI3Kδ is the main signal transducer of PI3K signaling downstream of TCR in human T cells (2). PI3Kδ is a heterodimeric enzyme consisting of the catalytic subunit p110δ, which most commonly assembles with the regulatory subunit p85α. Upon activation, PI3Kδ converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), an important second messenger that activates signaling pathways through AKT, TEC family kinases, and other PH domain proteins. These pathways in turn largely impact lymphocyte development, proliferation, metabolism, migration, and survival (3).

Enhanced PI3Kδ activity may contribute to the pathogenesis of SLE, as inhibition of the enzyme limits disease progression and improves survival in lupus mouse models. Heterozygous inactivation of the p110δ gene exerts an inhibitory effect on immune compartments, reducing serum IgG anti-nuclear antibodies (ANA) levels, dampening T-cell activity and attenuating glomerulonephritis (4). Pharmacologic inhibition of p110δ also decreased the production of proinflammatory cytokines and several lymphocyte populations, such as Th17 cells, CD3+CD4−CD8−B220+ cells, CD4+ effector memory T cells, as well as activated GL-7+IgG- germinal center (GC) B cells, IgG+ class-switched B cells, and plasma cells (5, 6). It also reduces de novo and memory recall responses in lupus mouse models (7).

Overactivation of PI3Kδ can lead to autoimmune manifestations and lupus-like phenotypes in humans and mice. A well-studied example of this is Activated PI3K Delta Syndrome (APDS), which is an inborn error of immunity caused by activating mutations in the genes PIK3CD and PIK3R1 encoding for PI3Kδ subunits p110δ and p85α respectively (8) (9). APDS patients suffer from immunodeficiency and recurrent upper respiratory tract infections, but also 34% of patients showed signs of autoimmunity or inflammatory disease (10, 11). The autoimmune features can resemble that of SLE patients to the level that at least 4 patients fulfilling ACR classification criteria for SLE turned out to have activating mutations in the PIK3CD gene (11–13), which demonstrates that APDS can manifest with an SLE-like phenotype. Additionally, elevated levels of anti-nuclear antibodies (ANA) have been observed in the sera of mice with the activating mutation in the Pik3cd gene (14). PI3Kδ inhibition was shown to be effective in patients with APDS. Leniolisib, an oral small-molecule selective inhibitor of the PI3Kδ enzyme, has successfully improved immune dysregulation and immunodeficiency in APDS patients and was recently approved for treating APDS (15, 16). Given the evidence for the elevated PI3Kδ activity in SLE pathogenesis, PI3Kδ inhibitors may also be beneficial in treating patients with SLE. However, owing to the high heterogeneity of the disease, this effect may not be observed in all patients, highlighting the need to identify a subgroup of SLE patients with elevated PI3Kδ activity.

The activity of the PI3Kδ enzyme can be determined ex vivo in leukocytes by measuring PIP3 levels using high-performance liquid chromatography-mass spectrometry (HPLC-MS) or by measuring intracellular mediators of PIP3 signaling, such as phosphorylated proteins AKT (p-AKT) or S6 (p-S6), using western blotting or flow cytometry. The advantage of the HPLC-MS assay is that it provides a direct and quantitative measure of PIP3, the product of the PI3Kδ enzyme (17).

Here, we assessed the activity of the PI3Kδ enzyme by studying PIP3 levels using HPLC-MS in T cells isolated from a cohort of patients with SLE and healthy individuals. We also investigated the relationship between levels of PIP3 in T cells and the frequency of different T-lymphocyte subpopulations present in the blood of these subjects. We found no difference in the T-cell PIP3 levels between SLE patients and healthy controls. However, some of the SLE patients show particularly strong increase of PIP3 in response to T-cell stimulation comparable to that of the APDS patient.

This is the first study to investigate PI3Kδ enzyme activity in a large cohort of SLE patients by measuring PIP3 levels in T cells. Here we used the HPLC-MS assay that allows direct PIP3 quantification (17). The inclusion of a patient with APDS, a disease known for its elevated PI3Kδ activity, provided a benchmark for assessing the increased activity of the enzyme in our study population.

We found that PIP3 levels measured in unstimulated T cells are not a reliable biomarker of increased PI3Kδ activity, as they do not differentiate the APDS patient from healthy controls. However, measuring PIP3 levels in stimulated T cells (after 90 minutes of stimulation) serves as an informative biomarker, which clearly distinguished the APDS patient. Similarly, the fold change of PIP3 levels in stimulated over unstimulated cells is also a suitable biomarker that provided additional information into the cells’ capacity to respond to stimulation and differentiated the APDS patient from healthy subjects.

Our results showed no difference in the PIP3 levels between the groups of SLE patients and healthy controls, suggesting that increased PI3Kδ activity in T cells is not generally associated with SLE. However, several SLE patients upregulated PIP3 levels after T-cell stimulation as strongly as the APDS patient. Previously, elevated PI3Kδ activity was reported in over half of SLE patients studied (20). This was determined using an in vitro kinase assay by assessing PIP3 production by PI3Kδ immunoprecipitated from lysed PBMCs, whereas we measured endogenous PIP3 production in T cells. Apart from the assay used, patients with active disease may have contributed to the higher frequency of elevated PI3Kδ activity observed in the study by Suárez-Fueyo et al. compared to our cohort.

In the present study, we found a strong correlation between normalized PIP3 levels in stimulated T cells and the frequencies of T-cell subtypes. In particular, PIP3 levels positively correlated with the frequency of CD4+ T cells and negatively correlated with the frequency of CD8+ T cells. This result is unlikely to be explained by the causative effect of PI3Kδ activity on the T-cell composition of the blood, given that in APDS patients hyperactivated PI3Kδ is known to lead to low CD4+ T cell counts and reduced ratio of CD4+/CD8+ T cells (8) (10, 11). More likely, our results indicate that after anti-CD3/CD28 stimulation CD4+ T cells show higher PI3Kδ activity than CD8+ T cells. This is consistent with the observation that PI3Kδ inhibition has a stronger effect on the proliferation of CD4+ than CD8+ T cells (21). Previous studies have shown that PI3Kδ mediates activation and proliferation of CD4+ T cells (22–24), which may explain its high activity in this cell type after stimulation. While the cytotoxic function of CD8+ T cells is also known to be regulated by PI3Kδ signaling (25, 26), CD8+ T cells become less dependent on PI3Kδ when they are differentiated into memory cells (27). Hence, the negative correlation between PIP3 levels and the frequency of CD8+ T cells in our data is likely to reflect lower PI3Kδ activity in CD8+ in comparison to CD4+ T cells. We also observed a negative correlation between PIP3 levels and the frequency of EMRA CD4+ and CD8+ T cells, suggesting that the presence of such T cells in the blood is associated with reduced PI3Kδ activity. EMRA T cells exhibit characteristics of replicative senescence and reduced proliferative capacity (28), which may be mediated by weakened PI3Kδ activation after stimulation. Overall, such intrinsic differences in PI3Kδ activity suggest that various T-cell subtypes will respond differentially to PI3Kδ inhibitors, which should be taken into consideration in the treatment regimens.

In conclusion, we show the distribution of basal and stimulated PIP3 levels in T cells from healthy subjects and a large cohort of patients with SLE measured for the first time using HPLC-MS and their correlation with T cell subsets. Overall, we found no differences in PI3Kδ activity between the groups of SLE patients and healthy controls. However, we identified individual SLE patients that showed a particularly strong upregulation of PIP3 levels after T-cell stimulation, comparable to that observed in the APDS patient. Given that PI3Kδ inhibitors can reduce responses to T-cell stimulation, this subgroup of SLE patients deserves further studies, as it may benefit from such a treatment.