Modulation of Tetraspanin 32 (TSPAN32) Expression in T Cell-Mediated Immune Responses and in Multiple Sclerosis

Abstract

Tetraspanins are a conserved family of proteins involved in a number of biological processes including, cell-cell interactions, fertility, cancer metastasis and immune responses. It has previously been shown that TSPAN32 knockout mice have normal hemopoiesis and B-cell responses, but hyperproliferative T cells. Here, we show that TSPAN32 is expressed at higher levels in the lymphoid lineage as compared to myeloid cells. In vitro activation of T helper cells via anti-CD3/CD28 is associated with a significant downregulation of TSPAN32. Interestingly, engagement of CD3 is sufficient to modulate TSPAN32 expression, and its effect is potentiated by costimulation with anti-CD28, but not anti-CTLA4, -ICOS nor -PD1. Accordingly, we measured the transcriptomic levels of TSPAN32 in polarized T cells under Th1 and Th2 conditions and TSPAN32 resulted significantly reduced as compared with unstimulated cells. On the other hand, in Treg cells, TSPAN32 underwent minor changes upon activation. The in vitro data were finally translated into the context of multiple sclerosis (MS). Encephalitogenic T cells from Myelin Oligodendrocyte Glycoprotein (MOG)-Induced Experimental Autoimmune Encephalomyelitis (EAE) mice showed significantly lower levels of TSPAN32 and increased levels of CD9, CD53, CD82 and CD151. Similarly, in vitro-activated circulating CD4 T cells from MS patients showed lower levels of TSPAN32 as compared with cells from healthy donors. Overall, these data suggest an immunoregulatory role for TSPAN32 in T helper immune response and may represent a target of future immunoregulatory therapies for T cell-mediated autoimmune diseases.

Keywords: T cell responses; TSPAN32; multiple sclerosis; tetraspanins.

Figure 1

Expression of TSPAN32 in murine immune cells. (A) Expression of TSPAN32 in murine immune lineages was evaluated by interrogating the GSE15907 microarray dataset. (B) Modulation of the transcriptomic levels of TSPAN32 during T cell development, as determined from the GSE15907 dataset. Data are shown as normalized mean ± SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction.

Figure 2

Expression of TSPAN32 during human T cell activation. (A) Expression of TSPAN32 was evaluated via real-time PCR, in CD4+ T effector cells stimulated at different time points with anti-CD3/CD28 antibodies or (B) with anti-CD3 alone or in combination with anti-CD28, anti-CTLA4, anti-PD1 and anti-ICOS antibodies for 36 h (n = 3 independent replicates). (C) TSPAN32 levels in CD4+ effector T cells upon anti-CD3/CD28 stimulation, in the presence or absence of rapamycin 200 nM, were evaluated by real-time PCR (n = 3 independent replicates). (D) TSPAN32 expression levels were evaluated at different time points upon activation of Treg cells via real-time PCR (n = 3 independent replicates). (E) TSPAN32 protein levels were determined by western blot upon activation of effector and regulatory T cells at different time points (pooled proteins of cells from 3 healthy donors). Data are shown as normalized mean ± SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction.

Figure 3

Expression of tetraspanins in human T cell polarization. (A) Expression of TSPAN32 was evaluated by real-time PCR in polarized Th1 and Th2 cells and unstimulated cells (n = 3 independent replicates). (B) Expression of CD9, CD37, CD53, CD63, CD81, CD82 and CD151 in polarized Th1 and Th2 cells and unstimulated cells as evaluated by real-time PCR (n = 3 independent replicates). Data are shown as normalized mean ± SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction.

Figure 4

Effect of TSPAN32 overexpression in Jurkat cells. Following transient transfection of Jurkat cells with a DNA plasmid encoding for TSPAN32 (pTSPAN32) or the empty plasmid, cells were stimulated with anti-CD3/CD28 for 24 h and the concentrations of TNF-alpha and IFN-gamma in the supernatant were determined by ELISA. Data are shown as normalized mean ± SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction.

Figure 5

Modulation of TSPAN32 in experimental allergic encephalomyelitis (EAE). (A) Expression of TSPAN32 was evaluated using real-time PCR in CD4+ T cells isolated from the brains and spinal cords of mice with EAE. Control cells are represented by CD4+ T cells isolated from the spleens of sham mice (n = 3 replicates, each of 3 pooled animals). (B) Expression of CD9, CD37, CD53, CD63, CD81, CD82 and CD151 was evaluated using real-time PCR in CD4+ T cells isolated from the brains and spinal cords of mice with EAE. Control cells are represented by CD4+ T cells isolated from the spleens of sham mice (n = 3 replicates, each of 3 pooled animals). (C) Expression of TNF-alpha, IL-6 and IL-2 was evaluated by real-time PCR in CD4+ T cells isolated from the brains and spinal cords of mice with EAE. Control cells are represented by CD4+ T cells isolated from the spleens of sham mice (n = 3 replicates, each of 3 pooled animals). Data are shown as normalized mean ± SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction.

Figure 6

Modulation of TSPAN32 in multiple sclerosis. Expression of TSPAN32 resting and activated CD4+ T cells from 14 relapsing-remitting MS patients and 14 healthy donors, as obtained from the GSE78244 dataset. Data are shown as normalized mean ± SD and statistical analysis performed using one-way ANOVA followed by Bonferroni multiple test correction. ** p < 0.01, *** p < 0.001.