T corpuscle therapies accept accustomed abiding adeptness and alleviative abeyant for the appraisal of some cancers. However, their use is apprenticed by accident to eyewitness tissues, as apparent in graft-versus-host ache afterwards donor lymphocyte infusion, or “on-target, off-tumor” toxicities incurred in some engineered T corpuscle therapies. All-embracing immunosuppression and irreversible T corpuscle aishment are currently the abandoned agency to ascendancy such deleterious responses, but at the amount of abrogating ameliorative allowances or causing accessory complications. On the base of the physiological archetype of accustomed inhibitory receptors, we advised antigen-specific inhibitory chimeric antigen receptors (iCARs) to preemptively constrain T corpuscle responses. We authenticate that CTLA-4– or PD-1–based iCARs can selectively absolute cytokine secretion, cytotoxicity, and admeasurement induced through the autogenous T corpuscle receptor or an activating chimeric receptor. The antecedent aftereffect of the iCAR is temporary, appropriately enabling T beef to action aloft a afterwards appointment with the antigen accustomed by their activating receptor. iCARs appropriately accommodate a dynamic, automated assurance about-face to prevent, rather than treat, the after-effects of bare T corpuscle specificity.
T corpuscle therapies accept apparent analytic adeptness in cartilage bottom and agency transplantation, blight immunotherapy, viral infections, and autoimmune diseases (1–6). Unfortunately, T beef may additionally appoint in deleterious ancillary effects. “On-target but off-tumor” adverse contest accept been appear in blight immunotherapy analytic trials application both T corpuscle receptor (TCR)– and chimeric antigen receptor (CAR)–engineered T cells. These accommodate B corpuscle aplasia in abiding lymphocytic leukemia patients advised with T beef cogent anti-CD19 CAR (7–9), baleful astute respiratory ache affection afterwards anti-ERBB2 CAR T corpuscle beverage anticipation to aftereffect from cross-reactivity on lung epithelium (10), and TCR-induced fatalities from cardiac myonecrosis or acoustic toxicity incurred in patients advised with TCRs acquainted cancer-testis antigens (11–13). Similarly, the alleviative assets of donor lymphocyte beverage (DLI) in allogeneic cartilage bottom transplantation are bedfast by the consecration of both astute and abiding graft-versus-host ache (GVHD) and cartilage bottom aplasia (14). Strategies to abstracted the benign furnishings of affix adjoin bump (GVT) from GVHD accept met with apprenticed success to date (15).
The accepted access to barrier T cell–mediated toxicities relies on the use of immunosuppressive regimens such as high-dose corticosteroid therapy, which apply cytostatic or cytotoxic furnishings on T cells, to arrest accustomed responses (16). Although effective, this access fails to discriminate amid benign and deleterious T corpuscle functions. Additionally, immunosuppressive drugs account abundant accessory ancillary effects, such as susceptibility to infections, and cardiac, kidney, and acoustic accident (14). Suicide gene engineering strategies, which may use accurate enzymatic metaizers of baneful agents, such as canker canker virus thymidine kinase (17) or inducible caspase-9 (18), or antibody-mediated burning strategies targeting ectopic epitopes engineered into T beef (19, 20), additionally annihilate T beef indiscriminately of their ameliorative efficacy. Furthermore, these approaches are acknowledging because they are implemented afterwards celebratory deleterious ancillary effects. Strategies that anticipate exceptionable T corpuscle acuteness are appropriately awful desirable.
Physiological adjustment of T corpuscle activation is able by several mechanisms that accommodate accustomed inhibitory receptors, which comedy a cardinal role in attenuating or absolute T corpuscle responses (21, 22). Inhibitory receptors can be up-regulated during T corpuscle conference to abate accustomed responses or basally bidding to adapt activation thresholds. Thus, mice amiss for the inhibitory receptor CTLA-4 affectation massive T corpuscle activation and admeasurement and eventually accede to astringent systemic autoimmune ache with aggression of activated T beef (23). Similarly, accident of PD-1, addition inhibitory receptor accurately bidding on activated T cells, causes accelerating arthritis and glomerulonephritis in C57BL/6 mice and accelerated insulitis in nonobese diabetic (NOD) mice (24, 25). Modulation of these receptors and their afterwards signaling pathways has abundant access on T corpuscle functions. In vitro articulation of CTLA-4 or PD-1 during T corpuscle dispatch blocks activation, cytokine release, and admeasurement (26). Notably, anti–CTLA-4 and anti–PD-1 antibodies accept apparent analytic affiance by derepressing anti–T corpuscle responses in some patients with melanoma, lung, and renal blight (22, 27, 28). Barricade of both CTLA-4 and PD-1 is additionally actuality actively advised for abandoning accustomed dysfunction and viral chain in abiding hepatitis B and HIV infection (29, 30). However, agnate to all-embracing immunosuppression, antibody-mediated inhibitory receptor checkpoint barricade is not antigen-specific and accordingly does not anticipate amid benign and deleterious T corpuscle populations.
Here, we acclimated a abiogenetic engineering action to accouter the accustomed T corpuscle inhibition appraisal and adapt T corpuscle responses in an antigen-selective manner. Conceptually, we accustomed to architecture an inhibitory CAR (iCAR) accepting a apparent antigen acceptance area accumulated with a able astute inhibitory signaling area to absolute T corpuscle admiration admitting circumstantial assurance of an activating receptor (Fig. 1A). We appearance here, in animal primary T cells, that PD-1– and CTLA-4–based iCARs reversibly bind analytical TCR or activating CAR functions, and appropriately acquiesce for bigotry amid ambition and astray beef in vitro and in vivo.
(A) T beef with specificity for both bump and astray tissues can be belted to bump abandoned by application an antigen-specific iCAR alien into the T beef to assure the astray tissue. (B) Schematic diagram of the bicistronic vectors acclimated for iCARs and Pdel. iCAR-P: a spacer, transmembrane, and intracellular appendage of anniversary inhibitory receptor were cloned into a ahead declared retroviral agent accepting a CD8 baton arrangement (LS). IRES, centralized ribosomal access site; hrGFP, humanized Renilla blooming beaming protein reporter. A Pdel ascendancy agent was advised with a spacer and CD8 transmembrane (TM) domain, and defective an intracellular tail. (C) Corpuscle apparent announcement of the iCARs was adjourned by breeze cytometry in transduced primary animal T cells. Dot plots are adumbrative of eight altered donors. GAM, dupe anti-mouse immunoglobulin G F(ab′)2 antibiotic that binds to the murine-derived extracellular area of the CAR.
We accepted that a single-chain capricious fragment (scFv) specific for an antigen alloyed to the signaling domains of immunoinhibitory receptors (CTLA-4 and PD-1) via a transmembrane arena would arrest T corpuscle action accurately aloft antigen recognition. As a aboriginal model, we acclimated an scFv specific for animal prostate-specific blur antigen (PSMA) (31). This scFv has been abundantly advised and is actuality advised in appearance 1 trials for immunotherapy of prostate blight (32). PSMA is overexpressed in metastatic prostate blight but is additionally begin in accustomed kidney, liver, colon, and academician astrocytes (33). Along with these two iCARs specific for PSMA (referred to as iCAR-P), we acclimated Pdel, which has the PSMA-specific scFv but lacks a cytoplasmic area (34), as a ascendancy receptor (Fig. 1B). Aloft transduction of animal primary T beef from borderline claret mononuclear cells, PD-1–iCAR-P and Pdel were bidding on the corpuscle apparent at agnate levels to the P28z receptor (34), a CD28/CD3ζ-based, dual-signaling PSMA-specific receptor that is currently acclimated in a analytic balloon (Fig. 1C). In the case of the CTLA-4–iCAR-P, we empiric able-bodied intracellular announcement by Western blemish and intracellular breeze cytometry, but apprenticed corpuscle apparent announcement (fig. S1, A and B, and Fig. 1C). This award is constant with the physiological trafficking of CTLA-4, which is constitutively internalized in comatose T beef and base through alternation with the endocytic adaptor circuitous AP-2 via its tyrosine accountability YVKM (35). Indeed, we begin up-regulation of the CTLA-4–iCAR-P to the corpuscle apparent afterwards T corpuscle activation (fig. S1C), and adequate basal apparent announcement application a tyrosine accountability Y165G aberrant to assemble mutCTLA-4–iCAR-P, which apparent corpuscle apparent announcement in comatose beef (Fig. 1C). PSMA acceptance by iCARs was accustomed application a cellular alliance appraisal in which iCAR-expressing T beef apprenticed abrasion thymoma EL4 beef cogent PSMA (fig. S2A).
To abstraction the aftereffect of iCARs on autogenous TCR-driven primary animal T corpuscle responses, we accustomed an alloreactivity archetypal application allogeneic blooming beef (DCs) as conference antigen-presenting beef and fibroblasts isogenic to the DCs as the targets (fig. S3A). In this model, the iCAR- or Pdel-engineered T beef were a with allogeneic monocyte-derived DCs (moDCs) and afresh evaluated adjoin fibroblasts cogent the PSMA antigen or not. To access replenishable fibroblasts isogenic to the DCs afterwards acute accepted bark biopsies, we accustomed induced pluripotent axis beef (iPSCs) from which we acquired abiding fibroblast corpuscle lines, termed iPS-fib (fig. S3, B to D). The iPS-fib displayed replicative adulteration and acquaintance inhibition, and could be calmly transduced, passaged, and built-in in NOD/severe accumulated immunodeficient (SCID)/γc− mice wherein they persisted for weeks afterwards basic tumors. To access almighty alloreactive T beef with autogenous TCR specificity adjoin the iPS-fib, we pulsed moDCs with lysates from the isogenic iPS-fib. This conference adeptness arrangement angry able-bodied cytotoxicity and cytokine beard from several T corpuscle donors, address both CD4- and CD8-driven responses (fig. S4, A to C).
To investigate the adeptness of the iCARs to bind alloreactivity adjoin PSMA cells, we sorted iCAR- or Pdel-expressing T beef a with two circuit of pulsed moDCs and afresh co-incubated them with iPS-fib or iPS-fib cogent PSMA (fig. S5A) (36). All groups of T beef calmly dead iPS-fib, demonstrating allogeneic cytotoxicity (Fig. 2, A and B), but the iCAR-positive T beef were decidedly inhibited in their adeptness to annihilate iPS-fib-PSMA beef (Fig. 2C). Cytotoxicity by T beef cogent the PD-1−based iCAR was bargain by up to 95% at low effector-to-target (E/T) ratios. Because cytotoxicity occurs rapidly and has a low activation beginning about to added T corpuscle responses, we additionally analyzed cytokine secretion. The PD-1 iCAR produced the stronger inhibition of cytokine beard (79 to 88% reduction), admitting the mutCTLA-4 iCAR elicited 55 to 71% abridgement (Fig. 2, D to F). These after-effects appropriate that iCARs could absolute acuteness in an antigen-dependent manner.
Control Pdel- or iCAR-transduced T beef a with allogeneic moDCs were incubated with iPS-derived fibroblasts (iPS-fib) cogent bang beetle luciferase (CBL), isogenic to the moDCs, application a ambit of E/T ratios. (A) Pdel-, PD-1–, or mutCTLA-4 iCAR-P–transduced T beef reacting adjoin ambition iPS-fib (n = 3 per condition). Killing of the iPS-fib was quantified with the Bright-Glo appraisal system. (B) Cytokine beard in corpuscle adeptness supernatants from (A) at 4:1 E/T arrangement was adjourned at 18 hours. GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-γ, interferon-γ; TNF-α, bump afterlife factor-α. (C) Pdel- or iCAR-positive T beef were incubated for 24 hours with astray iPS-fib cogent PSMA (iPS-fib-PSMA), and luciferase arresting (left) was quantified (right) (n = 3 for anniversary condition). (D to F) Cytokine beard abstinent at 24 hours in corpuscle adeptness supernatants from (C). Error bedfast represent ±SEM. *P < 0.01, ***P < 0.001 by appraisal of about-face (ANOVA) comparing iCARs to Pdel and column hoc appraisal with assorted t tests adapted with the Holm-Sidak method. Raw abstracts and P ethics are provided in the Supplementary Materials.
We advised whether the PD-1 iCAR-P could accommodate cogwheel levels of inhibition depending on its akin of announcement or that of the ambition antigen. We accordingly sorted a T beef for aerial or low levels of PD-1 iCAR-P or Pdel announcement and apparent them to iPS-fib-PSMA (fig. S6A). We begin a stoichiometric accord amid T corpuscle killing, cytokine release, and the akin of iCAR expressed. T beef sorted for low levels of announcement of the PD-1 iCAR-P could accommodate 50% inhibition abandoned up to E/T ratios of 1:1, but aerial levels of PD-1 iCAR-P announcement accustomed 80% inhibition up to E/T ratios of 8:1 and alike 50% inhibition at 16:1 (Fig. 3, A and B). To appraise the appulse of the iCAR antigen announcement level, we sorted iPS-fib for aerial or low PSMA announcement and apparent them to sorted PD-1 iCAR-P T beef (fig. S6B). iPS-fib with aerial PSMA announcement inhibited at atomic 80% of the killing and cytokine beard of PD-1 iCAR-P T beef beyond a ambit of E/T ratios (1:1 to 4:1), admitting iPS-fib with low PSMA announcement bootless to accommodate the aforementioned akin inhibition (Fig. 3, C and D).
(A) Pdel- or PD-1 iCAR-P–transduced alloreactive T beef were sorted for aerial or low announcement of anniversary corresponding receptor, as apparent in fig. S5A, and were seeded on iPS-fib-PSMA–expressing CBL. Killing of iPS-fib-PSMA about to basic beef was adjourned with the Bright-Glo appraisal arrangement (n = 3 for anniversary condition). (B) Cytokine secretion, abstinent at 24 hours in the corpuscle adeptness afloat from (A) at 4:1 E/T ratio. (C) PD-1 iCAR-P–transduced alloreactive T beef were incubated with iPS-fib-PSMA sorted for aerial or low levels of PSMA announcement as apparent in fig. S5B. Killing of anniversary citizenry about to basic beef was quantified with the Bright-Glo appraisal arrangement (n = 3 per condition). (D) Cytokines from (C) were adjourned at 24 hours. Error bedfast represent ±SEM. ***P < 0.001 by Student’s t test. Error bedfast represent ±SEM. *P < 0.01, ***P < 0.001 by ANOVA comparing to aerial Pdel accession and column hoc appraisal with assorted t tests adapted with the Holm-Sidak method. Raw abstracts and P ethics are provided in the Supplementary Materials.
To investigate whether an iCAR could assure a tissue from T cell–mediated aishment in vivo, we injected iPS-fib-PSMA beef (which additionally bidding CBL) intraperitoneally into NOD/SCID/γc− mice (fig. S6B). The beef accustomed nodules that could be monitored by bioluminescence imaging (BLI). Bristles canicule afterwards bang of 1 × 106 iPS-fib-PSMA cells, the mice were advised with 5 × 105 moDC-primed Pdel- or PD-1–iCAR-P–expressing T cells. The Pdel accession abandoned the iPS-fib-PSMA beef with a cogent abatement in the BLI arresting (7- to 22-fold), admitting the PD-1–iCAR-P accession was clumsy to bright the nodules with BLI agnate to ascendancy mice not advised with T beef (Fig. 4, A and B). These after-effects accommodate affirmation that an iCAR can absolute a TCR-driven acknowledgment in an antigen-specific appearance in vivo.
NOD/SCID/γc− mice were injected intraperitoneally with 1 × 106 iPS-derived fibroblasts cogent CBL/PSMA (iPS-fib-PSMA) and, 7 canicule later, were advised intraperitoneally with 5 × 105 PD-1 iCAR-P– or Pdel-transduced, sorted, alloreactive T cells. Basic mice (no T cells) were acclimated as control. (A) Survival of iPS-fib-PSMA was adjourned by BLI afore and at called time credibility afterwards T corpuscle infusion. Images of four adumbrative mice from anniversary accession are shown. (B) Absolute anatomy alteration (photons per second) for anniversary abrasion was quantified and averaged per accession (n = 5 per group). Error bedfast represent ±SEM. *P < 0.05, **P < 0.01 by ANOVA comparing to Pdel and column hoc appraisal with assorted t tests adapted with the Holm-Sidak method. Raw abstracts and P ethics are provided in the Supplementary Materials.
To abstraction the aftereffect of iCARs on modulating activating CARs, we acclimated 19-28z, an abundantly characterized second-generation CAR currently acclimated in analytic trials, which provides activation and CD28 costimulation in acknowledgment to the CD19 antigen (9, 34). Primary T beef were transduced with 19-28z CAR and the iCAR-P receptors, sorted for bifold expression, and seeded on ahead appear bogus antigen-presenting beef (AAPCs) cogent CD19 or both CD19 and PSMA, respectively, clay ambition and astray tissues (37) (figs. S5B and S7A). Although the T beef from the ascendancy groups (19-28z abandoned or 19-28z/Pdel) showed agnate cytokine beard on both AAPCs, the iCAR-expressing T beef showed a apparent abatement in cytokine beard back apparent to astray beef about to on-target beef (Fig. 5A and fig. S7B). PD-1 iCAR-P produced the arch abridgement of cytokine levels (71 to 89%), admitting mutCTLA-4 iCAR-P elicited a bottom abridgement (48 to 67%).
(A) Luminex circuitous cytokine appraisal of adeptness afloat 24 hours afterwards agriculture dual-sorted 19-28z/Pdel– or 19-28z/iCAR–transduced animal T beef on 3T3-CD19 (target) or 3T3-CD19-PSMA (off-target) AAPCs. The abstracts are represented as a arrangement of off-target/target ethics and affiliated from three absolute abstracts (n = 6 wells per condition). Error bedfast represent ±SEM. **P < 0.01, ***P < 0.001 by ANOVA comparing iCARs to Pdel and column hoc appraisal with assorted t tests adapted with the Holm-Sidak method. (B) Absolute counts of 19-28z/Pdel or 19-28z/iCAR T beef angry on canicule 0 and 7 with astray AAPCs. No exogenous cytokines were added. Abstracts are adumbrative of six absolute experiments. (C) Admeasurement of 19-28z/Pdel or 19-28z/iCAR T beef angry on canicule 0 and 7 with astray AAPCs about to admeasurement on ambition AAPCs. No exogenous cytokines were added. Abstracts are adumbrative of six absolute experiments. (D) T beef seeded at a 1:1 arrangement on ambition and astray mCherry AAPCs. Images at 38 hours and 5 canicule from one of bristles absolute abstracts are shown. Scale bars, 0.5 mm. (E and F) Quantification of mCherry arresting from (D) adjoin CD19 targets (E) or CD19-PSMA astray beef (F), as declared in Materials and Methods. Error bedfast represent ±SEM. **P < 0.01, ***P < 0.001 by Student’s t test. Raw abstracts and P ethics are provided in the Supplementary Materials.
19-28z provides a almighty admeasurement signal, induced by CD19-expressing AAPCs. Although 19-28z/Pdel T beef broadcast analogously on either AAPCs, T beef cogent mutCTLA-4 or PD-1 iCARs showed bargain accession in the attendance of the astray AAPCs, with the PD-1 iCAR-P causing a accumulative 90% abatement in T corpuscle accession afterwards the additional AAPC dispatch (Fig. 5, B and C, and fig. S7C). In this coculture system, we advised by quantitative microscopy the fate of these AAPCs, which we adapted to additionally accurate mCherry (Fig. 5D). Aural 38 hours, all groups of 19-28z/iCAR-P and ascendancy double-positive T beef lysed the ambition beef (Fig. 5E). Back apparent to CD19 PSMA astray cells, the mutCTLA-4– and PD-1–based iCARs acquired a 67 and 91% abridgement in cytotoxicity, appropriately (Fig. 5F). In the case of PD-1, AAPCs persisted for 5 days, admitting the aftereffect of the mutCTLA-4 iCAR was added apprenticed (Fig. 5D). We accordingly called the PD-1–based iCAR for added in vivo evaluation.
To appraise the action of the PD-1 iCAR in vivo, we transduced NALM/6, a CD19 B corpuscle leukemia corpuscle line, with PSMA, and compared ameliorative T corpuscle responses adjoin NALM/6 and NALM/6-PSMA beef in a ahead accustomed xenograft NOD/SCID/γc− abrasion archetypal (37, 38) (fig. S7D). Bristles canicule afterwards systemic bump infusion, the mice were advised with a distinct dosage of 3 × 105 19-28z/PD-1–iCAR-P–sorted double-positive T cells. BLI of bump accountability showed cogent differences (3- to 10-fold reduction) in the aishment of NALM/6-PSMA (off-target) as adjoin to NALM/6 (on-target) (Fig. 6, A and B). Although initially bedfast to cartilage marrow, NALM/6 leukemia eventually invades the spleen, the weight of which provides a late-stage base of ache burden. Afterwards appraisal with 19-28z/PD-1–iCAR-P T cells, NALM/6-PSMA mice showed no cogent aberration in annoyance weight from the ascendancy “no T cell” group, but the annoyance weights of the mice with NALM/6 were 2.6-fold lower (Fig. 6C). Breeze cytometric analyses accepted the decreased cardinal of NALM/6 beef in the annoyance and cartilage marrow, in adverse to the NALM/6-PSMA accession (Fig. 6, D and E). In parallel, we begin greater chain of T beef in the NALM/6 accession than in the NALM/6-PSMA accession (Fig. 6, D and F). These allegation accustomed that the PD-1–based iCAR selectively prevents the aishment of “off-target” NALM/6-PSMA beef in vivo while acceptance the ameliorative acknowledgment adjoin “on-target” NALM/6 beef to proceed.
(A) BLI depicting the bump advance of NALM/6 or NALM/6-PSMA in NOD/SCID/γc− mice advised with sorted 19-28z/PD-1 iCAR-P T cells. Basic mice (no T cells) were acclimated as control. (B) Bump accountability for anniversary abrasion was quantified, and boilerplate absolute alteration per accession is shown. (C) Annoyance weight of mice from (A) sacrificed at day 21. Anniversary dot represents one almsman mouse. (D) Breeze cytometric appraisal of the femur cartilage bottom from (C) for the attendance of bump beef (CD19 GFP ) and T beef (CD19−19-28z/GFP CD4 CD8 ). 19-28z announcement was adjourned by staining for LNGFR receptor whose commutual DNA (cDNA) is affiliated to 19-28z and is acclimated as a apprehension marker. (E and F) Absolute numbers of bump beef (E) and of CD19−19-28z/GFP CD4 CD8 T beef (F) in the spleens from (C) were quantified by breeze cytometry with CountBright chaplet (n = 4). Error bedfast represent ±SEM. **P < 0.01, ***P < 0.001 by Student’s t test.
An adorable aspect for the analytic account of iCARs is anatomic reversibility, that is, the reemergence of T corpuscle functionality afterwards antecedent acquaintance with an inhibitory astray tissue. Basal announcement of the iCARs did not blemish the T cells’ proliferative accommodation (post-CD3/CD28 bean or DC activation), cytokine secretion, or apparent brand announcement compared to ascendancy T beef (fig. S8, A to E). To appraise the banausic appearance of iCAR-medicated inhibition, we set up afterwards T corpuscle dispatch by ambition and astray cells, allegory the abeyant for killing, cytokine secretion, and admeasurement in four altered sequences. 19-28z/Pdel or 19-28z/PD-1–iCAR-P T beef were apparent to either the ambition (CD19 ) or astray (CD19 PSMA ) AAPCs as a aboriginal stimulation, followed by acknowledgment to either AAPCs in a additional dispatch (Fig. 7A). On the additional stimulation, both T corpuscle groups dead ambition beef appropriately able-bodied irrespective of the aboriginal dispatch ambition (Fig. 7B), acknowledging that the 19-28z/iCAR-P T beef apparent to astray beef in the aboriginal dispatch dead ambition beef and proliferated during the additional dispatch as able-bodied as the T beef that were apparent to ambition beef in both stimulations. Ascendancy T beef cogent the 19-28z/Pdel did not appearance bargain functionality beneath the aforementioned conditions. Additionally, T beef that were activated on the aboriginal dispatch could still be inhibited aloft acknowledgment to the iCAR ligand presented by the astray AAPCs on the additional stimulation, suggesting that iCARs could adapt an activated T cell. We additionally empiric that T beef apparent to astray beef on both stimulations had greater inhibition of their killing accommodation on the additional acknowledgment (Fig. 7, C and D).
(A) 19-28z/Pdel or 19-28z/PD-1 iCAR-P T beef were incubated with ambition (T) or astray (O) AAPCs for the aboriginal stimulation. Afterwards 3 or 7 days, the beef from anniversary accession were restimulated with either ambition [T→T (1) or O→T (2)] or astray [T→O (3) or O→O (4)] AAPCs in a bisect address to appraisal the furnishings of the aboriginal dispatch on afterwards T corpuscle function. (B) Killing of ambition (T) or astray (O) AAPCs at 24 hours afterwards evolution with anniversary T corpuscle accession (second stimulation) was analyzed with the Bright-Glo appraisal arrangement (n = 3 for anniversary condition). (C) Beard of effector cytokines in the corpuscle adeptness afloat from (B) was analyzed 24 hours afterwards the additional stimulation, and interferon-γ (IFN-γ) is apparent as a adumbrative aftereffect (n = 3 for anniversary condition). (D) T corpuscle admeasurement at day 7 afterwards the additional dispatch (n = 3 for anniversary condition). Error bedfast represent ±SEM. Statistical allegory was performed aural anniversary action (that is, T→T Pdel adjoin PD-1 iCAR-P). ***P < 0.001 by Student’s t test.
Corroborating these anatomic findings, we begin that the PD-1 iCAR, 19-28z/Pdel, and 19-28z/PD-1–iCAR-P double-positive T beef differentially phosphorylated the authoritative SHP-1 and SHP-2 phosphatases (fig. S9). Acknowledgment to CD19 ambition AAPCs showed lower SHP-1 and SHP-2 phosphorylation levels compared to the basal levels apparent afterwards acknowledgment to AAPCs defective CD19, constant with antecedent studies demonstrating dephosphorylation and consistent barricade of the suppressive furnishings of SHP-1/2 aloft T corpuscle activation (39, 40). In contrast, afterwards acknowledgment to astray AAPCs cogent CD19 and PSMA, SHP-1 and SHP-2 showed added levels of phosphorylation, acknowledging that the PD-1 iCAR recruits the aforementioned biochemical pathways as the autogenous PD-1 molecule.
Finally, we accustomed to appraise whether T beef cogent the PD-1–based iCAR could appraisal amid ambition beef in vitro and abnormally in vivo by attention astray beef in the attendance of ambition beef aural the aforementioned organism. We aboriginal addressed this book in an in vitro coculture arrangement bond GFP CD19 ambition AAPCs and mCherry CD19 PSMA astray AAPCs at a 1:1 ratio. We performed time-lapse microscopy to appraisal the aftereffect of 19-28z/Pdel or 19-28z/iCAR-P T cells. Both the ambition and the astray beef were abandoned at a agnate amount by 19-28z/Pdel T cells, but the 19-28z/iCAR-P T beef preferentially abandoned the ambition beef while sparing the astray beef (Fig. 8A and cine S1, A and B). Bisect abstracts with CBL-transduced versions of the AAPCs were acclimated to quantify this selectivity. At 38 hours, the 19-28z/iCAR-P T beef abandoned best (85%) of the ambition AAPCs but few (10%) of the astray cells, acceptance the after-effects from the time-lapse microscopy (Fig. 8B).
(A) 19-28z/Pdel or 19-28z/PD-1 iCAR-P T beef were incubated with a 1:1 mix of ambition (CD19 GFP , green) and astray (CD19 PSMA mCherry , red) AAPCs, and time-lapse microscopy was acclimated to anticipate real-time killing of anniversary citizenry for 38 hours. Adumbrative images are shown, and feature movies are accessible in cine S1 (A and B). Scale bars, 0.1 mm. (B) As in (A), 19-28z/Pdel or 19-28z/PD-1 iCAR-P T beef were incubated with a 1:1 mix of ambition (CD19 ) and astray (CD19 PSMA ) AAPCs. Killing of anniversary AAPC citizenry was adjourned in alongside abstracts area one of anniversary AAPC blazon was labeled with CBL (CD19 CBL /CD19 PSMA mix or CD19 /CD19 PSMA CBL mix). Killing was quantified with the Bright-Glo appraisal arrangement at 38 hours (n = 3 for anniversary condition). (C to E) NOD/SCID/γc− mice were injected with a 1:1 admixture of NALM/6 and NALM/6-PSMA beef and advised with 19-28z or 19-28z/PD-1 iCAR-P T cells. (C) Aloft sacrifice, the attendance of the ambition and astray NALM/6 beef in the cartilage bottom was analyzed by breeze cytometry. (D) Arrangement of target/off-target NALM/6 beef in the cartilage bottom of sacrificed mice was quantified by breeze cytometry. (E) Annoyance weight of advised mice was additionally recorded at sacrifice. Error bedfast represent ±SEM. ***P < 0.001 by Student’s t test.
To appraisal whether the aforementioned selectivity could be accomplished in vivo, we injected NOD/SCID/γc− mice with a admixture of NALM/6 and NALM/6-PSMA bump beef and advised these animals with 19-28z– or 19-28z/iCAR-P–transduced T cells. Aloft sacrifice, the mice advised with 19-28z T beef showed a threefold abridgement in the cardinal of PSMA beef in the annoyance and cartilage bottom compared to mice advised with 19-28z/iCAR-P T beef (Fig. 8, C and D). Accordingly, the iCAR-treated accession had a 3.3-fold access in annoyance weight and all-embracing added bump accountability (Fig. 8E). These abstracts authenticate that, in the attendance of a admixture of ambition and astray cells, an iCAR can selectively assure astray beef afterwards abrogating bounce of ambition cells.
Here, we booty a abiogenetic access to bind the specificity of T beef and authenticate that T beef can be engineered to accept an autogenous authoritative targeting apparatus to bear tumor-specific immunotherapy. We auspiciously accumulated an antigen acceptance area with the signaling domains of accustomed inhibitory receptors CTLA-4 and PD-1 to accomplish antigen-specific aishment of T corpuscle cytotoxicity, cytokine release, and proliferation. This proof-of-concept abstraction demonstrates the abeyant for iCARs as a action to absolute T corpuscle action at astray sites and appropriately alter accustomed responses abroad from adventitious ambition tissues.
The body of the iCAR action relies on three analytical properties. The aboriginal is that basal announcement of the iCAR does not arrest T corpuscle action in the absence of antigen. Autogenous CTLA-4 or PD-1 signaling requires the attendance of the corresponding ligands to apply their effect. Likewise, we did not acquisition announcement of the iCARs declared herein to affect basal T corpuscle functions. Added inhibitory receptors that are belted to T corpuscle subsets may act in concert to fine-tune the adjustment of T corpuscle responses (21, 22). Receptors such as LAG-3, 2B4, and BTLA and their aggregate (for example, as a distinct second-generation iCAR with assorted accumulated cytoplasmic domains) accreditation added investigation.
The additional key acreage is the aliment of T corpuscle functionality admitting antecedent assurance of the iCAR. We begin that iCAR-transduced T beef could still arise a acknowledgment adjoin a ambition antigen afterwards a antecedent acknowledgment to an inhibitory antigen. This reversibility is evocative of accustomed analgesic corpuscle behavior, in which the phosphorylation accompaniment of signaling molecules rather than transcriptional changes ascendancy accelerated anatomic responses, such as cytotoxicity (41). Anti–PD-1 and anti–CTLA-4 antibodies are able to about-face the broken action of anergized or beat T cells, afresh arguing for the adeptness to temporally adapt T corpuscle responses (22). Additionally, biochemical analyses of PD-1 and CTLA-4 furnishings on the TCR circuitous depend on phosphorylation states, afterwards kinases, and activity rather than apoptosis (40, 42–44). Although both our in vitro and in vivo after-effects authenticate inhibition in acknowledgment to astray beef with abiding ameliorative functionality, there is still the achievability that some of the beef may be anergized over time (42). Ultimately, a T corpuscle beverage is stochastic, with some T beef promptly encountering their ambition and eliminating it, admitting added T beef will aboriginal appointment the inhibitory cells. It is believable that T beef that again appointment astray beef will not expand—a satisfactory aftereffect for the iCAR strategy, which aims to acquiesce for ameliorative responses to advance while abbreviating the accustomed advance adjoin accustomed tissues. The all-embracing amplification of the alloyed T corpuscle citizenry will accommodate these altered paths occurring at the clonal level, with some T beef adeptness amplification while others are suppressed, possibly consistent in the dematerialization of all alloyed T beef over time. Beneath our beginning conditions, abundant T beef persisted over 3 weeks to annihilate the targeted tumor. Beneath such a circumstance, a additional or third T corpuscle beverage could be alloyed if needed, which may be clinically advantageous as discussed abroad (9). The closing consecration of anergy and clonal aishment as a agency to assure astray tissues while acceptance bump aishment to advance should be assorted to suicide gene strategies area adverse acuteness charge apparent itself afore T corpuscle aishment is triggered, which after-effects in absolute ameliorative responses as well.
Third, the iCAR access is antigen-specific and appropriately requires the adeptness to analyze tissue-specific ambition antigens that are absent or down-regulated on the bump but bidding by the astray tissues. This catechism has not been as broadly advised as the chase for bump antigens, although efforts, such as the Protein Atlas database, are beneath way to characterize the “surfaceome” of all animal tissues (45). One action is to use ample classes of apparent antigens that are down-regulated on bump cells. One archetype is represented by animal corpuscle antigen (HLA) molecules, which are begin in around all corpuscle types, but are down-regulated on tumors as a apparatus of bump escape from T corpuscle accustomed responses (46). Thus, allogeneic T beef cogent an iCAR adjoin a host HLA atom that is down-regulated on the bump may selectively advance the GVT effect. The iCAR access may be of accurate absorption in the ambience of DLI as a agency to assure GVHD ambition tissues afterwards impairing GVT responses. Addition chic of antigens affable to a agnate action includes corpuscle apparent bump suppressor antigens, such as OPCML, HYAL2, DCC, and SMAR1 (47–49). OPCML-v1, for example, is broadly bidding in all accustomed developed and fetal tissues but is down-regulated in lymphomas and and prostate cancer. Corpuscle apparent carbohydrates, lipids, and posttranslational modifications, such as mucin-type O-glycans (core 3 O-glycans) accept additionally been begin to be down-regulated by tumors (50). Addition applicant ambition is E-cadherin, which is awful bidding in accustomed skin, liver, and gut—the primary targets of GVHD (51)—but down-regulated by bump beef adeptness an epithelial to mesenchymal transition, advertence bump progression and alteration (52).
A above limitation of our abstraction is the abridgement of availability of a able-bodied clinically accordant animal “normal tissue” model, abnormally one that allows appliance of animal cells, animal antigens, and animal TCRs, CARs, and iCARs. We attempted to arch this gap by establishing iPS beef accumulated with DCs from the aforementioned donor to access an alloreactivity acknowledgment application animal T cells, animal ambition antigens, and animal iCARs. Simply co-incubating HLA altered allogeneic T beef with iPS or iPS-fib beef did not aftermath alloreactivity. The use of isogenic DCs was analytical to breeding almighty alloreactivity. However, we did not ascertain the attributes of this alloreactivity, and it is appropriately accessible that the responses we blocked accept no address on the mechanisms complex in GVHD.
We additionally showed that the akin of announcement of the iCARs is critical. In settings of aerial announcement akin of activating receptor or antigen and/or low announcement of iCAR or iCAR-targeted antigen, we could not accomplish acceptable blockade. In best of our analyses, the iCAR bargain T corpuscle action but did not abate it, rarely beyond 90% inhibition in any assay. In applying the iCAR action in a analytic setting, the functionality of every iCAR will charge to be optimized on the base of receptor affinity, receptor announcement akin (that is, apostle strength), and alternative of acceptable ambition antigens based, in part, on their announcement level. These will additionally charge to be counterbalanced adjoin the activating receptor to accomplish inhibition at astray sites. In the case of CAR-targeted therapy, an optimized CAR/iCAR arrangement could be accomplished through accurate agent design.
In conclusion, we accommodate a affidavit of abstraction that antigen-specific inhibitory receptors can auspiciously alter T corpuscle proliferation, cytokine secretion, and cytotoxicity aloft assurance of specific corpuscle apparent antigens, appropriately breach T corpuscle toxicity abroad from one tissue while application analytical effector action adjoin addition cogent the aforementioned antigen. We showed this in responses advised by either TCRs or CARs. This access prevents, or at atomic reduces, accident to adventitious ambition tissues and appropriately obviates the charge to irreversibly annihilate ameliorative T beef afterwards unacceptable toxicity has developed. It is a paradigm-shifting access that takes advantage of the able functionality of beef as drugs by application constructed receptors that adviser and brainwash T beef to abandoned accomplish benign functions. This activating assurance about-face may acquisition advantageous applications in a ambit of autologous and allogeneic T corpuscle therapies.
The purpose of this abstraction was to actualize a constructed receptor that could absolute T corpuscle toxicity adjoin a ambition corpuscle in an antigen-dependent and capricious manner. We advised two such receptors application intracellular cape of CTLA-4 or PD-1 and an scFv targeting area (against PSMA) and analyzed whether they could block (i) TCR- or (ii) CAR-driven T corpuscle functionality in vitro and in vivo. In vitro, the focus was on allegory (i) cytotoxicity, (ii) cytokine secretion, and (iii) T corpuscle proliferation. In vivo abstracts analyzed the chip adeptness of the iCAR to assure a cellular ambition application alive imaging and endpoint appraisal (dictated by the basic accession of mice). The beginning procedures were accustomed by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Blight Center (MSKCC). The accepted architecture of the abstracts was to betrayal T beef (expressing iCARs or the ascendancy Pdel receptor) to ambition beef (that bidding or lacked PSMA) and analyze the groups aggravating to catechize iCAR function, consistently in the attendance of centralized controls. We apprenticed T beef defective iCARs from communicable the after-effects by allocation T beef to be iCAR or iCAR/CAR double-positive (using anchorman genes). Anniversary agreement was performed assorted times application altered donor T beef (T beef were never pooled). In best cases, we present abstracts application a adumbrative agreement (with sample replicates of added than three) to abstain abashing variables such as differences due to transduction and allocation efficiencies, donor-related variability, and E/T ratios.
Each iCAR receptor was advised with the UniProt arrangement comment application two approaches. First, application bartering gene amalgam or cDNAs, the intracellular area of anniversary receptor was cloned in abode of the CD28/CD3ζ area of the ahead declared Pz1 receptor (53), appropriately application the CD8 transmembrane and articulation domains. Alternatively, we included the transmembrane domains and the amino acids up to the aboriginal annotated extracellular topological area (for PD-1, amino acids 145 to 288; for CTLA-4, amino acids 161 to 223) so as to use the autogenous articulation arena of anniversary receptor. These constructs were cloned into the P28z agent afterwards the PSMA scFv. We did not acquisition cogent anatomic differences amid the receptors generated by the two approaches. We additionally created versions of anniversary iCAR that lacked any targeting domain, but retained the transmembrane and intracellular portions of anniversary receptor. The ascendancy Pdel receptor was advised by excising the CD28/CD3ζ area of P-28z (34). iCARs should be acutely acclaimed from CARs, all of which activate T corpuscle activation, in abrupt adverse to iCARs.
Cell apparent announcement of anniversary iCAR was analyzed application the ahead declared dupe anti-mouse staining agreement (38). Cellular alliance appraisal was performed as ahead declared (54). Briefly, EL4 or EL4-PSMA beef were labeled with the lipophilic DiD dye (Invitrogen) and alloyed at a 1:1 arrangement with T beef in fluorescence-activated corpuscle allocation (FACS) tubes, incubated at 37°C for 5 min, and analyzed on a breeze cytometer. Western blemish appraisal was performed application accepted protocols with a Bio-Rad Mini-PROTEAN Tetra system. Intracellular appendage of CTLA-4 was detected application the polyclonal antibiotic C-19, which recognizes the CTLA-4 C aals (Santa Cruz Biotechnology).
Plasmids encoding the SFG retroviral agent were able application accepted atomic appraisal techniques. Amalgam of 19-28z–IRES–LNGFR, CD19, PSMA, GFP, mCherry, and CBL vectors has been declared (37, 38, 53). Retroviral producers were able from plasmid-transfected H29 corpuscle afloat as ahead declared (53).
EL4-CD19, EL4-PSMA, and the AAPCs NIH3T3-CD19 and NIH3T3-PSMA accept been declared (31, 34, 37, 38, 53). NIH3T3-CD19-PSMA, NIH3T3-CD19-mCherry, NIH3T3-CD19-GFP, and NIH3T3-CD19-CBL, as able-bodied as NALM/6-CBL and NALM/6-PSMA-CBL, were acquired afterwards transduction with corresponding retroviral supernatants of H29 ambassador cells. All allusive groups of corpuscle curve were sorted for agnate announcement of CD19, GFP, or mCherry with a MoFlo sorter.
Peripheral claret was acquired from advantageous donors afterwards a accord beneath a agreement accustomed by the MSKCC institutional appraisal board. Borderline claret leukocytes were abandoned with Ficoll-Paque and activated with phytohemagglutinin (PHA) for 48 hours. Activated T beef were transduced on three afterwards canicule by centrifugation in retronectin-coated (Takara), retroviral vector–bound plates. Beef were fed every 3 canicule with RPMI average supplemented with 20 U of interleukin-2 (IL-2). Ten canicule afterwards transduction, FACS alternative based on added GFP (marking the iCARs) and LNGFR (marking 19-28z) was acclimated to abstract absolute beef on a MoFlo sorter. Post-sort appraisal was performed to ensure agnate announcement of both reporters.
Peripheral claret lymphocytes were activated with PHA, transduced with retroviral supernatants (f-citrine-P2A-Myc-E2A-Sox2 and f-vexGFP-P2A-Okt4-T2A-Klf4), and argent afterwards 24 hours on abrasion beginning fibroblast agriculturalist beef (55). Average was afflicted to animal ES average with fibroblast advance agency (8 ng/ml) at day 5 afterwards transduction, and half-medium change was performed circadian afterwards that. T-iPS colonies appeared at about 22 to 25 canicule afterwards transduction. A subcutaneous xenograft teratoma appraisal was performed with the T-iPS-1.10 corpuscle line. At 3 months, the teratoma was removed and advised with collagenase (100 U/ml) (Invitrogen) and dispase (2 U/ml) (Invitrogen) for 2 hours at 37°C to accomplish a single-cell suspension. The beef were sorted for HLA-ABC–positive cells, and afterwards 1 anniversary in adeptness in RPMI supplemented with 1% l-glutamine, 1% penicillin, 1% streptomycin, and 10% fetal bovine serum (FBS), they reproducibly spontaneously generated the iPS-fib.
All breeze cytometric appraisal was done on an LSRII cytometer (BD Biosciences) and analyzed with FlowJo software adaptation 9.6 (Tree Star). Anti-human LNGFR, CD45, CD140b, CD10, HLA-ABC, HLA-DR, CD80, CD86, and CD62L were acquired from BD Biosciences; anti-human CD4, CD8, CD3, CD19, CD90, and 4′,6-diamidino-2-phenylindole (DAPI) were acquired from Invitrogen; anti-human PSMA was acquired from Medical & Biological Laboratories; anti-human CCR7 was acquired from R&D Systems; and anti-human Foxp3 (236A/E7) and Foxp3 isotype were acquired from eBioscience.
In general, for proliferation, effector cytokine assembly assays, and cytotoxicity assays, consecutive dilutions of sort-purified T beef were seeded on corresponding AAPCs (irradiated with 40 to 50 Gy and seeded 24 hours beforehand at 3 × 104 per well) in 96-well flat-bottomed plates. iPS-fib were not ablaze back acclimated as targets. Fresh average was added every 3 to 4 canicule or aloft average blush change. Cytokine assembly was quantified by either enzyme-linked immunosorbent appraisal kits (eBioscience) or Luminex assays (Invitrogen) as declared in the argument according to the manufacturer’s instructions. T corpuscle counts were affected application applicable corpuscle cardinal (DAPI) and CountBright chaplet (Invitrogen) on an LSR II breeze cytometer (BD Biosciences) by accession accomplished wells. All in vitro adeptness abstracts were done in RPMI supplemented with 1% l-glutamine, 1% penicillin, 1% streptomycin, and 10% FBS. No exogenous cytokines were administered at any time unless absolutely stated.
Cytotoxic T lymphocyte (CTL) assays application bioluminescence as the readout were performed as ahead declared (36). Briefly, all in vitro luciferase assays were performed with the Bright-Glo Luciferase Appraisal Arrangement (Promega) and 96-well Optical Bottom Black Microplates (Nunc) and were conducted according to the manufacturer’s agreement with accessory adjustments. All targets beef were engineered to accurate CBL with a GFP anchorman to ensure agnate levels of expression. Adeptness average was removed to leave 50 μl per well, 50 μl of able luciferase reagent was added to anniversary able-bodied of the 96-well plates, and the plates were incubated for 5 min to absolutely lyse the cells. Measurements were performed with the IVIS Imaging Arrangement 100 Series (Xenogen). Living Angel software adaptation 2.6 (Xenogen) was acclimated to quantify photon discharge intensities.
All microscopy imaging was performed with a Zeiss AxioVert 200M able with a alive imaging system. Time-lapse videos were acquired and aggregate with Multi Dimensional Acquisition in MetaMorph software (Molecular Devices). For CTL experiments, the arresting from mCherry-positive AAPCs was quantitated with the Chip Morphometric Appraisal action in MetaMorph.
Monocyte-derived DCs were generated with the Mo-DC Generation Tool Box (Miltenyi) from the aforementioned donor as the T-iPS cells. The moDCs were pulsed for 24 hours at the adolescent date (days 5 to 6) with lysates from iPS-fib, which were generated through six freeze-thaw cycles. The maturation of the DCs was accepted by breeze cytometry of CD80, CD86, and HLA-DR. Conference was performed as ahead declared (56). Briefly, the aboriginal annular of conference was done application a 1:30 T cell/moDC ratio, with the additional annular application a 1:10 to 1:30 ratio. RPMI supplemented with 1% l-glutamine, 1% penicillin, 1% streptomycin, 10% animal AB serum (CellGro), and animal IL-15 (5 ng/ml) (R&D Systems) was used. On day 3, IL-2 (20 U/ml) was added.
T beef were apparent to AAPCs at an E/T arrangement of 4:1 for 60 min, washed, lysed, and incubated (100 μg) on the Animal Phospho-Immunoreceptor Arrangement according to the manufacturer’s agreement (R&D Systems). All blots were detected application chemiluminescence on the aforementioned x-ray blur to assimilate acknowledgment levels. Scanned x-ray blur images were analyzed with angel appraisal software. All pixel body was normalized on anniversary arrangement with centralized pY controls.
For the NALM/6 studies, 6- to 12-week-old macho NOD/SCID/γc− mice (The Jackson Laboratory) were inoculated intravenously with 5 × 105 bump beef (same dosage for either distinct bump or alloyed bump experiments). NALM/6 beef were engineered to accurate CBL with a GFP reporter. Four canicule later, 3 × 105 sorted T beef were alloyed intravenously; corpuscle dosage was based on the percent GFP 19-28z as accepted by post-sort analysis. Mice were sacrificed at 21 canicule (no T corpuscle controls affectation hindlimb paralysis). For iPS-fib studies, 6- to 12-week-old macho NOD/SCID/γc (null) mice were inoculated intraperitoneally with 1 × 106 beef able in a 1:1 admixture of ice algid RPMI and Matrigel admixture (BD Biosciences). Eight canicule later, 5 × 105 alert moDC-primed GFP-sorted T beef were alloyed intraperitoneally; corpuscle dosage was based on the percent GFP as accepted by post-sort analysis. Additionally, an in vitro luciferase CTL appraisal was performed to authorize agnate allogeneic acuteness in all groups application iPS-fib as a target. In both models, d-luciferin (Xenogen, 150 mg/kg intraperitoneally) was acclimated as a substrate for bang beetle luciferase, and bioluminescence images were calm on an IVIS 100 Imaging System. Living Angel software adaptation 2.6 was acclimated to access and quantify the BLI abstracts sets as declared afore (38). Mice were cared for in accordance with the institutional guidelines of MSKCC.
Data are presented as agency ± SD or SEM as declared in the amount legends. After-effects were analyzed by unpaired Student’s t appraisal (two-tailed) or by ANOVA as declared in the text, and statistical acceptation was authentic at P < 0.05. Pairwise assorted comparisons were performed application assorted t tests adapted for assorted comparisons with the Holm-Sidak method. All exact P ethics are provided. All statistical analyses were done with Prism software adaptation 6.0 (GraphPad).
Fig. S1. CTLA-4 iCAR corpuscle apparent announcement is added afterwards T corpuscle activation.
Fig. S2. iCAR-P bind to PSMA-expressing cells.
Fig. S3. Allogeneic acuteness archetypal application iPS-derived fibroblasts and isogenic moDCs.
Fig. S4. Almighty acuteness of iCAR-transduced primary animal T beef adjoin allogeneic iPS-derived fibroblasts.
Fig. S5. Transduction and allocation action of iCAR or 19-28z/iCAR T cells.
Fig. S6. Allocation action of low/high iCAR–expressing T beef and PSMA-expressing iPS fibroblasts.
Fig. S7. iCARs arrest 19-28z–driven animal T corpuscle cytokine absolution and proliferation.
Fig. S8. Basal announcement of iCARs does not affect action of primary animal T cells.
Fig. S9. Signaling and biochemical alleyway assuming of the PD-1 iCAR.
Movie S1. iCAR- and CAR-expressing T beef anticipate targets in vitro.
Table S1. Raw abstracts and statistical acceptation testing for Fig. 2.
Table S2. Raw abstracts and statistical acceptation testing for Fig. 3.
Table S3. Raw abstracts and statistical acceptation testing for Fig. 4.
Table S4. Raw abstracts and statistical acceptation testing for Fig. 5.
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