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. 2006 Jun 12;203(6):1413-8.
doi: 10.1084/jem.20060210. Epub 2006 Jun 5.

Dysregulated T cell expression of TIM3 in multiple sclerosis

Ken Koguchi  1 , David E Anderson, Li Yang, Kevin C O'Connor, Vijay K Kuchroo, David A Hafler
Affiliations

Dysregulated T cell expression of TIM3 in multiple sclerosis

Ken Koguchi et al. J Exp Med. .

Abstract

T cell immunoglobulin- and mucin domain-containing molecule (TIM)3 is a T helper cell (Th)1-associated cell surface molecule that regulates Th1 responses and promotes tolerance in mice, but its expression and function in human T cells is unknown. We generated 104 T cell clones from the cerebrospinal fluid (CSF) of six patients with multiple sclerosis (MS) (n = 72) and four control subjects (n = 32) and assessed their cytokine profiles and expression levels of TIM3 and related molecules. MS CSF clones secreted higher amounts of interferon (IFN)-gamma than did those from control subjects, but paradoxically expressed lower levels of TIM3 and T-bet. Interleukin 12-mediated polarization of CSF clones induced substantially higher amounts of IFN-gamma secretion but lower levels of TIM3 in MS clones relative to control clones, demonstrating that TIM3 expression is dysregulated in MS CSF clones. Reduced levels of TIM3 on MS CSF clones correlated with resistance to tolerance induced by costimulatory blockade. Finally, reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-gamma secretion, directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-gamma secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel, intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases.

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Figures

Figure 1.
Figure 1.
Differential expression of T-bet, TIM3, and GATA-3 among Th1 and Th2 T cell clones. Expression levels of TIM3, T-bet, STAT1, and GATA-3 were examined by quantitative RT-PCR. Relative expressions of each transcript have been normalized to a single clone expressing the highest level of a transcript. (A) CD4+ T cell clones from the CSF of control subjects that secreted >100 pg/ml of IFN-γ and <100 pg/ml of IL-13 were defined as Th1 (n = 9), whereas those secreting >100 pg/ml of IL-13 and <100 pg/ml of IFN-γ were defined as Th2 (n = 6). Error bars represent the standard deviation of mRNA levels among the clones. (B) Highly polarized Th1 and Th2 clones were generated from the peripheral blood of a healthy normal individual. Levels of TIM3 and T-bet transcripts are depicted from representative Th1 and Th2 clones as described in A.
Figure 2.
Figure 2.
Cytokine profiles of CSF clones from MS and control subjects. (A) CD4+ T cell clones derived from the CSF of MS patients (n = 72) or control subjects (n = 32) were expanded and stimulated with 2.0 μg/ml of plate-bound anti-CD3 monoclonal antibody for 2 d. Supernatants were collected and IFN-γ and IL-13 were measured by ELISA. Levels of cytokine secretion in the absence of stimulation have been subtracted from all reported values. T cells from patients with MS secreted significantly higher amounts of IFN-γ (P = 0.033) and comparable amounts of IL-13 (P = 0.147) relative to those from control subjects. (B) T cell clones derived from both the peripheral blood (PBMC) (n = 23) and CSF (n = 27) of three MS patients were expanded and their cytokine profiles were characterized as described in A. CSF clones secreted significantly greater amounts of IFN-γ than did those from peripheral blood (P = 0.009).
Figure 3.
Figure 3.
Altered levels of TIM3, T-bet, and STAT1 among T cell clones from MS patients. (A) Expression levels of T-bet and TIM3 were examined by quantitative RT-PCR in the panel of CSF-derived T cell clones described in Fig. 2 A. Expression levels of each transcript have been normalized to a single clone expressing the highest level of a transcript. Clones from MS patients expressed significantly lower levels of T-bet (P = 0.014) and TIM3 (P = 0.029). There were no substantial differences in levels of STAT4, TIM1, and GATA-3 (not depicted). Note the greater percentage of clones in patients compared with control subjects that secrete high amounts of IFN-γ, yet express relatively low levels of TIM3 (bottom right regions). (B) Despite differences in levels of T-bet and TIM-3 expression between healthy subjects and MS patients, transcript levels were highly correlated among all T cell clones (R = 0.9651 and 0.6609, respectively).
Figure 4.
Figure 4.
Dysregulated TIM3 induction and function among MS CSF T cell clones. (A) Relative inability to induce TIM3 expression on MS CSF clones. CSF T cell clones from MS patients (n = 5) and healthy subjects (n = 6) that secreted comparable levels of IFN-γ (61.7 ± 22 and 58 ± 28 pg/ml, respectively, for control and MS CSF clones) were subjected to two rounds of Th1 polarization, at which point TIM3 expression and IFN-γ secretion after stimulation were determined. CSF T cells from patients with MS secreted significantly higher amounts of IFN-γ but not TIM3 (P = 0.01), demonstrating that they are impaired in their ability to up-regulate TIM3. (B) Reduced tolerance induction in MS CSF clones. Control and MS CSF clones were stimulated with plate-bound anti-CD3 mAb in the presence/absence of CTLA-4 Ig to induce tolerance. After 7 d, cells were collected and restimulated with anti-CD3 mAb, and supernatants were collected after 48 h for measurement of IFN-γ. On average, MS CSF clones expressed 3.3-fold less TIM3 than did control CSF clones and were resistant to tolerance induction (P = 0.01). (C) IFN-γ–mediated STAT1 signaling is functional in MS CSF clones. CSF T cell clones from MS patients and control subjects were cultured in the presence or absence of 0.1 or 1 ng/ml of recombinant IFN-γ for 3 h, after which time levels of total STAT1 protein and active STAT1 protein were determined by ELISA (R&D Systems). Error bars represent differences in levels of total or active STAT1 protein among three clones examined from MS patients and from healthy subjects.
Figure 5.
Figure 5.
TIM3 siRNA enhances T cell proliferation and IFN-γ secretion. (A) Ex vivo CD4+ T cells were transfected with TIM3 or negative control oligonucleotides and TIM3 mRNA levels were determined. After transfection, cells were collected and 2.5 × 104 cells/well were stimulated in triplicate with plate-bound anti-CD3/CD28 monoclonal antibodies. Proliferation measured using tritiated thymidine incorporation (B) and IFN-γ secretion (C) were measured after 48 h. Results are representative of three independent experiments using blood from three different donors. Similar observations were seen using a second TIM3 siRNA oligonucleotide and when transfecting polarized Th1 cell lines. Thus, RNAi inhibition of TIM3 expression induces a CD4 phenotype of the CSF-derived MS T cells. Error bars represent the standard deviation of triplicate wells in B and C.
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