Methotrexate Inhibits T Cell Proliferation but Not Inflammatory Cytokine Expression to Modulate Immunity in People Living With HIV

Abstract

Inflammation associated with increased risk of comorbidities persists in people living with HIV (PWH) on combination antiretroviral therapy (ART). A recent placebo-controlled trial of low-dose methotrexate (MTX) in PWH found that numbers of total CD4 and CD8 T cells decreased in the low-dose MTX arm. In this report we analyzed T cell phenotypes and additional plasma inflammatory indices in samples from the trial. We found that cycling (Ki67+) T cells lacking Bcl-2 were reduced by MTX but plasma inflammatory cytokines were largely unaffected. In a series of in vitro experiments to further investigate the mechanisms of MTX activity, we found that MTX did not inhibit effector cytokine production but inhibited T cell proliferation downstream of mTOR activation, mitochondrial function, and cell cycle entry. This inhibitory effect was reversible with folinic acid, suggesting low-dose MTX exerts anti-inflammatory effects in vivo in PWH largely by blocking T cell proliferation via dihydrofolate reductase inhibition, yet daily administration of folic acid did not rescue this effect in trial participants. Our findings identify the main mechanism of action of this widely used anti-inflammatory medicine in PWH and may provide insight into how MTX works in the setting of other inflammatory conditions.

Keywords: HIV; T cells; cell cycling; inflammation; methotrexate; proliferation.

Figure 1

Minimal effect of MTX on cytokine production and activation in vitro. Freshly isolated PBMCs from PWH (HIV+, blue triangles, n=6) or HIV-uninfected controls (HIV-, gold circles, n=6) were stimulated overnight with LPS (10ng ml^-1) (A), flagellin (500ng ml^-1) (B), or anti-CD3 (10μg ml^-1) and anti-CD28 (5μg ml^-1)(TCR)(C) in the presence or absence of MTX at indicated concentrations. At the end of the culture, supernatants were collected and analyzed by ELISA for indicated cytokines. (D) Cryopreserved PBMCs from PWH (blue triangles, n=6) or HIV-uninfected controls (gold circles, n=8-11) were stimulated for 4 days with anti-CD3 (10μg ml^-1) and anti-CD28 (5μg ml^-1)(TCR) in the presence or absence of MTX at indicated concentrations. At the end of the culture, CD4 (left) and CD8 (right) T cells were analyzed by flow cytometry for expression of activation markers CD69 and CD25. Data were normalized to medium-only control values. P-values in black indicate whether one curve fits both HIV- and HIV+ donors; variable slope non-linear regression, log(inhibitor) vs. normalized response. P-values in color indicate whether the slope of each curve is significantly non-zero; simple linear regression. ns, not significant.

Figure 2

MTX inhibits T cell proliferation in vitro. Cryopreserved PBMCs from PWH (blue triangles, n=6) or HIV-uninfected controls (gold circles, n=8-11) were labeled with CellTraceViolet (CTV) then stimulated for 4 days with anti-CD3 (10μg ml^-1) and anti-CD28 (5μg ml^-1)(TCR), IL-2 (100U ml^-1), IL-7 (100ng ml^-1), or IL-15 (24ng ml^-1) in the presence or absence of MTX at indicated concentrations. At the end of the culture, CD4 (A) and CD8 (B) T cells were analyzed by flow cytometry for CTV dilution. Data were normalized to medium-only control values. P-values in black indicate whether one curve fits both HIV- and HIV+ donors; variable slope non-linear regression, log(inhibitor) vs. normalized response. P-values in color indicate whether the slope of each curve is significantly non-zero; simple linear regression. ns, not significant.

Figure 3

MTX inhibits IL-15-induced proliferation at a step after cell cycle entry and mTOR activity. Cryopreserved PBMCs from PWH (blue triangles, n=2-4) or HIV-uninfected controls (gold circles, n=2-6) were labeled with CellTraceViolet (CTV) then stimulated for 4 days (A–E) or 45 minutes (F) with IL-15 (24ng ml^-1) in the presence or absence of MTX (100nM) or rapamycin (rapa; 250ng ml^-1). At the end of the culture, cells were harvested and analyzed by flow cytometry. (A) The proportion of CD4 (left) and CD8 (right) T cells expressing CD25. (B) The proportion of CD4 (left) and CD8 (right) CD25+ cells that diluted CTV dye. (C) The proportion of CD4 (left) and CD8 (right) cells that did not dilute CTV dye (CTV^hi) that expressed Ki67. (D) The mean fluorescence intensity (MFI) of mitochondrial TFAM among CD4 (left) and CD8 (right) CD25+ CTV^hi cells. (E) The MFI of mitochondrial COX IV among CD4 (left) and CD8 (right) CD25+ CTV^hi cells. (F) Normalized MFI of ribosomal S6 protein phosphorylated at S240 among CD4 (left) and CD8 (right) T cells. Statistical significance determined between IL-15+rapa and IL-15+MTX groups; Mann-Whitney test. ns, not significant; *P < 0.05; **P < 0.01.

Figure 4

Folinic acid restores proliferation inhibited by MTX. (A, B) Cryopreserved PBMCs from PWH (blue triangles, n=4) or HIV-uninfected donors (gold circles, n=5) were labeled with CellTraceViolet (CTV) then stimulated for 4 days with anti-CD3/anti-CD28 in the presence or absence of MTX (100nM) and folinic acid (0.05 – 1000μM). At the end of the culture, CD4 (A) and CD8 (B) T cells were analyzed by flow cytometry for CTV dilution. (C, D) Cryopreserved PBMCs from PWH (blue triangles, n=4) or HIV-uninfected donors (gold circles, n=9) were labeled with CellTraceViolet then stimulated for 4 days with anti-CD3/anti-CD28 (TCR) in the presence or absence of MTX (100nM), folinic acid (1mM), adenosine monophosphate (AMP, 1mM), or CD73 inhibitor (0.4mM). At the end of the culture, CD4 (C) and CD8 (D) T cells were analyzed by flow cytometry for CTV dilution. Statistical significance determined among all groups using nonparametric paired Friedman’s test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.