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

A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples

<div><p>Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an <i>E</i>. <i>coli</i> strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents.</p></div

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

THP-1 NF-κB-eGFP reporter cells are highly sensitive towards mycoplasma lipoproteins through engagement of TLR2/6.

<p>(A) THP-1 reporter cells pre-treated with a blocking TLR6 antibody or isotype control (30 min; both used at 5 μg/mL) were incubated with the indicated concentrations of the TLR2/6 ligands FSL-1 and MALP-2 or supernatants derived from mycoplasma infected cell cultures for 24 h. Standard LPS served as a negative control. NF-κB-driven eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from triplicates of three independently performed experiments (n = 3). (B) Fluorescent microscopy images of THP-1 reporter cells stimulated for 24 h with different dilutions of supernatants derived from mycoplasma infected cell cultures. Unstimulated cells served as negative control (left panel). Bright field images are shown for comparison (top row). Scale bar: 10 μm. (C) THP-1 NF-κB-eGFP reporter cells and a commercially available mycoplasma detection kit (MycoAlert) were probed with tissue culture supernatants from different cell sources and species: (1) mouse tail cells, (2) human mesotheliom, (3) human melanoma brain metastasis-derived cell line YDFR, (4) human LN229 glioblastoma, (5) human ovarian cancer cells, (6) COS-7 cell line and (7) human skin fibroblasts. Samples above red line are scored as positive. For the Mycoalert detection system the B/A ratio represents the ratio of the luminescence signals measured at two different time points (reading A and B). For THP-1 reporter assay mean and SE were calculated from duplicates. (D) K562 cells were infected with mycoplasma and then subjected to treatment regimens using commercially available mycoplasma removal agents (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178220#sec002" target="_blank">Material and Methods</a>). Supernatants were collected at various time points throughout the treatment course (day 2, 4, 7, 10, 15, 17, 21, 23, 25 and 29) and tested with the THP-1 NF-κB-eGFP reporter cells (upper panel). Indicated samples were also tested using the MycoAlert kit (lower panel).</p

Dose-dependent response of THP-1 NF-κB-eGFP reporter cells towards specific TLR ligands.

<p>(A-E) THP-1 NF-κB-eGFP cells were incubated with increasing concentrations of Pam3CSK4, FSL-1, Flagellin, standard LPS and MALP-2 as indicated. Untreated cells served as control. After 24 h, induction of NF-κB-driven eGFP was measured by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI, top panels). Mean and SE were calculated from triplicates of three independently performed experiments (n = 3). Flow cytometry histograms of a representative experiment are shown for comparison (bottom panels). Open histograms: control cells; filled histograms: TLR-activated reporter cells.</p