International Immunopharmacology

p38 inhibition enhances TCR-T cell function and antagonizes the immunosuppressive activity of TGF-β

Siyin Chen a, b, 1, Jing Zhang a, b, 1, Meiying Shen c, Xiaojian Han a, b, Shenglong Li a, b, Chao Hu a, b,
Wang Wang a, b, Luo Li a, b, Li Du a, b, Da Pang c,*, Kun Tao a, b,*, Aishun Jin a, b,*
a Department of Immunology, College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China
b Chongqing Key Laboratory of Basic and Translational Research of Tumor Immunology, Chongqing Medical University, Chongqing 400016, China
c Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin 150081, China

A R T I C L E I N F O

Keywords:
P38 inhibitor Adoptive cell therapy
Less differentiated T cells TGF-β
Cytokines

A B S T R A C T

The efficacy of adoptive cell therapy (ACT) relies on the abilities of T cells in self-expansion, survival and the secretion of effector molecules. Here, we presented an optimized method to generate T cells with improved functions by supplementing the culture medium with p38 inhibitor and the combination of IL-7 and IL-15 or IL-2 alone. The addition of p38 inhibitor, Doramapimod or SB202190, to IL-7 and IL-15 culture largely increased the capacity of T cells in the proliferation with enrichment of the naïve-like subsets and expression of CD62L.
Importantly, we found this regimen has generated complete T cell resistance to TGF-β-induced functional sup-
pression, with sustained levels of the IFN-γ and Granzyme-B productions. Such findings were also validated in the
melanoma-associated antigen recognized by T cells (MART-1) specific T cell receptor (TCR) engineered T cells, which were expanded in Doramapimod and IL-7 + IL-15 added media. In conclusion, we have established and optimized a protocol with the combination of p38 inhibitor, IL-7 and IL-15, rather than IL-2, for the generation of
functionally enhanced T cells applicable for ACT.

1. Introduction

Advances in adoptive cell therapy (ACT) has emerged as one of promising treatment methods for the solid tumors such as metastatic melanoma [1,2]. Genetically engineered human lymphocytes, T cell receptor T cells (TCR-T) and chimeric antigen receptor T cells (CAR-T), have been applied to treat a wide variety of cancer types. The in vitro generation of T cells plays a major role in exerting antitumor ability in vivo, such as strong proliferation capability [3,4], less differentiated
subsets and long-term survival [5–8].
Cytokines were the major factor not only for T cell proliferation and viability but also for T cell differentiation. The less differentiated T cell, which shown superior efficacy and longer survival, was thought to be an important phenotype for ACT, including naïve T cells (TN), central memory T cells (TCM) and stem-cell like T cells (TSCM) [8–11]. Specially, TSCM have a stronger therapy efficacy because of the self-renewal ability and differentiation potential [11,12]. IL-2 is the most commonly used cytokines for T cell culture, which plays a key role in lymphocyte

activation and proliferation. Recently, IL-7 and IL-15 have emerged as important cytokines for enhancing persistence of T cells in vivo [13–15]. A previous study has demonstrated that IL-7 and IL-15, rather than IL-2, are essentially involved in T cells homeostasis [16]. The optimal T cell expansion culture condition is not yet defined.
Another excited method is using small molecules that pharmaceuti- cally enhance T cell proliferation and antitumor ability. For example, p38 mitogen-activated protein kinase (MAPK) consist of four isoforms, α, β, γ, δ [17] and activated by a wide range of cellular stresses as well as in response to inflammatory cytokines [18]. The p38 MAPK is one of the major pathways triggered by TCR [19]. p38 MAPK was associated with regulation of inflammation and biosynthesis of pro-inflammatory cyto-
kines in human monocytes [20]. Inhibition of p38 MAPK promoted T cell proliferation and expression of CD62L which is a stem cell-like memory marker [21]. We selectively investigated the two most commonly used p38 inhibitors: SB202190 and Doramapimod [22]. SB202190 is an effective p38 inhibitor and targeting the p38 α and p38 β
MAPKs, and Doramapimod which is a more potent p38i targets p38 α, β,

Corresponding authors.
E-mail addresses: [email protected] (D. Pang), [email protected] (K. Tao), [email protected] (A. Jin).
1 These authors contribute equally to this study.

https://doi.org/10.1016/j.intimp.2021.107848

Received 29 April 2021; Received in revised form 28 May 2021; Accepted 1 June 2021
Available online 11 June 2021
1567-5769/© 2021 Elsevier B.V. All rights reserved.

γ, δ MAPKs [23]. High level of CD62L on the CCR7 positive subsets were considered to be the TSCM [24] which is a naïve-like T cell that could rapidly acquire effector functions following antigen stimulation. It re- mains unclear, however, whether p38 inhibitor could increase the expression of CD62L on the CCR7 positive subsets.
Associated tumor microenvironment (TME), the suppressive cyto- kines have been reported linked to poor therapeutic efficacy [25]. TGF-β
is an immunosuppressive cytokine which suppresses T cell proliferation and effector function [26–28]. In addition, TGF-β suppresses the CD8+ T cell maturation [29], proliferation [30] and the production of IFN-γ and
Granzyme-B [31,32]. Therefore, we need improve the T cell ability to resist the suppressive TME in ACT.

In this study, we compared T cells treated with different combination of two commonly p38 inhibitors, Doramapimod and SB202190, under supplementation of the culture medium with either IL-2 or IL-7 IL-15. The focus was to determine the effects of different combinations of p38 inhibitor and cytokine on T cell expansion, expression of CD62L on different subsets and antagonizing the suppressive effect of TGF-β.
2. Method and material
2.1. Cell culture

Lenti-X 293 T cell line was purchased from Takara Biomedical Technology, K562 cell line was purchased from American Type Culture Collection (ATCC). Lenti-X 293 T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, USA) sup- plemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin (Gibco, USA). K-562 cells were cultured in RPMI-1640 (Thermo Fisher Scientific, USA)
supplemented with 10% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin. All cells were maintained at 37 ◦C in an incubator with 5%CO2.

2.2. Culture of T cells from healthy donors

This study was approved by the Ethics Committee of Chongqing Medical University, and the written informed consent was obtained from all healthy donors. The peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation (Lymphoprep, STEM- CELL Technologies, Canada), and cultured in T cell medium containing RPMI-1640 with 10% FBS, 25 mM HEPES, 1 X GlutaMax, 100 IU/mL
penicillin, 100 μg/mL streptomycin, 55 µM β-Mercaptoethanol, 1 X non-
essential amino acid and 1 mM Sodium pyruvate. PBMCs were activated by 1 µg/mL plate-coated anti-CD3 (clone: OKT3) and 1 µg/mL anti-CD28 antibody with the 0.5 µM p38 inhibitors (p38i) Doramapimod (BIRB796, Selleck, USA) , SB202190 (Sigma, USA) or DMSO vehicle (veh) in 300 IU/mL IL-2 or 5 ng/mL IL-7 IL-15(PreproTech, USA) on day 1. Me- dium was changed every 2 days.
2.3. Detection of MART-1 specific T cells
Naïve CD8+ T cells were isolated from PBMC using naïve CD8+ T cell
positive isolation kit as per manufacture instructions (Stem Cell Tech- nologies). The purity of isolated population was confirmed to be >95%
using flow cytometry. CD14+ monocytes were isolated from PBMC using
CD14+ positive isolation kit (Stem Cell Technologies) and cultured in
the DC medium which was made with T cell medium plus 800 IU/mL GM-CSF (PeproTech, USA) and 800 IU/mL IL-4 (PeproTech, USA) for 6 days. On day 3, fresh DC media were added to the cultures. On the day 6, the monocytes were immature DCs. DCs were incubated in DCs medium and then 1 µM MART-1 peptide was added. After 4 h, the media were replaced to DC media supplemented with DC maturation cytokines including 10 ng/mL Lipopolysaccharide (LPS) (Sigma-Aldrich, USA) and
100 IU/mL IFN-γ (PeproTech, USA) incubated overnight. DCs were washed and co-cultured with naïve CD8+ T cells in T cell culture

medium supplemented 30 ng/mL IL-21 and 5 ng/mL IL-7 IL-15 for the first two days. On the third day, the medium was replaced to T cell culture medium supplemented with 5 ng/mL IL-7 IL-15. On day 10, the MART-1 specific T cells were obtained by tetramer staining and flow sorting.
2.4. Lentivirus production and transfection of human PBMC

The MART-1-TCR was cloned into the lentivirus vector pWPXL (Addgene Plasmid #12257). Lentiviruses were generated by co- transfecting Lenti-X 293 T cells with the lenti-vector and the pack- aging plasmids psPAX2 (Addgene Plasmid #12260) and pMD2.G (Addgene Plasmid #12259) at a ratio of 5:2:1, using the Xfect Trans- fection Reagent (Takara, Japan). The lentiviral supernatants were har- vested after 48 h. PBMCs were stimulated with 1 µg/mL OKT3 and 1 µg/
mL anti-CD28 antibody with the 0.5 µM p38 inhibitor in 300 IU/mL IL-2
or 5 ng/mL IL-7 IL-15 for 48 h. On the third day, the activated T cells were transduced with the MART-1-containing lentiviral supernatant and the supernatant was replaced with the fresh T cell medium contained
p38 inhibitor and IL7 + IL-15 or IL-2.
2.5. Rapid expansion of TCR-T cells

For rapid expansion, TCR-T cells were expanded using excess irra- diated (50 Gy) allogeneic PBMC feeder cells (a pool from 3 to 5 different donors) at a ratio of 1 to 100 with 30 ng/mL OKT3 and 0.5 µM Dor- amapimod, in the media consisted of a 1:1 miXture of the T cell media
and the AIM-V media (Gibco, USA). All cells were cultured at 37 ◦C with
5% CO2. Media were exchanged 3 days post-stimulation and then every other day. The cells were rapidly expanded in this way for two weeks.
2.6. ELISA assays

T cells were rested in cytokine-free and p38 inhibitor-free media overnight and restimulated by 0.5 µg/mL OKT3 for 24 h, and 30 ng/mL TGF-β1 (PreproTech, USA) was added simultaneously. The supernatant was added into ELISA plates coated with human IFN-γ capture antibody (Clone MD-1, 2 µg/mL, Biolegend) or human Granzyme-B capture antibody (Clone GB10, 2 µg/mL, Mabtech). The plates were developed with bio-human IFN-γ detection antibody (Clone 3D1D12, 1 μg/mL, Biolegend) or bio-human Granzyme-B detection antibody (Clone GB11,
1 μg/mL Mabtech), followed by incubation with Streptavidin-ALP (1:1000, Mabtech, Sweden) and pNPP. The plates were analyzed by the Varioskan LUX Multimode Microplate Reader for the OD405 value.
2.7. Flow cytometry analysis
For staining, 5 105 cells were re-suspended in PBS containing 2% FBS, and stained with the anti-human antibody cocktail for 30 min at room temperature in the dark. CD3-BV510 (Clone SK7, Biolegend, USA), CD4-PerCP-Cy5.5 (Clone RPA-T4, Biolegend, USA), CD8-FITC (Clone HIT8a, Biolegend, USA), CD45RA-APC (Clone HI100, Biolegend, USA), CCR7-BV605 (Clone G043H7, Biolegend, USA) were used for the T cell subsets marker assay. The expression of CD62L was detected by anti- CD62L-PE antibody (Clone DREG-56, Biolegend, USA). The tetramer/ BV421-HLA-A*02:01 MART-1 (ELAGIGILTV) (MBL, TB-0009-4, Japan)
and mouse TCRβ antibody (Clone H57-59, Biolegend, USA) used for the
identification of antigen-specific T lymphocytes. The CD69-PE (Clone FN50, Biolegend, USA) antibody was used for labeling activated T cells.
2.8. Statistical analysis

Statistical computations were done using the Graph Pad Prism 8.0 software. Data was presented as mean ± SEM. Statistic comparisons between groups were performed via a Student’s t-test for two groups, or a Paired Student t-test. A p-value of < 0.05 was considered significant.
Fig. 1. P38 inhibition enhances the proliferation of T cells and affects the proportion of different CD8+ T cell subtypes. (A-B) Cell number curves of PBMC with or without p38i treated was counted with an automatic cell counter on the 8th and 12th day in IL-7 + IL-15 and IL-2 (n = 3); statistical significance was calculated using
two-way Student t-test, *p < 0.05, **p < 0.01, ***p < 0.001; respresentative data of three independent and replicate experiments were shown. (C-D) Results from flow plots of one representative donor and summary data for indicated CD8+ T cells group were shown (left), and the bar plot of five donors were shown (right) in IL-
7 + IL-15 and in IL-2 (n = 5); statistical significance was calculated using two-way Student t-test, *p < 0.05, **p < 0.01, ***p < 0.001; respresentative data of three independent and replicate experiments were shown. (E-F) FACS-based quantification of the CD62L expression on total CD8+T cells (left), CD8+TN (middle) and CD8+TCM (right) after stimulation with anti-CD3/CD28 antibodies in vitro expansion for 10 days with Doramapimod or SB202190 in IL-7 + IL-15 (n = 5); statistical significance was calculated using Paired Student t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. TGF-β affects the secretion of IFN-γ and Granzyme-B. (A-B) The IFN-γ secretion of PBMCs was detected on day 2 after 0.5 µg/mL anti-CD3 antibody re- stimulation absence or presence of TGF-β by ELISA in IL-7 + IL-15 and in IL-2 (n = 3); statistical significance was calculated using two-way Student t-test, *p < 0.05, **p < 0.01, ***p < 0.001; respresentative data of two independent and replicate experiments were shown. (C-D) The Granzyme-B secretion of PBMCs was detected on day 2 after 0.5 µg/mL anti-CD3 antibody re-stimulation absence or presence of TGF-β by ELISA in IL-7 + IL-15 (C) and in IL-2 (D) (n = 3); statistical significance was calculated using two-way Student t-test, *p < 0.05, **p < 0.01, ***p < 0.001; respresentative data of two independent and replicate experiments were shown.
3. Results
3.1. SB202190 promotes the expansion and the differentiation of T cells in vitro
To investigate the differences between SB202190 and Doramapimod in promoting T cell expansion, we stimulated the bulk PBMCs from healthy donors with anti-CD3/anti-CD28 antibodies in IL-7 IL-15 or IL-2 conditioned culture in the presence and absence of p38i and analyzed T cell proliferation at day 8 and day 12. Although we did not observe any difference in T cell proliferation on day 8 in IL-7 IL-15 or IL-2 conditions, we found that Doramapimod and SB202190 could both significantly increase the total numbers of T cells 12 days post TCR stimulation, where SB202190 was shown to be more effective (Fig. 1A, B). Of note, such induction of T cell expansion was not observed when these p38i were added after 48 h in TCR-depended stimulation (Fig S1A- 1B). These results demonstrated that inhibition of p38 by Doramapimod or SB202190 could efficiently induce T cell proliferation during the phase of TCR-stimulation.
To test whether p38i affect the differentiation of T cells, we analyzed T cell phenotypes on day 10 after p38i culture via the markers of CD45RA and CCR7 for distinct T cell subsets. We found that the addition
of Doramapimod or SB202190 significantly increased the proportion of CD45RA+ and CCR7+ cells to approXimately 60%, in either IL-7 IL-15
or IL-2 conditioned media, compared with the control group (Fig. 1C). And an observation of preferred reduction in CD8+ effector memory re-
expressing CD45RA T cells (TEMRA)was made with media supplemented with IL-7 IL-15 (Fig. 1C), and CD8+ TEM with IL-2 (Fig. 1D). These
results demonstrated that p38 inhibition could retain T cells in less

differentiated phenotypes during expansion.
Given the importance of CD62L expressing T cells in antitumor functional activity, we determined whether p38i affected the expression
of CD62L in various T cell subsets. A significant increase in CD8+ T cells,
particularly the TN and the TCM subsets, was observed in IL-7 IL-15 conditioned media with the addition of Doramapimod (Fig. 1E and Fig
S1C) and SB202190 (Fig. 1F and Fig S1C). For the IL-2 condition, although the minimum effect observed in the frequency of CD62L+ cells in CD8+ TN subsets, Doramapimod effectively expanded total CD8+ T
cells, with a comparative induction of the TCM subsets (Fig S1E and Fig S1D). Similar results were found in SB202190 treated groups (Fig S1F and Fig S1D).
Taken together, inhibition of p38 by Doramapimod or SB202190 could increase the proliferative capability and prevent the differentia-
tion of T cells, with a preferred enrichment of CD62L expressing CD8+
TN and TCM subsets. And such improvement was better in IL-7 IL-15 than in IL-2 conditioned culture media.

3.2. P38 inhibition effectively antagonized the TGF-β mediated T cell functional suppression
Next, we tested whether the improved T cell characteristics brought by p38 inhibition would be affected by TGF-β. We cultured T cells following the same procedure as shown above, with the addition of 30 ng/mL of TGF-β (Fig S2A). Our results showed that TGF-β significantly reduced the IFN-γ secretion (Fig. 2A and B). When p38 was inhibited, we found that IFN-γ production was significantly elevated with either IL-7
+ IL-15 or IL-2 conditioned media (Fig. 2A and B). Interestingly, we
found that p38i could sustain the markedly enhanced IFN-γ secretion
Fig. 3. Transcriptome analysis of Doramapimod treatment CD8+ T cells by RNA-seq. (A) GSEA analysis of T cell proliferation upregulated gene set in Doramapimod
treatment versus veh control. (B) Volcano plot showing the aberrantly expressed genes between the Doramapimod treated samples and veh samples. Red dots indicated the genes showing an expression difference with a p < 0.05, while the green dots fail to meet the criteria. The Doramapimod treatment group upregulated DEGs are displayed on the right of the plot, and the downregulated DEGs are on the left side. Some genes were marked in the figure. The x-axis represents the log2 FC
score, and the y-axis shows the -log10 value (p value). (C) BoX plot of the CD62L FPKM in Doramapimod treatment and veh control in 3 donors; statistical sig- nificance was calculated using two-way Student t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

levels even in the presence of 30 ng/mL TGF-β (Fig. 2A and B). Also, the same phenomenon was found in Granzyme-B production (Fig. 2C and

treated T cells (Fig. 3B). IL-18 could sustain the cytolytic T cell attack in the long term and maintain the cytolytic CD8 + T cells in an earlyD).effector stage through alleviating T-bet and accumulating FoXO1
Taken together, p38 inhibition could enhance the secretion of IFN-γ and Granzyme-B of T cells upon TCR stimulation result in an enhance- ment of T cell functionality, and more importantly, maintain the capa- bility of T cells to release effector cytokines in the suppressive environment brought by TGF-β.

3.3. Gene expression profile with Doramapimod treatment is associated with T cell functional improvement
We next sought to obtain a more global assessment of the influence of Doramapimod on the gene expressions of CD8+ T cells. We performed a transcriptomic analysis of CD8+ T cells from 3 donors, following a 10-
day expansion in the presence of Doramapimod or the veh control. In total, we identified 3,511 and 3,986 significantly up- and down- regulated transcripts, respectively, associated with p38 inhibition (p
< 0.05, FDR < 0). Specifically, we found that the CD8+ T cells treated
with Doramapimod had stronger T cell proliferation gene enrichment than the control group (Fig. 3A). Furthermore, the volcano plot showed that the relative RNA expressions of genes involved in the early differ- entiated memory T cells, SPINT2, IRF8, MRPS26, CAPG, KCNK6, and the effector function genes, such as GZMB and GNLY, were elevated (Fig. 3B). The genes associated with the exhausted T cell population, TRAT1, TNFAIP8, MEOX1, ARL6IP5, CRTAM, PICALM, FABP5,
STAMBPL1, FLV6 and TSHZ2, were shown to be decreased in the analyzed samples (Fig. 3B). Besides, we found that two of IL-18 receptor genes, IL18R1 and IL18RAP, were both upregulated in Doramapimod

[33,34]. When we examined the specific transcripts of SELL, the gene encoding CD62L, we found that it was upregulated in all donors (Fig. 3C). These findings were consistent with our in vitro observations showed above (Fig. 1). Together, we have demonstrated that p38 inhi- bition with Doramapimod could promote a global gene profile change directly associated with T cell functional improvement in proliferation, differentiation and cytolytic potentials.

3.4. Doramapimod enhanced the function of MART-1 specific TCR- engineered T cells in vitro
To validate if Doramapimod could enhance the cytotoXic functions of TCR-engineered T cells, we engineered TCR-T cells recognizing the MART-1. Firstly, we screened MART-1-specific TCRs from the PBMC of a healthy donor with HLA-A*02:01 genotype (Fig S3A). The presence of
MART-1 specific CD8+ T cells were confirmed by detecting with MART-
1 tetramer (Fig. 4A). Then we detected MART-1 specific TCR-transduced CD8+ Jurkat cell line with MART-1 HLA-A*02:01 tetramer. (Fig. 4B). Moreover, the expression of CD69 on the CD8+ Jurkat cells that trans-
duced MART-1 TCR was elevated, which were co-cultured with HLA- A*02:01-transduced K562 cells loaded with MART-1 peptide, compared with the unloaded group (Fig S3B). These results confirmed the functional recognition of the MART-1 specific TCRs.
Next, we investigated the effect of p38 inhibition in regulating the memory phenotypes and functions of T cells engineered as above. As shown in Fig. 4C, the PBMCs derived from another 3 healthy donors
Fig. 4. Inhibition of p38 signaling promotes expansion of CD62L-expressing engineered human peripheral blood T cells and antagonized the TGF-β. (A) Flow
cytometry of naïve CD8+ T cells obtained from healthy donors PBMCs and cocultured with autologous DCs loaded with MART-1 peptide 10 days, then stained with CD8-antibody and MART-1-HLA-A*02:01 tetramer. (B) Specificity verification for MART-1 TCR in CD8-Jurkat cells. CD8-Jurkat cells were stained with MART-1- HLA-A*02:01 tetramer and anti-mouse TCR β chain (mTCR) antibody. (C) Schema of clinical scale lentivirus transduction and expansion of MART-1 TCR engi-
neered T cells with or without Doramapimod in IL-7 + IL-15. (D) Quantification of CD62L expression on MART-1 TCR+ CD8+ T cells after 24 days of clinical scale
transduction and expansion with or without Doramapimod detected by flow cytometry (n 3); statistical significance was calculated using Paired Student t-test, *p
< 0.05, **p < 0.01, ***p < 0.001. (E-F) Representative histograms showing IFN-γ and Granzyme-B secretion for MART-1 TCR-T and stimulated with 1X10-5 µM MART-1 peptide loaded K562-HLA-A*02:01 cells for 24 h absence or presence of 30 ng/mL TGF-β (n = 3); statistical significance was calculated using two-way Student t-test, *p < 0.05, **p < 0.01, ***p < 0.001; respresentative data of two independent and replicate experiments were shown.
were activated by anti-CD3/CD28 antibodies, transduced by the MART- 1 specific TCR and subjected to functional assays (Fig. 4C). We observed that Doramapimod had significantly increased the expression of CD62L
on CD8+ TCR-T cells (Fig. 4D). When we re-stimulated these TCR-T cells
with HLA-A*02:01-transduced MART-1 peptide loaded K562 cell line in the presence of TGF-β, we found that environmental TGF-β significantly inhibited their production of IFN-γ and Granzyme-B (Fig. 4E and F). Importantly, these suppressive effects generated by TGF-β could be robustly rescued by p38 inhibition (Fig. 4E). In fact, Doramapimod could induce more than 2-fold increase of the total level of Granzyme-B (Fig. 4F). These data thus establish the relevance and refusion therapy feasibility of T cell generation with p38 inhibitor containing IL-7 IL-15 in vitro.

4. Discussion

In this study, we reported that the addition of p38 inhibitor during TCR-T cell expansion culture could improve the quantity and quality of TCR-T cells, particularly using the combination of IL-7 and IL-15 as the activation stimuli. Importantly, we demonstrated that functionally blocking p38 kinase activity could antagonize the suppressive effect of
TGF-β treatment, improving the secretion of IFN-γ and Granzyme-B in
vitro.
CD45RA+ CCR7+ T cells have been characterized as naïve T cells which further differentiate into terminal T cells with the T cell expansion
culture in vitro. And the progressive differentiation of T cells to a ter- minal differentiated effector stage results in a series of phenotypic and functional changes that make them reduce the production of IFN-γ and Granzyme-B [35,36]. In our experiment, p38 inhibitor treatment
increased the proportion of CD45RA+ CCR7+ T cells and decreased the

proportion of TEMRA in IL-7 IL-15 and TEM in IL-2. Meanwhile, the
expression of CD62L on CCR7+ T cells was increased which supple- mentary with IL-7 and IL-15. Thus, we speculated that the increased
naïve T cells after p38 inhibitor treatment were naïve-like T cells which considered to be TSCM [24]. In the context of ACT, TSCM exhibit more cytotoXicity and long-lasting survival when encounters the cognate an- tigen [10]. The improved IFN-γ and Granzyme-B production provide a supporting evidence. Additionally, in our study, the early differentiated
memory genes and GZMB were upregulated in p38i treated CD8+ T cells
in RNA-seq.
IL-2 has been used as the primary modulator for the in vitro TCR-T expansion, and is important for T cell activation, proliferation and dif- ferentiation. In addition, IL-2 promoted the differentiation of effector T cells towards terminal subset, and eventually led to exhausted state [37]. The paring of IL-7 and IL-15 was a novel cytokine combination in ACT for the maintenance of TSCM [38], which result in better maintain T cell survival and cytotoXicity. We have confirmed the addition of p38i can further improve the enrichment of naïve-like T cells brough by IL-7 and IL-15. Thus, the combination of IL-7 IL-15 with p38i might be an optimal choice to elevate the proportion of functional T cell products for the transfer therapy.
TGF-β has been shown to inhibit T cell functions in at least two
distinct ways. On one hand, TGF-β affect terminally differentiated T cell subsets to a higher extent than the stem-like T cell populations [39,40]. On the other hand, it has been proven that TGF-β could inhibit T cell numbers and functions in a p38-dependent manner, by increasing the
proportion of CD8+ Treg cells [41] as well as inducing T cell apoptosis
[42]. Therefore, we speculated the addition of p38 inhibitor, particular the increase in naïve-like T cell numbers brought by p38i, could lead to a potential resistance of the T cells in expansion to the overall suppressive effect generated by TGF-β. Indeed, p38 inhibitor has completely rescued the inhibitive effect of TGF-β, as the post-stimulation secretions of IFN-γ and Granzyme-B have raised to the levels prior to encountering TGF-β. Moreover, the expressions of TGF-β receptors were not found to be downregulated in the RNA-seq results (data not shown), suggesting that the inhibition of p38 MAPK pathway did not affect the TGF-β signaling activity at the receptor levels, through the negative feedback loop [43]. It is worth mentioning that the specific inhibition of p38 α and β by SB202190 has demonstrated similar improvement in T cell expansion and IFN-γ and Granzyme-B secretions to those induced by Dor- amapimod, which targets a broad-spectrum of p38 subtypes. This is in line with the previous findings that p38 α and β are the dominant sub- units in the regulation of T cells functions [44]. Given the significance of p38 inhibitors and IL-7 IL-15 in the functional optimization for restimulated TCR-T cells, its effect in the regulation of tumor infiltrating lymphocytes (TIL) and CAR-T need to be addressed, preferentially in
combination with IL-2.
In summary, we have presented an optimal regimen for TCR-T expansion in vitro, with enhanced characteristics required for ACT. Such improvement may provide T cell products with the capability of long-term survival and facilitate the development of improved immu- notherapy of epithelial cancer, antagonizing immunosuppressive fac- tors, such as TGF-β, and increasing the therapeutic efficacy of Immune checkpoint inhibitors (ICIs) [45,46].
Funding

This work was supported by National Natural Science Foundation of China, grant number 81572807 and 81872329; and by Chongqing Municipal Education Commission of Science and Technology Research Project of China, grant number: KJZD-K201800403
CRediT authorship contribution statement
Siyin Chen: Investigation, Methodology, Writing – original draft.
Jing Zhang: Investigation, Methodology, Writing – original draft.

Meiying Shen: Investigation, Methodology, Writing – original draft, Methodology, Project administration. Xiaojian Han: Methodology, Project administration. Shenglong Li: Software, Data curation. Chao Hu: Software, Methodology. Wang Wang: Writing – review & editing. Luo Li: Formal analysis, Investigation, Resources. Li Du: Investigation, Formal analysis. Da Pang: Supervision. Kun Tao: Conceptualization, Supervision, Project administration. Aishun Jin: Conceptualization, Supervision, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements

We would like to thank all team members involved in tumor immunotherapy project at Chongqing Key Laboratory of Basic and Translational Research of Tumor Immunology, Chongqing Medical University for their technological assistance and help in developing this article. We also thank all healthy individuals participated in this study. We appreciate supporter Mr. Yuling Feng providing Chongqing Medical University fund (X4457).
Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi. org/10.1016/j.intimp.2021.107848.
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