Rituximab-induced direct inhibition of T-cell activation

Abstract

Background: Rituximab, an anti-CD20 monoclonal antibody, is reported to increase the T-cell-dependent infection risk. The current study was designed to investigate whether rituximab interferes with T-cell activation.

Patients and methods: Patients with non-Hodgkin lymphoma receiving 4-6 courses of 375 mg/m(2) rituximab underwent detailed assessment of T-cell activation pre- and post-rituximab. A similar analysis assessed the in vitro effect of rituximab on T-cell activation in response to allogeneic dendritic cells (allo-DCs) and other stimuli.

Results: Patients receiving rituximab exhibited a significant decline in IL-2 and IFN-γ levels in peripheral blood, most prominent after repeated rituximab courses. Evaluation at 3 months after rituximab therapy showed restoration of inflammatory cytokine production. Similarly, in vitro stimulation of peripheral blood mononuclear cells in the presence of rituximab resulted in a significant decrease in T-cell activation markers, inflammatory cytokine production and proliferative capacity. These effects were also observed using B-cell-depleted T cells (CD3(+)CD25(-)CD19(-)) and were accompanied with disappearance of CD3(+)CD20(dim) T-cell population.

Conclusion: Rituximab administration results in transient, dose-dependent T-cell inactivation. This effect is obtained even in B-cell absence and may increase the infection risk.

Fig. 1

Decrease in T-cell activation following rituximab administration in lymphoma patients. a Freshly isolated PBMCs from blood samples of NHL patients were obtained prior to and immediately after rituximab administration (+6 h). Following activation with PMA and ionomycin in the presence of protein transport inhibitor (GolgiStop™ containing monensin), the expression of intracellular cytokines was examined. The line series plots represent IFN-γ and IL-2 expression in paired samples obtained from 7 patients before and after treatment. Inflammatory cytokine production is shown out of gated CD3⁺ T cells. Expression rate of IFN-γ and IL-2 before and after rituximab administration was significantly different (P < 0.05). b PBMCs were obtained from blood samples of NHL patients treated with repeated doses of rituximab, used as a single agent. Inflammatory cytokines were measured on days 1 (prior to and immediately after rituximab), 7 14 and 120. The dot plots represent IFN-γ and IL-2 expression in T-cell population activated with PMA and ionomycin (one of the 3 evaluated patients)

Fig. 2

Decrease in T-cell activation markers following rituximab addition. PBMCs underwent CD25 depletion, using immunomagnetic beads. The CD25⁻ fraction was stimulated for 6 days with irradiated allogeneic mature DCs in a mixed lymphocyte culture (MLC), resulting in generation of inducible CD25⁺ T-cell population. a Rituximab was added to the culture in increasing concentrations (0.1–2 mg/ml). CD25⁺ T cells were detected with PE-conjugated CD25 monoclonal antibodies following 6 days of culture. The results are presented as a mean percent (±SD, n = 5) of CD25⁺ T cells out of the total number of gated lymphocytes. For negative control, the cells were incubated in parallel with trastuzumab or human IgG in the concentration of 2 mg/ml. b Activation markers were detected in the presence of rituximab or human IgG (2 mg/ml) with PE-conjugated anti-CD25 and anti-CD69, FITC-conjugated anti-CTLA-4, anti-GITR and APC-conjugated anti-CD40L monoclonal antibodies. The bar graphs represent mean expression of activation markers in the culture. The difference in the expression of CD25, GITR, CTLA-4 CD69 and CD40L following addition of rituximab versus IgG was statistically significant (P < 0.05, n = 5)

Fig. 3

Rituximab attenuates T-cell activation in the absence of B cells. CD3⁺CD25⁻CD19⁻ T cells were stimulated for 6 days with irradiated allogeneic mature DCs, in the presence or absence of 2 mg/ml rituximab. Subsequently, the cells were incubated with PMA, ionomycin and in the presence of protein transport inhibitor (GolgiStop™) for 5 h. a In addition to allogeneic DCs, T cells were stimulated with either CD3/CD28 monoclonal antibodies or pp65-CMV recombinant protein. The mean (±SD) proliferation rate is presented as an optical density (OD) output of 3 independent experiments performed using the tetrazolium salt proliferation assay. Each condition was performed in triplicate. Proliferation rate of T cells following rituximab addition was significantly different (P < 0.05). Non-stimulated cells were evaluated as a negative control. b Cells were stained for the inflammatory cytokine expression with PE anti-IL-2, anti-IFN-γ or anti-IL-12. Cells were additionally stained with Alexa Flour 647 antibody for the transcriptional factor T-bet or for the regulatory cytokine with PE anti-TGF-β. The bar graphs represent mean expression of 5 similar independent experiments. The expression rate with and without rituximab addition was significantly different (P < 0.05). c The dot plots represent IFN-γ and IL-2 expression in CD4⁺ and CD8⁺ T-cell populations in the presence or absence of 2 mg/ml rituximab, trastuzumab or IgG (n = 3)

Fig. 4

Characterization of CD3⁺CD20⁺ cell population. a PBMCs from blood samples of NHL patients were obtained prior to and immediately after rituximab administration (+6 h) (upper row). PBMCs from blood samples of various healthy donors were obtained and incubated in vitro with and without addition of 2 mg/ml rituximab for 30 min (lower row). CD3⁺CD20⁺ T-cell populations and CD3⁻CD20⁺ B-cell populations were identified using APC-conjugated CD3 and FITC-conjugated CD20 monoclonal antibodies (dot plot represents one of the 5 independent experiments). b Immunomagnetic CD25, CD19-depleted PBMCs were incubated with or without 2 mg/ml rituximab for 6 days. Cells were double labeled with PI and either FITC-conjugated anti-CD3 antibody (upper row) or APC-conjugated Annexin V (lower row) (dot plot represents one of the 4 independent experiments). c CD3⁺CD20⁺ T cells were enriched using magnetic bead isolation. Intracellular expression of IFN-γ, IL-2, TGF-β and FoxP3 was detected using monoclonal antibodies. The cells underwent flow cytometric analysis using PE-conjugated anti-CD3, CD25, CD69 and CD45RA monoclonal antibodies. The expression is presented out of gated CD20⁺ T cells (representative of 3 independent experiments). d Purified CD3⁺CD19⁻CD20⁻ T cells isolated by FACS sorting from PBMCs of healthy volunteers were incubated with 2 mg/ml rituximab or human IgG for 6 days. Subsequently, rituximab binding to T cells was detected using PE-conjugated goat-anti-human secondary antibody. Percent of goat-anti-human antibodies bound to T cells cultured with rituximab and human IgG was significantly different (n = 4, P < 0.5)