RAS guanyl-releasing protein 1 (RASGRP1) is a guanine nucleotide exchange factor and activator of the RAS-MAPK pathway initiated by diacylglycerol following TCR signaling (129). Mutations in RASGRP1 have been found in patients with a combined immunodeficiency (a ALPS-like disease) (59) presenting with recurrent respiratory infections in association with EBV-induced lymphoproliferative disease, chronic lymphadenopathy, hepatosplenomegaly, autoimmune hemolytic anemia, and immune thrombocytopenia (59–63). In addition to its availability as a screening tool to establish the diagnosis of RASGRP1 deficiency (59), flow cytometry has been widely applied to evaluate functional defects resulting from RASGRP1 mutations. For instance, this approach can be used to detect reduced T cell expansion by a cell proliferation kit (e.g., CellTrace), impaired T cell activation by CD69 staining, and markedly reduced phosphorylation of ERK. Diminished intracellular expression of active caspase 3 in lymphocytes associated with reduced apoptosis using annexin V (AV) and propidium iodide (PI) staining has been observed (59–61).

Disorders of T cell co-signaling pathways such as those caused by deficiencies in CD40L, SAP, OX40, or CD70/CD27 highlight the critical role of co-stimulation for host defense (135–137). Patients with mutations affecting the co-stimulatory molecules CD70 and CD27 (Figure 4), which are expressed on the surface of T, B and NK cells (138–140) present with similar clinical phenotypes. These patients exhibit impaired effector CD8⁺ T cell generation, hypogammaglobulinemia, lack of memory B cells, and reduced cytolytic and proliferative responses of T cells resulting in chronic EBV infections (EBV-associated lymphoproliferation, EBV-associated HLH, and B cell lymphoma). Additionally, affected patients might develop severe forms of other viral infections including influenza, herpesviruses (e.g., varicella-zoster virus), and cytomegalovirus (CMV) (64, 74, 134, 141–143). Cell-surface expression of both CD70 and CD27 are assessed by flow cytometry using specific monoclonal antibodies. Similar to other combined deficiencies, it is possible that a mutated non-functional protein is expressed on the cell surface (144, 145) in which case it is possible to analyse the ability of a CD27Fc fusion protein that binds to CD70, by flow cytometry (64).

The RLTPR (RGD motif, leucine-rich repeats, tropomodulin domain, and proline-rich containing) is a scaffold protein that bridges CD28 located on the cell-surface to the cytosolic adaptor called Caspase Recruitment Domain Family Member 11 (CARD11), enabling proper activation of the TCR-induced NF-κB signaling pathway (146, 147). Although human CD28 deficiency has not yet been characterized, RLTPR deficiency was recently reported as an autosomal recessive combined immunodeficiency highlighting the critical role of the CD28 pathway for T- and B-cell activation (66). RLTPR-deficient patients present with low numbers of memory CD4⁺ T cells, reduced numbers of T helper (Th)1, Th17, and T follicular helper cells, as well as reduced memory B cells, and show poor antibody responses to vaccines (67, 148). RLTPR deficiency causes susceptibility to a variety of pathogens, including bacteria, fungi, and viruses (e.g., EBV). RLTPR expression can be detected by flow cytometry in adaptive (B and T lymphocytes) and innate (monocytes and dendritic cells) immune cells. Moreover, NF-κB signaling defects (149, 150) in CD4⁺ and CD8⁺ T cells from patients with RLTPR mutations have been characterized by flow cytometry, primarily manifested by reduced NF-κB P65 phosphorylation and IκBα degradation following anti-CD28 stimulation (66). In this context, there is a debatable paradigm that CD28 co-stimulation is not necessary for the activation of memory T cells. In agreement, flow cytometric analysis of T cell proliferation has shown that the lack of RLTPR only impairs the proliferation of naïve, but not memory T cells (66). Flow cytometric analysis also points out a critical role of RLTPR in NK cells, since their degranulation capacity is impaired after K562 stimulation, depicted by reduced CD107a expression (151).

The cytidine nucleotide triphosphate synthase 1 (CTPS1) is a molecule involved in DNA synthesis in lymphocytes (152) and therefore plays a central role in lymphocyte proliferation (65, 131). Loss-of-function homozygous mutations in CTPS1 cause a combined immunodeficiency characterized by the impaired capacity of activated T and B cells to proliferate in response to antigen receptor-mediated activation (65). CTPS1-deficient patients are susceptible to life-threatening bacterial and viral infections, including those caused by EBV (e.g., EBV-related B-cell non-Hodgkin lymphoma). Flow cytometry has only been used to evaluate T lymphocyte proliferation in response to an anti-CD3 antibody or anti-CD3/CD28 coated beads, as well as B cells in response to anti-BCR plus CpG, which were found to be defective (65). However, patients with normal lymphoproliferative response have also been reported (131). There is no anti-CTPS1 fluorochrome-conjugated antibody commercially available. Therefore, CTPS1 expression is analyzed by western blot (65). CTPS1 expression by flow cytometry has been validated in HepG2 cells through incubation of primary unconjugated antibody followed by a dye-conjugated secondary antibody staining (153). This staining strategy represents a potential approach to screen patients with CTPS1 deficiency by flow cytometry.

Mutations in the IL-2-inducible T cell kinase (ITK) causes a life-threatening syndrome of immune dysregulation and therapy-resistant EBV-associated lymphoproliferative disease (154–156). ITK is a signaling molecule located proximal to the TCR (Figure 4). ITK is expressed in thymocytes and peripheral T cells, regulating the thresholds of TCR signaling and specific development of CD8⁺ T cells (131). Flow cytometry analysis has shown that ITK deficient patients exhibit a reduced TCR-mediated calcium flux in T cells (67) and an absence of NKT cells as determined by the lack of TCR Vβ11 and TCR Vα24 double-positive cells (156).

In addition to its essential role as a co-factor for nucleic acids and metabolic enzymes (157, 158), a critical role of magnesium ion (Mg²⁺) in immune responses has been demonstrated by disease-causing mutations in the magnesium transporter 1 gene (MAGT1). Li et al. (68) reported Mg²⁺ as an intracellular second messenger following TCR activation in patients with an X-linked inborn error of immunity characterized by CD4⁺ T cell lymphopenia, severe chronic viral infections (e.g., EBV infection associated with lymphoproliferative disease or lymphoma), and defective T lymphocyte activation. Flow cytometry was used by the authors to characterize several immunological defects, but not the expression of MAGT1, which was investigated by Western blots. A reduced CD69 expression by CD4⁺ T cells after anti-CD3 stimulation was identified, while the response to phorbol 12-myristate 13-acetate (PMA) plus Ionomycin was normal, thus suggesting a specific defective TCR signaling that was confirmed by impaired NF-κB and NFAT nuclear translocation using confocal microscopy. Reduced levels of naïve CD4⁺ T cells (CD27⁺, CD45RO⁻) expressing CD31, a cell surface marker of naive TREC-rich T cells, suggest a diminished thymic output (159–161). Kinetic analysis by flow cytometry also revealed abrogation of TCR-induced Mg²⁺ influx, which can be detected by the Mg²⁺-specific fluorescent probe, MagFluo4 (68). Another immunologic feature of the disease is the impaired cytotoxic function of NK and CD8⁺ T cells. Chaigne-Delalande et al. (162) elegantly demonstrated that decreased intracellular free Mg²⁺ causes impaired expression of the natural killer activating receptor NKG2D in NK and CD8⁺ T cells, impairing cytolytic responses against EBV.

Protein kinase C delta (PKCδ) (69–71, 163) belongs to a family of at least 11 serine/threonine kinase members involved in several pathological conditions (164, 165). Mutations in this gene cause a monogenic disease that presents either as SLE-like disease or as autoimmune lymphoproliferative syndrome (ALPS)-like disorder. PKCδ deficiency is associated with uncontrolled lymphoproliferation and chronic EBV infection. Immunologically, human PKCδ deficiency results in a B cell disorder characterized by B cell resistance to apoptosis, B cell hyperproliferation, increased production of autoantibodies, and decreased numbers of memory B cells (69–71, 163). A similar phenotype has been identified in PKCδ knockout mice (166–168), demonstrating the essential role of PKCδ in B cell tolerance. Flow cytometry applications to investigate this disease are designed to demonstrate increased B cell proliferation after anti-IgM stimulation, resistance to PMA-induced cell death (70), and the almost absence of CD27 expression on B cells (69), i.e., absence of memory cells.

X-linked lymphoproliferative syndrome (XLP) is a PID that presents with severe or fatal EBV infection, acquired hypogammaglobulinemia, malignant lymphoma, and HLH (72, 169). Most XLP cases are due to mutations in the SH2 domain protein 1A (SH2D1A) gene (XLP type 1), which encodes the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) (72). SAP is an adapter molecule that controls several signaling pathways involved in lymphocyte activation, proliferation, cytotoxicity, and also promotion of apoptosis [Figure 4; (170–172)]. The defect in antibody production exhibited by SH2D1A-deficient patients probably arise from impaired CD4⁺ T cell interaction with B cells rather than an intrinsic B cell failure (169, 173).

Mutations in the gene encoding the X-linked inhibitor of apoptosis (XIAP), which inhibits caspase-3,−7, and−9 by direct binding (174), are responsible for XLP type 2 syndrome (73).The clinical phenotype and the disease pathogenesis have been reviewed and compared in detail elsewhere (129, 172, 175, 176). Flow cytometry can be used to evaluate apoptosis, in order to distinguish both XLP forms. Due to the distinct physiological roles of SH2D1A and XIAP, enhanced apoptosis of T lymphocytes is observed in patients with XIAP-deficiency, while the absence of SAP in SH2D1A deficiency is consistently associated with impaired cell apoptosis (133, 170, 172). This might explain why cytopenia is common in XIAP but not in SH2D1A deficiency (129). The EBV-associated immune dysregulation in XIAP deficiency might, in part, be due to the combination of an intrinsic exacerbated proliferation of immune cells plus the incapacity to respond to EBV. The lymphoproliferative disease reported in SH2D1A deficiency seems to be more the consequence of extrinsic and constant stimulation induced by EBV that cannot be properly controlled. For both XLP forms, flow cytometry to test intracellular testing for SAP and XIAP protein expression is available [Figure 6; (34)]. In addition, flow cytometric testing has demonstrated that the absence of SAP or XIAP proteins results in reduced numbers of circulating NKT (Vα24TCR⁺/Vβ11TCR⁺) cells (73).

TCRα/β DN T cells are useful biomarkers, frequently elevated in children with autoimmune lymphoproliferative syndromes (ALPS) (179). The immunological functions of these cells have been reviewed in detail elsewhere (180). However, their precise role in the pathogenesis of autoimmune diseases is not well understood (179). ALPS is caused by mutations in five different genes: FAS, FASL, FADD, CASP8, and CASP10. The interaction between Fas (CD95) and Fas ligand or FasL (CD178), both expressed by activated T lymphocytes (the former also present on other cell types), triggers the formation of a death-inducing signaling complex (181, 182). This process involves the recruitment of Fas-associated death domain (FADD), cysteine-aspartic acid protease 8 (CASP8), and CASP10, initiating a cascade of signaling events that result in apoptotic cell death (183). This process regulates lymphocyte life span and promotes the elimination of autoreactive lymphocytes (Figure 7). The syndromes caused by mutations in these five genes have been classified by the National Institutes of Health (NIH) (177) as ALPS-FAS cell surface death receptor (the most frequent) (75), ALPS-FASL (76), ALPS-Caspase 8 (77), ALPS-Caspase 10 (78), and the FADD-deficiency (79). These disorders generally present as lymphadenopathy, splenomegaly, and autoimmune manifestations such as autoimmune hemolytic anemia, and severe recurrent thrombocytopenia (75–79, 184). Laboratory findings also include polyclonal hypergammaglobulinaemia, T lymphocyte apoptosis defect, and increased percentages of TCRα/β DN T cells (177).

Flow cytometry analysis demonstrates defective T cell apoptosis in response to anti-Fas antibody, recombinant FasL, or after phytohaemagglutinin (PHA)-/IL-2 stimulation by using FasT Kill assays or AV/PI or 7-AAD-staining. The technique of detecting increased percentages of TCRα/β DN T cells within peripheral blood mononuclear cells (PBMCs) is well established (76, 185, 186). Moreover, protein expression of FAS (187) and FASL (186) (both after T-cell blast generation by PHA plus IL-2) by flow cytometry is available to investigate ALPS-FAS and ALPS-FASL, respectively. Although the other ALPS forms (due to FADD (79), CASP8, or CASP10 deficiency) have not yet been studied by flow cytometry due to the unavailability of specific fluorescent conjugated antibodies, mutations in the FAS receptor is the most frequent disease form of ALPS found in ~70% of genetically defined ALPS (177, 178) thereby making flow cytometry an essential screening tool for patients suspected to have ALPS.

While heterozygous germline inactivating mutations in the signal transducer and activator of transcription 3 (STAT3) with dominant negative effect cause autosomal dominant hyper IgE syndrome (188), heterozygous gain-of-function (GOF) mutations in STAT3 result in an ALPS-like phenotype (81). Patients can develop early-onset poly-autoimmunity (e.g., type 1 diabetes), autoimmune hypothyroidism, enteropathy, pulmonary disease, cytopenias, arthritis, short stature, myelodysplastic syndrome, aplastic anemia, and lymphocytic leukemia (81, 189, 190). Increased percentages of TCRαβ⁺-DN T cells are occasionally identified (189). So far, STAT3 GOF mutations have been shown to enhance transcriptional activity and delay dephosphorylation of STAT3, without inducing constitutive phosphorylation as shown by flow cytometry studies. In agreement with the involvement of STAT3 in the inhibition of Tregs (191, 192) and enhancement of Th17 cell differentiation (193), flow cytometry has also shown increased Th17 levels while the FOXP3⁺CD25⁺ Treg population is reduced and the expression of CD25 (IL2RA) is decreased in patients with STAT3 GOF mutations (189). Due to its activity as a repressor of FAS-FASL activity, decreased FASL-induced apoptosis has been observed (190).

Mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause a common variable immunodeficiency (CVID)-like disease with predominant antibody deficiency (hypogammaglobulinemia) and autoimmunity (e.g., autoimmune hemolytic anemia as well as atrophic gastritis with autoantibodies against intrinsic factor, autoimmune enteropathy, hypothyroidism, myasthenia gravis, polyarthritis), and inflammatory bowel disease (80, 194, 195). The phenotype of LRBA deficiency has been well-characterized elsewhere (196). LRBA is highly expressed in immune cells such as T and B cells (80). The application of flow cytometry to screen patients with LRBA deficiency has been recently developed (197) as well as its application to evaluate several immunopathological mechanisms of this disease. More than 70% of the LRBA-deficient patients have reduced levels of Tregs (196) (CD4⁺CD25⁺FOXP3⁺), which may be related to the low surface expression of cytotoxic T lymphocyte–associated antigen 4 (CTLA4 or CD152) (198). CTLA4 is a cell surface molecule required for the proper suppressive function of Tregs (199–201). The reduced CTLA4 levels can be explained by the fact that LRBA is a regulator of CTLA4 vesicle trafficking [Figure 5; (197)]. Increased percentages of TCRα/β DN T cells have been found in up to 50% of LRBA-deficient patients (196). Several other defects associated with LRBA deficiency have been identified by flow cytometry (80). Among them are increased B cell apoptosis, low levels of IgG⁺IgA⁺CD27⁺ switched-memory B cells, reduced B cell proliferation, and impaired activation (as measured by CD138 expression).

Interleukin 10 (IL-10) is an important anti-inflammatory cytokine produced by cells like APCs. Early-onset (within the first months of life) of severe inflammatory bowel disease (EO-IBD), i.e., Crohn's disease and ulcerative colitis (UC), can be caused by IL-10 and IL10- receptor deficiencies (89, 90, 251). The expression of both IL-10 receptor alpha (IL-10RA) and IL-10 receptor beta (IL10RB) can be assessed by flow cytometry (90). Of note, IL-10 binds to its receptor, leading to the activation of the JAK1-STAT3 pathway [Figure 9A; (252)]. Normal or defective IL-10-induced phosphorylation of STAT3 in T cells has been evaluated by flow cytometry to distinguish patients with EO-IBD due to IL-10 or IL-10R deficiencies (Figure 9B). Recombinant IL-6 or IL-23 are used in parallel with IL-10 as stimuli to distinguish the specificity of IL-10 or IL10R deficiencies.