Dynamic changes in peripheral blood lymphocyte subset counts and functions in patients with diffuse large B cell lymphoma during chemotherapy

Abstract

Background: This study aimed to analyze the lymphocyte subsets, their activities and their dynamic changes during immunochemotherapy in patients newly diagnosed with diffuse large B cell lymphoma (DLBCL).

Methods: Patients with DLBCL (n = 33) were included in the present study. Their peripheral lymphocyte subsets, phenotypes and functions were detected using flow cytometry. The dynamic results of lymphocyte activities were available for 18 patients.

Results: Compared with healthy controls (HCs), the counts of CD3⁺, CD4⁺, and CD8⁺ T cells as well as those NK cells decreased in patients newly diagnosed with DLBCL, mainly attributed to patients with high risk of prognosis assessed by International Prognostic Index (IPI) score. Lymphocyte counts didn't present significant difference between high risk (IPI scores 3-5) and low risk patients (IPI scores 0-2), but CD4⁺ T cells and CD8⁺ T cells expressed higher levels of CD28 and HLA-DR, respectively, in patients with IPI score ranging from 3 to 5. Patients at high risk harbored higher percentage of regulatory T cells (Tregs), and their CD4⁺ and CD8⁺ T cells produced lower levels of IFN-γ, reflecting an impaired cellular immune response. The dynamic changes of lymphocyte numbers and functions during treatment were further investigated. Total counts of CD3⁺, CD4⁺, CD8⁺ T and NK cells progressively decreased because of the cytotoxicity of chemotherapy and then gradually recovered after six cycles treatment (rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone, R-CHOP). The functions of CD4⁺ and CD8⁺ T cells recovered by the end of two cycles R-CHOP treatment, although NK cell function was not significantly affected throughout treatment. These results suggest that the counts and functions of lymphocytes are significantly decreased in patients with DLBCL, particularly those of CD4⁺ and CD8⁺ T cells.

Conclusions: The absolute counts and functions of CD4⁺, CD8⁺ T cells, which were significantly lower in patients with DLBCL, gradually recovered after effective treatment. Therefore, combined detection of T cell counts and functions are critically important for administering effective personalized immunotherapy as well as for identifying new prognostic markers or DLBCL.

Keywords: CD4+ T cells; CD8+ T cells; Diffuse large B cell lymphoma; Lymphocyte subsets; NK cells.

Fig. 1

The lymphocyte subsets and immunophenotypic characteristics. Circulating lymphocytes in patients newly diagnosed with DLBCL and healthy controls (HCs) were analyzed using flow cytometry. A, B The percentages and absolute numbers of T cells, B cells and NK cells in different groups are shown in bar graphs (mean ± SD). C The expression of CD28 on CD4⁺ and CD8⁺ T cells, the expression of HLA-DR on CD3⁺ and CD8⁺ T cells, and the expression of CD45RO and CD45RA on CD4⁺ T cells in different groups are shown in bar graphs (mean ± SD). D The percentages of Tregs (CD4⁺ CD25⁺ CD127^low) in lymphocytes are shown in bar graphs (mean ± SD). (E) The percentages of CD69 in NK cells are shown in bar graphs (mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

The subsets and phenotypes of lymphocytes in patients with DLBCL according to IPI scores. Patients with DLBCL were stratified into low- to low-mediate-risk (IPI scores 0–2) and mediate-high- to high-risk (IPI scores 3–5) groups. The absolute counts of CD3⁺ T, CD4⁺ T, CD8⁺ T cells and NK cells (A), the percentages of CD28⁺ cells in CD4⁺ and CD8⁺ T cells (B), the percentages of HLA-DR⁺ cells in CD3⁺ T and CD8⁺ T cells (C), the percentages of CD45RO⁺ and CD45RA⁺ cells in CD4⁺ T cells (D) and percentages of Tregs in lymphocytes are shown in different groups (mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

The IFN-γ producing ability of CD4⁺ T cells, CD8⁺ T cells, and NK cells. PMA/ionomycin-stimulated lymphocyte function assay was performed in patients with DLBCL and HCs. A Representative FACS dot plots showing the production of IFN-γ in CD4⁺ T cells, CD8⁺ T cells and NK cells. B The percentages of IFN-γ⁺ cells in CD4⁺ T, CD8⁺ T cells and NK cells from patients with lymphoma and HCs are shown in bar graphs (mean ± SD). C The percentages of IFN-γ⁺ cells in CD4⁺ T, CD8⁺ T cells and NK cells from patients with low- to low-mediate-risk (IPI scores 0–2) and mediate-high- to high-risk (IPI scores 3–5) are shown in bar graphs (mean ± SD)

Fig. 4

Dynamic changes of CD3⁺ T, CD4⁺ T, CD8⁺ T cells and NK cells and the percentages of Tregs. The lymphocyte subsets in patients with DLBCL were detected at T0, T1, T2 and T3, respectively. All p values were the results of comparison with HCs. (A) Absolute numbers of CD3⁺ T, CD4⁺ T, CD8⁺ T cells and NK cells are shown in bar graphs at different time points (mean ± SD). (B) Percentages of Tregs are shown at different time points

Fig. 5

The dynamic changes of lymphocyte phenotypes. The expression of CD28, HLA-DR, CD45RO and CD45RA were detected at T0, T1, T2 and T3, respectively. The percentages of CD28⁺ cells in CD4⁺ and CD8⁺ T cells (A), the percentages of HLA-DR⁺ cells in CD3⁺ T and CD8⁺ T cells (B), and the percentages of CD45RO⁺ and CD45RA⁺ cells in CD4⁺ T cells (C) in patients during R-CHOP treatment are shown in bar graphs (mean ± SD)

Fig. 6

The dynamic changes of IFN-γ producing ability in CD4⁺ T cells, CD8⁺ T cells, and NK cells. The secretion capability of IFN-γ⁺ from CD4⁺ T, CD8⁺ T cells and NK cells were assessed at T0, T1, T2 and T3, respectively. Percentages of IFN-γ⁺ cells in CD4⁺ T (A), CD8⁺ T (B) and NK (C) cells are shown in bar graphs (mean ± SD)