Identification of protein kinases dysregulated in CD4(+) T cells in pathogenic versus apathogenic simian immunodeficiency virus infection

Abstract

Human immunodeficiency virus infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM) leads to a generalized loss of immune responses involving perturbations in T-cell receptor (TCR) signaling. In contrast, naturally SIV-infected sooty mangabeys (SM) remain asymptomatic and retain immune responses despite relatively high viral loads. However, SIV infection in both RM and SM led to similar decreases in TCR-induced Lck phosphorylation. In this study, a protein tyrosine kinase (PTK) differential display method was utilized to characterize the effects of in vivo SIV infection on key signaling molecules of the CD4(+) T-cell signaling pathways. The CD4(+) T cells from SIV-infected RM, but not SIV-infected SM, showed chronic downregulation of baseline expression of MLK3, PRK, and GSK3, and symptomatically SIV-infected RM showed similar downregulation of MKK3. In vitro TCR stimulation with or without CD28 costimulation of CD4(+) T cells did not lead to the enhancement of gene transcription of these PTKs. While the CD4(+) T cells from SIV-infected RM showed a significant increase of the baseline and anti-TCR-mediated ROR2 transcription, SIV infection in SM led to substantially decreased anti-TCR-stimulated ROR2 transcription. TCR stimulation of CD4(+) T cells from SIV-infected RM (but not SIV-infected SM) led to the repression of CaMKKbeta and the induction of gene transcription of MLK2. Studies of the function of these molecules in T-cell signaling may lead to the identification of potential targets for specific intervention, leading to the restoration of T-cell responses.

FIG. 1

Effect of SIV infection on the expression and phosphorylation of Lck. Purified CD4⁺ cells from SIV-negative (SIV–) or SIV-positive (SIV+) RM and SM were either not stimulated (NS) or stimulated with anti-CD3-coated beads (αCD3) or anti-CD3/anti-CD28-coated beads (αCD3/28), and cell lysates were assayed for Lck by Western blot. Western blots show unphosphorylated p56-Lck (lck) and phosphorylated p58-Lck (p-lck); β-actin expression is used as control for the equivalent loading. Representative data from one animal from each group (consisting of at least three animals) are shown.

FIG. 2

Protein kinase differential display from CD4⁺ T cells. Purified CD4⁺ cells from SIV-negative (A) and SIV-positive (B) RM and SM were incubated in medium alone (lanes 0), with anti-CD3 (lanes 1)- or anti-CD3/CD28 (lanes 2)-coated beads, and protein kinase display assays were performed as described in Materials and Methods. Amplification products were digested with restriction enzymes and electrophoresed with 10-bp ladder (M) as the molecular mass marker. Representative digests with BslI, HpaII, MnlI, and DdeI are shown.

FIG. 3

Select protein kinases are downregulated in pathogenic SIV infection. CD4⁺ cells from SIV-negative RM (○), SIV-infected RM (●), SIV-seronegative SM (▵), and SIV-seropositive SM (▴) were cultured in medium alone (NS) or stimulated with beads coated with anti-CD3 (CD3) or anti-CD3/anti-CD28 (CD3/28) antibodies. The transcription levels of MLK3 (A), GSK3 (B), PRK (C), and MKK3 (D) were assessed by real-time PCR, normalized to GAPDH, and expressed as copy numbers relative to the calibrator sample. Horizontal bars indicate mean of each group and P values for statistically significant differences (P < 0.05) are listed.

FIG. 4

Dysregulation of MKK3, GSK3, and ROR2 correlates with viral load in RM. CD4⁺ T cells from SIV-negative RM (○), SIV-infected RM with a viral load of <10⁶ vRNA/ml (□), and SIV-infected RM with a viral load of >10⁶ vRNA/ml (●) were cultured in medium alone (NS), stimulated with anti-CD3 (CD3), or stimulated with anti-CD3/anti-CD28 (CD3/28) antibody-coated beads. The transcription of selected protein kinases was assessed by real-time PCR, normalized to GAPDH, and expressed as copy numbers relative to the calibrator sample. Horizontal bars indicate the mean of each group, and P values for statistically significant differences (P < 0.05) are given.

FIG. 5

Complex effect of SIV infection on the transcription of ROR2, CaMKKβ, and MLK2. (A and B) CD4⁺ cells from SIV-negative RM (○), SIV-infected RM (●), SIV-seronegative SM (▵), and SIV-seropositive SM (▴) were cultured in medium alone (NS) or were stimulated with beads coated with anti-CD3 antibody with (CD3/28) or without (CD3) antibody. The transcription of selected protein kinases was assessed by real-time PCR, normalized to GAPDH, and expressed as copy numbers relative to the calibrator sample. Horizontal bars indicate the mean of each group, and P values for statistically significant differences (P < 0.05) are listed. (C) The levels of transcription of MLK2 in SIV-seropositive (RM+) or SIV-seronegative (RM–) RM and in SIV-seronegative (SM-) or SIV-seropositive (SM+) SM were determined as in panel A. The dotted line connects values from individual animals.