Differentiation and the subsequent clonal expansion of T helper precursor cells (Th0) can result into one of two major effector cell types (Th1 and Th2). These cells play an important role in the adoptive immune response, and provide protection against intracellular and extracellular pathogens, such as viruses and helminthes (Mosmann et al., 1989). The extent of T cell activation and differentiation is largely determined by the duration and strength of T cell receptor-mediated stimulation. In addition, several co-stimulatory molecules including the TNF receptor family members (CD154, CD134 and CD137) (Locksley et al., 1998), immunoglobulin superfamily members (CD28, CTLA-4, ICOS and PD-1) (Salomin et al., 2001) and TIM3 family members (Kane, 2010), as well as cytokines such as IL-2 and IL-15 regulate the extent of clonal expansion, deletion and anergy induction.
ב A. TIM3 and its ligand
The T cell immunoglobulin- and mucin-domain-containing molecule (TIM) proteins represent a previously unidentified group of molecules that act in concert with T cell receptor and co-stimulatory signal to regulate expansion and an effector function of Th1 and Th2 cell. TIM family members are type I membrane gylcoproteins expressed on T cells and containing Immunoglobulin V (IgV)-like domain, highly glycosylated mucin domain transmembrane domain and cytoplasmic domain. The mouse gene family includes 8 members (encoding Tim1, Tim2, Tim3 and Tim4 proteinsand the putative Tim5 to Tim8 proteins), while the human gene family includes 3 members encoding TIM1, TIM3 and TIM4 proteins (Kuchroo et al., 2003).
TIM3 is initially identified as a molecule expressed on Th1 but not Th2 cells in mice.
TIM3 protein is not expressed in naïve murine T cells. In contrast, repetitive in vitro re-stimulations in the presence of Th1-skewing conditions (IL-12 and anti-IL-4) lead to TIM3 expression in Th1 cells (Monney et al., 2002; Sanchez-Fueyo et al., 2003). Up-regulation of TIM3 in vivo also requires several rounds of cell division, and is closely associated with IFN-γ production, although all IFN- γ -producing murine T cells are not TIM3 positive (Sabatos et al., 2003; Snchez-Fueyo et al. 2003). Otherwise, the most of activated human CD4+ T cells in Th1-skewing conditions become TIM3+ cells after a few days of stimulation with anti-CD3/anti-CD28 (Hastings et al., 2009). TIM3 is also expressed by differentiated type 1 CD8+ T cells, Th17, regulatory T cells, monocytes, dendritic cells, microglia, and mast cells (Monney et al., 2002; Su et al., 2008).
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The putative Tim3 ligand is demonstrated on the surface of CD4+CD25- T cells (both naïve and memory), as well as on regulatory CD4+CD25+ T cells using Tim3-hIg and flow cytometry,. In addition, Tim3-hIg binds to spleenic CD11c+ and CD11b+ cell populations (Sabatos et al., 2003; Sanchez-fueyo et al., 2003). Recently galectin-9, a member of the S-type lectins, is identified as a TIM3 ligand by immunoprecipitation of cell surface proteins that binds to Tim3-hIg. Galectin-9-induced intracellular calcium flux, aggregation and death of Th1 cells are TIM3-dependent in vitro, and administration of galectin-9 in vivo resulted in selective loss of IFN-γ-producing cells and suppression of Th1 autoimmunity (Zhu et al., 2005). It appears that galectin-9 triggered activation of TIM3 pathway ensures an effective termination of Th1 driven immunity. One argument to these experiment is the fact that galectin-9 can have pleiotropic effects, through binding to multiple proteins with β-galactoside modifications, such as CD44 (Rabiovich et al., 2009). A recent study suggests the presence of other potential ligands for TIM3 based on the structure of TIM3 (Cao et al., 2007).
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B. The role of TIM3 in Th1 driven immune response
The function of TIM3 has been demonstrated in a variety of murine models as well as human disease by blockade of Tim3-Tim3 ligand interactions using blocking antibody (Ab) or soluble form of recombinant TIM3 molecules. For example, administration of anti-Tim3 monoclonal Ab can increase the severity of experimental autoimmune encephalomyelitis (EAE) in immunized SJL mice and lead to massive activation and clonal expansion of macrophages (Monney et al., 2002), indicating that Tim3 blockade may interfere with an interaction between Th1 cells and Tim3 ligand-expressing macrophages. In a human disease, T-cell clones isolated from the cerebrospinal fluid of patients with multiple sclerosis (MS) express lower levels of TIM3 and secrete higher levels of IFN-γ compared to normal subject and the result using siRNA to reduce TIM3 expression in human ex vivo CD4+ T cells demonstrated that reduction of T-cell expression of TIM3 resulted in enhanced T-cell proliferation and IFN-γ secretion after T-cell stimulation. Those data indicated that TIM3 functions to inhibit aggressive Th1-mediated auto- and allo-immune responses (Khademi et al., 2004).
TIM3 is involved in peripheral immune tolerance, in which long term survival and tolerance to MHC mismatched allografts are achieved through the administration of donor specific transfusion (DST) plus anti-CD154 (CD40L) co-stimulation blockade, administration of Tim3-hIg abrogates tolerance induction (Sanchez-fueyo et al., 2002 ; Sabatos et al., 2003). This effect suggested to be mediated, at least in part, by the immunosuppressive potency of CD4+CD25+ regulatory T cells, but the exact mechanism
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by which Tim3 influences T-cell tolerance is not known. Concordantly, in vivo 7administration of galectin-9 leads to not only down-regulation of Th1-mediated immune
responses but also an increase in regulatory T cells in a model of viral-induced immunopathology (Sehrawat et al., 2009). Furthermore, blocking the Tim3 pathway using Tim3-hIg during tolerance induction is sufficient to prevent tolerance and leads to increased proliferation and cytokine production. Moreover, Tim3-deficient mice could not be tolerized by treating with high-dose aqueous antigen (Sabatos et al., 2003).
TIM3 is associated with the phenomenon of immune exhaustion. It is previously described that subset of programmed death-1-expressing nonresponsive T cells were a largely overlapped the population of CD8+ T cells that express TIM3 in individuals with chronic human immunodeficiency virus (HIV) infection. Expression of TIM3 correlates with disease progression and is associated with a lack of activation potential. Most strikingly, soluble Tim3-hIg or a putative blocking Ab for human TIM3 can partially reverse the activation defect of these cells, and blocking both TIM3 and PD-1 leads to a cooperative or synergistic rescue of T cell activation (Anderson et al., 2007). Upregulation of TIM3 on CD4+ and CD8+ exhausted T cell was also recently reported in patients with chronic hepatitis C virus infection (Golden-Mason et al., 2009).
Furthermore, in HCV/HIV co-infection, both total and HCV-specific T cells co-express Tim3 and PD-1 in significantly higher frequencies, compared with HCV mono-infection.
Co-expression of these two markers on HCV-specific CD8+ T cells positively correlate with a clinical parameter of liver disease progression and were shown greater frequencies of Tim3/PD-1 co-expression than HIV-specific CD8+ T cells, which may indicate a greater
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degree of exhaustion in the former. Blocking Tim3 or PD-1 pathways restores both HIV- and HCV-specific CD8+ T-cell expansion in the blood of co-infected individuals (Vali et al., 2010). It remains to be seen whether a similar role exists for TIM3 in other case of infection or cancer where chronic stimulation may result in T cell exhaustion.
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C. The cytoplasmic tail of TIM3 and co-inhibitory receptors in T cell activation
An optimal balance between positive and negative signals delivered through co-stimulatory receptors on the surface of T cells is critical for the generation of an effective cellular immune response. T cell activation is controlled finely by co-stimulatory molecules on the surface of APCs and cytokines secreted by activated lymphocytes for the proper duration. After T cell activation, it is restricted by negative signals in order to maintain homeostasis of T cell population sequentially generated in activated T cells.
These negative signals depend on the co-inhibitory receptors, for example, cytotoxic T lymphocytes antigen-4 (CTLA-4) and programmed death-1(PD-1). It has been reported that these molecules are up-regulated by TCR signaling and then, inhibit proliferation or cytokine synthesis (Smith-Garvin et al., 2009).
Molecular mechanism for inhibitory effect by CTLA-4 have been revealed CTLA-4 blockade in vivo increases T cell responses to antigenic challenges, exacerbates autoimmune disease (Curiel et al., 2004), and enhances T cell-mediated tumor rejection (Leach et al., 1996). CTLA-4 shares the same ligands, namely 1 (CD80) and B7-2(CD86) between CD28. Because CTLA-4 is not readily detectable on resting T cells and is up-regulated after activation, there has been a prevailing idea that CTLA-4 terminates turn-on of T cell responses, possibly by opposing CD28-mediated co-stimulation by competing for the CD80/CD86 ligand and by actively blocking CD28-induced signals (Krummel et al., 1995). The cross-linking of CTLA-4 in conjunction with anti-CD3/CD28 mAb inhibits cell cycle progression, IL-2 secretion, and T cell proliferation (Fraser et al.,
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1999) through inhibition of exracellular signal-regulated kinase (ERK) 3/c-Jun N-terminal kinase pathways (Revilla-Calvo et al., 1997) and NF-κB and AP-1 activation (Olsson et al., 1999).
The cytoplasmic tail of CTLA-4 contains two tyrosine residues (Y) at positions 201 and 208 in mice and a proline (P)-rich region. Y201 is contained within a YVKM motif. In its unphosphorylated form, this motif allows association of CTLA-4 with AP50, the medium-chain subunit of the clathrin adaptor, AP-2. This interaction results in clathrin-dependent endocytosis of 4, therefore limiting 4 surface expression. CTLA-4 might also associate with the serine/threonine protein phosphatase 2A (PP2A)(Bradshaw et al., 1997; Chuang et al., 1997), although the functional consequence of this is not known.
After TCR stimulation, CTLA-4 might undergo tyrosine phosphorylation by SRC kinases, inducing surface retention (Miyatake et al., 1998; Chuang et al., 1999; Chikuma et al., 2000). Whether phosphorylationof Y201 requires for CTLA-4 to associate with phosphatidylinositol 3-kinase (PI3K) and SH2-domain containing protein tyrosine phosphatase (SHP-2) remains controversial (Alegre et al., 2001).
Currently, the molecular mechanisms for the inhibitory effect of TIM3 on T cell activation have not been well studied. TIM3 intracellular domain contains six tyrosine residues, including a tyrosine phosphorylation motif RSEENIY (Monney et al., 2002).
Phosphorylation of these tyrosine residues might be responsible for potentiating of signal transduction initiated by TIM3 activation. TIM3 appears to signal differently in DC and T cells since ligation of TIM3 with TIM3 mAb resulted in different patterns of phosphorylation in these cell types (Anderson et al., 2007). Otherwise, the intracellular
9 signaling of TIM3 is not identified yet.
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D. The effect of function of co-inhibitory receptors on antitumor immune response
It has been observed that a systemic or persistent presence of tumor antigen can impair memory CD8+ T cell function (Freeman et al., 2000; Den Boer et al., 2004). Continuous triggering of the TCR may lead to an up-regulation of negative regulatory molecules and an accumulation of inhibitory Treg cells. Programmed death-1 (PD-1; also referred to as CD279) on T cells binds to the programmed death ligand-1 (PD-L1; also referred to as CD274 or B7-H1) on antigen presenting cells (Dong et al., 1999; Iwai et al., 2002). PD-1 plays an important role in regulation of adaptive immune responses. CD8+T cells are more sensitive to the regulation of PD-1 signals, as demonstrated by the delayed hepatic deletion of activated CD8+ T cells and increased expansion and survival of CD8+ T cells in PD-L1-deficient mice (Dong et al., 2004; Latchman et al., 2004). Whereas PD-1and PD-L1 engagement results in T cell apoptosis, altered T cell cytokine production, diminished proliferation, and reduced cytotoxicity of effector T cells (Dong et al., 1999; Iwai et al., 2002; Blank et al., 2004), blocking PD-L1 signaling has been shown to improve antitumor immunity and immunotherapy (Strome et al., 2003; Hirano et al., 2005).
Tim3 is detected in CD8+ T cells and its ligand in regulatory T cells, raising possibility of modulation of the fate of tumor-specific CD8+ T cells through Tim3 and Tim3 ligand interaction. It is hypothesized that blocking Tim3 signaling may be shown to improve antitumor immunity and immunotherapy.
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E. Tumor-specific regulatory T cells in antitumor immunity
The regulatory T cells (CD4+CD25+Foxp3+ T cells, Tregs) are naturally present in the immune system as in a frequency of ~10% of CD4+ T cells and specifically express the transcription factor Foxp3 (forkhead box P3 transcription factor). Tregs play an essential role in sustaining self-tolerance and immune homeostasis by suppressing a wide variety of physiologically and pathological immune response against self antigens.
Many studies have revealed that Tregs are engaged in the control of antitumor immune responses. The role of Tregs in tumor immunity was first demonstrated by an experiment in which administration of cell-depleting anti-CD25 monoclonal antibody before tumor inoculation suppresses development of syngenic tumors (Onizuka et al., 1999; Shimizu et al., 1999). Several groups have reported that a large number of Tregs are present in tumors and draining lymph nodes of tumor-bearing mice and patients with a poor prognosis (Bromwich et al., 2003; Webster et al, 2006). Importantly, as the numbers of Tregs increase, the ratio of CD8+ T cells to Tregs decreases in tumors, which suggests that Tregs may impede antitumor T cell function (Chakraborty et al., 1991; Curiel et al., 2004;
Cesana et al., 2006). The removal of Tregs is moderately effective in raising immunity against established tumors. The in vivo depletion of CD4+ T cells or administration of anti-CD25 monoclonal antibody is suggested as a to remove Tregs but to simultaneously preserve antitumor CD8+ effector T cells (Tanaka et al., 2002;Yu et al, 2005). Furthermore, combination of CTLA-4 blockade and depletion of Tregs results in maximal tumor rejection (Sutmuller et al., 2001). However the impact of Tregs depletion on the recall
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response of antitumor memory T cells has not been systematically studied, particularly when combined with antitumor immunotherapy.
13 F. Purposes of this research
TIM3 has been shown to suppress Th1 cell function but the molecular mechanisms underlying TIM3-mediated suppression have not been well studied. Furthermore, the effect of blockade of TIM3 pathway in anti-tumor immunity has not been fully investigated. Therefore, I wanted to investigate the molecular mechanisms for inhibition of T cell function by TIM3 and the effect of blocking TIM3 pathway on tumor growth in mice.
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II. MATERIALS AND METHODS
A. Cell and cell stimulation
The human Jurkat T cell line was cultured in RPMI1640 medium (Gibco BRL, Paisley, Scotland) supplemented with 10% FBS (Gibco BRL, Paisley, Scotland), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco BRL, Paisley, Scotland) at 37℃ in 5% CO2. For Jurkat T cell stimulation, PMA (50 ng/ml, Sigma-Aldrich, St Louis, MO, U.S.A) and A23187 (0.5 μM, Sigma-Aldrich, St Louis, MO, U.S.A) were added to the cell suspensions and incubated for the indicating time at 37℃ in 5% CO2.
B. Mice
The C57BL/6 mice, 6 weeks old, were purchased from Deahan biolink (Chungcheongdo, Korea). All animals were kept under standard condition in a 12 h day/night rhythm with free access to food and water and received humane care in accordance with international guidelines and national law.
Study protocols were reviewed and approved by the Animal Care and Use Committee of Ajou University (Suwon, Korea).
15 C. RNA isolation and cDNA synthesis
The total RNA was isolated from the cultured cells using RNAiso (TAKARA Bio, Inc., Shiga, Japan). The cells were lysed using 500 μl of RNAiso and then incubated on room temperature (RT) for 10 min. After addition of 100 ul of chloroform, the cell lysates were incubated on RT and then spin at 13,000 rpm for 15 min at 4℃. The aqueous phase was transferred into the new tube and isopropanol was added into that tube together for total RNA precipitation. After centrifuge, total RNA pellet was washed by adding 500 μl of 75% ethanol, dried and the dissolved in diethyl pyrocarbonate (DEPC)-containing distilled water (DW). The 1.5 μg of total RNA isolated was used to synthesizing cDNA. The total RNA was mixed with 1ul of oligo dT (0.5 μg/ul, IDT, Coralvile, IA, USA), 1 μl of dNTP (each 2.5 mM, TAKARA Bio Inc., Shiga, Japan) and 13.9 μl of DEPC-DW, then incubated at 65℃ for 5 min. After chilling the tube on ice, 4 μl of 5x first stand buffer, 2 μl of 0.1 M DTT and 0.1 μl of reverse-transcriptase (200 u/l, Invitrogen, Carsbade, CA, USA) were added to the tube, then incubated at 42℃ for 1hr. For stopping enzyme activation, the samples were incubated at 72℃ for 15 min.
D. Construction of plasmids for Tim3-hIg fusion protein expression
The Tim3 gene was cloned from splenocytes of a BALB/c mouse. First, splenocytes (106) were stimulated with Concanavalin A (1 μg/ml) for 2 days, and then, total RNA was extracted by RNA-Bee RNA isolation reagent (Tel-Test, Friendswood, TX, USA). Total
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RNA was subjected to reverse transcription using RNase H- reverse transcriptase (Invitrogen, Carsbade, CA, USA). The cDNA was amplified using Tim3 specific primers (Tim3forward and Tim3reverse). The PCR products were cloned into pCR2.1-TOPO vector (Invitrogen, Carsbade, CA, U.S.A.) and sequenced. Tim3 gene sequences were registered in NCBI database. The gene encoding the extracellular domain of Tim3 was amplified with specific primers [Tim3-forward(NheI): 5’GCTAGCATGTTTTCAGGT CTTACCCTCAACTGTG-3’ and Tim3-reverse(BglII)): 5’AGATCTTCTGATCGTTTCT CCAGAGTCCTTAATTTCATCAG-3’], and the PCR product was inserted into pIRES2-EGFP vector (Clontech Laboratories, Inc, Mountain View, CA, U.S.A.) using the NheI and BglII site. The gene encoding IgG1 heavy chain CH2CH3 was amplified using
pTOPO-hIgG1 vector (kind gift of Dr. Kwon, Ajou University, Korea) and the specific primers [HIgC-foward(BglII): 5’-GAAGATCTGCACCTGAACTCCTGGGG-3’ and HIgC-reverse(BamHI): 5’-CGGGATCCTCATTTACCCTGCGACAG-3’]. Using the BglII and EcoRI site, the PCR product was ligated to pIRES2-EGFP downstream of Tim3 gene. The nucleotide sequences were verified.
E. Construction of plasmids for flag tagging TIM3 protein
The human TIM3 gene was cloned from peripheral blood mononcytes (PBMC). First, PBMC (106) were stimulated with Concanavalin A (1 μg/ml) for 2 days, and then, total RNA was extracted by RNA-Bee RNA isolation reagent (Tel-Test, Friendswood, TX, U.S.A.).
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Total RNA was subjected to reverse transcription using RNase H- reverse transcriptase (Invitrogen, Carsbade, CA, U.S.A.). The cDNA was amplified using human TIM3 specific primers [HTIM3F(KpnI): 5’-GGGGTACCGTTAAAACTGTGCCTAACAG-3’, HTIM3R(HindIII): 5’-CCCAAGCTTCAAAAATAAGGTGGTTGG-3’]. The PCR products were cloned into pGEM-T vector (Promega Co., Madison, WI, U.S.A.) and sequenced. To insert TIM3 gene into pIRES2-EGFP vector, The TIM3 gene was amplified with specific primers [TIM3(XhoI): 5’-CCGCTCGAGTACGAAGTGGAATACAGAGCG GAGG-3’and TIM3(XmaI): 5’-TCCCCCCGGGCTATGGCATTGCAAAGCGACAAC-3’]
and the PCR product was inserted into pIRES2-EGFP vector (Clontech Laboratories, Inc, Mountain View, CA, U.S.A.) using the XhoI and XmaII site. And for tagging the leader and flag amino acids to TIM3 protein, the sequence of leader and flag was inserted to pIRES2-EGFP upstream of TIM3 gene using NheI and XhoI sites. The nucleotide sequences were verified. The constructed vectors were named as pIRES2-flag/TIM3.
To construct of lentiviral vector, the gene of leader and flag-TIM3 from pIRES2-flag/TIM3 vector was inserted into the NheI and SwaI sites of pCDH-CMV-MCS-EF1-Puro vector. The constructed vectors were named as pDCH-CMV-TIM3.
To construct the expression vector of TIM3 cytoplasmic mutants, mutation of cytoplasmic tail of TIM3 gene of pIRES3-flag/TIM3 was performed by site-direct mutagenesis kit using specific primers (Stratagene, Santa Clara, CA, U.S.A.). The primers are as follows: hTIM3CPMut3F: 5’-GTAGCAGAGGGAATTCGCTCATAAGAAAAC ATCTATACC-3’, hTIM3CPMut4F: 5’-CATCTATACCATTTAAGAGAACGTATATGAA GTGGAGGAG-3’, hTIM3CPMut5F: 5’-GAAGTGGAGGAGCCCAATTAGTATTATTG
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vector. The constructed vectors were named as pDCH-CMV-TIM3ct36 (the expression vector of TIM3 cytoplasmic mutant containing 36 amino acid from K225 to S260), pDCH-CMV-TIM3ct43 (the expression vector of TIM3 cytoplasmic mutant containing 43 amino acid from K225 to I267), pDCH-CMV-TIM3ct54 (the expression vector of TIM3 cytoplasmic mutant containing 54 amino acid from K225 to N278) and pDCH-CMV-TIM3ct64 (the expression vector of TIM3 cytoplasmic mutant containing 64 amino acid from K225 to Q288).
F. Establishment of stable cells expressing TIM3 protein
For lentivirus production, the packaging cell line, 293TN cells (Invitrogen, Carsbade, CA, U.S.A.) were transfected with using lipectamine2000 (Invitrogen, Carsbade, CA, U.S.A.) with cloned vector, gag-pol expression vector and VSV-G expression vector. The lentiviral supernatants were collected on 48 hr after transfection, and 1ml of supernatants was used to infect Jurkat T cells adjusted to 106 cells/5 ml complete medium supplemented
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with polybrene (4 μg/ml). After infection one day, the cells were suspended with fresh medium and incubated for one day, and then, treated with puromycin (0.2 μg/ml, Invitrogen, Carsbade, CA, U.S.A.) for cloned cell selection.
G. Transfection of plasmids
In the case of Jurkat T cells, total 106 cells were transfected with 4.5 μg of plasmid and 0.5 μg of pEGFP-N1 vector by microporator (sigma-Aldrich, St Louis, MO, U.S.A.), using a single pulse of 40 ms at 1200 V. The pulsed cells were incubated in 2 ml complete medium before stimulation.
H. Preparation of enriched mouse and human CD4+ T cells
For enrichment of mouse CD4+ T cells, spleen and lymph nodes from mice were harvested and single cell suspensions were prepared. Lymphocytes were enriched by lympholyteTM (Cedarlane Lab. Ltd., Burlington, NC, U.S.A.) gradient centrifugation at 2,000 rpm for 20 min. For enrichment of human CD4+ T cells, blood was harvested from donators and peripheral blood cells were enriched by Ficoll-Plaque gradient centrifugation at 300 g for 40 min. CD4+ T cells were enriched by positive selection with the CD4+ T cell isolation kit (Miltenyi Biotec, Gladbach, Germany).
20 I. Flow cytometric analysis
TIM3-over expression on Jurkat T cells were analyzed with the following antibodies by FACSCantoII (Becton Dickinson, San Diego, CA, U.S.A.): anti-human TIM3-PE (R&D Systems, Minneapolis, MN, USA), anti-Flag (Sigma-Aldrich, St Louis, MO,
TIM3-over expression on Jurkat T cells were analyzed with the following antibodies by FACSCantoII (Becton Dickinson, San Diego, CA, U.S.A.): anti-human TIM3-PE (R&D Systems, Minneapolis, MN, USA), anti-Flag (Sigma-Aldrich, St Louis, MO,