**2. Evaluation of immunophenotype to aid in the differential diagnosis of PIDs**

Flow cytometry is a standard laboratory tool in the evaluation and identification of leukocyte populations and specific lymphocyte subpopulations. The analysis of the expression of cell surface markers [clusters of differentiation (CD)] allows to determine the cell lineage and to examine the stage of differentiation and cell activation. The clinical application is broad for routine use in diagnostic laboratories, which facilitates the assessment of patients with suspected primary immunodeficiencies. The circulating cellular compartment of the human immune system shows the most marked shifts during the first years of life, but is also dynamic during adult life, showing a tendency to a reduction in function and diversity with age [5]. When an immune system disorder is suspected, immunophenotyping of the patient's peripheral blood mononuclear cells (PBMCs) is one of the initial steps in the diagnostic work-up [6].

The basic immunophenotype analyzing T (CD3), B (CD19), and natural killer (NK) (CD56+CD16+) lymphocytes leads to a potential diagnosis of severe combined immunodeficiencies (SCID) resulting in four general categories of disease defined by the impact on lymphocyte populations: T−/B−/NK−, T−/B−/NK+, T−/ B+/NK−, and T−/B+/NK+. Overall, patients with adenosine deaminase (ADA) deficiency and reticular dysgenesis demonstrate the T−/B−/NK− immunophenotype and typically have the most profound lymphocytopenia among SCID patients [7]. Recombination defects usually show the phenotype T−/B−/NK+, and defects in cytokine signaling and T-cell receptor (TCR)-derived signals result in T−/B+/NK− and T−/B+/NK+ phenotypes. These phenotypes often differ depending on the type of mutation and protein affected. The expression of isoforms CD45RA, CD45RO in T cells should also be included in the study of patients with suspected SCID. An increase in the percentage of CD45RO memory helper T cells indicates the presence of autoreactive lymphocytes and reinforces the diagnostic suspicion of SCID, leaky SCID or Omenn syndrome (OS) (**Figure 1**). OS is an inherited disorder characterized by an absence of circulating B lymphocytes and an infiltration of the skin by activated T lymphocytes. Inherited mutations in RAG1 or RAG2, resulting in partial V(D)J recombinase activity, were shown to be responsible for OS [8].

The use of larger cytometric panels can include a higher number of lymphocyte subpopulations of interest in the diagnosis of other groups of PID, such as predominantly antibody deficiencies and immune dysregulation diseases among others. Below we describe the gating strategies for the study of a broader spectrum of subpopulations: T cell differentiation stages, recent thymic emigrants (RET), regulatory T cells (Tregs), T helper 1/2/17 cells, B cell differentiation stages, NK cells, dendritic cells and monocytes, and their application in the study of patients with PID.

**29**

**Figure 1.**

*are compared.*

cytometry.

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

*DOI: http://dx.doi.org/10.5772/intechopen.89743*

General immunophenotyping protocol:

3.Lyse the red cells using a lysing solution.

appropriate mix of antibodies in each situation).

4.Incubate for 10 min in the dark at RT.

revolutions per minute (rpm).

est, depending on the panel.

1.Stain 50 μL of EDTA whole blood with appropriate mix of antibodies of inter-

*Dotplot representing the expression of different isoforms CD45RA and CD45RO in subpopulations T CD4 and CD8. A healthy donor and a patient with severe combined immunodeficiency (SCID) of the same age range* 

5.Discard the lysing solution after 5 min of centrifugation at (324 g) or 1500

8.Add 200 μL of PBS, vortex and the sample is ready to be acquired by flow

(This protocol is used in all the immunophenotyping techniques, using the

2.Incubate for 20 min in the dark at room temperature (RT).

6.Wash with 2 mL of phosphate buffered saline (PBS).

7.Discard the PBS after 5 min of centrifugation at 1500 rpm.

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.89743*

### **Figure 1.**

*Innovations in Cell Research and Therapy*

rapid and sensitive result.

**diagnosis of PIDs**

the diagnostic work-up [6].

more subtle variations in the differentiated states of specific subpopulations [3]. For basic and advanced lymphocyte phenotype studies, the technical option is classical flow cytometry, although new approaches mainly used in research like mass cytometry or CyTOF, have emerged [4]. The following step in the diagnosis of patients with PID is the biochemical and functional characterization of the altered molecules, for example, perforin, CD40 ligand, etc. The advantage of using flow cytometry over other techniques such as western-blot in the case of protein expression studies and functional evaluation of different subpopulations is the

**2. Evaluation of immunophenotype to aid in the differential** 

Flow cytometry is a standard laboratory tool in the evaluation and identification

The basic immunophenotype analyzing T (CD3), B (CD19), and natural killer (NK) (CD56+CD16+) lymphocytes leads to a potential diagnosis of severe combined immunodeficiencies (SCID) resulting in four general categories of disease defined by the impact on lymphocyte populations: T−/B−/NK−, T−/B−/NK+, T−/ B+/NK−, and T−/B+/NK+. Overall, patients with adenosine deaminase (ADA) deficiency and reticular dysgenesis demonstrate the T−/B−/NK− immunophenotype and typically have the most profound lymphocytopenia among SCID patients [7]. Recombination defects usually show the phenotype T−/B−/NK+, and defects in cytokine signaling and T-cell receptor (TCR)-derived signals result in T−/B+/NK− and T−/B+/NK+ phenotypes. These phenotypes often differ depending on the type of mutation and protein affected. The expression of isoforms CD45RA, CD45RO in T cells should also be included in the study of patients with suspected SCID. An increase in the percentage of CD45RO memory helper T cells indicates the presence of autoreactive lymphocytes and reinforces the diagnostic suspicion of SCID, leaky SCID or Omenn syndrome (OS) (**Figure 1**). OS is an inherited disorder characterized by an absence of circulating B lymphocytes and an infiltration of the skin by activated T lymphocytes. Inherited mutations in RAG1 or RAG2, resulting in partial

V(D)J recombinase activity, were shown to be responsible for OS [8].

The use of larger cytometric panels can include a higher number of lymphocyte subpopulations of interest in the diagnosis of other groups of PID, such as predominantly antibody deficiencies and immune dysregulation diseases among others. Below we describe the gating strategies for the study of a broader spectrum of subpopulations: T cell differentiation stages, recent thymic emigrants (RET), regulatory T cells (Tregs), T helper 1/2/17 cells, B cell differentiation stages, NK cells, dendritic cells and monocytes, and their application in the study

of leukocyte populations and specific lymphocyte subpopulations. The analysis of the expression of cell surface markers [clusters of differentiation (CD)] allows to determine the cell lineage and to examine the stage of differentiation and cell activation. The clinical application is broad for routine use in diagnostic laboratories, which facilitates the assessment of patients with suspected primary immunodeficiencies. The circulating cellular compartment of the human immune system shows the most marked shifts during the first years of life, but is also dynamic during adult life, showing a tendency to a reduction in function and diversity with age [5]. When an immune system disorder is suspected, immunophenotyping of the patient's peripheral blood mononuclear cells (PBMCs) is one of the initial steps in

**28**

of patients with PID.

*Dotplot representing the expression of different isoforms CD45RA and CD45RO in subpopulations T CD4 and CD8. A healthy donor and a patient with severe combined immunodeficiency (SCID) of the same age range are compared.*

General immunophenotyping protocol:


(This protocol is used in all the immunophenotyping techniques, using the appropriate mix of antibodies in each situation).

## **2.1 T lymphocyte immunophenotyping**

T-lymphocytes mature in the thymus after going through positive and negative selection processes before migration into secondary lymphoid organs. The immune system at birth is largely dominated by truly naïve T cells, with very limited representation of memory cells and a relatively high proportion of recent thymic emigrants (RTEs). They are naïve peripheral T cells, which have only recently exited the thymus, have not undergone further peripheral proliferation and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naïve peripheral T cell pool [9]. Thymic function can be determined by evaluation RTEs, which are studied using CD3, CD4, CD27, CD31, and CD45RA, expression markers according to the gating strategy in (**Figure 2**). The assessment of the thymic function is performed also in a suspicion of severe combined immunodeficiency, DiGeorge syndrome, and other T-cell immune deficiency disorders.

Subsequently, the mature "naïve" T-lymphocytes circulate between the secondary lymphoid organs and the blood. In humans, naïve CD4+ T cells typically express CCR7, CD62L, and CD45RA, while they lack CD45RO expression [10]. CCR7 and CD62L are involved in the localization of T cells in secondary lymphoid organs (SLO) and interact with ligands expressed in high endothelial venules (HEVs). CD45RA and CD45RO play a role in the transduction of TCR signals, and their expression characterizes the different subsets of T cells, being CD45RA positive the naïve and CD45RO positive the memory [11]. Upon recognition of specific antigen complexes, naive CD4+ T cells proliferate and differentiate toward effector T cells, which provide immediate protection.

**Figure 2.**

*Gating strategy of recent thymic emigrants (RET). RTEs were studied using CD3, CD4, CD27, CD31, and CD45RA expression.*

**31**

**Figure 3.**

*gating on CD45RA− TCM and TEM cells.*

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

Most of these effectors T cells subsequently die from apoptosis, but a subset of antigen-specific T cells will persist as memory T cells [12]. There are two types of memory T cells in circulation, central memory T cells (TCMs) and effectors memory T cells (TEM): the former shows self-renewal potential with less effector functions, while the latter possesses immediate effector functions and can migrate rapidly to peripheral tissues. In addition, a distinct lineage of tissue-resident memory cells (TEMRA cells) is limited to different tissues. By using the characteristic expression markers CD45RA and CCR7 and a gating strategy for gated CD4+ and CD8+ T-cell subsets, we can identify the following populations CD45RA+/CCR7+ (naïve), CD45RA−/CCR7+ [central memory (TCM)], CD45RA−/CCR7− [effector memory (TEM)], and CD45RA+/CCR7− [terminal effector memory (TEMRA)]. Furthermore, based on CCR6 and CXCR3 expression markers, we can distinguish CD4+ T-helper (Th) populations at phenotypic level (Th1, Th2, Th17, and Th1-17) by gating on CD4+CD45RA− TCM and TEM cells (**Figure 3**). In several primary immunodeficiencies, there are alterations in different subpopulations. The distribution of naïve and memory T cells is particularly relevant for the diagnosis of severe combined immunodeficiency (SCID) and its variants in

In patients with antibody defects such as common variable immunodeficiency (CVID) has been described an increase abnormally differentiated T CD4+ effector cells showing a Th1-skewed profile and activated effector T CD8+ lymphocytes [13, 14].

*Gating strategy for differentiated CD4+ and CD8+ T cell subsets, based on CD45RA and CCR7 expression defining: CD45RA+/CCR7+ (naïve), CD45RA−/CCR7+ (central memory [TCM]), CD45RA−/CCR7− (effector memory [TEM]), and CD45RA+/CCR7− (terminal effector memory [TEMRA]). CD4+ T-helper (Th) populations (Th1, Th2, Th17, and Th1-17), based on CCR6 and CXCR3 expression, were analyzed by* 

*DOI: http://dx.doi.org/10.5772/intechopen.89743*

infants—Omenn syndrome and leaky SCID.

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.89743*

*Innovations in Cell Research and Therapy*

**2.1 T lymphocyte immunophenotyping**

which provide immediate protection.

T-lymphocytes mature in the thymus after going through positive and negative selection processes before migration into secondary lymphoid organs. The immune system at birth is largely dominated by truly naïve T cells, with very limited representation of memory cells and a relatively high proportion of recent thymic emigrants (RTEs). They are naïve peripheral T cells, which have only recently exited the thymus, have not undergone further peripheral proliferation and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naïve peripheral T cell pool [9]. Thymic function can be determined by evaluation RTEs, which are studied using CD3, CD4, CD27, CD31, and CD45RA, expression markers according to the gating strategy in (**Figure 2**). The assessment of the thymic function is performed also in a suspicion of severe combined immunode-

ficiency, DiGeorge syndrome, and other T-cell immune deficiency disorders.

Subsequently, the mature "naïve" T-lymphocytes circulate between the secondary lymphoid organs and the blood. In humans, naïve CD4+ T cells typically express CCR7, CD62L, and CD45RA, while they lack CD45RO expression [10]. CCR7 and CD62L are involved in the localization of T cells in secondary lymphoid organs (SLO) and interact with ligands expressed in high endothelial venules (HEVs). CD45RA and CD45RO play a role in the transduction of TCR signals, and their expression characterizes the different subsets of T cells, being CD45RA positive the naïve and CD45RO positive the memory [11]. Upon recognition of specific antigen complexes, naive CD4+ T cells proliferate and differentiate toward effector T cells,

*Gating strategy of recent thymic emigrants (RET). RTEs were studied using CD3, CD4, CD27, CD31, and* 

**30**

**Figure 2.**

*CD45RA expression.*

Most of these effectors T cells subsequently die from apoptosis, but a subset of antigen-specific T cells will persist as memory T cells [12]. There are two types of memory T cells in circulation, central memory T cells (TCMs) and effectors memory T cells (TEM): the former shows self-renewal potential with less effector functions, while the latter possesses immediate effector functions and can migrate rapidly to peripheral tissues. In addition, a distinct lineage of tissue-resident memory cells (TEMRA cells) is limited to different tissues. By using the characteristic expression markers CD45RA and CCR7 and a gating strategy for gated CD4+ and CD8+ T-cell subsets, we can identify the following populations CD45RA+/CCR7+ (naïve), CD45RA−/CCR7+ [central memory (TCM)], CD45RA−/CCR7− [effector memory (TEM)], and CD45RA+/CCR7− [terminal effector memory (TEMRA)]. Furthermore, based on CCR6 and CXCR3 expression markers, we can distinguish CD4+ T-helper (Th) populations at phenotypic level (Th1, Th2, Th17, and Th1-17) by gating on CD4+CD45RA− TCM and TEM cells (**Figure 3**).

In several primary immunodeficiencies, there are alterations in different subpopulations. The distribution of naïve and memory T cells is particularly relevant for the diagnosis of severe combined immunodeficiency (SCID) and its variants in infants—Omenn syndrome and leaky SCID.

In patients with antibody defects such as common variable immunodeficiency (CVID) has been described an increase abnormally differentiated T CD4+ effector cells showing a Th1-skewed profile and activated effector T CD8+ lymphocytes [13, 14].

### **Figure 3.**

*Gating strategy for differentiated CD4+ and CD8+ T cell subsets, based on CD45RA and CCR7 expression defining: CD45RA+/CCR7+ (naïve), CD45RA−/CCR7+ (central memory [TCM]), CD45RA−/CCR7− (effector memory [TEM]), and CD45RA+/CCR7− (terminal effector memory [TEMRA]). CD4+ T-helper (Th) populations (Th1, Th2, Th17, and Th1-17), based on CCR6 and CXCR3 expression, were analyzed by gating on CD45RA− TCM and TEM cells.*

Also with the suspected diagnosis of hyper-IgE syndrome (atopic dermatitis, recurrent otitis, pneumonias, and abscess caused by *Staphylococcus aureus*), the use of this gating strategy is very useful. The immunological phenotypes include high IgE levels, eosinophilia, low levels of Th17 cells, and memory B cell lymphopenia. T-cell differentiation, particularly Th17 cells is disrupted, for that reason, we use a flow cytometry panel to analyze the CD4+ T-helper (Th) populations (Th1, Th2, Th17, and Th1-17), based on CCR6 and CXCR3 expression, previously gated on CD45RA− TCM and TEM CD4+ T cells. It is important to compare the values of the patient with an age-matched healthy control [6].

Regulatory T-cells (Tregs) play a key role in maintaining peripheral tolerance under physiological and pathological conditions. Tregs are characterized by the constitutive expression of the critical transcription factor forkhead box P3 (Foxp3). Impairment of Treg function is the key pathogenic event leading to altered self-tolerance in patients with immune deregulation, poly-endocrinopathy, enteropathy, and X-linked syndrome (IPEX) [15]. The most common markers utilized for Treg typing by flow cytometry are CD25, forkhead box protein 3 (FoxP3), and CD127. Also for more accurate characterization, CCR4 is useful; a potential marker for effector-type regulatory T cells T-regulatory (Treg) cell populations: CD3+CD4+CD25+, CD127−, CCR4+, and CD45RO+.

See the immunophenotype protocol in Section 2*.*

### **2.2 B lymphocyte immunophenotyping**

Early development of human B cells from multipotent stem cells occurs in the bone marrow by antigen independent manner. Once the central development of B cells has been accomplished and the transitional B cells leave the bone marrow, maturation occurs in the secondary lymphoid organs, especially in the spleen.

B-cell subset phenotyping in peripheral blood has been used to classify and assess the prognosis of patients with humoral deficiencies such as common variable immunodeficiency (CVID). CVID is a heterogeneous disease, and most patients show defective differentiation of B cells into memory B cells or antibody-secreting cells. B cells can be identified in the lymphocyte gate by CD19 expression, a marker that is present in all circulating B cell populations. B-cell populations can be studied based on expression of IgD and CD27 [naïve (IgD+CD27−), pre-switched (IgD+CD27+), switched memory (IgD−CD27+), and exhausted (IgD−CD27−)]. In patients with CVID, the study of these subpopulations allows to identify severe defects of germinal center dependent B cell memory formation and early defects of peripheral B cell differentiation. The absence of switched memory B cells is very suggestive of an alteration of the germinal center reaction [16].

The combined staining for CD19, IgD, CD38, and CD24 allows the identification of transitional B cells (CD19+IgD+CD38highCD24high) and plasmablasts (CD19+IgD+CD38highCD24neg). CD27 and CD21 enabled study of the CD21low population. The expansion of circulating CD21lowB cells is associated with manifestations of a chronic immune activation and autoimmunity (**Figure 4**) [17].

Recently, germline heterozygous gain-of-function mutations in PIK3R1 and PIK3CD genes have been identified to cause a primary immunodeficiency in patients with node lymphoproliferation, splenomegaly, diarrhea, and dysgammaglobulinemia, initially diagnosed with CVID. In these patients, the extended immunophenotyping reveals high percentage of transitional B cells, low pre-switched memory, switched memory B cells, and skewing of CD4+ with CD8+ T cells toward terminally differentiated effector cells. This phenotype is suggestive of activated PI3K delta syndrome type 1 and 2 (APDS) [18].

**33**

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies*

Natural killer (NK) cells are one of the first lines of innate immune defense against pathogens and tumors, among other cells. NK cells are lymphocytes with cytotoxic capacity that provide rapid response to virus-infected cells and to tumor cells through the ability to recognize the absence of HLA class I and other signals on these cells. Innate cells can be analyzed by flow cytometry gating in the CD3<sup>−</sup>CD19<sup>−</sup> cells. Natural killer (NK) cells subpopulations can be divided into two

*Gating strategy used for the study of subpopulations of B lymphocytes. B-cell populations (naïve, pre-switched, switched memory, and exhausted) are distinguished depending on IgD and CD27 expression. The differing pattern of CD24+ and CD38+ expression identified transitional cells and plasmablasts. CD27 and CD21* 

and NKbright (CD16−CD56high). Characterizing the different subsets of NK is crucial in the diagnosis of NK cell deficiencies [19]. An example is the deficiency in MCM4, with autosomal recessive inheritance (AR), which may be suspected in the context of a higher percentage of CD56bright NK cells (of total NK cells) in individuals with

Monocytes are leukocytes that can differentiate into macrophages and myeloid lineage dendritic cells. As a part of the vertebrate innate immune system, monocytes also influence the process of adaptive immunity [21]. There are at least two subpopulations of peripheral monocytes in human blood based on their phenotypic receptors CD16 and CD14; classical monocytes (CD14highCD16<sup>−</sup>) and non-classical

Dendritic cells in periphery blood are responsible for the capture and processing antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs, and secrete cytokines to initiate immune responses [23]. Dendritic cells were studied into the population negative for the following markers: CD3, CD14, CD16, CD19, CD20, and CD56. High expression of HLA-DR, CD11c, and CD123

are markers commonly used to identify plasmacytoid DCs (HLA-DR+

deficiency is an autosomal dominant (AD) immunodeficiency classified in the

CD11c+

). These cells have been shown to exhibit distinct pheno-

) (**Figure 5**). GATA binding protein 2 (GATA-2)

CD56low)

CD123+

) and

subpopulations depending on CD56 and CD16 expression; NKdim (CD16+

*DOI: http://dx.doi.org/10.5772/intechopen.89743*

**2.3 Innate cells immunophenotyping**

*markers enabled study of the CD21 low population.*

endocrine and/or growth abnormalities [20].

*2.3.2 Monocytes and dendritic cells (DC)*

monocytes (CD14lowCD16+

type and function [22].

myeloid DCs (HLA-DR+

See the immunophenotype protocol in Section 2.

*2.3.1 Natural killer (NK) cells*

**Figure 4.**

See the immunophenotype protocol in Section 2.

*Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies DOI: http://dx.doi.org/10.5772/intechopen.89743*

### **Figure 4.**

*Innovations in Cell Research and Therapy*

patient with an age-matched healthy control [6].

CD3+CD4+CD25+, CD127−, CCR4+, and CD45RO+. See the immunophenotype protocol in Section 2*.*

suggestive of an alteration of the germinal center reaction [16].

of a chronic immune activation and autoimmunity (**Figure 4**) [17].

PI3K delta syndrome type 1 and 2 (APDS) [18].

See the immunophenotype protocol in Section 2.

**2.2 B lymphocyte immunophenotyping**

Also with the suspected diagnosis of hyper-IgE syndrome (atopic dermatitis, recurrent otitis, pneumonias, and abscess caused by *Staphylococcus aureus*), the use of this gating strategy is very useful. The immunological phenotypes include high IgE levels, eosinophilia, low levels of Th17 cells, and memory B cell lymphopenia. T-cell differentiation, particularly Th17 cells is disrupted, for that reason, we use a flow cytometry panel to analyze the CD4+ T-helper (Th) populations (Th1, Th2, Th17, and Th1-17), based on CCR6 and CXCR3 expression, previously gated on CD45RA− TCM and TEM CD4+ T cells. It is important to compare the values of the

Regulatory T-cells (Tregs) play a key role in maintaining peripheral tolerance under physiological and pathological conditions. Tregs are characterized by the constitutive expression of the critical transcription factor forkhead box P3 (Foxp3). Impairment of Treg function is the key pathogenic event leading to altered self-tolerance in patients with immune deregulation, poly-endocrinopathy, enteropathy, and X-linked syndrome (IPEX) [15]. The most common markers utilized for Treg typing by flow cytometry are CD25, forkhead box protein 3 (FoxP3), and CD127. Also for more accurate characterization, CCR4 is useful; a potential marker for effector-type regulatory T cells T-regulatory (Treg) cell populations:

Early development of human B cells from multipotent stem cells occurs in the bone marrow by antigen independent manner. Once the central development of B cells has been accomplished and the transitional B cells leave the bone marrow, maturation occurs in the secondary lymphoid organs, especially in the spleen. B-cell subset phenotyping in peripheral blood has been used to classify and assess the prognosis of patients with humoral deficiencies such as common variable immunodeficiency (CVID). CVID is a heterogeneous disease, and most patients show defective differentiation of B cells into memory B cells or antibody-secreting cells. B cells can be identified in the lymphocyte gate by CD19 expression, a marker that is present in all circulating B cell populations. B-cell populations can be studied based on expression of IgD and CD27 [naïve (IgD+CD27−), pre-switched (IgD+CD27+), switched memory (IgD−CD27+), and exhausted (IgD−CD27−)]. In patients with CVID, the study of these subpopulations allows to identify severe defects of germinal center dependent B cell memory formation and early defects of peripheral B cell differentiation. The absence of switched memory B cells is very

The combined staining for CD19, IgD, CD38, and CD24 allows the identifica-

(CD19+IgD+CD38highCD24neg). CD27 and CD21 enabled study of the CD21low population. The expansion of circulating CD21lowB cells is associated with manifestations

Recently, germline heterozygous gain-of-function mutations in PIK3R1 and PIK3CD genes have been identified to cause a primary immunodeficiency in patients with node lymphoproliferation, splenomegaly, diarrhea, and dysgammaglobulinemia, initially diagnosed with CVID. In these patients, the extended immunophenotyping reveals high percentage of transitional B cells, low pre-switched memory, switched memory B cells, and skewing of CD4+ with CD8+ T cells toward terminally differentiated effector cells. This phenotype is suggestive of activated

tion of transitional B cells (CD19+IgD+CD38highCD24high) and plasmablasts

**32**

*Gating strategy used for the study of subpopulations of B lymphocytes. B-cell populations (naïve, pre-switched, switched memory, and exhausted) are distinguished depending on IgD and CD27 expression. The differing pattern of CD24+ and CD38+ expression identified transitional cells and plasmablasts. CD27 and CD21 markers enabled study of the CD21 low population.*

## **2.3 Innate cells immunophenotyping**

### *2.3.1 Natural killer (NK) cells*

Natural killer (NK) cells are one of the first lines of innate immune defense against pathogens and tumors, among other cells. NK cells are lymphocytes with cytotoxic capacity that provide rapid response to virus-infected cells and to tumor cells through the ability to recognize the absence of HLA class I and other signals on these cells. Innate cells can be analyzed by flow cytometry gating in the CD3<sup>−</sup>CD19<sup>−</sup> cells. Natural killer (NK) cells subpopulations can be divided into two subpopulations depending on CD56 and CD16 expression; NKdim (CD16+ CD56low) and NKbright (CD16−CD56high). Characterizing the different subsets of NK is crucial in the diagnosis of NK cell deficiencies [19]. An example is the deficiency in MCM4, with autosomal recessive inheritance (AR), which may be suspected in the context of a higher percentage of CD56bright NK cells (of total NK cells) in individuals with endocrine and/or growth abnormalities [20].

See the immunophenotype protocol in Section 2.

### *2.3.2 Monocytes and dendritic cells (DC)*

Monocytes are leukocytes that can differentiate into macrophages and myeloid lineage dendritic cells. As a part of the vertebrate innate immune system, monocytes also influence the process of adaptive immunity [21]. There are at least two subpopulations of peripheral monocytes in human blood based on their phenotypic receptors CD16 and CD14; classical monocytes (CD14highCD16<sup>−</sup>) and non-classical monocytes (CD14lowCD16+ ). These cells have been shown to exhibit distinct phenotype and function [22].

Dendritic cells in periphery blood are responsible for the capture and processing antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs, and secrete cytokines to initiate immune responses [23]. Dendritic cells were studied into the population negative for the following markers: CD3, CD14, CD16, CD19, CD20, and CD56. High expression of HLA-DR, CD11c, and CD123 are markers commonly used to identify plasmacytoid DCs (HLA-DR+ CD123+ ) and myeloid DCs (HLA-DR+ CD11c+ ) (**Figure 5**). GATA binding protein 2 (GATA-2) deficiency is an autosomal dominant (AD) immunodeficiency classified in the

### **Figure 5.**

*NK/monocytes/DC dendritic cells (DC) gating strategy. NK cells and monocyte populations were analyzed in the CD3−CD19− gate. NK subpopulations (NKdim and NKbright) were studied using CD56 and CD16 expression markers. The markers CD16 and CD14 were used to identify classical monocytes (CD14+CD16−) and non-classical monocytes (CD16+CD14−). DCs were studied selecting the population negative for the following markers: CD3, CD14, CD16, CD19, CD20, and CD56. High expression of HLA-DR and CD11c and CD123 was used to identify plasmacytoid DCs (HLA-DR+CD123+) and myeloid DCs (HLA-DR+CD11c+).*

group of congenital defects of the number or function of phagocytes in which the following leukocyte populations are affected: CD56bright NK cell, B lymphocytes, monocytes and peripheral dendritic cells populations are affected. Patients with this PID have susceptibility to infections by mycobacteria and human papillomavirus (HPV), histoplasmosis, alveolar proteinosis, myelodysplastic syndromes such acute myelogenous leukemia and lymphedema [24].

See the immunophenotype protocol in Section 2.
