TIM-3 Does Not Act as a Receptor for Galectin-9

<div><p>T cell immunoglobulin and mucin protein 3 (TIM-3) is a type I cell surface protein that was originally identified as a marker for murine T helper type 1 cells. TIM-3 was found to negatively regulate murine T cell responses and galectin-9 was described as a binding partner that mediates T cell inhibitory effects of TIM-3. Moreover, it was reported that like PD-1 the classical exhaustion marker, TIM-3 is up-regulated in exhausted murine and human T cells and TIM-3 blockade was described to restore the function of these T cells. Here we show that the activation of human T cells is not affected by the presence of galectin-9 or antibodies to TIM-3. Furthermore, extensive studies on the interaction of galectin-9 with human and murine TIM-3 did not yield evidence for specific binding between these molecules. Moreover, profound differences were observed when analysing the expression of TIM-3 and PD-1 on T cells of HIV-1-infected individuals: TIM-3 was expressed on fewer cells and also at much lower levels. Furthermore, whereas PD-1 was preferentially expressed on CD45RA⁻CD8 T cells, the majority of TIM-3-expressing CD8 T cells were CD45RA⁺. Importantly, we found that TIM-3 antibodies were ineffective in increasing anti-HIV-1 T cell responses <i>in vitro</i>, whereas PD-L antibodies potently reverted the dysfunctional state of exhausted CD8 T cells. Taken together, our results are not in support of an interaction between TIM-3 and galectin-9 and yield no evidence for a functional role of TIM-3 in human T cell activation. Moreover, our data indicate that PD-1, but not TIM-3, is a promising target to ameliorate T cell exhaustion.</p> </div

Effect of antibodies to TIM-3 and PD-Ligands on T cell responses to HIV-peptides.

<p>Specific responses to HIV-peptides: Gag/Nef peptide treated samples that had at least 2-fold higher responses than mock treated samples from the same donor; Enhanced responses: HIV-peptide responses in samples treated with TIM-3/PD-L antibodies were at least 1.5-fold higher than both controls (Gag/Nef peptide samples without antibody or treated with isotype control antibody). Response versus controls: The mean response in presence of TIM-3/PD-L antibodies was divided by the mean response of the controls.</p>*<p>in two samples the IFN-γ production in presence of TIM-3 antibodies was also more than 1.5-fold lower than in both controls;</p>**<p>in two samples the number of CFSE^low cells in presence of TIM-3 antibodies was also more than 1.5 fold lower than in both controls.</p><p>n.d.: not determined.</p

TIM-3 antibodies do not affect human T cell activation.

<p>CD4 T cells were stimulated with plate-bound antibodies to CD3 and CD28 immobilized at the indicated concentration. The expression of TIM-3 (bold line; isotype control: thin line) following activation is shown (inset). TIM-3 antibody (clone 2E2) or control antibody (both at a final concentration of 10 µg/ml) were added to the cultures and following 48 h of stimulation, culture supernatants were harvested and methyl-³[H]-thymidine was added to the cultures. A) Upon 18 h of additional culturing, cells were harvested and methyl-³[H]-thymidine uptake was measured to assess T cell proliferation. B) The cytokine concentration in the supernatants was measured using a Luminex-based multiplex assay. The results of triplicate measurement of one experiment representative for four experiments are shown.</p

TIM-3 antibodies do not revert T cell exhaustion.

<p>PBMC from HIV-1-infected individuals were mock-treated or stimulated with Gag/Nef peptide pools. Control antibodies and antibodies to TIM-3 or PD-L were added as indicated. A) Methyl-³[H]-thymidine uptake was measured following seven days of culture. Shown is a representative aviremic and viremic patient. PD-L antibodies (p<0.001; n = 30) but not TIM-3 antibody (p = 0.59; n = 30) significantly enhanced methyl-³[H]-thymidine uptake compared to controls. B) INF-γ concentration of day 6 culture supernatants. Shown is a representative aviremic and viremic patient. The INF-γ concentration was significantly higher in stimulation cultures with PD-L antibodies (p<0.05; n = 23) whereas TIM-3 antibody (p = 0.92; n = 23) had no effect. C) CFSE dilution experiments with PBMC from suppressed (upper panels) and viremic patients (lower panels). Cells were analyzed on day 7 of culture and the experiments shown are representative of 22 independently performed (7 samples from viremic and 15 samples from suppressed patients). The number of CFSE^low CD8 T cells was significantly higher in stimulation cultures with PD-L antibodies (p<0.05; n = 22), whereas TIM-3 antibodies had no effect (p = 0.99; n = 22). A detailed summary of all experiments is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003253#ppat-1003253-t001" target="_blank">table 1</a>.</p

TIM-3 does not interact with galectin-9.

<p>A, D) Bw control cells (open histograms) or Bw cells transduced to express human (A) or murine (D) galectin-9 (grey histograms) were probed with galectin-9 antibodies or with immunoglobulin fusion proteins representing human TIM-3 (TIM3-Ig; A) or murine TIM-3 (m-TIM-3-Ig; D) or a control fusion protein (B7-H3-Ig). Bound antibodies were detected with PE-labelled donkey-anti-goat antibodies. Immunoglobulin fusion proteins were detected with PE-labelled goat antibodies to the Fc-part of human IgG. B, E) Bw control cells (open histogram) or Bw cells transduced to express human (B) or murine (E) TIM-3 (grey histograms) were probed with appropriate anti-TIM-3 mAbs or with biotin-labelled recombinant human (B) or murine (E) galectin-9. Bound anti-human TIM-3 mAb and anti-murine TIM-3 mAb were detected with PE-labelled goat-anti-mouse IgG and APC-labelled goat-anti-rat IgG, respectively. SA-PE was used as secondary reagent for biotin-labelled recombinant human and murine galectin-9. C, F) Recombinant human (C) or murine (F) galectin-9 was immobilized on ELISA plates and probed with immunoglobulin fusion proteins (Ig) representing the extra-cellular domain of human TIM-3 (TIM-3-Ig; C) or murine TIM-3 (m-TIM-3-Ig; F) at the indicated concentrations. HRP-conjugated goat-anti-human IgG-Fc-specific antibodies were used for detection. Binding buffer only (PBS) or the indicated immunoglobulin fusion proteins were used as controls. All binding experiments were repeated three times with similar outcome.</p

PD-L blockade but not TIM-3 antibodies increase allogeneic responses of human Th1 cells.

<p>Human MNC were stimulated under Th1 polarizing conditions. The expression of PD-1 (grey histogram) and TIM-3 (bold line) following Th1 polarization is shown (inset). Subsequently, polarized cells were co-cultured with immature and mature allogeneic DC (iDC and LPS-DC, respectively; numbers of DC/well are indicated) in presence of antibodies to TIM-3 or to PD-L1. Proliferation (A) and IFN-γ production (B) was measured following 6 days of cultures. The results of triplicate measurements of one experiment representative for four experiments are shown.</p

TIM-3 expression on CD8 T cells of HIV-1-infected individuals.

<p>A) TIM-3 and PD-1 expression on CD8 T cells from HIV-1-infected individuals. The percentage of positive cells in the CD45RA⁺ and CD45RA⁻ subsets are indicated. Correct assessment of TIM-3 and PD-1 positive cells was ensured by isotype control antibody (IgG-PE) staining for each sample. Representative samples from a suppressed (upper panels) and from viremic patients (middle and lower panels) are shown. B) Percentage of TIM-3⁺ (left graph) and PD-1⁺ (right graph) CD8 T cells from suppressed (S) and viremic (V) patients and from healthy individuals (H) are shown. Bars indicate median percentage. C) Percentage of TIM-3⁺ (left graph) and PD-1⁺ (right graph) CD8 cells in the CD45RA⁺ and CD45RA⁻ subsets. D) Percentage of TIM-3⁺ (left graph) and PD-1⁺ (right graph) CD8 cells in the CD57⁺ and CD57⁻ subset.</p