**3. The effect of the splenectomy on humoral immunity in patients with gastric cancer**

An analysis of the B cell subpopulation was performed in the gate of CD19+ cells. At the first stage of the analysis, the number of CD19+ B cells within the lymphocytes was cytometrically estimated (**Figure 1a**). Further, within the limits of only B cells [gate CD19+, **Figure 1b**], the expression of two antigens associated with B cells was analyzed. In these samples, there is an estimate of CD56 antigens concurrently with CD21 (**Figure 1c**).

The main indices of peripheral blood samples before and after surgery are given in **Table 2**. In general, in the group before the surgical intervention in comparison with the physiological norm, the average indices of both the relative quantity and the absolute content of B cells fit within the limits of the norm.

In 33% of patients with gastric cancer at the preoperative period, a decrease in the relative number of B cells (less than 5%) were detected. Moreover in 38% of patients decrease in the absolute number were revealed. Three months after the operation, in 52% of cases, the relative number of B cells was reduced, in 31% of the absolute content of B cells.

The average relative number of B cells, as well as their mean absolute content in 1 μl of peripheral blood, was slightly elevated 3 months after surgery in comparison with these parameters in preoperative assessment. The average percentage of B lymphocytes after the operation was slightly lower than before surgery. However, these differences were statistically unreliable (T-test when comparing two independent variables). This can be explained by a significant spread of the analyzed indicators both in patients before and after surgery.

### **3.1 B2 cells**

*Normal and Malignant B-Cell*

**2. Materials and methods**

ics (3 months after the operation).

tion (12 people).

absolute number of B cells.

To study the peculiarities of the B cell link in patients with gastric cancer after splenectomy, the subpopulation composition of B lymphocytes was analyzed. The study included two groups of patients who underwent surgery. The first group of patients received treatment in the volume of gastrectomy with spleen-protective D2 lymphodissection. In the second group, patients underwent gastrectomy with D2 lymphodissection and splenectomy. The study of subpopulations of B lymphocytes was carried out at the preoperative stage and 3 months after the surgical treatment.

We studied the subpopulation composition of B lymphocytes of peripheral blood in patients with gastric cancer. The possible influence of splenectomy on the subpopulation composition of B lymphocytes was investigated. The B cell link of peripheral blood lymphocytes in patients with gastric cancer was studied in dynam-

To assess the effect of the splenectomy on the B cell link, the subpopulation composition of B cells of peripheral blood after surgery was assessed both in patients after gastrectomy with standard D2 lymphodissection (splenectomy) (14 people) and in patients after gastrectomy with spleen-protective D2 lymphodissec-

The reaction was taken into account on flow cytometers (FACScan, Lysys II and FACSCanto II, FACSDiva program). Data processing: WinMDI 2.8 and FCS 3.0 applications. Evaluation of the expression of membrane antigens was carried out in the gate of CD19+ B cells. Cells were stained at the same time with three monoclonal antibodies labeled with different fluorochromes. In 42 peripheral blood samples before the operation and 23 samples after the operation on the hematological analyzer Micros 60, the hemogram was calculated, which allowed to estimate the

The expression of the following antigens is analyzed: CD20, CD21, CD23, CD38,

**Antigen Antibody clone Antibody isotype Manufacturer Fluorochrome**

HLA-DR, CD71, CD10, CD95, CD25, CD5, CD56, IgG-λ, and IgG-κ light chain immunoglobulins (**Table 1**). Direct conjugates of monoclonal antibodies with fluorochromes were used: FITC, fluorescein; PE, phycoerythrin; PerCP, peridinin

CD19 4G7, HIB19 IgG1-κ BD Biosciences PerCP CD20 2H7 IgG1-κ BD Biosciences PE CD23 LT23 IgG1-κ Sorbent, Russia FITC CD5 L17F12 IgG2а-κ BD Biosciences PE CD21 BL13 IgG1-κ BD Biosciences FITC CD10 HL10a IgG2а-κ BD Biosciences PE CD71 L01.1 IgG2а-κ BD Biosciences FITC CD95 DX2 IgG1-κ BD Biosciences PE CD25 2A3 IgG1-κ BD Biosciences FITC CD56 NCAM 16.2 IgG2а-κ BD Biosciences PE CD3 SK7 IgG1-κ BD Biosciences FITC IgG-λ/IgG-κ — — BD Biosciences PE/FITC

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**Table 1.**

*Monoclonal antibodies and antigens.*

Investigation of subpopulations of peripheral blood B lymphocytes in patients with gastric cancer at the preoperative stage established that all B cells had immunophenotype of naïve mature B2 cells: CD19+ CD20+ HLA-DR+ CD10-CD21low/+. The data of subpopulation composition of B lymphocytes of peripheral blood of gastric cancer patients are given in **Table 3**. B2 cells have a long life, localized mainly

#### **Figure 1.**

*(a) Estimating the total number of CD19+ B cells within the lymphocytes. The abscissa is the expression of pan-B cell antigen CD19+; along the ordinate axis—the parameter of lateral light scattering of the laser beam, reflecting the working cytometric concept—the granularity of the cells. (b) Cells that fall only in the region of CD19+, i.e., B cells accumulated during sample collection. (c) Evaluation of the expression of two antigens associated with B cells in the CD19+CD56 and CD21.*


#### **Table 2.**

*Parameters of peripheral blood of the general group of patients.*

in the spleen, bone marrow, lymph nodes, Peyer's patches, and individual follicles of the lymphoid tissue of the intestine. The histological unit, which is the site of the concentration of B2 cells, is the lymphoid follicle. These cells constitute the vast majority of circulating B lymphocytes. B2 cells undergo selection in the bone marrow and participate in the formation of an adaptive humoral immune response to thymus-dependent antigens.

Some patients noted the presence of transient T2B and T3B cells. They are characterized by pronounced expression of CD23 and CD21. The number of CD23+ cells ranged from 25 to 40% in different patients. In this case, as a rule, CD23+ B cells had a weaker expression of the antigen of mature B cells of CD20. **Figure 2a** shows samples of peripheral blood with a pronounced proportion of CD23+ B cells. In 40% of cases, CD23 coexpression was more than 25%, and in 22.4% of patients, the number of CD23+ B cells exceeded 40% of B cells. Some patients have an insignificant number of CD23+ B cells (6%) (**Figure 2b**).

Transient (immature) B cells were first characterized in mice [41]. Immature B cells migrate from the bone marrow to the spleen. Throughout their migration to the spleen and after spleen entry, they are considered T1 B cells; within the spleen, T1 B cells transition to T2 B cells. Under normal conditions, CD19+CD21lowCD23 B cells (T1-transient) pass positive selection in the course of B cell ontogeny: cells receive a signal through the B cell receptor without the participation of any


**139**

**Figure 3.**

*Splenectomy in Gastric Cancer: Influence of B Lymphocytes*

antigens, which ensures their survival. This process can occur both in the bone marrow and in the spleen [42, 43]. In the evaluation of subpopulations of peripheral blood B cells in patients with gastric cancer before surgery treatment, a significant number of B cells with a low level of CD21+ expression were detected [area R2, **Figures 3** and **4**]. This phenotype corresponds to the T1 transient stage of B cell development. Expression of CD21 antigen was characteristic of a larger number of

*Expression of CD23 antigen on peripheral B cells in cancer patients'stomach. (*а*) The population of CD20+CD23+ is the majority (59%) of B cells and represents as a separate discrete population. (b)* 

The selection of transient T2B and T3B cells, characterized by the expressed expression of CD23 and CD21 antigens, passes in the peripheral lymphoid organs (the spleen, lymph nodes) high levels of IgM, IgD, and CD23 and lower levels of CD21. The expressed expression of CD23 antigen is intrinsic to the B cells of the embryonic center of the follicle. The follicular B cells that make up the majority and B cells of the marginal zone are lining outside the marginal sinus and bordering the red pulp. Follicular B cells also express high levels of IgM, IgD, and CD23 and lower levels of CD21. These cells are absent in CD1 or CD5, which distinguishes them from

*DOI: http://dx.doi.org/10.5772/intechopen.80075*

B cells and averaged 82%.

*Insignificant number of CD23+ B cells.*

**Figure 2.**

B1 cells and B cells of the marginal zone.

*Тhe population of CD19+CD21low B cells (region R2).*

#### **Table 3.**

*Subpopulations of B lymphocytes of peripheral blood in patients' stomach cancer before surgery.*

*Splenectomy in Gastric Cancer: Influence of B Lymphocytes DOI: http://dx.doi.org/10.5772/intechopen.80075*

**Figure 2.**

*Normal and Malignant B-Cell*

Leucocytes (×103

**Table 2.**

to thymus-dependent antigens.

cant number of CD23+ B cells (6%) (**Figure 2b**).

CD19+CD5+ 14.2 ± 1.91 9.8 n = 46 CD19+CD23+ 24.5 ± 2.4 20.5 n = 49 CD19+CD20+ 97.2 ± 0.4 97.5 n = 48 CD19+CD71+ 9.6 ± 1.1 7.7 n = 48 CD19+CD10+ 0.48 ± 0.09 0.2 n = 45 CD19+CD38+ 19.8 ± 2.9 11.1 n = 45 CD19+HLA-DR+ 98.6 ± 0.3 99.1 n = 48 CD19+CD25+ 2.1 ± 0.82 0.5 n = 40 CD19+CD95+ 9.6 ± 1.96 7.8 n = 31 CD19+CD21+ 80.6 ± 1.43 82.1 n = 43

*Subpopulations of B lymphocytes of peripheral blood in patients' stomach cancer before surgery.*

*Parameters of peripheral blood of the general group of patients.*

in the spleen, bone marrow, lymph nodes, Peyer's patches, and individual follicles of the lymphoid tissue of the intestine. The histological unit, which is the site of the concentration of B2 cells, is the lymphoid follicle. These cells constitute the vast majority of circulating B lymphocytes. B2 cells undergo selection in the bone marrow and participate in the formation of an adaptive humoral immune response

Lymphocytes (%) 30.1 ± 1.8 n = 43 30.0 36.9 ± 3.06 n = 24 35.5 В lymphocytes (CD19+) % 6.44 ± 0.51 n = 48 5.5 6.0 ± 0.57 n = 28 4.8 В leucocytes (CD19+) % 2.12 ± 0.2 n = 42 2.04 2.5 ± 0.4 n = 22 1.86 В cells (CD19+)/μL 122.5 ± 10.8 n = 42 110.0 156.4 ± 21.5 n = 22 131.0

**Before operation After the operation**

/μL) 6.3 ± 0.34 n = 43 5.9 6.76 ± 0.49 n = 24 6.4

**Average Median Average Median**

Some patients noted the presence of transient T2B and T3B cells. They are characterized by pronounced expression of CD23 and CD21. The number of CD23+ cells ranged from 25 to 40% in different patients. In this case, as a rule, CD23+ B cells had a weaker expression of the antigen of mature B cells of CD20. **Figure 2a** shows samples of peripheral blood with a pronounced proportion of CD23+ B cells. In 40% of cases, CD23 coexpression was more than 25%, and in 22.4% of patients, the number of CD23+ B cells exceeded 40% of B cells. Some patients have an insignifi-

Transient (immature) B cells were first characterized in mice [41]. Immature B cells migrate from the bone marrow to the spleen. Throughout their migration to the spleen and after spleen entry, they are considered T1 B cells; within the spleen, T1 B cells transition to T2 B cells. Under normal conditions, CD19+CD21lowCD23 B cells (T1-transient) pass positive selection in the course of B cell ontogeny: cells receive a signal through the B cell receptor without the participation of any

**M ± m Median**

2.7 ± 2.0 0.15 n = 40, 1.5–2.0 n = 29, 1.0–1.3 n = 12

**138**

**Table 3.**

CD19+CD56+ (IgG-κ+/IgG-λ+)

*Expression of CD23 antigen on peripheral B cells in cancer patients'stomach. (*а*) The population of CD20+CD23+ is the majority (59%) of B cells and represents as a separate discrete population. (b) Insignificant number of CD23+ B cells.*

antigens, which ensures their survival. This process can occur both in the bone marrow and in the spleen [42, 43]. In the evaluation of subpopulations of peripheral blood B cells in patients with gastric cancer before surgery treatment, a significant number of B cells with a low level of CD21+ expression were detected [area R2, **Figures 3** and **4**]. This phenotype corresponds to the T1 transient stage of B cell development. Expression of CD21 antigen was characteristic of a larger number of B cells and averaged 82%.

The selection of transient T2B and T3B cells, characterized by the expressed expression of CD23 and CD21 antigens, passes in the peripheral lymphoid organs (the spleen, lymph nodes) high levels of IgM, IgD, and CD23 and lower levels of CD21. The expressed expression of CD23 antigen is intrinsic to the B cells of the embryonic center of the follicle. The follicular B cells that make up the majority and B cells of the marginal zone are lining outside the marginal sinus and bordering the red pulp. Follicular B cells also express high levels of IgM, IgD, and CD23 and lower levels of CD21. These cells are absent in CD1 or CD5, which distinguishes them from B1 cells and B cells of the marginal zone.

**Figure 3.** *Тhe population of CD19+CD21low B cells (region R2).*

#### **Figure 4.**

*Expression of CD21 antigen on B cells of blood of patient with stomach cancer. The abscissa axis is the expression of CD21; on the ordinate axis—CD19 expression, in the figure all cells of the sample, including granulocytes. Area R2, CD19+CD21+ cells, this region is located above the granulocytes, which indicates a weak expression of CD21 B cells*.

Thus, in patients with gastric cancer, a violation of the immunological repertoire of B2 cells was established: the presence of a pronounced proportion of CD21+ B cells with weak expression, a significant number of CD23+ cells, and cases of clonal B cells. In most of the studied samples, B cells were polyclonal with a predominance of Ig-κ (**Figure 5**). When the thymus-dependent pathway of the immune response is realized, these patients will have a disruption in the synthesis of antibodies.

It is known that a B cell can be activated without the involvement of a T lymphocyte, which leads to the full development of B lymphocytes to antibody-producing forms. It is a characteristic that in this case the immunoglobulin M is synthesized predominantly [5–7]. The process of activation of B cells without T lymphocyte is provided by thymus-independent antigens of type I and II [4]. They are lipopolysaccharides, polysaccharides, and proteins.

Antibodies of IgM class are the earliest in immunogenesis and make up about 6% of all immunoglobulins; their half-life is 5–6.5 days. Approximately half of the

**141**

*Splenectomy in Gastric Cancer: Influence of B Lymphocytes*

ing the affinity for antigens do not affect or minimize B1 cells.

serum IgM is secreted by a pool of B1 lymphocytes. Many of the antibodies are polyspecific, i.e., are able to interact with several antigens, including autologous ones. These antibodies have a low affinity for antigens, including autoantigens, and are not capable of causing damage to tissues. B1 cells are constantly circulating between the spleen and the abdominal cavity but do not enter the follicles, since they do not express the chemokine receptor. Therefore, the processes of "improving" the humoral immune response in the form of switching isotypes and increas-

The population of B1 cells is the most phylogenetically ancient branch of antibody producing cells found in humans and mice. B1 lymphocytes develop in the liver of the fetus from progenitor cells. The B1 precursor, which differs from the B-line progenitor that develops primarily in the B2 population, is identified in the murine bone marrow to a lesser degree in the adult bone marrow [34]. B1 cells are considered as key players of the early humoral response against pathogens and considered primary antibody producers in response to T cell-independent type 2 antigens [44]. A specific thymus-independent response is realized by two subpopu-

Both subpopulations of B1 cells are characterized by an "activated phenotype," which is manifested in the expression on their surface of costimulatory molecules CD80 and CD86. This property provides the ability of B1 lymphocytes to function as antigen-presenting cells. B1b cells are phenotypically similar to B1a cells, but they are characterized by the absence of CD5 expression. B1b lymphocytes realize an adaptive immune response, and B1a lymphocytes produce natural antibodies that are specific to microbial agents and opsonize pathogen-mediated innate immunity

Such a B1a population of lymphocytes has the phenotype CD19+CD21lowCD23-

CD5+IgM++. A number of experimental studies have revealed that immunity disorders after splenectomy primarily affect the B cell immune response, including thymus-independent (TN) type 2 antigens, which is provided by the population of

Among B lymphocytes of peripheral blood of patients with gastric cancer, expressed subpopulation of CD5+ cells was noted. The number of CD19+CD5+ B cells averaged 17.7%. In 23% of patients, CD19+CD5+ lymphocytes were more than 20%, and in three patients, more than 40% of CD19+CD5+ lymphocytes were detected. Normally, the population of CD19+CD5+ B cells is no more than 10% of the total number of peripheral blood B lymphocytes. Among the B cells of the group with a higher CD19+CD5+ B lymphocyte count (more than 15%), the percentage of cells expressing CD25+ and CD21+ antigens was significantly higher; differences in the number of CD38+ B cells were found to be significant, but the range of values for one group of samples with respect to this antigen was higher. The

The presence of CD38 and CD25 antigens on circulating peripheral B cells indicates their activation. It is possible in coexpression of CD23 antigen, which was observed in a group of samples containing more than 25% of B1a lymphocytes.

A high probability of the presence of the CD19+CD23+CD38+CD5+ population (the presence of a reliable correlation relationship for B cells with the expression of CD38 and CD23 antigens, R = 0.885, p = 0.008) was established. There was also a high probability of the presence of the CD19+CD5+CD25+ population (significant

*DOI: http://dx.doi.org/10.5772/intechopen.80075*

**3.2 B1 cells**

[45–47].

lations of B1 cells: B1a and B1b.

B1a lymphocytes [31, 32].

data are presented in **Table 4**.

**Figure 5.** *Polyclonality of B cells with predominance of Ig-κ.*

serum IgM is secreted by a pool of B1 lymphocytes. Many of the antibodies are polyspecific, i.e., are able to interact with several antigens, including autologous ones. These antibodies have a low affinity for antigens, including autoantigens, and are not capable of causing damage to tissues. B1 cells are constantly circulating between the spleen and the abdominal cavity but do not enter the follicles, since they do not express the chemokine receptor. Therefore, the processes of "improving" the humoral immune response in the form of switching isotypes and increasing the affinity for antigens do not affect or minimize B1 cells.
