**2. Analysis of inflammatory and regulatory cell profiles in PIDs**

Immune dysregulation with autoimmunity is observed in many PIDs such as LRBA, CTLA4, STAT3 GOF, PIK3CD deficiencies as well as IPEX syndrome caused by loss or dysfunctional FOXP3 expression [4–18]. Disrupted T helper cell plasticity is pointed out as a prominent feature of the autoimmunity in PIDs. Deregulated numbers and functions of Treg cells are observed in most of the patients with IPEX or IPEX-like (such as in patients with LRBA deficiency) [6, 7, 19–21]. Decreased Treg cell numbers or loss of Treg cell functions are related to severe form of autoimmunities in PIDs. In contrast, deregulated inflammatory cell numbers/ratios and the inflammatory cytokines produced by inflammatory cells are observed as autoimmune manifestations of PIDs such as LRBA and STAT3 LOF deficiencies. In LRBA deficiency, increased number of circulating T folicular helper (Tfh) is associated with autoimmune manifestations of the disease [5]. Moreover, decreased Th17 cell numbers are related to inflammatory response to Candida infections observed in patients with LOF mutations in STAT3 deficiency [22–24].

In these cases, the first attempt is to analyze regulatory and inflammatory cell ratios in the clinical immunology laboratory to clarify the cellular background of autoimmunity.

## **2.1 Analysis of Treg cells in PIDs**

Treg cells are unique subset of T helper cells through its equilibrating functions on immune response to self and foreign antigens. Tregs suppress inflammatory T cell function and proliferation, therefore it plays critical roles to prevent autoimmune disorders. In PIDs with autoimmunity, impaired functions of Treg cells in parallel with decreased number of Treg cells are observed. IPEX is a well-known syndrome affecting Treg cell development due to mutations of FOXP3 which is a main transcription factor in the development of Treg cells. In patients with IPEX syndrome, loss of circulating and tissue associated Treg cells are thought to cause the multi-organ autoimmune manifestations [6, 20, 21]. Patients with CD25 (IL-2Rα) deficiency have IPEX-like phenotype as well as in patients with LRBA deficiency. Decreased Treg ratio is a significant laboratory characteristics in these PIDs [7, 25]. In patients with AIRE deficiency which is related to Autoimmune Poly Endocrinopathy, Candidiasis and Ectodermal Dystrophy (APECED) syndrome, decreased Treg cell ratio and function are associated with the occurrence of the disease [26].

Investigating Treg cell ratio by flow cytometry provides an important insight to understand autoimmunity from the benchside to bedside.

Below, it was described the Treg staining protocol and the gating strategy for human peripheral blood Treg cells (**Figure 1**).

#### *2.1.1 Treg staining protocol*

• Peripheral blood mononuclear cells (PBMCs) are separated by ficoll density gradient protocol from 4 ml of whole blood in tube with EDTA.

*Flow Cytometric Approach in the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.96004*

#### **Figure 1.**

*Representative image of CD4+ CD127loCD25hi FOXP3+ Treg cells in peripheral blood of healthy control and a patient.*


#### **2.2 Analysis of circulating Tfh and TH17 cells in PIDs**

Tfh cells are specialized Th cell subset which plays important role in B cell differentiation in lymph nodes, in producing high affinity antibodies and the

development of memory cells. Therefore, Tfh provides help germinal center (GC) formation and selection of plasma cells [27–30]. Tfh cells have unique molecules that are expressed in cell surface and have special functions such as CXCR5. CXCR5 is a chemokine receptor and provides migration of Tfh cells to GC zone. Besides, Tfh expresses B Cell Lymphoma (BCL-6) and (Inducible T Cell Costimulator) ICOS or CD278 on their surfaces. Increased Tfh cell numbers in peripheral blood are investigated as an inflammatory marker of some PIDs such as LRBA deficiency [5].

Th17 cells are also a subset of helper T cells which are responsible for producing IL-17, a pro-inflammatory cytokine recruiting neutrophils to infection site to combat infection [22, 23, 31, 32]. IL-6 expression and STAT3 activation are required for the differentiation of Th17 cells from CD4+ T lymphocytes. Therefore in STAT3 deficiency caused by autosomal dominant loss of function mutations of STAT3 gene, decreased number of circulating Th17 cells are associated with susceptibility to Candida infections in STAT3 LOF deficiency which is a type of Autosomal Dominant- hyper IgE Syndrome (AD-HIES) [24].

Detection of Tfh and Th17 cell ratios in the peripheral blood of the patients with designated PIDs in clinical immunology laboratory by flow cytometry using various surface and intracellular markers which are unique to circulating Tfh and Th17 cells is important step to understand the inflammatory background of the autoimmune manifestations (**Figures 2** and **3**). See the Section 2.1.1. for the staining protocol.

Below, it was demonstrated Tfh and Th17 gating strategy.

#### **Figure 2.**

*Analysis of cTfh cells in a healthy control (top) and a patient with PID (below). In the patient, increased ratio of cTfh is observed.*

*Flow Cytometric Approach in the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.96004*

**Figure 3.**

*Th17 gating strategy. Increased ratio of Th17 cells expressing IL17A and IL17F are observed in a patient (below) compared to healthy control (top).*
