Preparation of PD-1 knockdown CART-19 cells
We constructed dual-promoter vectors those could express PD-1 targeting shRNA and CAR simultaneously, to which a ZsGreen motif was linked by a T2A motif. The expression of shRNA was driven by U6 promoter, and elongation factor 1-alpha (EF1-α) was used to initiate the expression of CD19-targeting CAR (CAR19) (Fig. 1a).
The expression of CAR19 was confirmed by WB and immunofluorescence (Additional file 1: Figure S1A and B). As shown in Fig. 1b, a strict one-to-one correspondence between the expression of CAR and ZsGreen was presented, which allowed us to track and purify CAR-T cells by ZsGreen. The infection efficiency was between 15 and 25% on the third day after infection, and no significant difference between different constructs was demonstrated.
Six different PD-1 targeting shRNA sequences were synthesized to screen for valid ones, and a scramble sequence (SCR) was used as control. The PD-1 silencing efficiency was firstly analyzed in Jurkat cells by qRT-PCR, WB and flow cytometry (Additional file 1: Figure S1C, D and Fig. 1c) to exclude invalid shRNA sequences. Finally, we screened out two valid shRNA sequences, shRNA-3 (S3) and shRNA-4 (S4).
The functions of S3 and S4 were further confirmed in T cells. The expression of PD-1 was not significantly inhibited by S3 or S4 until the fourth day after lentivirus infection. On the seventh day of culture, which was the fifth day after viral infection, the PD-1 positive rates in S3 and S4 modified CART-19 (S3-CART19 and S4-CART19) cells decreased by about 72 and 88% respectively compared with that in the SCR modified CART-19 (SCR-CART19) cells (Fig. 1d).
In addition, we confirmed that the expression of PD-1 in SCR-CART19 cells would be significantly up-regulated by target cell-induced immune response (TCIIR) after 24 h co-culture with Raji cells (Additional file 1: Figure S1F). And the up-regulations could be effectively inhibited by S3 and S4 (Fig. 1e). The in vivo CAR and PD-1 expression in different CAR-T cells were also detected 7 days after CAR-T infusion (Additional file 1: Figure S3A). Most of the CD3-positive T cells expressed CAR molecule, and S3 and S4 could effectively inhibit the expression of PD-1.
Due to the more pronounced PD-1 silencing efficiency, S4 was chosen for the following functional tests.
PD-1 knockdown did not enhance the cytotoxicity of CAR-T cells
To evaluate the TCIIR potential, the expression of IFN-γ and CD107a of CAR-T cells cultured for 7 days were detected after co-culture with Raji and CD19 positive A549 cells (A549–19). As shown in Fig. 2a and b, co-culture induced a higher positive rates of IFN-γand CD107a in SCR-CART19 cells than that in S4-CART19 cells, and the strong expression of PD-L1 significantly inhibited CAR-T cells CD107a expression (Fig. 2b and Additional file 1: Figure S2). This indicated that PD-1 knockdown might impair CAR-T cells TCIIR in this short-time co-culture experiment. Moreover, the residual PD-1 could still exert significant inhibitory effect, and this was further confirmed by CD107a expression test with A549–19 cells expressing moderate level of PD-L1 (Additional file 1: Figure S5D).
Inflammatory cytokines secretion is another important factor in determining anti-tumor activity. Multi-factor flow assay demonstrated that more IL-2 was secreted by S4-CART19 cells (cultured for 10 days). However, the secretions of the other tested factors were decreased by PD-1 knockdown. In this experiment, PD-1 knockdown significantly increased CAR-T cells resistance to PD-L1-mediated immunosuppression (Fig. 2c).
To test the cell lysis directly, Raji and A549–19 cells expressing firefly luciferase (Raji-luc and A549–19luc) were established. Purified CAR-T cells (cultured for 10 days) were co-cultured with target cells for 24 h. It was demonstrated that S4-CART19 and SCR-CART19 cells presented similar CAR-specific lysis of Raji-luc cells at higher E:T ratios. But at lower E:T ratios, 0.2:1 and 0.1:1, S4-CART19 cells was slightly, but with significance, less effective than SCR-CART19 cells (Fig. 2d). However, no significant difference in lysis of A549–19luc or IFN-γ-stimulated A549–19luc cells between SCR-CART19 and S4-CART19 was demonstrated (Fig. 2e and f). We speculated that this might be because the cytokines released by T cells during co-culture up-regulated the expression of PD-L1 in A549 cells, and this was proved true by the results presented in Additional file 1: Figure S5C.
Taken together, these results indicated that PD-1 knockdown did not enhance the cytotoxicity of CAR-T cells, but instead impaired it under certain conditions.
Long-lasting PD-1 knockdown would impair the in vivo anti-tumor function of CAR-T cells
To evaluate the effect of long-lasting PD-1 knockdown on T cells, we conducted an in vivo experiment (Fig. 3a). In this xenograft model, 5 × 106 A549–19luc cells were implanted subcutaneously 2 weeks before CAR-T infusion. Tumor burdens across groups were equalized by BLI before CAR-T treatment. For CAR-T treatment, 1 × 106 sorted CAR-T cells cultured for 10 days were administered, and non-infected T cells were used as control. It was found that SCR-CART19 cells could rapidly eradicate tumors, and no tumor recurrence was detected during the following 2 months observation. Better anti-tumor function of S4-CART19 cells was not observed, on the contrary, the tumor clearance was significant slower and weaker (Fig. 3a, b and Additional file 1: Figure S3B). All the treated mice exhibited little T cells expansion, except for one receiving SCR-CART19 cells developed significant T cells amplification 3 weeks after CAR-T cells infusion (Fig. 3c and Additional file 1: Figure S3C). The survival statistics showed that the mice treated with S4-CART19 cells did not have prolonged survival compared with those treated with SCR-CART19 cells (Fig. 3d).
These results suggested that long-lasting PD-1 silencingmight impair the in vivo anti-tumor function of CAR-T cells.
PD-1 knockdown impaired CAR-T cells in vitro proliferative potential
Proliferation is a key factor in determining CAR-T cells’ anti-tumor potential [24]. In previous in vivo experiments, the effect of PD-1 knockdown on T cell proliferation was not demonstrated due to the insufficient expansion of CAR-T cells. Therefore, we conducted more specific in vitro experiments to study the proliferative capacity of CAR-T cells.
First, 0.4 × 104 purified CAR-T cells cultured for 10 days were co-cultured with Raji-luc cells at a low E:T ratio (0.1:1) for 3 days. The tumor lysis by S4-CART19 cells was found to be significantly impaired compared with SCR-CART19 cells (Fig. 4a). Meanwhile, the amplification times of S4-CART19 cells was only about one-third of that of SCR-CART19 cells (Fig. 4b), indicating that the amplification driven by TCIIR was impaired.
Next, we tested the expression of ki67 in CAR-T cells to further elucidate the proliferative potential. It was confirmed that the ki67 positive rate of S4-CART19 cells was lower than that of SCR-CART19 cells not only after but also before TCIIR (Additional file 1: Figure S4A). This suggested that CAR-T cells proliferation driven by cytokines might be also impaired by PD-1 silencing. This conclusion was conflicting to the widely accepted view that PD-1 receptor was detrimental to T cells proliferation [25]. We doubted whether the shRNA4 sequence mistakenly targeted other genes involved in cell proliferation. To exclude this possibility, another PD-1 targeting shRNA sequence, S3, was tested to verify the results’ authenticity.
We examined the expression of ki67 in CAR-T cells those were cultured for fourteen days. The results showed that S3 could produce similar effects on CAR-T cells as S4, although the effect was a little weaker (Additional file 1: Figure S4B).
By continuously tracking the ZsGreen positive rates during cultivation, we further confirmed that the proliferations of S3-CART19 and S4-CART19 cells were both damped, but the attenuation in S3-CART19 cells was delayed and more moderate than S4-CART19 cells (Fig. 4c). By calculating the absolute number of CAR-T cells, it was found that on the ninth day of culture (day 7 after viral infection), the proliferation multiples of S3-CART19 and S4-CART19 began to be significantly lower than SCR-CART19 (Fig. 4d). The daily doubling time was also calculated, and the proliferation of PD-1 silenced CAR-T cells became slower and slower with the prolongation of cultivation (Fig. 4e). Compared with SCR-CART19, the obvious prolongation of doubling time in S4-CART19 and S3-CART19 first appeared on day 8 to day 9 and on day 10 to day 11, respectively (Fig. 4f). Cell cycle assays also demonstrated that the proliferative potential during cultivation was impaired by PD-1 silencing, and the effect of S3 was weaker than that of S4 (Additional file 1: Figure S4C and D).
The 7-AAD staining results indicated that the decrease of PD-1 silenced CAR-T cells in total population was not due to increased apoptosis (Additional file 1: Figure S5A). We detected several genes most likely to be mistargeted by S3 or S4, and the qRT-PCR data further confirmed the specificity of S3 and S4 (Additional file 1: Figure S6).
The proliferation of S3-CART19 and S4-CART19 driven by co-culture with A549–19 cells (PD-L1 would be significantly up-regulated) were also tested. 0.5 × 104 purified CAR-T cells were co-cultured with A549–19 cells at the E:T ratio of 01:1 for 72 h. The absolute T cell numbers were recorded daily, and SCR-CART19 presented more significant proliferation than S3-CART19 and S4-CART19 (Fig. 4g). The 72 h lysis analysis (E:T at 0.1:1 for Raji-luc cells and E:T at 0.05:1 for A549–19luc cells) demonstrated that SCR-CART19 presented higher lysis ability than S3-CART19 and S4-CART19 (Fig. 4h and i).
Taken together, these results demonstrated that PD-1 silencing in CAR-T cells would impair their proliferative potential, as well as the authenticity of this finding.
T cells’ differentiation kinetics was altered by PD-1 knockdown
Differentiation status plays a decisive role in T cell proliferation. We assessed CAR-T cells’ senescence by detecting the expression of TIM3 and LAG3. It was found that PD-1 knockdown did not up-regulate the expression of TIM3 or LAG3, but decreased them. Meanwhile, this effect was more pronounced in S4-CART19 cells compared with S3-CART19 cells (Fig. 5a and b). During cultivation, T cells would gradually differentiate from early-differentiation status into late-differentiation status, and the proliferative capacity would also gradually decrease [26]. We labeled CD62L and CD45RO for CD8-positive T cells to analyze their differentiation status. Typically, CD62L+CD45RO− T cells are considered to be naive T cells. As differentiation progresses, T cells would become CD62L+CD45RO+ central memory T cells and CD62L−CD45RO+ effector T cells. Although there are some different opinions about the status of CD62L−CD45RO− T cells, we tend to believe that these cells are effector memory T cells derived from effector T cells.
As shown in Fig. 5c and Additional file 1: Figure S5E and F, during the cultivation, knockdown of PD-1 significantly accelerated CAR-T cells’ differentiation into CD62L−CD45RO+ T cells. In particular, the naive T cells in S3-CART19 and S4-CART19 faded rapidly. Meanwhile, PD-1 knockdown appeared to prevent further differentiation of CD62L−CD45RO+ T cells into CD62L−CD45RO− T cells. By analyzing ZsGreen negative T cells (non-infected T cells) in the same cell populations (cultured for ten days), we excluded the possibility that different culture conditions caused this difference (Additional file 1: Figure S5B).
Next, the effect of PD-1 silencing on exhausted CAR-T cells was studied. Raji-luc cells were added at E:T = 1:3 on the 5th day of culture (single stimulation, SST) or on the 5th and 8th day of culture (double stimulation, DST) to induce T cell exhaustion. As shown in Fig. 5d, e and f, DST induced more significant exhaustion phenotype than SST, proofed by higher up-regulation of TIM-3 and LAG-3 and lower TCIIR induced CD107a expression. Of note, PD-1 knockdown seemed to retard T cell exhaustion during SST and DST. However, after DST, the antitumor functions of PD-1 knockdown CAR-T cells were still weaker than that of SCR-CART19 cells (Fig. 5g).
The effect of long-lasting PD-1 blockade by antibodies was also observed. We found that the antibodies barely altered the expressions of TIM-3 or LAG-3, differentiation kinetics and proliferative ability (Fig. 5h, i and Additional file 1: Figure S7). This suggested that simply blocking PD-L1/PD-1 interaction (T cells also express PD-L1, Additional file 1: Figure S1E) was quite different from intrinsic PD-1 silencing.
Taken together, we confirmed that PD-1 knockdown but not antibody-mediated blockade altered CAR-T cells’ differentiation kinetics.
PD-1 knockdown impaired in vivo persistence and proliferation of CAR-T cells
Persistence is another key factor determining CAR-T cells’ function [24]. To evaluate the persistence, we administered 1 × 106 purified CAR-T cells or non-infected T cells per mouse. After 2 weeks of feeding, these mice were inoculated with 2 × 107 A549–19luc cells subcutaneously. As shown in Fig. 6a, little tumor growth was observed in each group during the first 2 weeks after inoculation. During the next 2 weeks, tumors in control group grew rapidly. In comparison, the residual S3-CART19 and S4-CART19 cells limited tumor growth effectively, but the limitation was significantly less effective than that of SCR-CART19 cells (Fig. 6a and b). This suggested the persistence of CAR-T cells might be impaired by PD-1 knockdown.
In previous in vivo experiments, CAR-T cells did not show significant expansion, which might be due to the limited contacts between CAR-T cells and transplanted tumors. Therefore, we planned to use a hematological tumor model in which the contacts were sufficient to study the T cells proliferation. In this model, 1 × 106 Raji-luc cells were inoculated 1 week before the CAR-T treatment (Fig. 6c). After equalization by BLI, 5 × 105 CAR-T cells that were cultured for 10 days were given. As shown in Fig. 6d, the infused T cells reached a numerical peak on the third day after treatment (including the non-infected T cells) and then gradually fell back. In the fourth week after treatment, significant secondary amplifications of CAR-T cells were observed in two SCR-CART19 treated and one S3-CART19 treated mice. The statistical results showed that the expansion of S4-CART19 cells was significantly lower than that of SCR-CART19 and S3-CART19 cells. Tumor cells could be rapidly erased in almost all the CAR-T treated mice during the first week of treatment. During the second week, tumor clearance persisted in the SCR-CAR-T group, meanwhile, significant tumor growth was observed in both S3-CART19 and S4-CART19 groups. During the following 2 weeks, tumor burdens increased rapidly in all groups, but SCR-CART19 cells presented better anti-tumor function than S3-CART19 and S4-CART19 cells (Fig. 6c and e). And the survival statistics was consistent this view (Fig. 6f).
Considering that PD-L1-mediated immunosuppression could be partially rescued by PD-1 blockade, different amplification potential might be presented by solid tumor infiltrating CAR-T cells. To study the intratumoral expansion, 1 × 107 A549–19luc cells were implanted subcutaneously. Three weeks later, the tumors, of which the diameters were about 1 cm, were equalized by BLI. And then 1 × 106 purified CAR-T cells which were cultured for 10 days were given. After infusion, the qRT-PCR results showed that infiltrating CAR-T cells showed a significant proliferation on day 7 after a decline on day 3. The mean copy number of SCR-CART19 cells was significantly higher, about five times, than that of the S3-CART19 and S4-CART19 cells (Fig. 6g). This suggested that the intratumoral amplification was also impaired significantly by PD-1 silencing.
The effects of PD-1 knockdown were prevalent in different culture conditions and CAR-T systems
Limited proliferation potential is one of the hallmarks of T cell exhaustion and this could be promoted or rescued by many factors [27].
It has been reported that cytokines such as IL-7, IL-15 and IL-21 can retard T cell senescence, and promote differentiation into memory phenotype and proliferation [28,29,30]. We confirmed that the combined use of these cytokines did enhance the proliferative activity, but the proliferation inhibition caused by PD-1 knockdown could not be rescued (Fig. 7a). Meanwhile, the alteration of differentiation kinetics caused by PD-1 knockdown was still present here (Fig. 7b).
It has been reported that T cells exhaustion could be triggered by unique conformation of CAR19 molecule mediated autonomous activation [31]. To verify whether the impairment of proliferation caused by PD-1 knockdown was also present in other CAR-T systems, we constructed and tested epidermal growth factor receptor (HER-1) targeting CART (SCR-CART-HER1, S3-CART-HER1 and S4-CART-HER1) cells. As shown in Fig. 7c and d, the proliferation potential of CART-HER1 cells was also inhibited by intrinsic PD-1 blockade, and the alteration of phenotypes was similar to that observed in CART-19 cells.