Abstract
Background
T-cell longevity is undermined by antigen-driven differentiation programs that render cells prone to attrition through several mechanisms. CD8 + T cells that express the Tcf-1 transcription factor have undergone limited differentiation and exhibit stem-cell-like replenishment functions that facilitate persistence. We engineered human CD8 + T cells to constitutively express Tcf-1 and a TCR specific for the NY-ESO-1 cancer-associated antigen. Co-engineered cells were assessed for their potential for adoptive cellular immunotherapy.
Methods
Tcf-1 mRNA encoding TCF-1B and TCF-1E isoforms, along with GzmB expression were assessed in CD62L + CD57 −, CD62L − CD57 −, and CD62L − CD57 + CD8 + T cells derived from normal donor lymphocytes. The impact of stable Tcf-1B expression on CD8 + T-cell phenotype, anti-tumor activity, and cell-cycle activity was assessed in vitro and in an in vivo tumor xenograft model.
Results
TCF-1B and TCF-1E were dynamically regulated during self-renewal, with progeny of recently activated naïve T cells more enriched for TCF-1B mRNA. Constitutive TCF-1B expression improved the survival of TCR-engineered CD8 + T cells upon engagement with tumor cells. Tcf-1B prohibited the acquisition of a GzmB High state, and protected T cells from apoptosis associated with elicitation of effector function, and promoted stem cell-like characteristics.
Conclusions
Tcf-1 protects TCR-engineered CD8 + T cells from activation induced cell death by restricting GzmB expression. Our study presents constitutive Tcf-1B expression as a potential means to impart therapeutic T cells with attributes of persistence for durable anti-tumor activity.
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Background
In order for adoptively transferred CD8+ T cells to mediate durable regression of advanced cancers, they must have sufficient longevity; as not all CD8+ T-cell differentiation subsets are capable of long-term persistence as cell-therapy T cells [1,2,3,4]. Naïve CD8+ T cells exist in a quiescent state in which epigenetic programs imprinted by thymopoiesis restrain activation and potentiate both persistence and readiness for entry into cycling states required for the propagation of large numbers of clonal cells. With activation, naïve CD8+ T-cells undergo successive rounds of cell-division to propagate memory T cells with stem-cell-like replenishment functions as well as more differentiated effector cells which are short-lived and function to destroy neoplastic targets via the mobilization of granzymes and IFN-γ. Progressive differentiation through naïve, memory, and effector programs is facilitated by the silencing the locus of T Cell Factor 1 (Tcf-1), a master regulator of T-cell stemness [5, 6]. CD8+ T cells in the Tcf-1+ stages of differentiation have advanced anti-tumor activity in cellular immunotherapies; Tcf-1-enriched subsets out-persist and out-perform Tcf-1-low/negative subsets in adoptive cell transfer (ACT) [7]. Tcf-1 expression is heterogenous among tumor-infiltrating lymphocytes, and further limited in peripheral blood T cells available for manufacture into CAR or TCR-gene engineered cells [8, 9]. Cell-therapy T cells that do express Tcf-1 are susceptible to Tcf-1 silencing in vivo, and a consequential loss of cell persistence [10]. A natural question is whether genetic approaches to enforce Tcf-1-mediated programs could prolong the persistence of TCR-engineered CD8+ T cells.
TCRs harnessed from naturally occurring tumor-specific T cells allow engineered cell-therapy T cells efficacy against advanced cancers [11], via recognition of HLA-complexed peptide antigenic targets and the transduction of signals that direct cytolytic activity and expansion over attrition. Antigen recognition is understood to impose significant pressure on CD8+ T cells. For example, tumor-reactive T cells infiltrating melanoma tumors express Tcf-1 at reduced levels relative to bystander cells lacking tumor specificity [8]. Experiments with TCR-transgenic murine T cells have revealed the potential for differentiation and dysfunction to occur as a result of tumor-recognition rather than just as a result of effects of the tumor microenvironment [12]. With TCR cross-linking, CD3-directed signal transduction cascades elicit activated states which sustain granzyme synthesis [13]. Low intracellular granzyme content is an attribute of stemness and CD8+ T cells acquire peak granzyme B (GzmB) expression and cytotoxicity via serial antigen encounter [14]. Granzymes predispose T cells to apoptosis via their leakage from lysosomal granules to the cytoplasm and subsequent cleavage of caspase-3 or disruption of mitochondrial integrity [15,16,17,18,19]. The propensity for granzymes to limit cell longevity is underlined by the finding that GzmB−/− CD8+ T-cells have enhanced anti-leukemia activity in pre-clinical models as a result of their being better able to persist to use IFN-γ and FAS-ligand to neutralize cancer cells [17]. Epigenetic regulation of granzyme expression could be a critical determinant of CD8+ T-cell longevity. The GzmB locus of Tcf-1-expressing stem-cell-like CD8+ T-cells is inaccessible and further regulated in memory T cells relative to effector cells [20].
In order to mitigate T cell death and enhance anti-tumor activity, T cells transferred into patients may be supported with exogenous IL-2, but off-target activation of self-directed CD8+ T-cells and regulatory CD4+ T-cells limit the efficacy of IL-2 and there is a recognized need for alternative methods to support cells in vivo [21]. T cell subsets differ in IL-2 receptivity [22], production [23], and sensitivity to deprivation [24]. The potential for a relationship between differentiation subsets and IL-2 requirements compel inquiry into whether Tcf-1 sustains viability in contexts where exogenous IL-2 is not provided. Further compelling is the question of whether Tcf-1 influences cell-cycle activity supported by IL-2.
Herein, we examined the kinetics of Tcf-1 expression in relation to primary activation and found that activation resulted in upregulation of a specific isoform of Tcf-1; TCF-1B. We assessed the impact of constitutive TCF-1B expression on the functions of human CD8+ T-cells also engineered to express a therapeutic TCR specific to NY-ESO-1, a particularly immunogenic and widely expressed cancer-associated-antigen. Tcf-1 transgenic T-cells demonstrated resistance to apoptosis associated with the elicitation of cytotoxic functions, modestly enhanced survival in the absence of IL-2 support, and stem-cell-like cycling characteristics of early lineage T-cells. These findings elucidate mechanisms by which Tcf-1-expressing CD8+ T-cells persist and implicate Tcf-1 overexpression as a potential strategy for imparting cells with Tcf-1-programmed attributes for adoptive cellular immunotherapy.
Materials and methods
Flow cytometry
To detect Tcf-1, surface-marker labelled cells were fixed and permeabilized with True Nuclear Transcription Factor Buffer Set (BioLegend #424,401) and labelled intracellularly with αTcf-1 clone C63D9 (Cell Signaling Technologies #6709). LSR-II and Fortessa cytometers were used. In experiments detecting apoptosis, cells were labelled for fifteen minutes at room temperature with Annexin-V:APC and Zombie UV viability dye in annexin-binding buffer(Biolegend #640,919)(Biolegend #42,307). Data were analyzed with BD Biosciences Flowjo software. Information regarding all antibodies can be found in the Supplemental Methods.
Quantitative PCR
RNA was extracted using phenol–chloroform extraction and converted to cDNA with Iscript Reverse Transcription Supermix(BioRad #1,708,840). cDNA was assayed by real-time PCR with custom primers from Integrated DNA Technologies and iQSYBR Green Supermix(BioRad #1,708,880), using a BioRadCFX96-Touch-Detection-System: (i) 3-min at 95 °C (ii) 15-s at 94 °C, detection (iii) annealing and extension for 1-min (iv) repeat ii-iii 39 times. Data were analyzed with BioRad CFX Manager. Expression was evaluated using Ribosomal Protein L4 (RPL4) as a control gene. Relative expression values (y) used for comparison were generated by subtracting the Cq of RPL4 from the Experimental Cq and log-transforming the difference with y = 2−ΔCq. Primer information and annealing temperatures are found in Supplemental Methods.
Cloning TCF-1-GFP transgenes
cDNA from Jurkat cells was used as a template for PCR to generate amplicons for assembly. Primers were designed for TCF-1B (NM_003202.5) (NP_003193.2). dnTCF-1B (NM_201632.5)(NP_963963.1) was amplified from exons 2–10 of TCF-1B. TCF-1E (EAW62279.1) was generated by assembly of exons 2–8 with a gBlockTm gene fragment for exons 9–10. Detailed methods are in Supplemental Methods.
Tcf-1 and TCR transduction
A high-affinity NY-ESO-1 specific HLA-A*02-restricted TCR gene was transduced, 19305DP (19,305-TCR) [25]. CD8+ T cells were isolated from cryopreserved healthy-donor blood mononuclear cells (PBMC) using a Naive CD8 + T-cell Isolation-Kit (Miltenyi Biotec 130–093-244). The isolated cells (2.0 × 106 cells/well) < space removed were plated in complete RPMI supplemented with recombinant human IL-2* 300 IU/ml (Peprotech 200–02) and anti-CD3/CD28 dynabeads (1:3 to 1:1, bead:cell) (Thermo Fisher Scientific 11161D) in 6-well culture plates. After 48 h, T cells were harvested and seeded onto plates coated with recombinant retrovirus from PG13 supernatant. After transduction, T cells were de-adhered from dynabeads and re-suspended at (0.5–0.7 × 106/ml) in complete RPMI with IL-2. *Media supplemented with IL-2, IL-7, and IL-15 (10 ng/ml, Peprotech 200–02, 200–07, 200–15) was used for transduction of PBMC and naive CD8+ T-cells.
PBMC GzmB content and Cytokine production
PBMC were transduced and expanded 9-days and stained to detect GzmB (BD Bioscience 560,213). PBMC were expanded an additional 9-days with media supplemented with solubilized anti-CD3 antibodies (OKT3) (50 ng/ml) and IL-2 (300 IU/ml). PBMC were re-stimulated with Cell Activation Cocktail with BFA for detection of IL-2 and IFN-γ (Biolegend).
In vivo Anti-tumor Efficacy
NSG mice (NOD.Cg-PrkdcScid IL2rgTm1wjl/SzJ Jackson Laboratory #005,557) were used. HLA-A*02:01+ NY-ESO-1+ SK-MEL-37 melanoma cells (1 × 106) were injected subcutaneously into the left rear flank. Mice with tumors that reached 40 mm3 were injected intravenously with human CD8+ T cells (0.75 × 106) transduced to express either [19305-TCR and GFP] or [19305-TCR and TCF-1B-GFP]. IL-2 was delivered via intraperitoneal injection on the day of transfer and 24- and 48-h after (50,000 IU GoldBio IL-2). Tumors were measured with calipers, measuring the longest dimension (L) and perpendicular width (W):Volume (mm3) = (L × W2)/2. Tumor-infiltrating lymphocytes were extracted from tumors via enzymatic digestion with type IV-collagenase(Sigma#c5138-500 mg) and typeΙV-DNAse1(Sigma#D5025-15ku). Methods for analysis of tumor-infiltrate are in Supplemental Methods.
Cytotoxicity assays
TCR-engineered CD8+ T-cells (4 × 105 cells) were plated on SK37 (5 × 104 cells) in the 48-well format. After 24-h cells were harvested for flow-cytometry. Adherent cells were further harvested using 0.25% trypsin/EDTA solution. In the second format, used to study GzmB upregulation, T cells (2 × 105) were seeded onto SK37 (1.5 × 104) in 96 well plates. Cultures were collected after 15-h. Some cultures were supplemented with BFA at 3 µg/ml (ThermoFisherScientific#00–4506-51).
Survival and expansion co-cultures
19,305-TCR engineered CD8+ T-cells (2.0 × 105 cells) expressing GFP or Tcf-1 were suspended in 200 µL media and passaged onto SK37 (1.5 × 104) pre-seeded in the 48-well format. The media in which T cells were suspended contained either no IL-2 (survival cultures) or IL-2 at concentrations of 300 or 1500 IU/ml (expansion cultures). After 72-h, media was replenished. After 7-days, cells were collected for cytometry. Countbrite Absolute Counting beads were used to determine relative numbers of CD45+ T cells(ThermofisherScientific#C36950).
Statistical analyses
Graphpad Prism.v9 was used to assess statistical significance. Student t-tests were used to compare significance, with p-values of less than 0.05 represented by an asterisk. All graphs include error-bars which indicate one standard deviation about the mean.
Results
Dynamic regulation of Tcf-1 and GzmB in relation to CD8+ T-cell differentiation
CD8+ T-cell differentiation is tightly regulated to ensure some cells persist to fulfill progenitor roles while others function to engage in immuno-surveillance and cytotoxic activity [26]. While antigen-experience is understood to be pre-requisite for expression of GzmB, it remains unclear whether regulation of GzmB also occurs in the stages of differentiation subsequent to primary activation, in which memory T-cells with progenitor functions are maintained by Tcf-1 mediated programs [26]. Therefore, to begin our study, we examined the degree to which subsets of CD8+ T-cells in different stages of differentiation co-express Tcf-1 and GzmB. CD8+ T-cells of normal donor blood formed discrete populations that expressed Tcf-1 and GzmB at different levels (Fig. 1A, left plot). We stratified three subsets for comparison of Tcf-1 and GzmB enrichment: (i) CD62L+CD57− T-cells which include naïve and central memory T-cells expected to express Tcf-1 at peak levels (ii) CD62L−CD57− T-cells which include effector and effector-memory T cells (iii) CD62L−CD57+ T-cells which include senescent effector and effector-memory T cells (Fig. 1A, right plot). These subsets differed with respect to their enrichment for populations expressing Tcf-1 versus GzmB at peak levels (Fig. 1B): CD62L+CD57− cells were enriched for a population with peak expression of Tcf-1 and little GzmB. Senescent CD62L−CD57+ cells formed a discrete population with minimal Tcf-1 content and peak GzmB content. CD62L−CD57− cells were heterogenous, containing populations with peak Tcf-1 expression and peak GzmB content. Since GzmB was found at significantly reduced levels in CD62L− CD57− cells relative to CD62L−CD57+ cells (Fig. 1C), sub-maximal GzmB content may be an attribute of antigen-experienced T-cell subsets that express Tcf-1.
Tcf-1 is expressed as one of a number of isoforms which vary as a result of dual promoter usage and alternative splicing [27]. TCF-1B and dnTCF-1B have near-identical sequences except the latter lacks an amino-terminal domain thought to bind β-catenin. Compared to TCF-1B, TCF-1E recognizes a wider range of gene-targets via an extended C-terminus [28]. It remains unclear whether these isoforms are of variable importance to naïve versus memory stages of differentiation, and whether they differ in utility to our aims. Therefore, we characterized their expression in relation to primary activation, during which naïve T-cells propagate progeny with peak per-cell Tcf-1 protein levels in the initial rounds of cell division [5]. We isolated naïve human CD8+ T-cells and used flow-cytometry to assay Tcf-1 content before activation, after 24-h of activation, and after 72-h of activation. T cells assayed after 24-h had twice the Tcf-1 content of those assayed at baseline and were composed of a single population which had not divided (Fig. 1D). After 72-h, four generations of cells with homogenous Tcf-1 content could be observed. Tcf-1 was found at peak levels in cells that had undergone one division and was found at reduced levels in cells that had divided two to three times, but remained homogenous among cells of a given generation (Fig. 1E). The pattern of expression observed is consistent with a model where naïve cell activation results in upregulation of Tcf-1 protein levels in advance of cell-division, suggestively so sufficient Tcf-1 content exists for assortment into memory T-cells generated by successive rounds of division. This mechanism would effectively prevent cell-division from out-pacing production of Tcf-1 required for daughter cells. We used qPCR to assess changes in isoform abundance in relation to primary activation: TCF-1E-transcripts peaked in naïve cells and were found at significantly reduced levels after activation (Fig. 1F). TCF-1B-transcripts were minimally expressed in naïve cells and reached peak levels in cells activated for 72-h (Fig. 1G). These results suggest that TCF-1E primarily serves naïve cell gene-expression programs and that TCF-1B is upregulated to accommodate Tcf-1 expression in memory T cells.
Tcf-1 overexpression antagonizes human CD8+ T-cell GzmB expression
We cloned GFP transgenes for constitutive expression of TCF-1B, TCF-1E, and dnTCF-1B (Fig. 2A). We reasoned that one isoform could be advantaged over others and that the activity of each could be assessed via detection of GzmB, IFN-γ, and IL-2 after expanding cells via standard protocols. We assayed CD8+ T cell GzmB content after expanding transduced PBMC for a total of nine days, and assayed cytokine-induction with mitogens after an additional nine days of expansion with solubilized anti-CD3 antibodies and IL-2 (Fig. 2B). The transduction efficiency of each isoform was variable, with TCF-1B, dnTCF-1B, and TCF-1E expressed by 90%, 56%, and 37% of CD8+ T cells (Fig. 2C-D). Each isoform repressed GzmB, reducing levels to as little as 1/5th of that of GFP transduced control cells on a per cell basis (Fig. 2E), resulting in large but non-significant differences (Fig. 2F). Surprisingly, each isoform was observed to antagonize cytokine production (Fig. 2G, H). Since TCF-1B was the isoform relevant to antigen-experienced stages of differentiation, it was selected for further characterization, and cells transduced with it will hereafter be referred to as Tcf-1 transgenic (Tcf1Tg).
CD28 is not only expressed by naïve T cells but also effector and effector-memory T cells which have not undergone terminal differentiation [29]. Since CD28 is increasingly being used to define T cells which have yet to reach senescence, we examined the ability of Tcf-1 to maintain CD28+ cells [30, 31]. Naïve CD8+ T cells were (i) transduced and expanded nine days in IL-2 supplemented media (ii) labelled with proliferation dye (iii) further expanded via culture with media supplemented with solubilized anti-CD3 antibodies and IL-2. We compared the CD28 and GzmB content of GFP+ Tcf1Tg T-cells with peak Tcf-1 expression to GFP+ control T cells (GFPTg) with similar division history. Tcf-1 maintained a CD28+ population that had low GzmB content relative to CD28− cells (Fig. 2I, J). CD28− GFPTg T-cells had three-fold greater GzmB levels than CD28− Tcf1Tg T-cells (Fig. 2K), indicating that Tcf-1 could limit the cytotoxic potential of CD8+ T cells. CXCR3 promotes efficient trafficking to tumors and its expression could be a desirable attribute for cell-therapy T cells [32]. Further, CXCR3 is expressed in a hierarchical manner by early lineage differentiation subsets [33]. Therefore, we assayed CXCR3 in Tcf1Tg CD8+ T-cells after nine days of cell manufacture from PBMC, the earliest time point at which gene-edited cells are typically injected after in vitro expansion. CXCR3 was significantly enriched in Tcf1Tg CD8+ T-cells relative to control cells (Fig. 2L).
Tcf-1 allows TCR-engineered T cells to resist death associated with elicitation of cytotoxicity
We proceeded to assess the impact of Tcf-1 on the cytolytic activity of CD8+ T-cells engineered to express an anti-tumor TCR derived from tumor-infiltrating-lymphocytes: the 19,305-TCR, one which confers recognition of NY-ESO-1 presented in the context of HLA-A*02:01 [25]. CD8+ T cells were engineered to overexpress Tcf-1 and the 19,305-TCR and afterward implemented in co-cultures with SK-MEL-37 melanoma cells (SK37), a cell line that expresses NY-ESO-1 and HLA-A*02:01. Briefly, CD8+ T cells were isolated from PBMC and (i) co-transduced to express the 19,305-TCR and Tcf-1 or the 19,305-TCR and GFP (Fig. 3A) (ii) conditioned to advanced cytotoxicity via serial propagation over monolayers of SK37 in media supplemented with IL-2 at 300 IU/ml or 1,500 IU/ml, with passage once every seven days for 21-days (iii) implemented in 24-h co-cultures with SK37 for assessment of cytotoxicity and susceptibility to AICD. Engagements between T-cells and SK37 resulted in substantial reductions in T-cell viability which were mitigated by Tcf-1 (Fig. 3B, C). Tcf1Tg T cells were less cytotoxic than control cells and underwent reduced rates of AICD: Tcf1Tg T cells conditioned with 1500 IU/ml IL-2 were two-thirds as cytotoxic as control cells but retained three-fold greater viability (Fig. 3D, E). We speculated that the observed resistance to AICD could be due to differences in GzmB synthesis in response to conjugation of SK37: that TCR-directed GzmB synthesis could result in increased rates of GzmB leakage into the cytoplasm. Therefore, we assayed GzmB content and viability at baseline and after 15-h of co-culture with SK37. Whereas GFPTg T cells assayed at baseline had substantial GzmB content and viability, T cells assayed after co-culture contained tenfold greater per cell GzmB levels than cells assayed at baseline and were significantly more apoptotic (Fig. 3F–H). Tcf1Tg CD8+ T-cells did not upregulate GzmB as robustly as GFPTg CD8+ T-cells and retained significantly greater viability. We set up co-cultures with Brefeldin-A (BFA) with the expectation that BFA-mediated inhibition of translation would antagonize synthesis of proteins like GzmB [34]. BFA prevented T-cells from upregulating GzmB and acquiring an apoptotic profile (Fig. 3F–H). These results indicate that (i) upregulation occurs as a result of target-cell engagement and increases CD8+ T cell susceptibility to AICD (ii) Tcf-1 mediated GzmB restriction limits AICD but does not prevent T cells from engaging in cytotoxic activity, although cytotoxicity is limited.
Tcf-1 mitigates apoptosis and promotes stem-cell-like cycling activity
We used extended co-cultures to compare the survival of Tcf1Tg TCR-engineered CD8+ T-cells to that of control cells. Since IL-2 deprivation is a standard test of T cell survival, T cells were cultured without IL-2;[35] equal numbers of CD8+ T cells were cultured with SK37 (without IL-2) and counted at concurrent time-points via flow-cytometry, using CD45 alone to identify T-cells (because CD8 surface levels change in relation to activation) [36]. Tcf1Tg T cells were collected in greater numbers at all time points and were more viable than GFPTg T cells (Fig. 4A, B). We set up additional co-cultures and made multiparameter assessments with cells cultured 48-h with and without support by exogenous IL-2. Mitochondrial polarization was indexed with MitoTrackerDeepRedFM(MTDR), via labelling with a 10 nM solution. MTDR-labelled Tcf1Tg T-cells were significantly brighter than controls (Fig. 4C). Ki67 was highly expressed by Tcf1Tg T cells but not GFPTg T cells. IL-2 allowed control T cells to resemble Tcf1Tg T cells with respect to cell-cycle activity, mitochondria-polarization, and viability (Fig. 4D–F).
CD8+ T cell biomass, viability, and protein content is halved by just 48 h of IL-2 deprivation in vitro [37]. Since TCR or CAR transgenic T cells are typically expanded in IL-2 for 14 days before therapy, there is an opportunity for cells to be conditioned to a state of differentiation more susceptible to apoptosis from IL-2 deprivation, something understood to limit the efficacy of cell-therapy T cells, especially those expanded from tumor-infiltrating-lymphocytes [35, 38, 39]. To examine the effect of Tcf-1 on this process, T cells were (i) conditioned via expansion in SK37 co-cultures with IL-2, (ii) seeded into co-cultures with SK37 without IL-2, and (iii) counted after 7 days via flow-cytometry-based detection of T cells and counting beads. This was done with the expectation that T cells would be subject to a test of survival which would be influenced by prior conditioning. Non-conditioned T cells were collected in similar numbers, although more Tcf1Tg T cells could be collected (Fig. 5A). Tcf1Tg T cells were collected in significantly greater numbers than GFPTg T cells when implemented after 7 or 14 days of prior conditioning (Fig. 5B, C). To assess the impact of Tcf-1 on expansion, we did similar experiments but with IL-2 support. The addition of IL-2 changes the assay kinetics. When the assay is performed with IL-2, T cell expansion is more pronounced. Further, SK37 monolayers are depleted: IL-2 supports population dynamics and effector functions which allow T cells to clear SK37 and continue to expand. Tcf1Tg T cells underwent limited expansion in these co-cultures when implemented without prior conditioning, and when implemented after just 7 days of conditioning (Fig. 5D, E). However, with 14 days of conditioning, Tcf1Tg cells were collected in four-fold greater numbers (Fig. 5F), likely as a result of their enhanced survival rather than cell division activity.
Since Tcf-1-transgenic T cells from syngeneic models have been reported to incorporate BrdU less readily than wild-type T-cells, [40] we decided to examine the effect of Tcf-1 on cycling activity at a higher resolution. T-cell expansion occurs via proliferative bursts; TCR signaling instructs the duration of time a T-cell and its immediate progeny undergo successive rounds of division before becoming static [41]. Activation states initiated by TCR-signaling are maintained by IL-2 such that T cells can be expanded for extended periods in vitro via aggressive replenishment of IL-2 support. After the cessation of activation, cells return to quiescence, a state understood to leave T cells poised for secondary activation. When this trajectory is studied in vivo in the context of antigen depletion, memory precursors are observed to cycle at reduced rates relative to more differentiated cells. Intrinsic regulation of cell-cycle activity is evidenced by findings that FoxO1 constrains cell activation and could have implications for cell manufacture [9]. Therefore we studied cell-cycle activity in the context of expansion after transduction, when activation states initiated by anti-CD3/CD28 dynabeads are maintained by IL-2. We assayed Ki67 in the final days of a 14-day protocol in which dynabeads are removed after 5-days. GFP+ Tcf1Tg T-cells had reduced Ki67 levels relative to GFP+ control cells and levels further declined with time (Fig. 5G). When re-stimulated, GFP+ Tcf1Tg T-cells divided robustly relative to control cells, and were observed to undergo more rounds of division in the same amount of time (Fig. 5H, I). These results are consistent with a model where Tcf-1 checks cell-cycle activity via a regulatory mechanism that can be overcome by TCR signaling. Since Tcf-1 transgenic PG13 fibroblast cell-lines expand at reduced rates relative to parental cell lines (data not shown), the effect is likely independent of input from IL-2 receptors and T cell receptors.
Effect of Tcf-1 overexpression on the anti-tumor activity of TCR-engineered CD8+ T-cells
We carried out a xenograft model of adoptive cell transfer for the purpose of assessing the influence of Tcf-1 overexpression on the efficacy of TCR-engineered cell-therapy products (Fig. 6A): NSG mice with subcutaneous SK37 melanoma tumors were intravenously injected with CD8+ T cells transduced to express Tcf-1 and the 19,305-TCR. This cell-therapy product had identical activity as the control product (GFP and 19,305-TCR transduced) despite it containing an artificial memory compartment composed of Tcf1Tg cells with limited cytotoxicity (Fig. 6B–D). Whereas 43% of cells from the Tcf1Tg cell product were GFP+, GFP was expressed by the majority of cells recovered from tumors, for reasons that remain unclear but correlate with expression of Tcf-1. T cells recovered from the tumors of animals injected with Tcf1Tg T cells expressed Tcf-1 at 2.7-fold greater levels than GFPTg T cells (Fig. 6E–G). Tcf-1 checked T-cell effector function; Tcf-1 (i) prevented T cells from acquiring a GzmBhigh phenotype (ii) checked TNF-α production in re-stimulated cells (iii) did not have a significant effect on IFN-γ production (Fig. 6H–L). These results indicate that the inclusion of a Tcf-1 enforced memory compartment does not hamper the anti-tumor activity of TCR-engineered CD8+ T-cells.
Discussion
Since Tcf-1 is the prototypical identity factor for potent immunotherapy CD8+ T cells with attributes of stemness [1, 10, 40], we interrogated its ability to promote stem-cell-like functions and persistence in TCR-engineered T-cells. Tcf-1 promoted functions (i) that have the potential to synergize with programs imparted on cell-therapy T cells (ii) which provide understanding as to the mechanisms by which stem-cell-like T-cells persist. Naïve T cells express Tcf-1 at peak levels and persist as quiescent cells which have yet to acquire expression of granzymes, effector cytokines, or IL-2. The Tcf-1+ memory T-cells they give rise to are often described as having optimal potential for cell-therapy because of their intrinsic longevity [1]. The mechanisms by which these cells persist remain elusive as do strategies to manufacture therapeutic T cells with memory attributes. Here, we reduced cell persistence to survival of discrete engagements with cancer cells and observed T cells to undergo apoptosis as a result of the elicitation of their cytotoxicity. We found that the main function of Tcf-1 is to protect T-cells from apoptosis and that this occurs via regulation of GzmB. We also found evidence of regulation of GzmB expression in post-naïve stages of differentiation where Tcf-1 is abundantly expressed, consistent with findings by other groups [20, 29]. Accordingly, we conclude GzmB to be an intrinsic checkpoint on persistence that contributes to the fragility of effector cells. This is consistent with previous reports in murine T cells indicating that GzmB is expressed at notably lower levels by T-cells imparted with genetic enhancement of Tcf-1 or upstream-regulator FoxO1 [9].
While cytotoxic lymphocytes are capable of tumor control in the absence of GzmB, [17, 42] the GzmB-exocytosis pathway is significant for cytotoxic function. Therefore, Tcf-1 overexpression presents a tradeoff. This tradeoff may or may not influence ACT-performance, depending on the ability of the cells to utilize non-granzyme centered pathways against tumor cells. The effect of Tcf-1 on in vivo IFN-γ producing capabilities was insignificant, and limited in magnitude relative to its effect on GzmB. Tcf-1-engineered cells may primarily enact anti-tumor activity via IFN-γ, TNF-α, and FAS-ligand. In line with this, CD8+ T-cells from syngeneic mice containing constitutive Tcf-1 transgenes were recently reported to have potent anti-tumor activity because of their ability to persist and retain polyfunctional cytokine production [40]. Here, we report a proof-of-concept approach to applying constitutive Tcf-1 expression to TCR-engineered human T cells. Differences in the results obtained between our study and the one discussed may be attributed to the methodologies used; constitutive Tcf-1 expression would ideally be studied in the context of intact and immunogenic tumor microenvironments.
Tumor-infiltrating lymphocytes (TIL) are typically effector cells with peak GzmB expression. TIL are susceptible to attrition because of their extended ex-vivo preparation with anti-CD3 antibodies and IL-2 [43]. Their use is somewhat limited by their requirement for exogenous IL-2 and susceptibility to apoptosis in its absence [35]. Signals transmitted by IL-2 receptors maintain anti-apoptotic Bcl-2 proteins and ultimately antagonize mitochondria permeabilization and cytochrome-C release, the dominant axis of GzmB-mediated apoptosis [19]. Since Tcf-1-transduced T-cells had a modest in vitro survival advantage in the absence of IL-2, and this advantage was potentiated by conditioning that made T cells more cytotoxic, it is possible that the limited GzmB content of Tcf1Tg T cells could translate to a decreased reliance on IL-2 for maintenance of mitochondrial-integrity and survival. Such would assume the existence of a stoichiometric tug-of-war at mitochondria between IL-2-supported anti-apoptotic Bcl-2 proteins and GzmB-activated pro-apoptotic BID. This mechanism could explain the elevated IL-2 requirements of effector cells which generally express GzmB at peak levels and would be consistent with a model where IL-2 exists primarily to reinforce mitochondrial stability in Tcf-1 deficient stages of differentiation where T-cells have peak GzmB expression. In the context of the resolution of an endogenous immune response, this axis would allow the selective deletion of effector cells (over GzmB-low memory cells) when IL-2 levels decline.
An unresolved question in CD8+ T-cell biology is whether the tendency of Tcf-1 expressing T-cells toward quiescence is programmed or rather simply results from depletion of signals which maintain a heightened state of activation. We observed evidence of Tcf-1 mediated checks on cell cycle activity consistent with an entry into quiescence, as has been reported by others, and which is strikingly similar to effects mediated by FoxO1, the upstream regulator of Tcf-1 [9]. We propose that Tcf-1 promotes cell-cycle egress via a modest effect that is penetrant when cycling activity initiated by TCR-signaling is maintained by IL-2. IL-2 driven cell-cycle activity promotes terminal differentiation, and synthetic alternatives to IL-2 which are less expansive are touted for their potential for manufacture of therapeutic T-cells with increased in vivo activity [44].
There are potential limitations to pursuing methods to engineer persistent T cells. A principal concern is whether durable T cell responses could perpetuate off-tumor on-target toxicity. Transferred T cells may theoretically contain endogenous TCRs capable of self-recognition. Transferred cells could elicit pathologies via endogenous TCRs or alternatively perpetuate underlying T cell responses against self-antigen via secreted cytokines. Although the preclinical model studied here was inadequate to address these issues, each should be considered in the context of the specificity of the TCR or CAR. In this regard, the specificity of the NY-ESO-1 TCR utilized in our studies has been demonstrated by extensive testing for cross-reactivity to HLA-A*02:01+ and non-HLA-A*02:01+ targets pulsed with NY-ESO-1 or irrelevant peptides [25]. While insertional oncogenesis represents another potential concern which can occur in gene-modified cells, [45] these events are relatively rare in T cells modified using gamma retrovirus [46]. To address these potential limitations, constructs delivered to engineered T cells could be addressed via suicide genes which instigate apoptosis in response to administered agents [47].
In conclusion, within the context of cell-manufacture protocols in which cells are expanded in vitro after engineering, Tcf-1 mediated checks on post-activation cycling activity could provide extended instructions to divide, and limit GrzB mediated self-destruction. In the context of an endogenous immune response, a return to quiescence after antigen-experience could allow cells to engage in surveillance activity, mitigate bioenergetic requirements associated with excessive activation, and resist stress from DNA-damage associated with cell division.
Availability of data and material
The datasets used in this manuscript are available upon request.
Change history
06 June 2022
A Correction to this paper has been published: https://doi.org/10.1007/s00262-022-03233-1
Abbreviations
- AICD:
-
Activation-induced-cell-death
- dnTCF-1B:
-
B isoform of Tcf-1 lacking trans-activation domains, beta-catenin domain
- GzmB:
-
Granzyme-B
- PBMC:
-
Peripheral blood mononuclear cells, collected from leukoreduction chambers
- Tcf-1:
-
The T Cell Factor One (TCF-1) gene 5Q31.1 and protein
- TCF-1B:
-
B isoform of Tcf-1
- TCF-1E:
-
E isoform of Tcf-1
- Tcf1Tg :
-
Tcf-1 transgenic, cells infected to express the TCF-1B-GFP transgene
- TCR:
-
T cell receptor
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Acknowledgements
This work utilized the following shared resources at Roswell Park Comprehensive Cancer Center supported by P30CA016056: Genomics, Flow Cytometry, Immune Analysis and Division of Laboratory Animal Resources. We thank E. Repasky, S. Gollnick, S. Dasgupta, A. Stablewski, T. Chodon, R. Muthuswamy, K. Dejong, M. Ryszka, J. Prendergast.
Funding
This work was supported by RPCI-UPCI Ovarian Cancer SPORE P50CA159981-01A1 (KO), NCI Cancer Center Support Grant to Roswell Park P30CA016056, U01CA233085 (K.O.), and the University of Chicago Medicine Comprehensive Cancer Center Support Grant P30CA014599 (K.O.).
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BZ and KO established the conceptual design of the study. BZ performed experiments including (i) design of recombinant DNA (ii) molecular cloning (iii) establishment of virus producing cell lines (iv) T cell transductions (v) flow cytometry (vi) in vitro and in vivo assays; and wrote the manuscript. KO, TT and JM advised on approaches, methodology, and contributed materials. RM, RK, FI, HM, and CE advised on approaches, methodology, and concept. SB, KO, RM, RK, TC, TT and JM contributed to revising and editing.
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JM, TT, TC and KO are inventors of a patent application by Roswell Park Comprehensive Cancer Center regarding the TCR genes. KO and RK are cofounders of Tactiva Therapeutics and receives research support from AstraZeneca and Tesaro.
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Animal experiments were carried out with protocols approved by the Institute of Animal Care and Use Committee at the Roswell Park Comprehensive Cancer Center. PBMC were collected as byproducts of apheresis of platelets from consenting donors.
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Zangari, B., Tsuji, T., Matsuzaki, J. et al. Tcf-1 protects anti-tumor TCR-engineered CD8+ T-cells from GzmB mediated self-destruction. Cancer Immunol Immunother 71, 2881–2898 (2022). https://doi.org/10.1007/s00262-022-03197-2
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DOI: https://doi.org/10.1007/s00262-022-03197-2