Peripheral T Cell Populations are Differentially Affected in Familial Mediterranean Fever, Chronic Granulomatous Disease, and Gout

Both innate errors of immunity, such as familial Mediterranean fever (FMF) and chronic granulomatous disease (CGD), and the common inflammatory disease gout are characterized by episodes of sterile inflammatory attacks in the absence of an infection. While these disorders encompass distinct pathologies due to differentially affected metabolic pathways and inflammasome activation mechanisms, their common features are the excessive production of interleukin (IL)-1ß and innate immune cell hyperreactivity. On the other hand, the role of T cells and innate-like lymphocytes such as gamma delta (γδ) T cells in these pathologies is ill-defined. In order to widen our understanding of T cell involvement in CGD, FMF and gout pathology, we developed multicolour immunophenotyping panels for flow cytometry to characterize γδ T cells as well as CD4 and CD8 T cell populations in terms of their cytokine production, activation status, memory or naive phenotypes, exhaustion status, homing receptor expression, and cytotoxic activity. Our study is the first deep immunophenotyping analysis of T cell populations in CGD, FMF, and gout patients. We found that CGD affects the frequencies and activation status of T cells, while gout impairs the cytokine production capacity of Vδ2 T cells. FMF was characterized by decreased percentages of regulatory T cells in circulation and attenuated IFN-γ production capacity by Vδ2 T cells. Autoinflammatory syndromes and congenital defects of phagocyte differentially affect T cell compartments. Future studies are warranted to assess whether these phenotypical changes are relevant for disease pathology. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-023-01576-7.


Introduction
Inborn errors of immunity (IEI) are caused by genetic variants which alter the function of individual genes and compromise innate and/or adaptive immunity [1].To date, this large group of disorders encompasses 485 diseases [2].Systemic autoinflammatory diseases (SAID), a subgroup of IEI, are described as distinct heritable disorders, mostly affecting the skin, joints, gut, and eyes, and are characterized by episodes of sterile fever and inflammation, mediated predominantly by cells and molecules of the innate immune system [3,4].Several SAIDs, including familial Mediterranean fever (FMF), are mediated by excessive interleukin (IL)-1β production.IL-1β maturation and secretion are under control of inflammasomes, multiprotein complexes containing the cysteine protease caspase-1 that are activated both by infections as well as endogenous stimuli (e.g., metabolic stimuli, and stress).
Alterations in inflammasome activation [5] and in various metabolic pathways [6] can lead to dysregulation of IL-1β secretion and cause pathology.Gout, which is not an IEI, is another autoinflammatory disease with dysregulated IL-1β biology.Congenital defects of phagocyte are another group of IEI in which phagocytes are unable to function properly to kill the invading pathogens.CGD is one of the diseases in this subgroup with a particular defect of respiratory burst.While many of the SAIDs have been originally characterized by the absence of auto-antibodies or auto-reactive T cells, CGD-causing mutations result in the presence of lupus-like auto-antibodies and auto-reactive T cells [7][8][9].Therefore, the lymphoid compartment might still be affected and contribute to the pathology of the diseases [10,11].Furthermore, dysregulated IL-1β production likely affects the T cell compartment in these diseases, as the effect of this cytokine on lymphocytes has been reported [12][13][14][15][16]. Indeed, many autoinflammatory conditions, including FMF and gout, display upregulation of T helper type 17 (Th17)-related cytokines [17][18][19].Despite of this, a systematic analysis of the T cell compartment, including innate-like gamma delta (γδ) T cells, has not been performed in theis group of diseases.
Our study focuses on two diseases with known dysregulation of IL-1 cytokine production: FMF, the most prevalent monogenic autoinflammatory disease worldwide, and gout, which is the most common form of inflammatory arthritis, as well as chronic granulomatous disease (CGD) which is a rare inherited primary immunodeficiency disorder with hyperinflammatory characteristics [20][21][22].FMF is recessively inherited and caused by gain-of-function mutations in the MEFV (Mediterranean FeVer) gene encoding the pyrin protein [23].Pyrin is mainly expressed in innate immune cells [24] where the activated form of the protein promotes oligomerization of apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) and inflammasome formation resulting in IL-1β production [25].Although the expression of MEFV gene has not been detected in T cells according to The Human Protein Atlas [26], it has been found in other source databases such as BloodSpot (https:// serve rs.binf.ku.dk/ blood spot/).Furthermore, numerous early studies have postulated the activation of the T cell compartment during inflammatory attacks in FMF patients [10,27].Another inborn error of immunity, CGD, is caused by mutations in genes encoding components of the reduced nicotinamide dinucleotide phosphate (NADPH) oxidase complex: gp91 phox , p22 phox , p67 phox , p40 phox , or p47 phox , which generates reactive oxygen species (ROS) [28,29].As a result, phagocytes such as neutrophils, monocytes, and macrophages cannot properly clear phagocytized microorganisms, leaving the body vulnerable to frequent infections and chronic inflammation [28].This leads to life-threatening bacterial and fungal infections.Interestingly, ROS defects can also lead to a defective regulation of IL-1β production and granuloma formation [30].In contrast, gout is not associated with monogenic mutations but is caused by increased concentrations of urate in the serum, which leads to the formation of monosodium urate (MSU) crystals in the joints [31].Genetic factors, however, might play a role in the gout pathogenesis.Genome-wide association studies link high serum urate levels and gout to defective gene variants involved in the renal urate-transport system including: ABCG2, SLC2A9, SLC17A1, SLC22A12, CGKR, PDZK1, and others [32].The pathology of gout is known to be driven mainly by innate immune cell responses, in which inflammasomes play a crucial role [33].As such, innate immune cells produce an excess amount of IL-1β upon engulfing MSU crystals [34].It has been shown that uric acid and MSU crystals have stimulatory effects also on T cells and can enhance T cell responses to secondary stimuli [35,36].Moreover infiltrated T cells were found in the tissues of gout patients [37].The involvement of T cells in the pathogenesis of gout is however poorly known.
Therefore, the aim of our study is to assess the lymphoid compartment in patients with CGD, FMF and gout, with a focus on innate-like unconventional gammadelta (γδ) T cells as well as CD4 and CD8 conventional alphabeta (αβ) T cells.

Patient Recruitment
All gout, CGD, and FMF patients gave informed consent to use leftover blood for research purposes.Blood draw from healthy volunteers were approved by the Ethical Committee of the Radboud University Medical Center (no.NL32357.091.0 and no.NL42561.091.12).

PBMCs Staining for Flow Cytometry
PBMCs were isolated by density gradient centrifugation on Pancoll (Pan Biotech).PBMCs were washed with PBS and incubated with Fc block solution (BioLegend) for 10 min.The antibody mixes (Table S1) in staining buffer (BD Bioscience) were added, and cells were incubated for 30 min at 4 °C in the dark.Samples were washed and stored at 4 °C in the dark until the reading.

Intracellular Cytokine Staining for Flow Cytometry
PBMCs were incubated with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL, Sigma-Aldrich) and ionomycin (1 μg/ mL, Sigma-Aldrich) in the presence of Golgi Plug and Golgi Stop (BD Biosciences) in RPMI 1640 complete medium (10% fetal bovine serum, 1 mM sodium pyruvate (Gibco), 2 mM glutamax (Gibco), 100 U/ml Penicillin, and 100 ng/ml Streptomycin (Pan Biotech)) for 4 h at 37 °C.Then, the cells were washed with cold PBS, and Fc blocking followed by surface marker staining were performed as described above.Cells were washed with PBS and incubated in cytofix/cytoperm permeabilization solution (BD Bioscience) for 30 min at 4 °C in the dark.The cells were washed with the washing solution (BD Bioscience), and antibody mix in the washing solution was added.After 30 min of incubation at 4 °C in the dark, the cells were washed and stored at 4 °C in cell fixation solution (BD Bioscience) until analysis.

Data Analysis
Flow cytometry data was analyzed using FlowJo (version 10.0) software.The graphs were generated using the GraphPad Prism (version 8.4.3) software.Non-parametric Mann-Whitney test was applied to calculate statistical significance.Two-tailed p values were considered statistically significant if below 0.05.Significant p values were shown with asterisks as follows: * < 0.05, ** < 0.01, *** < 0.001.

Results
To characterize the T cell populations in patients with CGD, FMF, and gout in steady state, we applied several flow cytometry multi-color immunophenotyping panels on peripheral blood mononuclear cells (PBMCs) isolated from patients in between febrile episodes and without known ongoing infections and from healthy controls (Supplementary Table 1).We hypothesize that T cell populations can undergo phenotypical and functional changes caused by recurrent inflammation, deficiencies in metabolic pathways, or hyperuricemia in gout.T cell subpopulations were investigated in-depth for their (1) activation status and naïve/memory phenotype, (2) susceptibility to apoptosis, (3) exhaustion status, (4) cytokine production ability, (5) homing potential, (6) adhesion, and (7) cytotoxic markers.

CGD Affects the Distribution of Peripheral Immune Cells
First, we determined whether CGD, FMF, and gout affect immune cell distribution in peripheral blood.Our gating strategy enabled discrimination of γδ, CD4, and CD8 T cell subpopulations as well as B cells, CD56 + natural killer (NK) cells, regulatory T cells (Tregs), and NKT-like cells (Fig. 1 and Figure S1).Two main subsets of γδ T cells have been described in humans: Vδ1 and Vδ2, which mainly reside within epithelial tissues or are found in peripheral blood, respectively [38].Because Vδ2 T cells are the most prevalent γδ T cell population in human peripheral blood, consisting up to 90% of the total γδ T cells [39], they were the main focus among γδ T cells in this study.We found significantly increased percentages of regulatory T cells and decreased percentages of CD56 + NK cells in CGD patients compared to healthy controls (Fig. 1f, h).We have also observed lower percentages of Vδ2 and CD4 T cells in CGD patients compared to healthy controls (Fig. 1b, c).However, statistical analysis did not reach significance possibly due to the small size of the cohort.The distribution of cell populations within PBMCs remained largely similar in FMF and gout patients compared to healthy controls, with the exception of almost significantly reduced percentages of regulatory T cells in FMF patients (Fig. 1f).

Naive and Memory T Cell Compartments are Not Affected by CGD, FMF, and Gout
To further scrutinize T cell populations in CGD, FMF, and gout, we analyzed the distribution of naïve (T Naive ), effector memory (T EM ), central memory (T CM ), and terminally differentiated T cells (T EMRA ) based on CD45RA and CD27 expression (Figure S2) [40].These distinct T cell subsets differ in their effector function such as, T Naive cells (CD45RA + CD27 + ) do not mediate effector immune responses effectively, T CM (CD45RA − CD27 + ) cells have a higher sensitivity to antigenic stimulation and proliferative potential, and T EM (CD45RA − CD27 − ) cells exhibit rapid effector function and lack of proliferative capacity, while T EMRA (CD45RA + CD27 − ) cells represent the most differentiated type of memory cells and express high levels of cytotoxic molecules [40].We observed a large variability in distribution of different T cell subpopulations between individuals, and no significant changes could be detected between patients and healthy controls (Figure S2b, c, d).Consistently, we did not observe significant changes in the expression of other naïve and memory markers: CD127 and CD45RO, respectively (data not shown).Thus, the distribution of different effector subpopulations among T cells is not affected in gout, CGD, and FMF.

Vδ2 and CD8 T Cells Exhibit Increased Activation Status in CGD
CD38 and CD69 are induced on T cells upon activation and are therefore commonly used as markers of activated or differentiated cells [41,42].We observed elevated proportions of CD38-and CD69-expressing Vδ2 T cells and CD69-expressing CD8 T cells in CGD patients (Fig. 2 and  S3).Overall, this data suggests that the activation status of these T cells is increased in CGD.We further assessed the expression profile of the death receptor CD95 (Figure S4a), which is known to induce apoptosis [43].The number of CD95-expressing Vδ2 T cells was elevated in CGD (Fig. 3a).The number of CD95-expressing CD4 and CD8 T cells was also increased in gout patients but did not reach statistical significance, possibly due to the small number of donors (Fig. 3c).Increased activation of T cells can lead to the exhaustion phenotype.Our results show that the Fig. 1 The distribution of circulating lymphocyte populations is significantly affected in CGD patients.a-h Percentages of different cell populations: CD3 + TCRVδ1 + cells (a), CD3 + TCRVδ2 + cells (b), CD3 + CD4 + cells (c), CD3 + CD8 + cells (d), CD3 + CD56 + NKTlike cells (e), CD3 + CD4 + CD25 hi CD127 − regulatory T cells (f), CD3 − CD19 + B cells (g), and CD3 − CD56 + NK cells (h) in freshly isolated PMBCs from healthy controls and patients determined by flow cytometry.CGD chronic granulomatous disease (n = 5), FMF familial Mediterranean fever (n = 7), gout (n = 6).Gray circle: FMF patient with Behcet disease.Non-parametric Mann-Whitney test was applied.*p ˂ 0.05, **p ˂ 0.01 expression of the exhaustion marker PD-1 is significantly decreased in FMF patients in comparison to healthy controls (Fig. 3f), while numbers of CTLA-4-expressing CD8 T cells have tendency to increase in FMF and gout patients (Fig. 3g-i and S3c).It is, however, important to mention that the numbers of CTLA-4-expressing cells were very low, questioning a functional relevance of this observation.This data indicates that FMF condition may affect the exhaustion status of CD8 T cells.

Cytokine Production by Vδ2 T Cells, but Not Conventional αβ T Cells, is Impaired in Gout and FMF Patients
The effector function of T cells is largely driven by their cytokine production potential.Previous clinical studies demonstrated that cytokine production patterns are disrupted in patients with inborn errors of immunity, and these changes might help to distinguish different syndromes and their severity [44].We assessed whether the capacity to produce cytokines by T cells is also affected in patients with CGD, FMF, and gout (Fig. 4).Our analysis revealed a significant reduction of IFN-γ-and TNF-α-producing Vδ2 T cells in gout (Fig. 4a, d, and S5).Furthermore, the percentages of IFN-γ-producing Vδ2 T cells were also significantly reduced in FMF patients (Fig. 4a).There were no significant changes in cytokine production among CD4 and CD8 T cells in all patients.We also analyzed the production of other cytokines, including the following: IL-4, IL-9, and IL-17α; however, we could not detect significant production of these cytokines (Figure S5c and data not shown).Thus, Vδ2 T cells are more susceptible to metabolic alterations in cytokine production than conventional T cells in examined conditions.

CGD, FMF, and Gout Do Not Significantly Alter the Homing Receptor Expression on T Cells
Immune cell migration to inflamed tissues is an important component of inflammatory processes.We therefore examined the expression profile of homing receptors to better understand the migratory status of Vδ2, CD4, and CD8 T cells in CGD, FMF, and gout (Fig. 5, S6, and S7).The expression of the following chemokine receptors was assessed by flow cytometry: CCR2 (Fig. 5a-c and S7a), which induces cell recruitment to sites of inflammation [45]; CCR5 (Fig. 5d-f and S7b), which regulates cell trafficking to the site of inflammation but also retention in tissues [46]; CCR7 (Fig. 5g-i and S7c), which mediates T cell migration from the blood to secondary lymphoid tissues [47]; CCR8 (Fig. 5j-i and S7d) and CCR4 (Figure S6a-c and S7e), which are skin homing receptors [48,49] as well as CXCR3 (Figure S6d-f and S7f), which functions as a homing receptor to sites of infection and inflammation [50].We observed increased numbers of CCR5-expressing CD4 T cells and reduced numbers of CCR7-expressing CD4 and CD8 T cells in gout patients (Fig. 5e, h, i).However, these numbers did not reach statistical significance, likely because of the sample size of the groups.Our results also show a trend for the increased number of CCR7 + Vδ2 T and CD8 T cells in CGD patients as well as the increased number of CCR8 + Vδ2 T, CD4 and CD8 T cells in FMF patients (Fig. 5g, i, j, k, l).Other homing receptors, CCR4 and CXCR3, did not show alterations in expression among T cell subpopulations in CGD, FMF, and gout patients compared to healthy controls (Figure S6).Altogether, no significant changes in the homing receptor expression pattern were detected in comparison to healthy controls, suggesting an unaltered migratory potential of T cells.

The Adhesion Potential of T Cells is Not Affected by CGD, FMF, and Gout
Adhesion potential of T cells is critical for cell migration, activation, and cytotoxic function.LFA-1 and CD54 interaction determines the strength and duration of cell-to-cell contact and therefore cell function [51].This LFA-1/CD54 interaction on T cells influences their cytokine production profile, efficiency of activation, migration through tissues, and cytotoxic properties.Our assessment of LFA-1 and CD54 expression on T cells did not reveal any significant changes in all examined disorders (Fig. 6a-c, S8a-c, and  S9a, d).Among T cells, CD8 and Vδ2 T cell can directly kill target cells [52,53].We examined whether this cytotoxic property is also affected in T cells by analyzing expression of CD56, NKG2D, and CD16 [54] (Fig. 6d-g, S8d, e, and S9b, c, e).The expression of the three molecules correlates well with each other as well as with a high content of perforin and granzyme B and cytotoxic CD8 T cell function [55,56].They have therefore been suggested to mark cells with cytotoxic properties.We did not observe significant differences in cell frequencies expressing the three markers between patient groups and controls (Fig. 6d-g and S8d, e).

Discussion
In this study, we characterized three populations of blood lymphocytes: innate-like Vδ2, CD4, and CD8 T cells in CGD, FMF, and gout patients.Our comprehensive immunophenotyping revealed differential alterations in the lymphoid compartment in terms of (i) distribution of circulating immune cell types, and (ii) expression of cell surface markers and cytokines in the examined disorders.
We found that the distribution of lymphoid cells was the most affected in CGD patients, in whom CD56 + NK cell percentages were significantly lower and regulatory T cell percentages were significantly higher than in healthy controls (Fig. 1).Furthermore, in our study, the percentages of Vδ2 and CD4 T cell populations tend to be lower in CGD patients compared to healthy controls (Fig. 1).Previous studies reported contradictory observations, where increased, reduced, or not affected T cell numbers were shown [57][58][59][60].The discrepancy between findings might be due to the differential treatment of patients at the time of analysis, the different distribution of patients' age, or causative mutations in p91 phox or p47 phox coding genes within cohorts.Indeed, age-related differences in the distribution of T cells in CGD patients were reported, where individuals older than 3 years displayed reduced numbers of CD4 and CD8 T cells [57].This is consistent with our observation that focuses on adult subjects.The cause of abrogated T cell numbers might result from the reduced proliferative capacity [59]; but whether this is intrinsic, due to deficiency of the NADPH oxidase complex, or extrinsic, as a result of disturbed immune homeostasis, remains to be determined.
T cells have been shown to express the functional phagocyte-type NADPH oxidase that is activated upon TCR stimulation [61].Deficiency in its activity in mice results in enhanced activation of MEK-Erk pathway and augmented Th1 and Th2 responses [62] as well as reduced differentiation and suppressive activity of regulatory T cells [62,63].Although we did not observe significant alterations in IFN-γ production by T cells in CGD, our data revealed a trend towards increased numbers of TNF-α-and IFN-γ-producing CD4 T cells (Fig. 4), in line with mouse data [61] and with human CGD biopsies from inflamed tissues [64].Contradictory results were found in a study of human cohort where reduced IFN-γ production by CD4 T cells, but enhanced Th17 differentiation were reported [65].We did not find elevated numbers of IL-17α-producing T cells in CGD (Fig. 3 and data not shown).This discrepancy might originate from the different mutations causing the disease in our cohort.While Horvath et al. investigated the X-linked form of CGD, caused by p91 phox mutations, our cohort is mainly composed of patients with p47 phox mutations (Table S1).These two forms of disease might have distinct pathophysiology [66,67].While animal models suggest ROS-dependency of Tregs [63,68], CGD patients with p47 phox mutations have been shown to have similar numbers of circulating Treg in comparison to healthy controls [68,69].These findings are not consistent with our observations of increased regulatory T cell numbers in CGD patients.The discrepancy in results might stem from the differences in age between the studies: our study focuses on adults (median age 36 years), while van de Geer et al. examined children (median age 13.6 years) (Table S1) [69].A study with an increased number of inclusions will reveal if there is indeed a difference in T cell phenotype between these subgroups of patients.
Our analysis revealed an increased number of CD69expressing CD8 and Vδ2 T cells in CGD patients (Fig. 2).CD69 is considered as an early activation and a tissue retention marker [70].Increased expression on circulating T cells in CGD patients is less likely to be due to recent activation of these cells since the patients did not display any signs of Fig. 5 Homing receptors expression on peripheral T cells is not affected in CGD, FMF and gout.a-l Percentages of Vδ2 (a, d, g, j),  CD4 (b, e, h, k), and CD8 T cells (c, f, i, l) expressing CCR2 (a, b, c), CCR5 (d, e, f), CCR7 (g, h, i), and CCR8 (j, k, l) assessed by flow cytometry in freshly isolated PMBCs from healthy controls and patients.CGD chronic granulomatous disease (n = 5), FMF familial Mediterranean fever (n = 7), gout (n = 6).Gray circle: FMF patient with Behcet disease.Non-parametric Mann-Whitney test was applied.*p ˂ 0.05 ◂ acute attacks at the time of examination but could rather be due to the retention of these cells in the inflamed tissues.Yet, the numbers of CD69 + T cell are, as expected, very low within circulating lymphocytes.The significance of CD69 overexpression in CGD needs to be determined, especially at the sites of inflammatory attacks.
While most studies of CGD focus on conventional αβ T cells, this is the first report to our knowledge characterizing unconventional γδ T cell populations using human material.Apart from reduced numbers of these cells, the expression of activation markers CD38 and CD69 (Fig. 2, S3) as well as death receptor CD95 (Fig. 3a) was significantly elevated on Vδ2 T cells.This suggests an increased activation status and susceptibility to undergo apoptosis, possibly explaining the reduced numbers of Vδ2 T cells in CGD (Fig. 1b).Our analysis has also revealed a reduced number of CD56 + NK cells (Fig. 1h).Although early studies show a normal cytotoxic function of NK cells in CGD patients [71], a more recent cohort study revealed an association of NK cell lymphopenia with the more severe granulomas in patients with immunodeficiencies [72], indicating an involvement of these cells in granuloma formation.This suggests that development, rather than the functions of NK cells, is affected by NADPH complex deficiency.The analysis of a larger cohort is necessary to confirm these speculations.
FMF is a widely studied monogenic autoinflammatory disease, in which the T cell compartment has been best characterized so far.Early reports revealed an increased number of CD4 and CD8 T cells [10] as well as an increased number of IFN-γ-producing T cells in the asymptomatic phase and during acute inflammatory attacks [73].The enhanced Th1 polarization in FMF patients was also suggested based on increased serum concentrations of IL-18, IL-12, and IFN-γ irrespective of the attack-free or acute inflammation phases [73][74][75].However, reduced, rather than increased, IFN-γ concentrations were found in PBMC cultures from FMF patients at different stages of the disease [76].We did not observe any significant differences in CD4 and CD8 T cell frequencies (Fig. 1) between FMF patients and healthy volunteers, but we observed lower percentages of IFN-γ-producing Vδ2 cells (Fig. 4).The discrepancy in T cell frequencies between earlier studies and our findings might be due to differences in age: while our cohort included only adults (ages 35 to 55 years old) (Table S1), earlier studies investigated pediatric patients (ages 2 to 17 years old) [10,73].It is well known that the T cell compartment changes during a lifespan; therefore, the differences in T cell status in children with FMF might disappear over time in adult patients due to the maturation of the T cell compartment.We observed decreased percentages of regulatory T cells in our FMF cohort compared to healthy controls, suggesting that the reduced suppressive activity of Tregs might contribute to the severity of inflammatory attacks (Fig. 1).On the other hand, a small study of 6 FMF patients [77] reported unchanged Treg frequencies in FMF patients at different stages of febrile attacks compared to the healthy control group [77] despite the increased concentration of Treg-related cytokines such as IL-10 and TGF-ß in the circulation of FMF patients [75,78].The discrepancy between the findings might be due to the different medication history of the patients (Table S2) or different timing of the blood sampling.Moreover, our results suggest a reduced exhaustion status of CD8 T cells in FMF patients based on a significant decrease in the expression of PD-1 (Fig. 3).These results suggest that CD8 T cells might be more active in FMF and contribute to the inflammatory flares.Indeed, increased percentages of the activation markers CD69 and CD25 expressed on CD8 T cells were found in FMF patients during the inflammatory attack [56].The speculation on the role of CD8 T cells in the pathology of FMF warrants further functional validation.We also found a tendency toward increased expression levels of the CCR8 homing receptor by all examined T cell populations in FMF patients (Fig. 5).CCR8 expression points to changes in the migratory patterns of T cells.Yet, the frequencies of CCR8 + cells in peripheral blood are very low, in accordance with other studies [79,80] but enriched in the skin [79].Examination of the immune cells in the skin of FMF patients will reveal whether T cells are involved in inflammatory reactions at the site of rashes, for example.
Of note, one of our FMF patients also has Behçet disease (Table S1).As this patient exhibits a huge increase in Vδ2 T cells, the observation also reported in previous studies [81] it did not cause any other outliers in our analysis.Yet, this data point needs to be interpreted with caution.
While inflammatory attacks in gout are mainly driven by cells of the innate immune system, T cells have also been found in the gouty tophi [37].Furthermore, a recent discovery of the NLRP3 assemblage in T cells [82], which is known to be activated by MSU crystals in innate immune cells and to drive inflammatory flares in gout [33], suggests that adaptive immune cells can also be involved in gout pathology.Our analysis revealed some trends in the expression of homing receptors CCR5 and CCR7 as well as CD38 on CD4 T cells in gout patients (Figs. 5 and  2) indicating that the migratory pattern of T cells can be affected.While CCR7 regulates trafficking to lymph nodes and intestinal Peyer's patches [83], CCR5 mediates migration and effector function of T cells to sites of inflammation [46].Increased frequency of CCR5 + CD4 T cells and reduced numbers of CCR7 + CD4 T cells in gout (Fig. 5) suggests an enhanced recruitment of these cells to inflamed tissues.Consistently, gout patients have elevated concentrations of the CCR5 ligand: regulated upon activation normal T cell expressed, and presumably secreted (RANTES) [84][85][86][87].CCR7 signaling has been shown to influence the Th1/Th2 balance by skewing CD4 T cell differentiation towards Th1 fate [88][89][90][91].The Th1 cells potentiate inflammatory responses to MSU crystals [19,92].However, we did not find increased numbers of circulating IFN-γ + T cells in gout condition (Fig. 4), consistent with recent reports [93,94].Similarly, we did not find significant differences in IL-17-production by T cells (data not shown), despite previous studies reporting increased levels of Th17-related cytokines in gout [18,19].However, our study shows significantly reduced numbers of cytokine-producing Vδ2 T cells (Fig. 4).Despite their innate-like character, γδ T cells are understudied in autoinflammatory disorders, but they are a major source of IL-17 production during the early onset of acute gout arthritis [18].The exact role of various T helper and especially unconventional γδ T cell subsets in gout remains to be determined.Especially, further research is needed to evaluate whether the observed changes in Vδ2 T cells are due to hyperuricemia or MSU crystal deposition and how these cells act during gout flares.
As there is currently no cure for FMF, gout, and CGD current treatment strategies are aimed at reducing symptoms and preventing inflammatory attacks (in case of FMF and gout) or at preventing and managing bacterial and fungal infections and granulomas (in case of CGD).As such, the prescribed medications specifically target immunological dysfunctions, and therefore we cannot exclude the possibility that they have collateral effects on the peripheral T cell compartment.FMF and most gout patients from our cohort are on colchicine treatment which, by inhibiting the microtubule function, can affect various cell types including T cells (Table S2).Indeed, early studies reported that colchicine normalizes the CD4 to CD8 T cell ratio in FMF patients, while in healthy individuals, it reduces the total T cell numbers [95].CGD patients, apart from receiving antibiotics (trimethoprim, sulfamethoxazole, flucloxacillin, metronidazole cream) and anti-fungals (ketoconazole, posaconazole), are also prescribed steroids (triamcinolonacetonide, prednisone, prednisolone, ciclesonide aerosole, Emovate) (Table S2).Consistent with the notion of a general immunosuppressive effect, steroids have been reported to exhibit numerous direct effects on T cells via increasing the expression of immunoregulatory proteins, inhibitory receptors, and apoptotic genes and decreasing the expression of pro-inflammatory cytokines, co-stimulatory molecules, and cell cycle mediators [96].Other immunomodulatory medications that might influence the T cell compartment in FMF, gout, and CGD patients are IL-1β inhibitors (canakinumab or anakinra, used by two of the FMF patients) and TNF blockers (etanercept, used by one of the FMF patients), which by targeting these cytokines might modulate the polarization signals for T cells; non-steroidal antiinflammatory drugs (NSAID) (diclofenac, ibuprofen; five FMF patients); statins (fluvastatin, simvastatin, taken by two gout patients); and allopurinol (four gout patients) (Table S2).The effect of these treatments on T cellshas either not been well investigated or has been reported contradictory.TNF blockers, for example, have been shown to either suppress cytokine production by circulating T cells in some inflammatory conditions [97][98][99][100] or to increase T cell responsiveness [101,102].Whereas in vitro treatment of PBMC cultures with allopurinol, a xanthine oxidase inhibitor which is used to reduce uric acid levels and treat gout, impairs cytokine production capacity by T cells [103], yet the in vivo effect is not well defined.Apart from known immunomodulators, pain medications such as opioids (oxycodone, tramadol), proton pump inhibitors reducing the amounts of stomach acid (omeprazole, pantoprazole), or even vitamins (vitamin D, folic acid) might modulate the T cell compartment in our cohort (Table S2).Yet, we cannot exclude vitamin uptake by healthy volunteers.As there is a possible impact of ongoing medical interventions on T cell populations in studied patients, it is important to report the immune aberrations in patients undergoing treatments as a potential target for further improvement of the disease burden.
Overall, our findings indicate that IEI are complex diseases of immune dysregulation in which not only myeloid but also lymphoid cell compartment is impacted.Despite the small size of our cohort, we were able to unravel significant changes in T cell populations.However, we cannot exclude the possibility that the observed alterations in the lymphoid compartment are influenced by patient treatment, genetics, clinical history, or other factors.Performing a broader examination of the T cell compartment over the course of the attack and resolution phase and at sites of inflammation, for example, synovial fluid in gout, is necessary to reveal the involvement of these cells in the pathology of the diseases.Furthermore, this is the first study to our knowledge characterizing unconventional γδ T cells in FMF and CGD patients.Our findings point to the involvement of the adaptive immune system in the pathology of certain IEI and prompt a broader assessment of T cell involvement in gout, CGD and FMF.

Fig. 2 Fig. 3
Fig. 2 CGD results in increased expression of activation markers on Vδ2 and CD8 T cells.a-f Percentages of Vδ2 (a, d), CD4 (b, e), and CD8 T cells (c, f) expressing CD38 (a-c) and CD69 (d-f) assessed by flow cytometry on freshly isolated PMBCs from healthy controls

Fig. 4
Fig. 4 FMF and gout patients show reduced numbers of cytokineproducing Vδ2 T cells.a-f Percentages of IFN-γ-(a-c) and TNFα-(d-f) producing Vδ2 (a, d), CD4 (b, e), and CD8 T cells (c, f) assessed by flow cytometry in freshly isolated PMBCs from healthy

Fig. 6 T
Fig. 6 T cells exhibit no change in expression of cytotoxicity markers in FMF, CGD, and gout.a-g Expression levels of CD54 (a-c), and CD56 (d, e) and NKG2D (f, g) on peripheral Vδ2 (a, d, f), CD4 (b), and CD8 T (c, e, g) assessed by flow cytometry on freshly isolated PMBCs from healthy controls and patients.Dashed lines in