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

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.

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

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.).

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

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

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 lympholyte^TM (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, U.S.A.) and secondary antibody, anti-mouse IgG-PE (ebioscience, Inc., San Diego, CA, U.S.A.).

To confirm of purified CD4⁺ T cell from mouse lymphocytes and human PBMC, the following antibodies were analyzed: anti-mouse CD4-APC (ebioscience, Inc., San Diego, CA, U.S.A.), anti-human CD4-APC (BD biosciences, Franklin Lakes, NJ, U.S.A.).

Stained cells were analyzed by a BD FACSCantoII system (Becton-Dickinson Co.

Franklin Lakes, NJ, U.S.A.).

To sorting of TIM3^highCD4⁺ T cell and TIM3^lowCD4⁺ T cell from human CD4⁺ T cell, the cells were stained with anti- human CD4-APC and anti-human TIM3-PE. Stained cells were sorted by a BD AriaII system (Becton-Dickinson Co.Franklin Lakes, NJ, U.S.A.).

The frequency of Tregs in mice was analyzed with the following antibodies by FACSCantoII: anti-mouse CD4-APC, anti-mouse CD25-PE, anti-mouse Foxp3-FITC (ebioscience, Inc., San Diego, CA, U.S.A.).

All antibodies were bound at 4℃ for 30 min. After incubation, cells were washed twice in PBS containing 2% BSA. All data were analyzed with FACSDiva software (Becton-Dickinson Co.Franklin Lakes, NJ, U.S.A.) or WinMD2.8.

21 J. SDS-PAGE and Western blotting

Jurkat T cells (3 x 10⁵) were lysed in 80 μl of lysis buffer (50 mM HEPES, 250 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% Triton X-100) containing protease inhibitor (Calbiochem, La Jolla, CA, U.S.A.). The concentration of protein was measured by Bradford protein assay. The total lysate was mixed with 20 μl of 5x SDS sample buffer, denaturized at 100℃ for 5 min. The 15~100 μg of protein was loaded in each lane.

Proteins were separated by SDS-PAGE and transferred onto Polyvinylidene difluoride (PVDF) membranes. The indicated antibodies used to probe the membranes are as follows:

Anti-Flag monoclonal antibody (Sigma-Aldrich, St Louis, MO, U.S.A.), anti-TIM3 polyclonal antibody (R&D Systems, Minneapolis, MN, U.S.A.), anti-c-Jun Ab (Abcam, Cambridge, UK) and anti-NFAT-1 Ab (BD Biosciences, Franklin Lakes, NJ, U.S.A.). The blots were probed with anti-rabbit IgG or anti-mouse IgG Ab linked to horseradish peroxidase and developed by using chemiluminescence. To ensure equal loading, blots were stripped and then reprobed with anti-β-actin Ab or anti-α-tubulin Ab (Cell Signal Tech, Inc., Danvers, MA, U.S.A.).

K. Real-time reverse-transcript (RT)-PCR

The expression of particular gene in Jurkat T cell lines stimulated with PMA (50 ng/ml) and A23187 (0.5 μM) was examined by real-time PCR. This relative quantification was performed using the ABI PRISM 7000 Sequence Detection System (Applied

Biosystems, Foster, CA, U.S.A.). SYBR green Ex Taq Premix (TAKARA Bio, Inc., Shiga, Japan) containing primer sets was used in the PCR. The levels of GAPDH mRNA or CD4 mRNA were measured as an internal standard for calibration. The primers are as follows:

human IL-2: forward-5’CAACTCCTGTCTTGCATTGCACTAA-3’ and

revese-5’AATGTGAGCATCCTGGTGAGTTTG-3’, human IFN-γ:

forward-5’CTTTAAAGATGACCAGAGCATCCAA-3’ and reverse-5’GGCGACAGTTCAGCCAT CAC-3’, human c-Jun: forward-5’GGGAACAGGTGGCACAGCTTA-3’ and reverse-5’GCAACTGCTGCGTTAGCATGA-3’, human c-Fos: forward-5’GGGAGCTGACTGAT ACACTCCAAG-3’ and revese-5’TGGCAATCTCGGTCTGCAA-3’, human GAPDH:

forward-5’AGGGCTGCTTTTAACTCTGGTAAA-3’ and reverse-5’CATATTGGAACAT (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 for 36 hr before stimulation. Cells were stimulated with PMA (50 ng/ml) and A24187 (0.5 μM) for 12 hr. After stimulation, the cells were washed with cold PBS, lysed and assayed luciferase activity (Luciferase Assay System Kit; Promega Co., Madison, WI, U.S.A.). Cells were lysated with 130 μl of lysis

buffer and 100 μl of cell lysates were used for luciferase activity assay. The percentage of cells expressing GFP protein was measured by FACSCantoII for calibration of transfection efficiency.

M. ELISA

Culture supernatants were collected for 2 or 3 days following T cell stimulation and IL-2 or IFN-γ level was measured in a sandwich enzyme-linked immunosorbent assay (ELISA; R&D systems, Inc., Minneapolis, MN, U.S.A.) following the manufacturer’s recommendations.

III. RESULTS

A. IL-2 transcription is reduced in T cells expressing TIM3

Previously, the function of TIM3 has been explored in autoimmune diseases and severe virus-mediated diseases. Those studies showed that TIM3^high T cells failed IL-2 or IFN-γ production (Jones et al., 2008; Hastings et al., 2009). To confirm these results, enriched CD4⁺ T cells were stimulated with anti-CD3 and CD28 mAb for 1 week and sorted into TIM3^high and TIM3^low cell population (Fig. 1A). Each cell population was re-stimulated with PMA and A23187 for 4 hr and the IL-2 expression was assessed by real-time RT-PCR.

Consistent with the previous report, TIM3^high CD4⁺ T cells produced lower amount of IL-2 than TIM3^low CD4⁺ T cells (Fig. 1B).

To study molecular mechanisms that TIM3 expression inhibits IL-2 and IFN-γ production upon CD4⁺ T cell activation, I established Jurkat T cells-over expressing flag-tagging TIM3 by lentiviral gene delivery method and then examined whether inhibitory effect of TIM3 expression on IL-2 production can be reproduced in these cells. The TIM3 expression in these cells was demonstrated by flow cytometry using antibodies against to flag tag and TIM3 (Fig. 2A). These cells stimulated with PMA and A23187 for 4 hr were analyzed by real-time RT-PCR and found to be significantly reduced compared to parental Jurkat T cells (Jurkat) and control cells infected with empty lentivirus (ConV) (Fig. 2B).

Inhibitory effect of TIM3 expression on IL-2 production was further evaluated by measuring the amount of IL-2 secreted into culture supernatant after stimulation these cells

with PMA and A23187 for 2 or 3 days (Fig. 2C). Compatible to transcription level, the amount of secreted IL-2 profoundly decreased in Jurkat T cells-over expressing TIM3. The reduction of IFN-γ secretion could not be demonstrated since IFN-γ was not detected in culture supernatant of any cell line by ELISA. These results indicate that this cell line system is appropriate to explore the mechanism for the inhibitory effect of TIM3 expression on IL-2 and IFN- γ production.

26 A

Fig. 1. IL-2 transcription is reduced in primary CD4⁺ T cells expressing TIM3. CD4⁺ T cells were purified from human peripheral blood (N=3) and stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) monoclonal Ab one week after stimulation. Cells were stained with APC-anti-human CD4 and PE-anti-human TIM3 Ab and sorted into TIM3^high and TIM3^low population by flow cytometry (A). After sorting, each cell population was stimulated for 4 hr with PMA (50 ng/ml) and A24187 (0.5 μM). IL-2 transcript level was analyzed by real-time RT-PCR and normalized to CD4 transcript level. Relative IL-2 transcription level of stimulated cells to unstimulated cells is shown.

28 A

Fig. 2. IL-2 and IFN-γ transcription is reduced in Jurkat T cells expressing TIM3.

TIM3 expression on the cell surface was analyzed in cell lines: parental Jurkat T cells (Jurkat), Jurkat T cells infected with control lentivirus (ConV), and Jurkat T cells infected with lentivirus expressing flag tagged T cells. These cells were labeled with PE-conjugated anti-TIM3 Ab (left) and anti-Flag Ab (right), respectively and then analyzed by flow cytometry (A). Transcription levels of IL-2 (left) and IFN-γ were analyzed in cells stimulated with PMA (50 ng/ml) together with A23187 (0.5 μM) for 4 h by real-time RT-PCR. Transcript level of IL-2 and IFN-γ, respectively, was normalized to GAPDH transcript level. Relative transcript level in stimulated cells to non-stimulated cells in shown. Data is shown as mean ±SD of three independent experiments (B). IL-2 secretion by cells stimulated with PMA (50 ng/ml) together with A23187 (0.5 μM) for 2 or 3 days were analyzed by ELISA. Data is shown as mean ±SD (C).

B. Transcriptional activities of AP-1 and NFAT are reduced in TIM3-over expressing jurkat T cells

Upon T cell activation, IL-2 production is promoted via three major pathways;

calcium-NFAT pathway, NF-κB pathway and Ras-MAPK-AP-1 pathway. To determine which of these three pathways are affected by TIM3-over expression, luciferase reporter assay was performed. Luciferase activities were increased in cells transfected with plasmid containing DNA element responsive to NFAT, AP-1 and NF-κB, respectively. Notably, NFAT activity was reduced by 30% and AP-1 activity by 80% in TIM3-over expressing Jurkat T cells compare to controls. However, NF-κB activity was similar between TIM3-over expressing Jurkat T cells and control cells (Fig. 3). These results indicate that TIM3 expression affects activation of NFAT and AP-1, but not of NF-κB.

Fig. 3. Transcriptional activities of AP-1 and NFAT are decreased in TIM3-over expressing Jurkat T cells. Cells were transfected with the indicated luciferase activity plasmid together with pEGFP-N1 for normalization of the transfection efficiency. Thirty-six hr after transfection, the cells were stimulated with PMA (50 ng/ml) and A23187 (0.5 μM) for 12 hr. A part of cell was subjected to flow cytometry for the EGFP expression and the rest of cells to the assay for luciferase activity. Basic: pGL3-Basic luciferase control vector, NFAT: NFAT driven luciferase plasmid, AP-1: AP-1 driven luciferase plasmid, κB: NF-κB driven luciferase plasmid. All data are presented as fold induction relative to pGL3-basic transfector without stimulation. Data is shown as mean±SD of three independent experiments.

C. TIM3 expression attenuates the induction of c-Jun in stimulated T cells but not of c-Fos

The finding that AP-1 transcriptional activity is suppressed in TIM3-over expressing Jurkat T cells prompted us to investigate the expression of c-Jun and c-Fos protein, components of AP-1, in these cells after stimulation. First, induction of c-Jun and c-Fos mRNAs was analyzed by real-time RT-PCR. The mRNA levels of c-Fos and c-Jun were increased 30 min after stimulation and declined 1h after stimulation in all cells. The transcript level of c-Fos was similar in TIM3-over expressing Jurkat T cells and control Jurkat T cells 30 min after stimulation but significantly higher in TIM3-over expressing Jurkat T cells than control cells 1hr after stimulation. In contrast the mRNA induction of c-Jun was significantly repressed in TIM3-over expressing Jurkat T cells compared to control ConV cells at all time points after stimulation and to Jurkat T cells 30 min after stimulation (Fig. 4A). C-Jun expression was further evaluated by Western blotting. In accordance with transcriptional level of c-Jun, its protein level was increased 1h after stimulation, and less in TIM3-over expressing Jurkat T cells than control cells. These results imply that TIM3 expression represses c-Jun induction, which then leads to reduced AP-1 activating (Fig. 4B).

33 A

Fig. 4. TIM3 expression attenuates the expression of c-Jun but not of c-Fos. Cells were stimulated with PMA (50 ng/ml) and A24187 (0.5 μM) for the indicated time. The mRNAs levels of c-Jun and c-Fos was analyzed by realtime RT- PCR. All data are presented as fold induction according to transcript level of unstimulated cells, respectively (A). The expression of c-Jun protein in cells stimulated with PMA and A23187 for indicated time was analyzed by Western blotting. β-actin expression was used as a loading control (B). Jurkat:

parental Jurkat T cells, ConV: Jurkat T cells infected with control lentivirus, TIM3: Jurkat T cells infected with lentivirus expressing TIM3. Data is shown as mean±SD. *, P<0.02 compared with Jurkat. **, P<0.02 compared with ConV.

D. TIM3 expression affects the phosphorylation status of NFAT in stimulated T cells

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. When cells are activated, these proteins are dephosphorylated by the calcium/calmodulin-dependent phosphatase calcineurin, translocate to the nucleus, and activate target genes (Kiani et al, 2000; Okamura et al, 2000). To examine whether the defective NFAT transcriptional activity in TIM3-over expressing Jurkat T cells was due to reduced dephosphorylation, the kinetics of NFAT dephosphorylation was analyzed in cytoplasmic fraction of cells stimulated with PMA and A23187 stimulation by Western blotting. A major NFAT band with an apparent molecular weight of 140 kDa was observed in unstimulated cells, indicating that the predominant fraction of NFAT is present in the cytoplasm in a phosphorylated form (Fig. 5 lane 1, 6, 11). After stimulation, smear of bands smaller than NFAT in a phosphorylated form appeared as the band of

문서에서 The Suppressive Mechanisms of TIM3 on T Cell Cytokine Production and The Anti-Tumor Effect of TIM3-Pathway Inhibition (페이지 21-0)