Construction of macrophage specific CARs and development of EGFRvIII-targeting CAR-iMACs
The CD3ζ signal transduction domain obtained from T cell specific CAR has been proved to be capable of stimulating the first generation of human CAR-iMACs29,31,49. However, the domain cannot be helpful to maintain the M1 state of macrophages in TME29. Therefore, we designed macrophage specific CARs containing the intracellular TIR domain, whose activation can drive M1 polarization of macrophages via interplaying with TIR domain-containing adaptors. Meanwhile, we employed EGFRvIII-targeting scFv as the extracellular domain. We designated it as TIR-CAR to distinguish it from the previous CD3ζ-CAR (Figure 1a). Inspired by the evolutionary process of different generations of CARs in T cells (T-CARs), we simultaneously designed a second generation macrophage specific CAR (M-CAR) by integrating intracellular CD3ζ and TIR domains in tandom designated as CD3ζ-TIR-CAR (Figure 1a). A truncated CAR without an intracellular domain was designed as the negative control (Figure 1a). We then transduced the above CARs into human iPSCs with lentivirus. CAR-expressing iPSCs were subsequently differentiated into CAR-iMACs through the periods of mesoderm induction, hematopoietic specification, myeloid expansion, and macrophage maturation (Figure 1b). Flow cytometric (FCM) analysis showed that all types of CAR-iMACs shared about 80% transgene expression (Figure 1c). Thus, we successfully developed four types of EGFRvIII-targeting CAR-iMACs.
We subsequently analyzed expression of TLR4 signaling pathway genes at different stages of myeloids/macrophages differentiation with RNA-seq data. We found that the key factors such as IRF7, NF-κB/P65, MYD88, TRAP6, TRAM, and TAB2/3 were highly expressed on day 18 and declined on day 28 (Figure 1d). This data suggested that most of the downstream modulators of TLR4 are higher when the iMACs are still in a monocyte-like state, compared with the later stage when the iMACs are more matured. Furthermore, in vitro M1 polarized iMACs expressed higher level of the same TLR4 signaling pathway genes compared to the naive or M2 polarized iMACs (Figure 1e). Together, the data suggested that the early differentiated iMACs resembled the M1 polarized iMACs in terms of higher expression of the TLR4 pathway adaptor and signal transduction genes, and therefore might possess more potent signaling effects from the activated TIR domain.
In order to make a target cell line for the EGFRvIII CAR expressing iMACs, we cloned the human EGFRvIII sequence into a lentivirus vector (Supplemental Figure 1a), and overexpressed the gene in U87MG cells to develop a stable EGFRvIII-expressing tumor cell line (U87MGEGFRvIII). While basic expression of total EGFR was observed in both wild-type (WT) and transgenic U87MG cells, only the latter had strong expression of EGFRVIII protein (Supplemental Figure 1b and c). With immunofluorescence, we observed the location of EGFRVIII on the cell membrane, as well as in cytoplasm due to overexpression (Supplemental Figure 1d). Thus we have successfully constructed an U87MGEGFRvIII cell line for testing effector functions of EGFRvIII CAR-iMACs.
Both TIR and CD3ζ domains enhanced antigen-dependent functions of CAR-iMAC in vitro
Previous work demonstrated that the first generation of CAR conferred antigen-dependent functions of CAR-iMACs29,31,49. In order to evaluate whether the TIR signal domain can also enhance phagocytosis of CAR-iMACs against tumor cells in an antigen-dependent manner, we first performed experiments to incubate the single intracellular domain-containing CAR-iMACs cells with U87MGEGFRvIII cells in vitro. We first stimulated WT-iMACs, truncated CAR-iMACs, CD3ζ-CAR-iMACs, and TIR-CAR-iMACs with IFN-γ and LPS respectively, and then co-cultured the cells with tdTomato-expressing (tdTomato+) U87MGEGFRvIII cells with different effect/target ratios (E/T) of 3/1, 5/1, and 10/1. After 12 hours of co-culturing, we observed enhanced adhering property of CAR-iMACs to the antigen-expressing cancer cells compared to WT-iMACs (Supplemental Figure 1e), signifying that the CAR enabled iMACs with targeting capacity. Moreover, immunofluorescence assays showed that both CAR-iMACs and WT-iMACs engulfed tdTomato labeled tumor cells (Supplemental Figure 1f), demonstrating that the iMACs possess basic phagocytosis like primary macrophages, and overexpression of CAR did not impair this feature. Next, we collected all the cells and assessed the number of residual tumor cells by FCM analysis (Supplemental Figure 2a). Compared to WT-iMACs or truncated CAR-iMACs, U87MGEGFRvIII cells co-incubated with CD3ζ or TIR-CAR-iMACs suffered more killing and remained fewer cells at the same condition of E/T ratio (Supplemental Figure 2a and b). At the same time, all types of CAR-iMACs showed strengthened cytotoxicity with increased E/T ratios (Supplemental Figure 2b). Moreover, we did not observe obvious disparity of cytotoxicity between CD3ζ-CAR-iMACs, and TIR-CAR-iMACs at the 12 hours incubating time point (Supplemental Figure 2b).
To demonstrate progressive cytotoxicity of CAR-iMACs against their target cells, we next performed the same experiments at 24 hours of co-incubating. Compared to 12 hours, 24 hours co-incubating significantly improved tumor cell killing efficacy of CAR-iMACs at all tested E/T ratios (Figure 2a and b). Although both CD3ζ-CAR-iMACs and TIR-CAR-iMACs showed powerful anti-tumor competence compared to truncated iMACs, it appeared the TIR domain conferred stronger killing capacity than CD3ζ at this time point (Figure 2b). When we compared the trend of killing effect of the three types of CAR-iMACs from 12 hours to 24 hours co-incubating with U87MGEGFRvIII cells, we found that TIR-CAR-iMACs had greater killing persistence than CD3ζ-CAR-iMACs as time goes on (Figure 2c). To validate the above findings, we over-expressed a firefly luciferase (FFluc) gene in U87MGEGFRvIII cells, and performed more killing assays to further test the anti-tumor cell capacity of the CAR-iMACs. Consistently, the bioluminescence signal from tumor cells was evidently attenuated by both CD3ζ-CAR-iMACs and TIR-CAR-iMACs comparing to truncated CAR-iMACs, and the efficiency achieved optimum at the E/T ratio of 10/1 (Figure 2d). Furthermore, in order to investigate whether the pro-inflammatory activity of CAR-iMACs is relied on CAR activation, we measured release of TIR downstream cytokines. ELISA tests revealed that when encountering U87MGEGFRvIII cells for 24 hours at an E/T ratio of 10/1, TIR-CAR-iMACs secreted considerable pro-inflammatory cytokines including IL6 (Figure 2e), IL12A (Figure 2f) and IL23 (Figure 2g), indicating more pro-inflammatory activity of TIR-CAR-iMACs. Interestingly, the CAR containing T cell-specific CD3ζ domain also promoted milder expression of these cytokines in CAR-iMACs (Figure 2e-g), and the expression of TNF-α, another TLR4 regulated immune factor, was unexpectedly higher in CD3ζ-CAR-iMACs than in TIR-CAR-iMACs (Figure 2h), suggesting combining the two might achieve the maximal pro-inflammatory activity. Taking together, these results demonstrated that both TIR and CD3ζ domains enhanced CAR-dependent cytotoxic activity of CAR-iMACs, but TIR-CAR-iMACs exhibited more potential of persistent and potent pro-inflammatory activity.
TIR-CAR-iMACs possess stronger anti-tumor potency than CD3ζ-CAR-iMACs in vivo
We next investigated anti-tumor activities of CD3ζ-CAR-iMACs and TIR-CAR-iMACs in tumor-bearing animals. We first established the tumor model in abdominal cavity of immunodeficient mice with FFluc+U87MGEGFRvIII cells. IFN-γ/LPS polarized truncated CAR-iMACs, CD3ζ-CAR-iMACs, and TIR-CAR-iMACs were labeled by the DiR dye, and then injected intraperitoneally into the tumor-bearing mice with an E/T ratio of 10/1. Animal imagings were performed 2 hours after immune cells injection (day 0), and at the same time of day 1, day 3 and day 7 (Figure 3a). All the three types of CAR-iMACs exerted anti-cancer cell activity against the tumor at day 1 immediately (Figure 3b and c). However, comparing with truncated CAR-iMACs and CD3ζ-CAR-iMACs, TIR-CAR-iMACs exhibited stronger anti-tumor activity, and achieved optimum efficacy at day3 (Figure 3c). Of note, even though the tumor started to relapse at day 7, the tumor signals from TIR-CAR-iMACs-treated mice were still maintained at a lower level than that in PBS and CD3ζ-CAR-iMACs-treated mice, suggesting that introducing the TIR domain into CAR-iMACs might have conferred resistance to reprogramming by the tumor microenvironment over time. Further survival analysis showed that TIR-CAR-iMACs markedly prolonged the life time of tumor-bearing mice (Figure 3d). Collectively, the data demonstrated that although both TIR-CAR-iMACs and CD3ζ-CAR-iMACs showed antigen-dependent anti-tumor activity in vivo, the TIR-CAR conferred more persistent killing capacity of CAR-iMACs against cancer cells.
The combination of TIR and CD3ζ domains strengthened M1 polarization and anti-tumor potency of CAR-iMACs
In the above work, we have verified that TIR-CAR-iMACs exhibited the M1 phenotype and both TIR and CD3ζ domains enabled CAR-iMACs with antigen-dependent phagocytosis capacity. Based on the design of second generation of T-CAR, we hypothesized that integration of TIR and CD3ζ domains into one CAR structure could further promote anti-tumor potency of CAR-iMACs. Thus we developed a new M-CAR consisting of intracellular CD3ζ and TIR domains in tandom (Figure 1a), and then generated CD3ζ-TIR-CAR-iMACs (Figure 1c). We first compared expression of pro-inflammatory or M1 polarization genes supposedly driven by TIR activation. As expected, qPCR analysis revealed that, when incubating the three types of CAR-iMACs with U87MGEGFRvIII cells for 24 hours, expression of IL1A/B, IL6, IL12A, IL23, CCL8, CXCL8 and TNF-α were elevated significantly in CD3ζ-TIR-CAR-iMACs compared to TIR- or CD3ζ-CAR-iMACs at the condition of no IFN-γ/LPS pretreatment (Figure 4a-h), revealing a synergistic effect of TIR and CD3ζ domains. Correspondingly, ELISA testings confirmed that CD3ζ-TIR-CAR-iMACs improved production of IL6, IL12A, IL23, and TNF-α (Figure 4i-l). These results further demonstrated that the TIR domain contributed to M1 polarization of CAR-iMACs. Subsequently, we examined cytotoxicity of the integrated CD3ζ-TIR-CAR-iMACs confronting FFluc+U87MGEGFRvIII cells. Measurement of bioluminescence from FFluc+U87MGEGFRvIII cells indicated that comparing to single CD3ζ or TIR domain-containing CAR-iMACs, 24 hours incubation with CD3ζ-TIR-CAR-iMACs led to the lowest number of residual tumor cells under the condition of E/T of 10/1(Figure 4m), revealing that CD3ζ-TIR-CAR-iMACs conferred much more lethality against EGFRvIII positive tumor cells than single domain-containing CAR-iMACs.
Previous reports have illustrated that the TME leads to exhaustion of T cells and promotes M2-like polarization of macrophages12,38,72−75. To explore whether the TIR based CAR-iMACs were equipped with the ability of withstanding immunosuppressive effects from tumor cells, we examined the polarization state of the four types of CAR-iMACs co-cultured with tdTomato+U87MGEGFRvIII cells under the condition of no IFN-γ/LPS pre-treatment and an E/T ratio of 3/1. Subsequent FCM analysis showed that 24 hours incubation with the tumor cells stimulated high expression of CD80 in all types of CAR-iMACs, and the two TIR-based CAR-iMACs showed more CD80-positive (CD80+) populations compared to those in truncated CAR-iMACs and CD3ζ-CAR-iMACs (Supplemental Figure 3a). Of note, prolonged periods of exposure to tumor cells resulted in marked reduction of CD80+ populations to about 40-50% for truncated CAR-iMACs and CD3ζ-CAR-iMACs (Supplemental Figure 3a, and Figure 4n and p), whereas the percentage of CD80+ cells in both TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs persisted at a higher level of ~80% (Supplemental Figure 3a, Figure 4n and p), indicating that TIR contributed to stimulation of the M1 state. On the other hand, we also determined M2 populations of the four types of CAR-iMACs by measuring CD163, a typical M2 marker, and found that long time exposure to the tumor cells led to various level of CD163 expression in the first three types of CAR-iMACs, whereas CD3ζ-TIR-CAR-iMACs exhibited robust potency against M2 polarization (Supplemental Figure 3b, Figure 4o and q). Indeed, the percentage of CD163+ CD3ζ-TIR-CAR-iMACs declined to about 36.93% at day 3 of co-incubation, and remained to be low at day 7 (Supplemental Figure 3b and Figure 4o and q). Based on the above results, we concluded that the combination of the TIR and CD3ζdomains supported M1 polarization of CAR-iMACs exposing to tumor cells, and contributed to prevent reversion of M1 to M2 state.
We next investigated anti-tumor potency of CD3ζ-TIR-CAR-iMACs in vivo. Operated as above, truncated CAR-iMACs, TIR-CAR-iMACs, CD3ζ-CAR-iMACs, and CD3ζ-TIR-CAR-iMACs with no IFN-γ/LPS pretreatment were injected intraperitoneally into FFluc+U87MGEGFRvIII cells-bearing mice at the condition of an E/T ratio of 15/1 (Supplemental Figure 4a). Bioluminescence imagings were performed at day 0, day 1, day 3, and day 7. As expected, treatment by TIR-CAR-iMACs or CD3ζ-TIR-CAR-iMACs markedly alleviated tumor cell growth at day 1 compared to truncate CAR-iMACs or CD3ζ-CAR-iMACs (Supplemental Figure 4b and c). Noteworthy, CD3ζ-TIR-CAR-iMACs showed the strongest anti-tumor potency in the living body environment (Supplemental Figure 4b and c), and prolonged survival time of the tumor-bearing mice most prominently (Supplemental Figure 4d). In order to assess whether TIR contributed to M1 polarization of CAR-iMACs in the course of treatment inside the living body, we isolated the immune cells from abdominal cavities of the mice after two days of treatment, and measured CD80+ and CD163+populations by FCM. As observation in vitro, more TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs exhibited the CD80 positive feature than that in CD3ζ-CAR-iMACs (Supplemental Figure 4e and f), and much lower CD163 positive M2 feature was present in CD3ζ-TIR-CAR-iMACs (Supplemental Figure 4e and f). In summary, we confirmed that integration of intracellular CD3ζ and TIR domains into one CAR structure could enhance cytotoxic activity against tumor cells and potency of maintaining M1 polarization of CAR-iMACs.
The CAR promotes M1 polarization of CAR-iMACs via TIR domain-mediated canonical NF-κB/P65 transcription activity
The above data demonstrated that the TIR domain enhanced M1 polarization and activation of CAR-iMACs in an antigen-dependent manner. TIR domain is responsible for signal transduction from TLR4, and the downstream signaling liberates NF-κB/P65 from IκB-α-mediated degradation followed by the translocation of NF-κB/P65 into nucleus to transcriptionally activate pro-inflammatory target genes70,76. In order to investigate whether NF-κB/P65 mediated the pro-inflammatory activity of TIR-CAR-iMACs encountering the antigen, we first tested expression of NF-κB/P65 gene RELA with qPCR analysis and found its higher expression in both TIR domain-containing CAR-iMACs (Figure 5a). Whereas ERK, another gene involved in macrophage activation had no distinction of expression in all CAR-iMACs (Figure 5b). Next, we monitored nucleus translocation of NF-κB/P65 by immunofluorescence when the control truncated CAR-iMACs were treated with LPS for 20 minutes (Supplemental Figure 5a). Afterwards, we co-cultured truncated CAR-iMACs, TIR-CAR-iMACs, CD3ζ-CAR-iMACs, and CD3ζ-TIR-CAR-iMACs with tdTomato+ U87MGEGFRvIII cells respectively with an E/T ratio of 3/1, followed by immunoflurescence to detect NF-κB/P65 subcellular localization. Confocal imaging showed that sufficient contact between CAR-iMACs and U87MGEGFRvIII cells for 4 hours initiated expression of NF-κB/P65 in CD3ζ-TIR-CAR-iMACs (Supplemental Figure 5b). When the incubating time was prolonged to 10 hours, NF-κB/P65 exhibited evident expression in both TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs, and weak expression in CD3ζ-CAR-iMACs, and it aggregated in the nucleus of the two types of TIR-based CAR-iMACs (Figure 5c and d). Nevertheless, after 24 hours of incubation, the aggregation of NF-κB/P65 in the nuclei of TIR-CAR-iMACs or CD3ζ-TIR-CAR-iMACs was abrogated (Supplemental Figure 5c), resembling the outcome of LPS-induced tolerance in macrophages77–81. Notably, phagocytosis of tumor cells also led to nuclear export and cytoplasmic accumulation of NF-κB/P65 in TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs (Supplemental Figure 5c), indicating inactivation of the immune cell functions after completing tumor-killing process. Besides, we didn’t detected obvious expression and nuclear localization of NF-κB/P65 in the TIR-CAR-iMACs that did not touch U87MGEGFRvIII cells (Figure 5c), consistent with an antigen-dependent activation mechanism. Together, these results demonstrated that the NF-κB signal pathway was activated in the TIR domain-containing CAR-iMACs.
To further confirm the role of NF-κB/P65 in CAR-iMAC, we treated the CAR-iMACs co-cultured with FFluc/tdTomato+ U87MGEGFRvIII cells with a NF-κB/P65 inhibitor JSH23. As expected, blocking of nuclear location of NF-κB/P65 by JSH23 abrogated cytotoxicity of CAR-iMACs against FFluc/tdTomato+ U87MGEGFRvIII cells (Figure 5e). Similarly, FCM analysis of residual tumor cells indicated that blocking NF-κB/P65 into nucleus increased residual tdTomato positive tumor cells in TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs groups (Figure 5F). Collectively, we confirmed that immune activation and M1 polarization of TIR-based CAR-iMACs were through the NF-κB pathway.
Single cell RNA-sequencing revealed robust molecular features and mechanisms of M1 polarization of TIR-based CAR-iMACs
We next attempted to examine to what extent the TIR-based CAR-iMACs could promote the pro-inflammatory state when exposed to tumor cells through single cell RNA-sequencing analysis (Figure 6a). In this experiment, we pretreated the CAR-iMACs with IFN-γ/LPS for 24 hours, and then incubated each of them with U87MGEGFRvIII cells for another 24 hours. After that, we performed 10×genome single cells RNA-sequencing analysis with all the cells, and found that they could be clustered into six sub-populations (Supplemental Figure 6a, c and e). According to the in vivo single cell RNA-seq data of human hematopoietic development, C1, C2 and C3 clusters were mainly matched to the Macrophage cell phenotype (Supplemental Figure 6b, d and f). Meanwhile, C4 and C5 clusters were primarily matched with the U87MG cancer cell phenotype in all the three types of CAR-iMACs groups we examined. Interestingly, the C5 cluster also showed evident macrophage feature in the CD3ζ-TIR-CAR-iMACs group, indicating these were the cells that engulfed the tumor cells and thus assumed features of both macrophage and tumor cells (Supplemental Figure 6b, d and f). According to the single cell analysis, compared with truncated CAR-iMACs and TIR-CAR-iMACs groups, CD3ζ-TIR-CAR-iMACs treatment resulted in fewer remaining tumor cells (Supplemental Figure 6a-f, and Figure 6b). In addition, even though the C6 cluster of all the three groups were mainly matched with macrophage, C6 in TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs showed more evident and incremental DC characteristics compared to truncate CAR-iMACs (Supplemental Figure 6b, d and f), implying that TIR-based CAR-iMACs might have acquired enhanced antigen-presenting competence as dendritic cells.
As in the above results, we have demonstrated the M1 polarization-promoting effect of TIR-CAR. Next, we performed more quantitative analysis of M1 or M2 markers using single cell RNA-seq data. Normalized gene expression showed that more TIR-CAR-iMACs exhibited high expression of CD80 and CD86, and CD3ζ-TIR-CAR further boosted the expression levels of the two M1 marker genes, (Figure 6c-e). Accordingly, expression of M2 marker CD206 was reduced in TIR-CAR-iMACs, and to the lowest level in CD3ζ-TIR-CAR-iMACs, compared to that in truncated CAR-iMACs (Figure 6c-e). Moreover, the percentage of CAR-iMACs that expressed other M1 state-associated genes including CD83, CCL8, CXCL9, and CXCL11, increased incrementally from truncated CAR-iMACs to TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs (Figure 6f). Accordingly, the normalized expression level of these genes exhibited the same trend (Figure 6g-j). On the contrary, fewer percentage of TIR-CAR-iMAC and CD3ζ-TIR-CAR-iMACs expressed M2 state-associated genes such as CCL13 and CD163 (Figure 6k), and even though only a handful of cells had these genes being detected, TIR-CAR-iMAC and CD3ζ-TIR-CAR-iMACs presented much lower expression compared to truncated CAR-iMACs (Figure 6l and m). Collectively, all the results illustrated that integration of the TIR and CD3ζ domains facilitated M1 genes expression and suppressed M2 gene expression in the same time.
Next we further investigated whether the global gene expression signature resembled that of the M1 macrophages. We divided CAR-iMACs to CD80-expressing and CD80-absent (non-CD80) populations, as the latter represented the iMACs that might not encounter tumor cells, and evaluated the discrepancy of their expression profile. Principle component analysis (PCA) using M1-related genes identified in our previous study indicated that CD80-expressing TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs were positioned closer to the IFN-γ/LPS-stimulated M1 iMACs29, compared to CD80-positive truncated CAR-iMACs and non-CD80 CAR-iMACs from all the types (Figure 6n). Furthermore, pathway analysis revealed that CD80-expressing CAR-iMACs showed lower level of M2-related pathways such as Oxidative phosphorylation and Respiratory electron transport, than that in non-CD80 CAR-iMACs, but higher level of M1-related pathway Interferon gamma response (Figure 6o). More importantly, within the CD80-expressing groups, TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs showed evidently higher Interferon gamma response and lower Oxidative phosphorylation (Figure 6o), indicating that TIR based CAR conferred the CAR-iMACs with strengthened pro-inflammatory activity. Lastly, via hierarchical clustering analysis based on a defined set of M1 and M2-associated genes29, we found that both TIR-CAR-iMACs and CD3ζ-TIR-CAR-iMACs clustered with IFN-γ/LPS- polarized iMACs, whereas in contrast, truncated CAR-iMACs clustered with M2 polarized iMACs (Supplemental Figure 7a and b). The same trend of clustering results was observed when using the CD80-expressing populations (Supplemental Figure 7c and d). To sum up, the above single cell RNA-seq analysis strongly demonstrated that TIR-based CARs contributed to the M1 polarization of CAR-iMACs in an antigen-dependent manner when encountering tumor cells, and improved immune activity against target tumor cells.