Comprehensive phenotyping of human peripheral blood B lymphocytes in pathological conditions.

Several diseases are associated with alterations of the B-cell compartment. Knowing how to correctly identify by flow cytometry the distribution of B-cell populations in the peripheral blood is important to help in the early diagnosis. In the accompanying article we describe how to identify the different B-cell subsets in the peripheral blood of healthy donors. Here we show a few examples of diseases that cause dysregulation of the B-cell compartment.

INTRODUCTION

Several published reports have reported the use of complex and effective staining for B cell phenotyping [1, 2, 3, 4, 5]. Here we show the method we use to distinguish the B‐cell subpopulations in the peripheral blood of patients with different diseases associated with B‐cell perturbations. B‐cell phenotyping can be useful to address diagnosis and follow‐up patients during therapy. As already described (accepted doi: 10.1002/cyto.a.24507), B cells are identified as CD19pos and can be further classified as transitional, mature‐naïve, memory, atypical memory, activated B cells and plasmablasts according to their maturation stage and function. By flow‐cytometry the different subpopulations can be recognized by the regulated expression of different combinations of cluster of differentiation (CD) markers on their surface, as reported in Table 1.

TABLE 1

Cluster of differentiation expressed on the different B‐cell population

	Transitional	Mature‐Naïve	Memory	Plasmablasts	Atypical memory	Activated memory
CD45	++	++	++	++	++	++
CD19	++	++	++	++	+++	++
CD24	+++	++	+++	−	−/+	+ and ++
CD21	+ and ++	++	++	+	−	−
CD27	−	−	+ and +++	+++	−	++
CD38	+++	+	−	+++	−	−
Igs	IgM+IgD+	IgM+IgD+	IgM+IgD+ IgG+ IgA+ IgM only IgE+	intracellular staining	IgM+IgD+ IgG+ IgM only	IgM+IgD+ IgG+

To identify each B‐cell population we used a combination of nine antibodies (BD, Biosciences) labeled with different fluorochrome: CD45 V500‐C (2D1), CD19 PE‐Cy7 (SJ25C1), CD24 PE (ML5), CD27 APC (L128), CD38 PerCP‐Cy5.5 (HIT2), CD21 BV605 (B‐ly4), IgM FITC (G20‐127), IgG APC‐H7 (G18‐145), and IgD V450 (IA6‐2) (Table 2). Our staining is performed on whole blood following red blood cell lysis. Bulk lysing is performed to lyse the entire blood sample to be stained. Briefly, 500 μL of fresh total peripheral blood (EDTA) are incubated for 10 min at room temperature with 9.5 mL of the lysing solution Pharm Lyse 1X (BD Biosciences) to remove red blood cells. Afterwards, cells are washed twice with 10 mL of phosphate‐buffered saline (PBS) containing 1% of bovine serum albumin (BSA). Two washing steps after lysis are essential because they wash‐off free IgG immunoglobulins in the plasma that, by sequestering the labeled anti‐IgG antibody, prevent the identification of memory B cells expressing IgG on the surface. Cells are re‐suspended in 200 μL of PBS and added to a B‐Cell lyotube (BD, Bioscience). CD21 BV605 is added as a drop‐in. After 20 min incubation at room temperature in the dark, the samples are washed in PBS 1% BSA and finally re‐suspended in 300 μL of PBS 1% BSA. Data are acquired on a BD FACSLyric™ cytometer (BD Biosciences) and analyzed by FlowJo ver. 10.7 (Becton, Dickinson & Company) (See supporting informations).

TABLE 2

Antibodies used for the staining of the peripheral blood and the identification of the B‐cell population

	Fluorochrome	Clone
CD45	V500‐C	2D1
CD19	PE‐Cy7	SJ25C1
CD24	PE	ML5
CD27	APC	L128
CD38	PerCP‐Cy5.5	HIT2
CD21	BV605	B‐ly4
IgM	FITC	G20‐127
IgG	APC‐H7	G18‐145
IgD	V450	IA6‐2

Live cells are identified based on the FSC/SSC lympho‐monocyte gate and then selected as CD45posCD19pos B cells [6]. Briefly, we first discriminate transitional B cells (CD24posCD38bright) and plasmablasts (CD24negCD38bright). CD21 expression separates transitional B cells into CD21dull transitional 1 B cells (T1, corresponding to recent bone marrow emigrants), and CD21brigh transitional 2 (T2). Plasmablasts are CD21neg. We create a not transitional/not plasmablasts gate that includes all other B cell populations. We plot CD27 versus CD21 in order to identify mature‐naïve (CD27negCD21pos), memory (CD27posCD21pos), atypical memory (CD21negCD27neg), and activated memory B cells (CD21negCD27pos). Based on the intensity of CD27, memory B cells can be further divided into CD27dull and CD27bright [7]. CD27dull are the first population of memory B cells that develop in children and can be generated independently of T cells and the germinal center reaction [7, 8], CD27bright cells are detected only when the cooperation between T and B cells is perfectly functional [7]. In the CD27pos memory B‐cell population, we find IgDpos and IgDneg memory B cells. IgDneg memory B cells can be further divided into IgGpos, IgGnegIgMneg (that are mostly IgApos), [9] and IgM‐only [10]. IgDpos memory B cells also express IgM and are called IgM memory B cells (IgMposIgDposCD27pos) [11, 12]. IgM memory B cells are an heterogeneous population composed of innate memory B cells [8] and IgM memory B cells that have been remodeled in the germinal centers [13, 14, 15].

Among atypical memory B cells, IgM, and IgD expression identifies different subtypes. Atypical memory B cells increase during autoimmune disease, aging, and in viral infections [16, 17, 18, 19, 20, 21]. These cells are the product of an extrafollicular or of an impaired germinal center reaction [21, 22].

A correct staining allows to identify the different B‐cell populations and is of importance and utility in the diagnosis of diseases associated with these cells (Table 3).

TABLE 3

Immunodeficiencies with B‐cell alterations [23]

		Circulating B cells	Disturbances of B cell maturation/differentiation steps
SCID B+	X‐linked SCID	Normal	Decreased memory B cells and absence switched memory B cells
	JAK3	Normal or increased	Decreased memory B cells and absence switched memory B cells
	IL7R	Normal or increased	Decreased memory B cells and absence switched memory B cells
	CD45 deficiency	Normal	Decreased switched memory B cells
	CD3δ deficiency	Normal	Decreased switched memory B cells
	CD3ε deficiency	Normal	Decreased switched memory B cells
	CD3ζ deficiency	Normal	Decreased switched memory B cells
	LAT deficiency	Normal	Decreased memory B cells, increased transitional B cells
	IkBa GOF (NFKBIA/IkB)	Elevated B cells	Low memory B cells
SCID B‐	RAG1	Significantly decreased	Not detectable
	RAG2	Significantly decreased	Not detectable
	Artemis deficiency	Significantly decreased	Not detectable
	DNA‐PKcs deficiency	Significantly decreased	Not detectable
	NHEJ1 deficiency	Absent	Not detectable
	DNA ligase IV deficiency	Significantly decreased	Not detectable
	AK2 deficiency (Reticular Dysgenesis)	Decreased	Not detectable
	ADA deficiency	Absent	Not detectable
	BLNK deficiency	Absent	Not detectable
	BTK deficiency	Absent	Not detectable
	IKAROS deficiency	Significantly decreased or absent	B cell acute lymphoblastic leukemia (B‐ALL)
	BAFF receptor deficiency	Decreased	Decreased switched memory B cells
	Wiskott Aldrich syndrome (WAS)	Normal or decreased	Decreased memory B cells and increased transitional B cells
	CVID	Normal or decreased	Absence of memory B cells
	TACI deficiency	Normal or decreased	Decreased switched memory B cells
	ICOS deficiency	Normal (children)/ Decreased (adults)	Decreased switched memory B cells and naive B Cells
	Hyper IgM syndromes	Normal	Absence of switched memory B cells
	IgA deficiency	Normal	Absence of IgA memory B cells
	CD19 deficiency	Normal	Decreased switched memory B cells
	AT (gene ATM/ATM)	Normal	Low naive B cells, transitional B cells and memory B cells, increased atypical memory B cells
	CD40 L deficiency	Normal	Absence of switched memory B cells
	CD40 deficiency	Normal	Absence of switched memory B cells
	Di George syndrome	Normal	Decreased switched memory B cells
	DOCK8 deficiency	Normal	Significant reduction of memory B cells; switched memory B cells low
	Fischer Evans syndrome	Normal	Increased transitional B cells and atypical memory B cells, reduction of memory B cells

MODIFICATION OF B‐CELL SUBSETS IDENTIFIED BY FLOW CYTOMETRY IN PERIPHERAL BLOOD OF PATIENTS WITH IMMUNE DISORDERS

In this brief review we provide selected examples of alterations of the B‐cell phenotype in different diseases.

Bone marrow transplantation

After hematopoietic stem cell transplantation cells of the B lineage first appear in the bone marrow where the early precursors differentiate and proliferate. Around day 30 after transplantation, rare B cells become detectable in the periphery. They are transitional B cells of the CD21dull type (Figure 1A) and very few plasmablasts. At day 60, the frequency of B cells increases. Most of them are still transitional B cells, now both CD21dull and CD21pos. Plasmablasts are measurable indicating the ability of transitional B cells to differentiate into antibody producing cells (Figure 1A). In the following months mature‐naïve B cells start to increase. The reconstitution of the memory B‐cell pool, representing the progressive establishment and increase of antigenic experience, can take years [24].

FIGURE 1

(A–B) FACS plots show the B‐cell reconstitution in one patient after hematopoietic stem cell transplantation and in one patient following anti‐CD20 treatment. In both patients the reconstitution starts from transitional B cells. In (C) we show FACS plots of two SCID patients, one with JAK3 deficiency and the other with CD3ε deficiency. In both patients most B cells are of the transitional and mature‐ naïve B‐cell type and the memory B cells are strongly reduced [Color figure can be viewed at wileyonlinelibrary.com]

Anti‐CD20 treatment

CD20 is a marker expressed on cells of the B lineage first at the pre‐B cell stage, increased in mature‐naive and memory B cells and down‐regulated in plasmablasts and plasma cells. Since the FDA approval in 1997, anti‐CD20 monoclonal antibodies have been successfully used in the management of B‐cell lymphoma and to deplete the B‐cell compartment in inflammatory and autoimmune diseases [25] . Anti‐CD20 monoclonal antibodies can directly and transiently deplete all the CD20+ B‐cells, including transitional, mature‐naïve, and memory B‐cell subsets, but can also indirectly eliminate short‐lived plasma cells, which lack CD20 expression but have a limited lifespan. Peripheral CD20+ B cells usually tend to reappear after 6 months, starting with transitional and mature‐naïve B cells that normalize within 12 months, whereas memory B‐cell subsets remain significantly reduced for more than 2 years from anti‐CD20 administration [26, 27, 28, 29]. Of note, the time of the B‐cell subset reappearance is comparable following treatment with anti‐CD20 monoclonal antibodies directed against different epitopes of CD20 such as rituximab and ofatumumab [30].

In order to evaluate the duration of B‐cell depletion and follow‐up the reconstitution of the B‐cell compartment in treated patients, a combined B and T cell staining has been proposed [31]. Our staining can be also used to monitor B cell depletion and regeneration (Figure 1B). Here we show the phenotype of peripheral B cells in the same individual before and up to 15 months after one single anti‐CD20 monoclonal antibody dose. The patient was a 13 year‐old child with steroid‐dependent nephrotic syndrome. As after hematopoietic stem cell transplantation, the reconstitution starts from transitional B cells. Also in this case, the regeneration of the B‐cell memory pool is very slow.

Severe combined immunodeficiency

Severe combined immunodeficiency (SCID) includes a group of rare disorders caused by mutations in different genes involved in the development and function of the immune system. Based on the immunological phenotype and the underlying genetic defect, different types of SCID can be classified (Table 3): without T cells but with B cells (T‐B+ SCID) or SCID without T and B cells (T‐B− SCID). Both of these groups include several forms with or without natural killer (NK) cells [32].

SCID typically presents in infancy with early‐onset severe respiratory infections and failure to thrive and, without therapy, leads to death within the first year of life. Hematopoietic stem cell transplantation is the curative treatment. In Figure 1B, we show two examples of B‐cell development in JAK3 and CD3ε deficiency, two autosomal recessive forms of SCID [32].

The first patient, a 1‐year‐old boy, had a mutation of the JAK3 tyrosine kinase that is indispensable for signals generated by all interleukin receptors containing the gamma chain (IL‐2, IL‐4, IL‐7, IL‐9, IL‐15, and IL‐21) [33]. As IL‐7 is indispensable for the development of T cells and IL‐15 is necessary for the production of NK cells, patients with JAK3 mutations have no T and NK cells. IL‐7, which regulates B cell development in the mouse, does not have this function in humans. The mutation abolishes the T and NK cell compartments, whereas B cells are generated in the bone marrow, resulting in a T‐B+NK− phenotype. In the peripheral blood most B cells are of the transitional and mature B‐cell type. Memory B cells are all CD27dull (Figure 1C) and only express IgM [7, 8].

The T‐cell antigen receptor (TCR) and pre‐TCR complexes are composed of multiple signal‐transducing subunits (CD3γ, CD3δ, CD3ε, and ζ). Signaling by the TCR and its precursor, the pre‐TCR, are required for T‐cell development and selection. Patients with CD3ε deficiency have no T cells, but B and NK cells are in normal or increased numbers (T‐B+NK+ phenotype) [32].

Also in this patient, analyzed and diagnosed at 8 months of life, most of the B cells have the transitional or mature‐naïve phenotype (Figure 1C). Also in this patient, the few memory B cells developing in absence of T cells are CD27dull IgM memory B cells [7, 8].

Common variable immunodeficiency

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency (PID). It is characterized by hypogammaglobulinemia and impaired production of specific antibodies. Patients with CVID present a broad range of clinical manifestations, including recurrent bacterial infections, autoimmunity, interstitial lung disease, enteropathy, lymphoproliferation, malignancy, and allergic diseases [34]. In most cases the genetic cause of disease remains unknown, but monogenic disorders [35] and epigenetic modifications have been identified as responsible for CVID [36, 37]. Since CVID includes a heterogeneous group of primary antibody immunodeficiencies with various clinical and immunological features, in recent years, several classifications have been proposed including a discrimination based on the phenotype of B‐cell populations [38]. In Figure 2A we show the B‐cell phenotype of a 14 years old CVID patient with a significant reduction of transitional and mature‐naive B cells and an increased frequency of atypical memory B cells. In this particular patient all memory B cells are CD27dull suggesting an impairment of the germinal center reaction [7]. CD27dull memory B cells are either IgMposIgDpos or IgM only with a complete absence of switched memory B cells thus confirming the inability of this patient to carry on and complete the germinal center reaction [7, 8]. The absence or the significant reduction of memory B cells is an indicator of a subgroup of CVID patients with a severe prognosis [39], and predicts clinical prognosis more accurately than serum immunoglobulin concentrations [40]. In this regard it is interesting to note that the reduction of memory B cells might be a predictive marker of outcome in hypogammaglobulinemia during infancy [41].

FIGURE 2

FACS plots show the B‐cell population in four examples of immune disorders. CVID (A) and ataxia‐telangiectasia (B) patients have both a significant reduction of transitional B cells and mature‐naïve B cells and a higher frequency of atypical memory B cells. The CVID patient only has few CD27dull memory B cells that express either IgD and IgM or IgM only, thus lacking switched memory B cells. Patients with Fisher‐Evans Syndrome (C) and with Wiskott Aldrich syndrome (D) present a significantly higher number of transitional B cells, an increased frequency of atypical memory B cells and a strong reduction of memory B cells [Color figure can be viewed at wileyonlinelibrary.com]

Ataxia telangiectasia

Ataxia telangiectasia (AT) is an autosomal‐recessive primary immunodeficiency disorder caused by genetic mutation in the ataxia telangiectasia mutated (ATM) gene, located on chromosome 11q22‐23, which is involved in the recognition and repair of damaged. AT is characterized by ataxia, neurologic decline, oculocutaneous telangiectasia, a wide range of immune function abnormalities [42, 43] and increased risk of developing various types of cancer [44]. AT patients present with recurrent viral and bacterial infections, which cause significant morbidity and are a frequent factor or cause of death [45]. Signs of T and B immunodeficiency of variable severity have been reported: decrease of naïve CD4+ and CD8+ T lymphocytes, increase of NK cells and reduced numbers of B cells [46, 47]. Among the dysfunctions in immunoglobulin production, increased levels of IgM, reduction of IgG, IgA deficiency, and lack of response to polysaccharide antigens are the most frequent [46]. DNA repair activity is indispensable in the bone marrow for immunoglobulin recombination and in the germinal centers for the mechanisms of somatic mutation and class switch recombination. All these processes are severely impaired in AT [47]. The FACS‐plots of a 18‐year‐old AT patient are depicted in Figure 2B. The reduction of transitional and naive B cells is the consequence of the poor production of B cells in the bone marrow due to the inefficient process of immunoglobulin rearrangement, whereas the low number of switched memory B cells and the increased frequency of atypical memory B cells can be explained by the impairment of somatic mutation and class‐switch recombination in the germinal centers [48].

Fisher‐Evans'syndrome

Fisher‐Evans'syndrome (ES) is defined as the concomitant or sequential association of auto‐immune haemolytic anemia (AIHA) with immune thrombocytopenia (ITP), and less frequently autoimmune neutropenia. ES is a rare situation that represents up to 7% of AIHA and around 2% of ITP [49, 50]. The determination of the primary or secondary nature of ES is important, since the association of this syndrome with other diseases, such as hematological malignancies, systemic lupus erythematosus, infections, or primary immune deficiencies (PID) can interfere with the management or alter the prognosis [50]. Inborn errors of immunity were reported in about 70% of ES patients and recently the new sequencing techniques allowed the discovery of several novel mutations [51, 52]. In these patients specific immunophenotypic characteristics were identified, such as T and B lymphopenia, decrease in naїve T cells, switched memory B cells, plasmablasts, and/or immunoglobulins, increase in effector/central memory T‐cells and increase of CD21low atypical B memory cells [53]. In Figure 2C we show the immune phenotype of a 17‐year‐old patient with ES characterized by high frequencies of transitional B cells, that are all CD27dull, and of atypical memory B cells, and a significant reduction of memory B cells.

Wiskott–Aldrich syndrome

Wiskott–Aldrich syndrome (WAS) is an X‐linked disease caused by mutations in the WAS gene characterized by a triad of thrombocytopenia, eczema, and immunodeficiency [54]. Without curative therapies, affected patients have diminished life expectancy and reduced quality of life. The WAS protein (WASP) is constitutively expressed in all hematopoietic stem cell‐derived lineages and is located predominantly in the cytoplasm with the highest protein expression toward the cell membrane [55]. WASP is a member of a distinct family of proteins that link signaling pathways to actin cytoskeleton reorganization and localize in the immunologic synapse [56], the site of the interaction between various leukocyte subpopulations. The immunological synapse allows the physical contact between antigen presenting cells, T and B cells and it is required for an adaptive immune response, including class switch recombination and somatic hypermutation [55, 57]. WAS is associated with autoimmunity and increased incidence of hematologic malignancies, especially B‐cell lymphomas. Affected patients are susceptible to bacterial, viral, and fungal infections, secondary to defects in both cellular and humoral immunity. In the Figure 2D the B‐cell phenotype of a 6‐year‐old child shows a prevalence of transitional B cells and atypical memory B cells associated with a reduction of the memory pool. The altered distribution of the B‐cell populations might be explained by the impairment of adaptive immune responses in the germinal centers.

Plasmodium falciparum infection

B cells and antibodies are critical for control of parasitemia in malaria, caused by P. falciparum infection. Antibodies also provide protection to reinfection. However, it has been demonstrated that in the absence of constant re‐exposure, Plasmodium‐specific serum antibody levels rapidly decrease, and full protection from clinical symptoms is lost, suggesting that B‐cell memory is functionally impaired [58]. Some studies attribute this to the expansion of atypical memory B cells, which express multiple inhibitory receptors and activation markers, and are hyporesponsive to B‐cell receptor (BCR) restimulation in vitro [59]. In Figure 3 the B‐cell phenotype of a 3‐year‐old child diagnosed with P. falciparum malaria shows expansion of plasmablasts, transitional B cells and atypical memory B cells in the peripheral blood. The phenotype of this patient is also characterized by the absence of IgGpos and IgApos switched memory B cells.

FIGURE 3

FACS plots showing the B‐cell population in a patient with malaria. This patient shows an increased frequency of transitional B cells, plasmablasts and atypical memory B cells. All CD27posIgDneg memory B cells are IgM‐only [Color figure can be viewed at wileyonlinelibrary.com]

In conclusion, our staining identifies different B‐cell populations that have distinct phenotype and precise functions. A proper B‐cell flow cytometric analysis, that correctly identifies the different B‐cell populations in the peripheral blood, may address the diagnosis in immunodeficiency and be useful in the follow‐up in different clinical conditions.

AUTHOR CONTRIBUTIONS

Francesco Corrente: Formal analysis (equal); methodology (equal). Claudia Capponi: Formal analysis (equal); methodology (equal). Mattia Mirabella: Methodology (equal). Simona Cascioli: Methodology (equal). Patrizia Palomba: Methodology (equal). Valentina Bertaina: Methodology (equal). Manuela Colucci: Formal analysis (equal); methodology (equal).

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/cyto.a.24518.

Appendix S1. Supporting Information.

Minimum Information About A Flow Cytometry Experiment (MIFlowCyt) Checklist

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Figure S1. TSNE and FLOWSOM.

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