PD‐1‐induced T cell exhaustion is controlled by a Drp1‐dependent mechanism

Programmed cell death‐1 (PD‐1) signaling downregulates the T‐cell response, promoting an exhausted state in tumor‐infiltrating T cells, through mostly unveiled molecular mechanisms. Dynamin‐related protein‐1 (Drp1)‐dependent mitochondrial fission plays a crucial role in sustaining T‐cell motility, proliferation, survival, and glycolytic engagement. Interestingly, such processes are exactly those inhibited by PD‐1 in tumor‐infiltrating T cells. Here, we show that PD‐1pos CD8+ T cells infiltrating an MC38 (murine adenocarcinoma)‐derived murine tumor mass have a downregulated Drp1 activity and more elongated mitochondria compared with PD‐1neg counterparts. Also, PD‐1pos lymphocytic elements infiltrating a human colon cancer rarely express active Drp1. Mechanistically, PD‐1 signaling directly prevents mitochondrial fragmentation following T‐cell stimulation by downregulating Drp1 phosphorylation on Ser616, via regulation of the ERK1/2 and mTOR pathways. In addition, downregulation of Drp1 activity in tumor‐infiltrating PD‐1pos CD8+ T cells seems to be a mechanism exploited by PD‐1 signaling to reduce motility and proliferation of these cells. Overall, our data indicate that the modulation of Drp1 activity in tumor‐infiltrating T cells may become a valuable target to ameliorate the anticancer immune response in future immunotherapy approaches.

Programmed cell death-1 (PD-1) signaling downregulates the T-cell response, promoting an exhausted state in tumor-infiltrating T cells, through mostly unveiled molecular mechanisms. Dynamin-related protein-1 (Drp1)-dependent mitochondrial fission plays a crucial role in sustaining T-cell motility, proliferation, survival, and glycolytic engagement. Interestingly, such processes are exactly those inhibited by PD-1 in tumorinfiltrating T cells. Here, we show that PD-1 pos CD8 + T cells infiltrating an MC38 (murine adenocarcinoma)-derived murine tumor mass have a downregulated Drp1 activity and more elongated mitochondria compared with PD-1 neg counterparts. Also, PD-1 pos lymphocytic elements infiltrating a human colon cancer rarely express active Drp1. Mechanistically, PD-1 signaling directly prevents mitochondrial fragmentation following T-cell stimulation by downregulating Drp1 phosphorylation on Ser616, via regulation of the ERK1/2 and mTOR pathways. In addition, downregulation of Drp1 activity in tumor-infiltrating PD-1 pos CD8 + T cells seems to be a mechanism exploited by PD-1 signaling to reduce motility and proliferation of these cells. Overall, our data indicate that the modulation of Drp1 activity in tumor-infiltrating T cells may become a valuable target to ameliorate the anticancer immune response in future immunotherapy approaches.

Introduction
Programmed cell death-1 (PD-1) is a T-cell surface receptor that downregulates T-cell activation and the immune response [1]. PD-1 signaling is activated by PD-1 interaction with its ligands PD-L1 and PD-L2, expressed on adjacent cells [2], and it dampens signals originating from T-cell receptor (TCR) and CD28, such as the activation of mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways [3,4]. Besides being frequently observed in T cells during chronic infections [5], activation of PD-1 signaling has also been widely reported in tumor-infiltrating T cells, contributing to their functional exhaustion and poor antitumor response [6]. Consistently, antagonistic PD-1 antibodies efficiently reinvigorate tumor-infiltrating T cells, thereby ameliorating antitumor response [7,8].
Mitochondria are central modulators of cellular bioenergetics, and their dynamic morphology is tightly linked to cell functions. Among the mitochondrialshaping proteins, dynamin-related protein-1 (Drp1) is the main profission protein and it is recruited from the cytosol to mitochondria thanks to post-translational modifications and to several receptors, such as Mff and Fis1 [9,10]. Interestingly, mitochondrial morphology is tightly linked to an optimal T-cell functionality [11]. Particularly, Drp1-dependent mitochondrial fragmentation sustains T-cell motility and proliferation, and effector T (T eff ) cell apoptosis following TCR engagement [12,13]. Also, Drp1-dependent mitochondrial relocation at the immunological synapse controls the influx of calcium upon T-cell activation [14], sustaining the cMyc-dependent upregulation of glycolytic enzymes [13,15], thus allowing the metabolic reprogramming required to cope with the increased bioenergetic demand of an activated T cell [16,17]. All these processes contribute to an optimal antitumor T-cell response, which is indeed defective in T cells lacking Drp1 [13].
Of note, most of these Drp1-dependent processes are also downregulated by PD-1 co-inhibitory signaling, especially when considering tumor-infiltrating T cells. Indeed, PD-1 signaling reduces T-cell proliferation and motility (both of them requiring Drp1) [4,13,18], and it also promotes a shift from a glycolysis-based metabolism (supported by Drp1) toward an OXPHOS-based metabolism (requiring mitochondrial fusion) [19]. Given this striking inverse correlation, we asked whether PD-1 signaling may modulate Drp1 activity, and to what extent this modulation may downregulate several processes in T cells. This point is of extreme importance, since the molecular mechanisms by which PD-1 regulates the aforementioned processes in T cells are not yet completely understood.
We here show that tumor-derived PD-1 pos CD8 + T cells exhibit a significant downregulation of Drp1 activity and a more fused mitochondrial network. Mechanistically, PD-1 signaling prevents Drp1 activation following T-cell stimulation by regulating its phosphorylation on Ser616 through the modulation of extracellular-regulated kinase 1/2 (ERK1/2) and mTOR proteins. Also, we provide evidence that Drp1 downregulation contributes to the reduced proliferation and motility of PD-1 pos tumor-infiltrating T cells, and, as a consequence, we identify Drp1 as a possible target for future therapeutic approaches aiming at restoring antitumor response in PD-1 pos exhausted CD8 + T cells.

Human samples
Peripheral blood samples were purified from buffy coats of healthy volunteer blood donors (independently of sex and age) under procedures approved by Institutional Review Board of Bambino Ges u Children' Hospital (Approval of Ethical Committee No 1314/2020 prot. No 19826), including informed and written consensus for research purpose. The study methodologies conformed to the standards set by the Declaration of Helsinki. Blood cells were isolated as previously reported [12]. Briefly, cells were incubated with RosetteSep Human T-Cell Enrichment Cocktail antibody mix (StemCell 15061). Unlabeled human peripheral blood T (hPBT) cells were isolated by density gradient over Lymphoprep (StemCell 07811), with centrifugation for 20 min at 1200rcf. Then, T cells have been collected, washed, and used for subsequent analyses. Human colon adenocarcinoma tissue sections were collected from the archives of the Tumor Immunology Laboratory, Department of Health Science according to the Helsinki Declaration and under the approval of the University of Palermo Ethical Review Board (Approval No 09/2018).

Mice
WT and Drp1 fl/fl Lck:cre+ c57BL/6 mice were bred and maintained under conventional conditions at the Plaisant Srl (Castel Romano) Animal Facility. Mouse strains have been previously described [13] and are in-house stocks. Mice were kept in cages of no more than 5-6 mice each, divided by sex, under 12-h/12-h light/dark cycles, with standard temperature, humidity, and pressure conditions according to FELASA guidelines. Small red squared mouse house and paper were used for cage enrichment. Mouse health was monitored daily by veterinary staff, and health analysis for pathogens was performed every 3 months according to FELASA guidelines. All mice were sacrificed by neck dislocation at 2-3 months of age. All efforts were made to minimize animal suffering and to reduce the number of mice used, in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). The mouse protocol has been approved by the Allevamenti Plaisant Srl Ethical Committee as well as by the Italian Ministry of Health (Authorization #186/2020-PR). It has been written following the ARRIVE Guidelines, and the numeric details have been chosen following the criteria described in The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (http://www.nc3rs.org.uk/). Sample size for the experiments performed has been established using power analysis method. Experiments involving growth of tumor cells in mice were performed using male mice.
To induce isolated murine na€ ıve CD8 + T cells into an exhaustion-like state in vitro, cells have been stimulated up to 4 times with 5 µgÁmL À1 anti-CD3 (platecoated) (eBioscience 14-0031-86) and 1 µgÁmL À1 anti-CD28 (Invitrogen 14-0281-86) for 24 h in 96-well plate. Between each stimulation, cells have been expanded using 20 ngÁmL À1 mouse IL-2 (R&D System 402-ML). Cells were considered into exhaustion-like state after 4 stimulations (T ex ) and were compared with effector-like (T eff ) cells isolated from sibling mice and stimulated only once. For in vitro migration experiments, T ex and T eff cells were finally expanded in vitro for additional 6 days in IL-2-containing medium and then used for the assays.

Immunofluorescence
Immunofluorescence staining has been performed as previously described [12]. Anti-TOM20 (Santa Cruz, Dallas, TX, USA, sc-11415) primary antibody was used to identify the mitochondrial network. Nunc Lab-Tek Chamber Slides (Thermo Fisher 154534) have been used to culture in vitro T cells directly on slides before fixation and were coated with 10 ngÁmL À1 fibronectin (Millipore, Burlington, MA, USA, FC010) for 1 h at RT before adding the cells. Images were acquired using a Perkin Elmer Ultraview VoX microscope. The mitochondrial network has been always evaluated upon 0.4 mm slices z-stack reconstructions.

Flow cytometry
To evaluate glucose uptake, 30 lM 2-NBDG (Thermo N13195) has been added to the cells for 20 min in DPBS. Then, cells have been washed and analyzed by flow cytometry (BD Accuri C6).
For the evaluation of the mitochondrial membrane potential, 100 nM TMRE (Thermo Fisher T669) and 100 nM MitoTrackerGreen (Thermo M7514) have been added for 20 min, and then, the cells were washed and analyzed. As a positive control for mitochondrial depolarization, cells have been pretreated with 50 lM FCCP (Sigma-Aldrich C2920).

Tumor induction
Tumor inoculation was performed as previously reported [12]. Briefly, 5*10 5 MC38 cells were injected subcutaneously into the right flank of two-month-old male WT or Drp1 fl/fl and Drp1 fl/fl Lck:cre+ mice. Mice were kept for up to 17/18 days in animal facility, and tumor growth was monitored twice or three times per week and recorded as [longest diameter]*[shortest diameter] 2 in cubic millimeters. At days 7, 9, 11, 14, and 16 from tumor inoculation, mice were inoculated intraperitoneal with 150 µg of InVivoMab anti-mouse PD-1 (CD279), clone RMP1-14 (Bioxcell, BE0146) or 150 µg InVivoMab rat IgG2a isotype control, clone 2A3 (Bioxcell, BE0089) antibodies (in 150 µL of saline). Mice were randomly subdivided into each experimental group (a-IgG or a-PD-1) before inoculation of antibodies (no specific randomization method was used). At day 17, mice were sacrificed and tumors were collected. Tumor tissues were mechanically dissociated over 70 mm-cell strainers, and mononuclear cells were enriched from tumor-derived cell suspensions by 40%/ 80% Percoll (GE Healthcare GE17-0891-01) density gradient, by collecting cells at the interface between 40% and 80% Percoll solution.
Isolated TILs have been used for subsequent proliferation and migration in vitro analyses or stained for flow cytometric measurements.

Seahorse analysis
Basal OCR has been measured in T cells during acute phase (unstimulated or stimulated for 12 h) or after 48 h of stimulation and 4 days of in vitro expansion with IL-2, as previously described [13]. ECAR and OCR have been measured in control and Drp1-KO T cells at 24 h and 48 h of stimulation with aCD3/28and aCD3/28+PD-L1 beads, and the relative metabolic parameters (basal glycolysis rate and basal respiration rate) were estimated as previously described [13].

Electroporation
Jurkat cells have been electroporated using Neon Transfection System (Thermo Fisher) following manufacturer instructions and kept on in antibioticfree medium before being washed and used for protein extraction. The following siRNA has been used: siERK1/2 (Cell Signaling 6560), siCTRL (Santa Cruz sc-37007), and simTOR (Cell Signaling 6381).

Statistical analysis
In the Figure legends, 'n' indicates either the number of independent experiments (in vitro primary cells) or the number of mice used. Data are expressed as mean AE SEM from at least three independent experiments unless specified otherwise (Microsoft Office Excel and SigmaPlot v12.5 have been used for analysis). The number of mice used has been estimated using the power analysis method. All the acquisitions of the experiments have been performed blinded without knowing the specific conditions of each sample. Comparisons between groups were done using twotailed Student's t-test (two groups) or one-way and two-way ANOVA (multiple groups and repeated measurements, adjustments for pairwise comparisons were performed using Holm-Sidak method). Mann-Whitney rank-sum test or ANOVA on ranks has been used if samples did not meet assumptions of normality and/ or equal variance. Chi-square test has been used to evaluate data in Figure 1B. P-values are indicated in the Figures as follows: * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

PD-1 pos CD8 + T cells from MC38-derived murine tumors show a reduced mitochondrial fission
To investigate whether PD-1 signaling modulates the morphology of the mitochondrial network in tumorinfiltrating T lymphocytes (TILs), we looked at PD-1 neg and PD-1 pos CD8 + T cells infiltrating an 18-dayold solid tumor mass derived from s.c. inoculation of MC38 cells (murine adenocarcinoma) in c57BL/6 WT mice. We took advantage of this tumor model, since it is characterized by (a) a high level of T-cell infiltration [13], (b) the presence of PD-1 pos CD8 + (but largely not CD4 + ) TILs (Fig. S1A), (c) the expression of PD-1 ligand PD-L1 (but not PD-L2) in tumor cells (Fig. S1B), and (d) the expression of PD-L1 and PD-L2 in ca. 10% and 2% of tumor-infiltrating CD45 pos non-T (i.e., CD4 neg CD8 neg ) cells (Fig. S1C). By comparing the expression levels of different mitochondrialshaping proteins in CD8+ TILs, we observed that PD-1 pos CD8 + TILs do not show altered levels of total Drp1 compared with PD-1 neg counterparts (Fig. 1A). However, we observed a specific downregulation of Drp1 phosphorylation on its activating residue Ser616 ( Fig. 1A and Fig. S1D), while the inhibitory phosphorylation on Ser637 did not vary. Also, we did not observe any differences in the expression of other main profusion (Mfn1, Mfn2, Opa-1) or profission (Fis1, Mff) proteins (Fig. 1A).
Last, we extended these observations to a homologue human tumor context, by staining moderately differentiated (G2) human colon carcinoma sections with anti-CD8, anti-PD-1, and anti-Drp1-pSer616 antibodies. Of note, we observed a reduced percentage of phospho-Drp1 pos elements in PD-1 pos CD8 + T cells compared with PD-1 neg CD8 + T cells ( Fig. 1D and Fig. S1G). This suggests that also in a human tumor context, PD-1 pos CD8 + T cells have a downregulated Drp1 activity.
In sum, tumor-infiltrating PD-1 pos CD8 + T cells show a tendency toward a more interconnected mitochondrial morphology, associated with a reduced activation of Drp1.

PD-1 signaling prevents Drp1 activation and mitochondrial fragmentation in both murine and human T cells, upon in vitro stimulation
To investigate whether such an altered Drp1 expression in PD-1 pos CD8 + TILs is directly caused by PD-1 activation, we switched to an in vitro system to specifically modulate PD-1 signaling. To this aim, we stimulated in vitro T cells isolated from spleen of WT mice with beads coated with anti-CD3/28 plus BSA (thereafter aCD3/28 beads) or anti-CD3/28 antibodies plus PD-L1 (thereafter aCD3/28-PD-L1 beads) for 48 h. Of note, T-cell stimulation with aCD3/28 beads leads to the activation of mTOR, ERK1/2, and Drp1 by phosphorylation ( Fig. 2A) and to the upregulation of PD-1 surface expression (Fig. S2A). As expected, concomitant activation of PD-L1/PD-1 axis during T-cell stimulation dampens activation of both mTOR and ERK1/2 ( Fig. 2A) [3,4]. Interestingly, we also observed that Drp1 phosphorylation on Ser616 is strongly reduced by the engagement of PD-1 signaling ( Fig. 2A), while no significant difference was observed for other mitochondrial-shaping proteins (Fig. 2B). In line with this, while T cells engage the fragmentation of mitochondria upon activation [12,13,20], in the presence of PD-L1 the same cell type retains a mitochondrial morphology more similar to unstimulated counterparts (Fig. 2C). Of note, mitochondria of murine activated T cells might characteristically appear slightly swollen when fragmented (Fig. 2C), still being fully functional (Fig. S2B, C). In addition, such an absence of fragmented mitochondria in PD-L1stimulated T cells is not due to ongoing mitophagy (which could hypothetically promote the clearance of the small and fragmented mitochondria) (Fig. S2D).
Next, we asked whether such PD-1-dependent regulation of Drp1 is restricted or not to some specific Tcell subpopulation. Therefore, we looked more closely at na€ ıve (CD44 neg ) and antigen experienced (CD44 pos ) CD8 + T-cell subpopulations, which, once activated, may undergo exhaustion within the tumor microenvironment. Of note, although na€ ıve CD8 + T cells do not express PD-1 on their cell surface, they rapidly acquire PD-1 expression as early as 12 h after stimulation with aCD3/28 beads (as also observed for na€ ıve CD4 + T cells), and the concomitant presence of PD-L1 ligand does not affect such upregulation (Fig. S2E). We thus isolated both subpopulations from the spleen of WT mice and stimulated them in vitro with aCD3/28-or aCD3/28-PD-L1 beads for 48 h, which is the optimal time point to detect Drp1 phosphorylation, especially in na€ ıve CD8 + T cells (Fig. S2F). Interestingly, we found that engagement of PD-1 signaling during cell activation dampens Drp1 phosphorylation and mitochondrial fragmentation in both CD44 pos and CD44 neg CD8 + subsets (Fig. 2D, E), in parallel with a reduced activation of ERK and mTOR pathways (Fig. 2D).
Overall, these data indicate that the engagement of PD-1 signaling during T-cell activation prevents Drp1 phosphorylation and mitochondrial fragmentation both in mice and in humans.

PD-1 signaling downregulates Drp1 activation by modulating mTOR and ERK pathways
Next, we aimed at better investigating the molecular pathways linking PD-1 activation to the downregulation of Drp1 activity. It is well established that PD-1 dampens the signaling pathways originating from TCR and CD28 activation [3,4], such as PI3K/Akt/mTOR [3] and MAPK/ERK pathway [4], as indeed confirmed by our results (Fig. 2A, D, F). Since both these pathways have been reported to modulate Drp1-dependent mitochondrial fragmentation [21,22], we tested whether their inhibition is sufficient to reduce Drp1 activation upon T-cell stimulation. Of note, ERK1/2 inhibitor FR180204 (ERKi) prevents both Drp1 phosphorylation and mitochondrial fragmentation in activated T cells (Fig. 3A, B), this without affecting mTOR phosphorylation (Fig. 3A). Further, we rescued ERK1/2 activity downstream of PD-L1/PD-1 engagement by using low doses of C6-ceramide (to avoid apoptosis induction), a known activator of the ERK pathway [23]. Ceramide, which slightly rescues ERK phosphorylation without affecting that of mTOR (Fig. 3C), also rescues Drp1 phosphorylation and mitochondrial fragmentation in PD-1-engaged hPBT cells during activation (Fig. 3C, D). Next, also the mTOR inhibitor RAD-001 [24] prevents both Drp1 phosphorylation and mitochondrial fragmentation during T-cell activation (Fig. 3E, F), without affecting ERK phosphorylation (Fig. 3E). Furthermore, we confirmed also in Jurkat cells that the specific silencing of both ERK1/2 and mTOR by siRNA downregulates Drp1 phosphorylation on Ser616 (Fig. S3A). In addition, Drp1 phosphorylation upon T-cell activation is reduced also when inhibiting the activity of Akt, the kinase upstream of mTOR (Fig. S3B). Interestingly, while ERK1/2 is known to directly phosphorylate Drp1 on Ser616 [21], it is currently unknown how mTOR may regulate Drp1 activity. An extensive crosstalk between mTOR and ERK pathways has been frequently reported [25,26]. Therefore, mTOR may regulate Drp1 in T cells via the modulation of ERK1/2. However, we here observed that a low dose (10 nM) of mTOR inhibitor is sufficient to prevent Drp1 phosphorylation without affecting ERK1/2 activity (Fig. 3E). In line with this, pharmacological inhibition of Akt prevents both mTOR and Drp1 phosphorylation without affecting ERK1/2 upon T-cell activation (Fig. S3B). Therefore, at least in T cells, mTOR seems to regulate Drp1 in a ERK1/2-independent way.
In sum, the activation of PD-1 signaling during Tcell stimulation reduces Drp1 phosphorylation and mitochondrial fragmentation presumably through an inhibition of both ERK and mTOR pathways downstream of TCR/CD28 signaling.
Recently, an in vitro protocol based upon subsequent cycles of CD3/CD28 stimulation (see Methods for details) has been developed to induce a TCR/CD28hyporesponsive state in CD8 + T cells, thus mimicking an exhausted-like condition of diminished proliferative and cytotoxic potential without the need of a concurrent engagement of inhibitory coreceptors, such as PD-1 [27]. Therefore, we decided to take advantage of this protocol to understand whether Drp1 could be modulated also in this context. To this aim, we compared Drp1 phosphorylation and mitochondrial morphology in TCR/CD28 responder T eff -like cells (i.e., stimulated up to two times with aCD3/CD28 Abs) and nonresponder T ex -like cells (i.e., the same cells stimulated three or more times). Interestingly, we observed that Drp1 phosphorylation and fragmentation of mitochondria are strongly reduced in T ex -like cells compared with T eff -like ones (Fig. S3C, D).
Collectively, these data argue that Drp1 downregulation could be a common feature observed during the T-cell exhaustion induced either by the activation of inhibitory coreceptors (such as PD-1) or by a chronic hyporesponsive state due to TCR unresponsiveness.

Drp1 is required for an efficient reduction of tumor growth mediated by anti-PD-1 therapy
Given the importance of Drp1 in regulating multiple processes in T cells, we asked whether the ability of anti-PD-1 therapy to reduce solid tumor growth requires the restoration of Drp1 activity in tumorinfiltrating PD-1 pos CD8 + T cells. To answer this point, we analyzed s.c. MC38-derived tumors in mice, whose growth is significantly reduced by (a) treatments with antagonistic anti-PD-1 Abs [28] and (b) by a functional Drp1 in T cells [13]. We inoculated s.c. MC38 cells into both control (Drp1 fl/fl ) and Drp1 conditional-KO mice (Drp1 fl/fl Lck:cre+, T-cellrestricted Drp1 ablation, indicated as Drp1-cKO) (Fig. S4A), which we previously characterized [13]. After one week, we treated these mice with either anti-IgG (control) or antagonistic anti-PD-1 antibody every 2/3 days for up to 10 days (Fig. 4A). Of note, we found that anti-PD-1 treatment is much more effective in reducing tumor growth in control mice than in mice where Drp1 was absent in T cells (Drp1-cKO) (Fig. 4B). Even when correcting for the larger tumor volume in Drp1-cKO mice compared with control mice [13], we still observed a from 60% to ca. 20% in the efficacy of anti-PD-1 therapy in Drp1-cKO mice (Fig. 4C). Of note, Drp1 ablation in T cells does not affect per se PD-1 expression following T-cell activation (Fig. S4B), thus excluding that the impaired effect of anti-PD-1 therapy in Drp1-cKO mice can be due to an altered expression of PD-1. In addition, we found that the strong reduction in tumor growth by anti-PD-1 treatment in control (Drp1 fl/fl ) mice correlates with a rescue of Drp1 phosphorylation in PD-1 pos CD8 + T cells to a level comparable to PD-1 neg CD8 + T cells (Fig. 4D). On the contrary, in Drp1-cKO mice (where Drp1 is absent and therefore cannot be 'rescued'), anti-PD-1 treatment is much less efficient in reducing tumor growth. Overall, these data suggest that the restoration of Drp1 activity in PD-1 pos CD8 + TILs is a key step for the effectiveness of the anti-PD-1 therapy.
Next, we tried to get more insight into the cellular processes requiring the rescue of Drp1 activity during anti-PD-1 therapy. Of note, while anti-PD-1 treatment increases the number of CD8 + TILs recovered from the tumor mass (per mm 3 ), this effect is not observed in Drp1-cKO mice (Fig. 4E), indicating that Drp1 is required to mediate such anti-PD-1-dependent increase in CD8 + TILs accumulation. On the contrary, PD-1dependent regulation of IFNc production does not involve Drp1 (Fig. 4F). We previously reported that Drp1 is also required to sustain T-cell clonal expansion after stimulation, both in vitro and in vivo [13]. Therefore, we asked whether the inability of Drp1-KO CD8 + TILs to increase their number within the tumor mass upon anti-PD-1 treatment may depend or not on their impaired proliferation. To this aim, we isolated TILs from MC38-derived tumor masses grown in control or Drp1-cKO mice (treated with anti-IgG or anti-PD-1) and let them expand in vitro in the presence of IL-2, IL-7 and IL-15 cytokines. Interestingly, while the anti-PD-1 treatment significantly increases the fold expansion of control CD8 + T cells, this effect is completely lost in Drp1-KO CD8 + T cells (Fig. 4G). These data suggest that Drp1 inhibition may be at least one of the mechanisms by which PD-1 signaling reduces the proliferation of PD-1 pos CD8 + TILs.
In sum, a functional Drp1 is required for the efficacy of the anti-PD-1 treatment in reducing MC38derived tumor growth in mice. Also, PD-1 signaling may mediate the reduction in CD8 + TILs proliferative potential via Drp1 downregulation.

Reduced motility of PD-1 pos and in vitro exhausted-like T cells correlates with altered Drp1-dependent mitochondrial remodeling
Besides controlling T-cell proliferation, Drp1 is required to sustain T-cell motility by favoring mitochondrial repositioning at the uropod, and it is directly phosphorylated on Ser616 in response to chemokine stimulation [13]. Since T-cell motility is another process dampened by PD-1 signaling [18], we asked whether PD-1 signaling may exploit the downregulation of Drp1 to reduce T-cell motility. To answer this point, we activated murine T cells in vitro for 48 h, and expanded them for additional 4d with IL-2 (a timepoint in which we still observed PD-1 surface expression, data not shown). Then, cells were preincubated for 2 h with BSA (as control) or PD-L1 and then stimulated with CCL19 and CCL21 chemokines. Interestingly, while the chemokine stimulation promotes both Drp1 and ERK1/2 phosphorylation (Fig. 5A) and the mitochondrial fragmentation and relocation at the uropod (Fig. 5B), as expected [13], the concomitant engagement of the PD-1 signaling prevents both Drp1 activation and mitochondrial relocation (Fig. 5B, C). Collectively, these data indicate that PD-1 signaling engagement deprives T cells of a key cellular event (i.e., the Drp1-dependent mitochondrial repositioning upon fragmentation) required to sustain motility.
Furthermore, we found that Drp1-mediated motility is lost also in an in vitro generated exhausted-like (T ex ) state (obtained through induction of TCR hyporesponsiveness and without PD-1 involvement, as described for Fig. S3C, D). Indeed, these cells, which migrate less than control functional T eff cells (Fig. 5C), show an impaired Drp1 phosphorylation (Fig. 5D) and a lack of mitochondrial relocation at the uropod upon chemokine stimulation (Fig. 5E).
Last, we asked whether the rescue of Drp1 activity in tumor-derived exhausted T cells is important for the effectiveness of anti-PD-1 immunotherapy in rescuing T-cell motility. To answer this point, we isolated CD8 + TILs from MC38-derived tumor-bearing control and Drp1-cKO mice treated with either anti-IgG or anti-PD-1 antibodies. Interestingly, we found that anti-PD-1 treatment significantly increases motility of control PD-1 pos CD8 + TILs (as assessed by Transwell assay), when compared to the motility of the same cells from anti-IgG-treated control mice (Fig. 5F). However, this effect is completely lost when looking at PD-1 pos CD8 + TILs from Drp1-cKO mice, whose T cells lack Drp1 (Fig. 5F). Of note, these data suggest that (a) the rescue of Drp1 activity by anti-PD-1 treatment is required to restore TIL motility and (b) Drp1 inhibition may be one of the mechanisms by which PD-1 signaling reduces motility of PD-1 pos CD8 + TILs.
Overall, these data indicate that PD-1 signaling may mediate the reduction in CD8 + TILs motility via Drp1 downregulation, thus preventing the Drp1-dependent mitochondrial fragmentation upon chemokine stimulation, a key step normally required for T-cell motility.

PD-1 does not modulate T-cell metabolism through the regulation of Drp1 activity
Several works have shown that both the engagement of PD-1 signaling and the downmodulation of Drp1 activity, during T-cell activation, modulate both glycolysis and OXPHOS [13,19,29]. Given the here reported downregulation of Drp1 activation by PD-1 signaling, we asked whether PD-1 may exploit such a Drp1 downregulation to also modulate T-cell metabolism. To answer this point, we analyzed the metabolism of both control and Drp1-KO T cells upon activation in the presence of aCD3/28 or aCD3/ 28+PD-L1 beads.
Interestingly, we found that upon 24 h of lymphocyte activation PD-1 signaling, but not Drp1 ablation, reduces glycolysis, while the opposite is observed at 48 h (Fig. S5A, B). Therefore, although both PD-1 engagement and Drp1 ablation can reduce glycolysis during T-cell activation, their effects occur with different kinetics, Moreover, both PD-1 engagement and Drp1 ablation alone partially reduce glucose uptake upon T-cell activation, but the combination of the two further reduces glucose uptake (Fig. S5C). Regarding OXPHOS, while we observed no effects of PD-1 engagement on basal respiration up to 48 h upon activation, this parameter is instead increased by Drp1 ablation at this timepoint (Fig. S5D, E), without a concomitant modulation of the mitochondrial membrane potential (Fig. S5F).
Overall, these data suggest that although both PD-1 and Drp1 can modulate the T-cell metabolism upon cell activation, and even if PD-1 directly regulates the Drp1 activation status, the effects of PD-1 signaling on metabolism likely do not depend on the concomitant modulation of Drp1 activity.

Discussion
It has been reported that tumor-infiltrating T cell shows an altered mitochondrial functionality and morphology when compared with T cells from peripheral blood [30]. However, the modulation of mitochondrial morphology in different subpopulations of TILs has never been investigated before. Here, we report that in MC38-derived tumors PD-1 pos tumor-infiltrating murine CD8 + T cells display a reduced activation of Drp1 and a more fused mitochondrial network when compared with PD-1 neg counterparts. Of note, these data are shared also in a corresponding human context of colon tumor, in which tumor-infiltrating lymphocytic elements almost never coexpress PD-1 and active Drp1. Mechanistically, we provided evidence that PD-1 signaling downregulates Drp1 activating phosphorylation on Ser616 (and consequently mitochondrial fragmentation) via the inhibition of ERK1/2 and mTOR kinases.
Also, we explored the functional consequences of such PD-1-dependent downregulation of Drp1 activity in the tumor context. Of the highest importance, altogether, our data suggest that PD-1 signaling may exploit the downregulation of Drp1 activity to dampen some of the processes required for an optimal Tcell functionality. In line with this, the restoration of Drp1 activity in TILs is strictly required for the effectiveness of the anti-PD-1 therapy. Specifically, Drp1 seems to play an important role in controlling both motility and proliferation of PD-1 pos CD8 + TILs. Mechanistically, we observed that the Drp1-dependent mitochondrial relocation at the cell rear-edge during cell migration, a phenomenon occurring in healthy effector CD8 + T cells [13], is completely lost upon PD-1 engagement in CD8 + T cells, which are unable to activate Drp1 upon chemokine stimulation. Our data are thus consistent with previous observations made in a persistent infection mouse model, in which the recovered motility of PD-1 pos T cells upon anti-PD-1 treatment was associated with an increased activation of ERK [18], a kinase known to regulate Drp1 in T cells [12,13]. Therefore, downregulation of ERK/ Drp1 axis may be exploited by PD-1 signaling to dampen T-cell motility. Regarding the role of Drp1 in T-cell proliferation, we previously reported that, in the absence of Drp1, T cells show an abnormal length of mitosis, due to the acquisition of aberrant centrosome morphologies [13], as observed also in cancer cells [31]. On the contrary, we here provided data suggesting that PD-1 signaling does not regulate T-cell metabolism via Drp1 downregulation. PD-1 has been reported to dampen both glycolysis and OXPHOS upon cell activation in both murine and human T cells, although a detailed kinetic analysis of the metabolic modulations caused by PD-1 engagement is still lacking. Although we here demonstrated that PD-1 prevents Drp1 activation upon T-cell stimulation, we found that in murine T cells early upon activation (i.e., up to 48 h) the effects on metabolism observed upon PD-1 engagement and Drp1 ablation are either different in direction (PD-1 does not modulate OXPHOS while Drp1 ablation increases it) or in timing (both PD-1 and Drp1 ablation reduce glycolysis but at different timepoints). plate-coated anti-CD3 (5 µgÁmL À1 ) and soluble anti-CD28 (1 µgÁmL À1 ) and then expanded for 3 days with IL-2. In (A), cells have been incubated for 2 h with BSA-or PD-L1-coated beads (10 µg for 20 mln beads), and then, 200 nM CCL19 and CCL21 have been added for 20 min, proteins have been extracted, and the expression level of the indicated (phospho)-proteins has been evaluated by western blot (A, n = 1 experiment representative of three independent experiments). In (B), cells have been left to adhere for 30 min to fibronectin-coated slides and were incubated with either 5 µgÁmL À1 BSA or PD-L1 for 2 h. Then, 200 nM CCL19 and CCL21 have been added for 20 min, and cells were fixed and processed for immunostaining to analyze mitochondrial morphology by immunofluorescence (TOM20, green) (B, n = 3). (C-E) Murine exhausted T cells (Tex) have been generated in vitro through 4 cycles of aCD3/28-mediated stimulation (24 h) and IL-2-mediated expansion (6 days) and compared to effector T cells (T eff ) generated through a single cycle of stimulation and expansion. After the last 6 days in IL-2-containing medium, cells have been starved from serum for 2 h, and then, the following assays were performed. In (C), the migration index in response to 50 nM CCL19/CCL21 gradient for 2 h has been calculated using Transwell migration assay (n = 6). In (D), the cells have been stimulated with 50 nM CCL19 and 50 nM CCL21 chemokines for 15 min in an Eppendorf tube, and then, proteins were extracted. The expression level of the indicated (phospho)-proteins has been evaluated by western blot (n = 3). In (E), cells have been left to adhere for 30 min to fibronectin-coated slides. Then, cells have been stimulated with 50 nM CCL19 and 50 nM CCL21 chemokines for 15 min and then fixed and processed for immunostaining. Representative images showing the mitochondrial network (anti-TOM20 staining) in effector T (T eff ) and exhausted T (T ex ) cells are shown on the left. Quantification of the percentage of cells showing fragmented mitochondria in each condition is reported in the graph on the right (n = 3). (F) TILs have been isolated from MC38-derived tumor masses grown for 17 days in control Drp1 fl/fl or conditional-KO Drp1 fl/fl Lck:cre+ mice inoculated with anti-IgG or anti-PD-1 antibodies as indicated in Fig. 5A. Then, TILs were starved from serum for 2 h and allowed to migrate in response to 10% fetal bovine serum for 2 h using Transwell migration assay. The graph indicates the relative (anti-PD-1 / anti-IgG) migration index calculated for PD-1 neg and PD-1 pos CD8 + TILs isolated from tumor-bearing control Drp1 fl/fl or conditional-KO Drp1 fl/fl Lck:cre+ mice inoculated with anti-IgG or anti-PD-1 antibodies as indicated in Fig. 4A (n = 7). Data are shown as mean AE SEM. Scale bar: 5 µm in B and E. Significance is indicated as follows: *=P < 0.05; **=P < 0.01; ***=P < 0.001. Statistical tests used: two-way ANOVA with Holm-Sidak post hoc (B, D, E); unpaired t-test (C), one-way ANOVA with Holm-Sidak post hoc (F).
Moreover, we could speculate that the downregulation of Drp1 by PD-1 signaling may represent a mechanism exploited by exhausted TILs to modulate mitophagy, too. A defective mitophagy in PD-1 positive T cells has been reported to contribute to the exhausted phenotype of these cells [32]. Interestingly, Drp1 may facilitate dismissal of small mitochondria via mitophagy [33]. Therefore, the downregulation of Drp1 activity in PD-1 pos CD8 + TILs may provide a mechanism to reduce the mitophagy rate in these cells, preventing the generation of small mitochondria that can be targeted to degradation. This is consistent with the observation of a higher mitochondrial mass in CD8 + TILs [30].
Of note, a recent publication by Ogando et al. [29] shows an unaltered rate of mitochondrial fragmentation in stimulated T cells (independently of the presence of PD-1 engagement) compared with unstimulated cells. This finding can be accounted for by significant differences in the methodology applied for the analysis. Indeed, they chose a specific parameter to estimate the mitochondrial fragmentation (i.e., circularity). At variance with that work, we considered here that stimulated T cells, compared to unstimulated (na€ ıve) counterparts, are significantly larger, as a consequence of their activation in vitro, and also that the organelle circularity might consequently be altered, becoming a nonuseful parameter. Also, fused mitochondria in T cells appear more tangled due to the round shape of this nonadherent and small cell type. In addition, Ogando et al. only analyzed total levels of Drp1, without focusing on its specific phosphorylated residues [29], which are more reliable indicators of Drp1 activation compared with the total protein amount.
In sum, our data indicate that downregulation of Drp1 mediated by PD-1 signaling may be required to attain an efficient inhibition of T-cell response. Therefore, we dare to propose Drp1 as a therapeutic target to ameliorate exhausted T-cell functionality during anticancer approaches, although drugs able to activate this protein need to be developed yet. Interestingly, CAR T-cell-based approaches are currently being exploited for the treatment of solid cancers [34], but they frequently fail to confer long-term tumor regression due to a poor ability of CAR T cells to survive and infiltrate within a solid tumor mass. This may be partially explained by the tendency of CAR T cells to undergo functional exhaustion, similar to endogenous T cells [35]. However, whether a PD-1-dependent downregulation of Drp1 activity is also present in exhausted CAR T cells is still not known. Should such modulation be observed, targeting Drp1 activity in CAR T cells (either pharmacologically or genetically) could represent a new strategy to ameliorate CAR Tcell survival or infiltration.

Conclusion
In sum, the modulation of Drp1 in tumor-derived exhausted T cells may represent a valuable target to ameliorate anticancer immune response in a number of instances, and the manipulation of CAR T system to this aim may represent a valid future strategy.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.