Expression of the scavenger receptor CD36 on lymphocytes is intriguing. We observed that a minor subpopulation of lymphocytes expressed CD36 on the cell surface. We investigated the source of CD36 and also the proliferation and cytokine production of these CD36⁺ CD4⁺ lymphocytes. Flow cytometry analysis and immunofluorescence microscopy showed that CD36⁺ platelets were responsible for CD36 detection on lymphocytes. CD36 was then used as a tool to characterize lymphocytes with bound platelets. Activation-induced proliferation was lower in CD4⁺ lymphocytes with bound platelets than lymphocytes without bound platelets. IL-17 and IFN-γ production was also reduced in lymphocytes with bound platelets. We then studied the presence of CD36⁺ CD4⁺ lymphocytes in RA patients. We observed that the percentage of CD4⁺ lymphocytes with bound platelets was higher on RA patients than in healthy donors. RA patients with higher titers of anti-CCP, RF levels, and cardiovascular risk index presented a lower percentage of CD4⁺ lymphocytes with bound platelets. These patients also had higher IL-17 and IFN-γ production. These results suggest that platelet-binding modifies lymphocyte function. This binding could be a regulatory mechanism in RA that confers a less severe phenotype.

Soluble factors released from platelets can modulate the immune response of leukocytes. We and others have recently found that T lymphocytes with bound platelets have reduced proliferation and IFN-γ and IL-17 production. Thus, we speculate that if we induce the binding of platelets to lymphocytes, we will be able to regulate the inflammatory response. When we cocultured platelets with lymphocytes at different ratios, we were able to increase the percentage of lymphocytes with bound platelets. The coculture of platelets with lymphocytes in the presence of stimulation decreased the production of IFN-γ and TNF-α, T cell proliferation, and the expression of CD25, PD-L1, and SLAM. However, this coculture increased CD39 expression. All of these effects were dependent on the dose of platelets and operated indistinctly with platelets from different healthy donors. When platelets were cocultured in the same compartment with lymphocytes, we observed less IFN-γ and TNF-α production and T lymphocyte proliferation than in cultures with platelets separated from lymphocytes by a 0.4-μm pore size filter. The binding of platelets to lymphocytes was blocked with anti–P-selectin Abs, and when this occurred we observed higher IFN-γ and TNF-α production than in nonblocked conditions. The cocultures of platelets with synovial fluid cells from rheumatoid arthritis patients reduced inflammatory cytokine production and increased IL-10 production. These results suggest that platelet binding to lymphocytes effectively regulates T lymphocyte function. This mechanism could be easily applied to reduce inflammatory responses.

Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5±2.9%) whereas 32.8±6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4±7.5% and 57.1±5.5%; Ficoll: 29.5±2.2% and 5.4±4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5±0.4 and Ficoll: MFI 3.3±0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2±4.5% and Ficoll: 55±4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured.

Cytotoxic T lymphocyte (CTLs) activation is an independent predictor of response to neoadjuvant chemotherapy (NACT) in breast cancer (BC) patients. Here, we go deeper into the function of CD8+ HLA-DR+ T cells from NACT treated HER2 negative BC patients. Flow cytometry analysis revealed that CD8+ HLA-DR+ T cell percentage was increased in NACT responder (R) compared to non-responder (NR) patients. R patients with ER-/PR- hormone receptors had the highest CD8+ HLA-DR+ T cell frequencies, while no differences were found when patients were classified according to cancer stage or menopause status. Interestingly, the cytotoxicity and production of anti-tumor cytokines were enhanced when CD8+ HLA-DR+ T cells from healthy donors were cultured with plasma from R, but not from NR patients. The induced anti-tumor profile of CD8+ HLA-DR+ T cells was associated with plasmatic IL-12 and IFN-γ levels, increased cytokines in R patients. IL-12 or IFN-γ neutralization decreased cytotoxic activity and TNF-α production by cultured CD8+ HLA-DR+ T cells in R plasma presence. All these data suggest that an effective response to NACT in BC patients is associated with increased IL-12 or IFN-γ levels involved in the induction of cytotoxic and pro-inflammatory mechanisms in CD8+ HLA-DR+ T cells.

Introduction: Adalimumab is a fully human anti–tumor necrosis factor α (anti-TNFα) monoclonal antibody that specifically blocks the interaction of TNFα with its receptors. It binds both soluble and transmembrane TNFα. We hypothesized that blocking these TNFα signals regulates the altered TNFα production in rheumatoid arthritis (RA) patients. Methods: We compared, by flow cytometry, Toll-like receptor induction levels of membrane and intracellular TNFα in monocytes (iTNFα + CD14+ cells) from 12 patients before and after adalimumab treatment with those from 5 healthy donors. Results: Before starting the treatment, the percentage of iTNFα+ CD14+ cells in the RA patients was significantly lower than that in healthy donors (mean ± SEM = 33.16 ± 4.82% vs 66.51 ± 2.4%, P < 0.001). When we added in vitro TNFα to healthy donor culture cells, levels of iTNFα+ CD14+ cells decreased, suggesting that the TNFα signal was responsible for the iTNFα+ CD14+ cell downregulation observed in the RA patients. After 2, 6 and 12 adalimumab injections, we observed significant blocking of membrane and soluble TNFα and a progressive increase in iTNFα+ CD14+ cells in ten patients with a good to moderate response as defined by the European League Against Rheumatism (EULAR) criteria. Levels of iTNFα+ CD14+ cells after 12 injections in these 10 patients were comparable to levels in healthy donors. In two patients, iTNFα+ CD14+ cell upregulation was not observed, and their EULAR-defined responses had not improved. The first patient developed antiadalimumab antibodies, explaining why adalimumab was not able to block membrane and soluble TNFα. In the second patient, adalimumab was discontinued because of adverse effects, which led to a decrease in iTNFα+ CD14+ cells to levels measured before treatment. Conclusions: Our findings suggest that adalimumab treatment in RA patients can return iTNFα levels to those of healthy donors. This effect was not observed in the presence of neutralizing antiadalimumab antibodies.