Establishment of a novel platform cell line for efficient and precise evaluation of T cell receptor functional avidity

Adoptive T-cell therapy with T cell receptor (TCR) -engineered T cells is an attractive strategy for cancer treatment and the success in this therapy is dependent on the functional avidity of the transduced TCRs against targeted tumor antigens. Therefore, the establishment of the methodology of the efficient and precise evaluation of TCR functional avidity has been awaited. Here, we show a novel platform cell line, named 2D3, which enables the functional avidity of transduced TCRs to be evaluated efficiently and precisely. In the 2D3, the precise TCR functional avidity of transduced TCRs is easily evaluable by the expression of green fluorescent protein (GFP) reporter gene driven by nuclear factor of activated T cells (NFAT) activation via TCR signaling. Four different TCRs of HLA-A*24:02-restricted Wilms’ tumor gene 1 (WT1)-specific CD8+ cytotoxic T lymphocytes (CTLs) were transduced into 2D3 cells and the functional avidities of these four TCRs were evaluated. The evaluated functional avidity of these TCRs positively correlated with cell proliferation, cytokine production, and WT1-specific cytotoxicity of the TCR-transduced CD8+ T cells in response to WT1 antigen. These results showed that 2D3 cell line was a novel and stable tool useful for the efficient and precise evaluation of the functional avidity of isolated and transduced TCRs in developing TCR-based immunotherapy.


INTRODUCTION
Adoptive immunotherapy using tumor-associated antigen (TAA)-specific CD8 + cytotoxic T lymphocytes (CTLs) and/or CD4 + helper T (Th) cells can induce the regression of large established tumor in not only mouse models but also cancer patients [1][2][3]. These preclinical and clinical evidences encourage us to develop T-cell adoptive immunotherapy using genetically engineered T cells that are transduced with a T-cell receptor (TCR) gene specific for TAA. Furthermore, more recent studies have demonstrated that neo-antigens, which are generated from passenger mutations, would be promising targets for the engineered TCR-T cell therapy [4,5].

Research Paper www.oncotarget.com
In parallel with seeking for good targets from TAAs and neo-antigens by genome-wide approaches [6][7][8], novel methods analyzing huge number of TCR repertoire [9,10] and efficiently isolating TCR gene from a singlecell [11] have been developed. Unfortunately, not all isolated TCRs can sufficiently elicit anti-tumor immunity. Hence, development of a new method for the precise and efficient evaluation of the isolated TCRs has been awaited for the prediction of clinical response in the engineered TCR-T cell adoptive therapy.
TCR affinity, TCR avidity, and functional avidity are known as an indicator to predict the in vitro/in vivo properties and behavior of the TCR-transduced T cells [12][13][14]. TCR affinity, which is defined as the binding-strength of TCR molecules to peptide-major histocompatibility complex (pMHC), is often used for this prediction beyond TCR's specificity because it can standardize the strength of TCR binding to pMHC by using a numerical value (ie, K D value). However, purified soluble TCR α/β complex is needed for calculating TCR affinity. It is, therefore, not feasible for screening a large number of candidate TCRs. In addition, it has been shown that TCR affinity is sometimes not consistent with actual T cell function [12,14]. On the other hand, both TCR avidity (which is usually measured by pMHC tetramers) and functional avidity (which is assessed using a titrated concentration of antigen peptide with antigenpresenting cells) are correlated with in vitro cytotoxicity and in vivo anti-tumor activity in TCR-transduced T cells [12,15]. Since preparation of large sets of tetramer for candidate TCRs is difficult in terms of cost, time, and effort, assessment of functional avidity must be the most adequate and feasible approach for screening of TCRs capable of provoking a good clinical response in engineered T-cell adoptive immunotherapy.
Functional avidity is assessed by phosphorylation of linker for activation of T cells (LAT) and extracellular signal-regulated kinase (ERK), calcium influx, and cytokine release after the stimulation with a titrated concentration of antigen peptide. Compared to TCR affinity, functional avidity is a relative indicator and easily influenced by various factors such as CD8/CD4 coreceptors and TCR clustering (ie, quantity of TCR/CD3 molecules and where and how TCR-pMHC interaction are formed) [13,16]. Therefore, the use of primary T cells for the assessment of precise functional avidity is inappropriate because they are heterogeneous and express endogenous TCRs that cause incorrect TCR clustering by mispairing with transduced TCRs [17] and competing for CD3 molecules [18].
In this study, we describe a novel platform cell line, named 2D3, for efficient and precise evaluation of TCR functional avidity. 2D3 cells are endogenous TCRnull and CD8-positive and can express green fluorescent protein (GFP) through transcription factor nuclear factor of activated T cells (NFAT) that is activated by TCR signaling. Therefore, the establishment of 2D3 cells enabled us to selectively analyze the functional avidity of appropriately transduced TCRs by using GFP expression as a marker.
Thus, 2D3 cell line should be a good tool useful for the evaluation of the functional avidity of isolated and transduced TCRs and prediction of the TCR-transduced T cell function in developing effective adoptive T-cell immunotherapy against cancer.

Establishment of 2D3 cell line by the transduction of hCD8 and NFAT-GFP reporter genes
We established a 2D3 cells in which the signals from transduced TCRs activated the NFAT, followed by the GFP expression as a selection marker for appropriately TCR-transduced cells ( Figure 1A). Jurkat-76, a TCR α/βnegative sub-line of Jurkat (CD8 − T lymphoma cell line) was thought to be an ideal candidate as a source of the platform cell line because it could not produce endogenous TCRs and thus because transduced TCRs would be well expressed without competition with endogenous TCRs. Therefore, we transduced Jurkat-76 cells with hCD8 gene and established J76.7 cell line, and finally established CD8 + 2D3 cell line by the transducing the J76.7 cells with NFAT-GFP reporter gene. 2D3 cells did not express CD3 molecules on the cell surface because of lack of their endogenous TCR expression ( Figure 1B), and strongly expressed GFP in the majority of cells when they were stimulated with Phorbol 12-myristate 13-acetate (PMA)/ Ionomycin to activate NFAT ( Figure 1C). Both expression of hCD8 and NFAT-GFP reporter genes was stable and long-lasting (data not shown). Thus, we succeeded in the establishment of 2D3 cell line suitable for evaluating the expression and function of CTL-derived TCRs.

2D3 is a platform cell line for efficient and precise evaluation of the expression and function of transduced TCRs
To confirm that 2D3 cell line could be a platform cell line suitable for the evaluation of the expression and function of transduced TCRs, 2D3 cells were transduced with lentiviral vector encoding B10-TCR, which was the TCR that was isolated and cloned from an HLA-A * 24:02-restricted, WT1 235 peptide-specific CTL clone, B10 [19]. B10-TCR-and mock-transduced 2D3 cells could be monitored by the expression of Venus fluorescent protein. As expected, B10-TCR-transduced 2D3 cells expressed both CD3 and TCR α/β molecules on their surface and were WT1 235 tetramer-positive. In contrast, mock-transduced 2D3 cells expressed neither CD3 molecules nor B10-TCR ( Figure 2A). Furthermore, www.oncotarget.com B10-TCR-transduced 2D3 cells showed GFP expression in WT1 235 peptide-concentration dependent manner ( Figure 2B). This WT1 235 peptide concentration-response curve showed that Effective concentration 50 (EC50), which was often used to describe the functional avidity of TCR, was 52.7 nM (95% confidence interval (CI), 42.3-64.8 nM) for B10-TCR. In general, since TCR functional avidity is determined by several factors such as TCR affinity and quantities of TCR, CD3, and CD8/CD4 molecules, it is easily influenced by cell types used for experiments, especially by TCR constructs that regulate expression efficacy [20]. Therefore, to confirm that B10-TCR functional avidity was stably evaluable by the 2D3 cells regardless of the difference in B10-TCR constructs, we examined the response of 2D3 cells transduced with codon-optimized α-p2A-β or β-p2A-α B10-TCR that was different from original B10-TCR construct but specific to WT1 235 peptide ( Figure 2C). As expected, EC50 was 50.8 nM and 53.7 nM for codon-optimized α-p2A-β and β-p2A-α B10-TCRs, respectively, and similar to that obtained from original B10-TCR construct (α-p2A-β)transduced 2D3 cells ( Figure 2C). Thus, 2D3 cell line was thought to be a platform cell line suitable for efficient and precise evaluation of the expression and function of transduced TCRs.
Next, we determined that the TCR functional avidity evaluated by the 2D3 cells also correlated positively with cytotoxicity of the TCR-transduced CD8 + T cells against HLA-A * 24:02-positive, WT1expressing leukemic cells. Since the WT1-expressing leukemic cells expressed natural WT1 235 peptide (nWT1 235 ), the functional avidity of the B10-TCR-or TM-H2-TCR-transduced CD8 + T cells was evaluated by the 2D3 cells in response to the nWT1 235 , instead of mWT1 235 ( Figure 3D). The EC50 of B10-and TM-H2-TCRs was 62 nM and 180 nM, respectively, and that of B10-TCR was approximately three times higher than that of TM-H2-TCR. As shown in Figure 3E, B10-TCR-transduced CD8 + T cells could lyse HLA-A * 24:02positive, WT1-expressing leukemic cells, while TM-H2-TCR-transduced CD8 + T cells could not lyse them. These results showed the positive correlation between Taken together, these findings indicated that TCR functional avidity evaluated by 2D3 cells was clearly and positively correlated with the effector functions such as proliferation, cytokine production, and cytotoxicity of the TCR-transduced CD8 + T cells regardless of whether the TCR specificity was for natural or modified WT1 235 peptide.

DISCUSSION
In the present study, we successfully established 2D3 cell line as a platform cell line to efficiently evaluate the function of TCRs. Actually, we demonstrated the clear correlation between TCR functional avidities evaluated by the 2D3 cells and effector functions such as cell proliferation, cytokine production, and cytotoxicity of the TCR-transduced CTLs.
To our knowledge, there is no standard method to evaluate simply the functional avidity of human TCRs. T cell lines (ex. 58αβ -, TG40, or Jurkat) and T cells are often used as a platform cell for the evaluation of functional avidities of transduced TCRs, such as cytokine production, killing activity, and phosphorylation of proteins downstream of TCR signaling [21,22]. Here is a question-are functional avidities evaluated by using these cells? Since mouse T cell lines, 58αβand TG40 express CD3 molecule, human transduced TCRs are expressed on the cell surface with mouse CD3 molecule. However, it is unknown whether the complex of human TCRs and mouse CD3 can induce normal TCR signaling. Indeed, Cohen et al. reported that binding stability between human TCRs and mouse CD3 differed from that between human TCRs and human CD3 [23]. Nagai et al. used TCR-negative Jurkat/MA cells [24] that expressed only CD8 α molecule to monitor TCR signaling [25] because CD8 α could be expressed as CD8 α/α homodimers, which could bind to MHC class I molecule, without CD8 β on the cell surface. On the other hand, it is well-known that CD8 β associates with only CD8 α and cannot be expressed alone on the cell surface. Although both CD8 α/α and CD8 α/β could express on the surface, there is difference in the binding ability to MHC class I molecules between CD8 α/α and CD8 α/β [26]. In addition, CD8 β intracellular domain promotes association of lymphocytespecific protein kinase (Lck) and LAT with surface CD8 complexes [27]. Of course, almost all mature CD8 + T cells express CD8 α/β heterodimer in vivo. Therefore, 2D3 cell line, which expresses CD8 α/β heterodimer, is a suitable platform cell line to assess TCR functional avidity. Furthermore, since 2D3 cell line is deficient in endogenous TCR expression, only transduced TCRs can be expressed on 2D3 cell surface without mispairing with endogenous TCRs. Interestingly, it has been shown that T cell recognition of pMHC can be increased up to 50-fold after priming with the same pMHC [28]. In addition, functional avidity maturation of CTLs can occur through the change of TCR clustering of various molecules such as lipid raft, Lck, and CD3 without the selection of higher affinity TCR during early stage of viral infection [29]. In addition, the same mechanism can also induce the inability of CD8 + T cells for the recognition of pMHC [30]. Taken together, TCR functional avidity of human T cells is variable in response to antigen stimulation. Therefore, human T cells are not suitable for platform T cells to evaluate TCR functional avidities, whereas the 2D3 cells are convenient for the evaluation of TCR functional avidities because of its functional stabilities.
Previous studies demonstrated that TCR functional avidity determined T cell fate. It is well-known that in Th1/Th2 polarization, weak TCR signaling favors Th2 differentiation and stronger one induces Th1 differentiation [31][32][33]. In addition, the difference in TCR functional avidity to self-antigens also has an effect on memory/effector T cell development. Allen PM and his colleagues reported that the magnitude of secondary response in Listeria-specific T cells was determined by the strength of TCR functional avidity to self-antigen [34,35]. Furthermore, we previously demonstrated that WT1-specific CD8 + T cells with high-avidity TCR to WT1 peptide easily differentiated into effector T cells in TCR-retrogenic mice [36]. However, it remains unclear how TCR avidity controls T cell responses and their fate, especially memory/effector differentiation. Since TCRstimulation of quiescent T cells such as naïve and memory T cells induces metabolic shift from catabolic to anabolic energy production, it may be speculated that the strength of TCR avidity finely regulates the metabolic condition that determines T cell differentiation [37]. Primary human T cells are not suitable for the evaluation of the role of TCR avidity in the T cell functional differentiation because they are heterogeneous and are difficult to keep the cells viable for long term after transduction of TCR genes. On the other hand, Jurkat cell line, which is a parent cell line of 2D3 cell line, is stable to viability and can respond to TCR signals. Jurkat cells can form lipid raft [38], like primary human T cells, and increase CD3 ζ and ERK phosphorylation through cholesterol removal [39]. Therefore, it appears that Jurkat cells functionally mimic primary human T cells, and thus 2D3 cell line should be useful for the study of how TCR avidity controls T cell responses and their fate.
Since 2D3 cells has a GFP, instead of luciferase, as a reporter gene, we can easily sort the activated TCRtransduced 2D3 cells by using GFP-positivity as an indicator and examine the molecules associated with the signals from the transduced TCR.
In conclusion, we demonstrate a novel platform cell line as a useful tool to evaluate efficient and precise TCR functional avidity for developing TCR-based immunotherapy.

Establishment of 2D3 cell line
Two hundred thousand Jurkat-76 cells were subjected to electroporation with hCD8 α-E2A-hCD8 β-encoding pcDNA3.1/Zeo (+) using the Neon transfection system (Thermo Fisher Scientific Inc., MPK5000) according to the manufacturer's guidelines. hCD8 α-E2A-hCD8 β construct was kindly provided by Prof Hans J Stauss. CD8-transduced Jurkat-76 cells were single-cell sorted into 96-well round-bottom plates and stably hCD8 α/β-expressing Jurkat-76 cell line, named J76.7 was successfully established. Two hundred thousand J76.7 cells were transduced with NFAT-GFP reporter plasmid by electroporation followed by single cell sort. A single-cell-derived cell line capable of highly expressing GFP protein only when stimulated with PMA/Ionomycin was established as 2D3 cell line.

Transduction of WT1-specific TCR genes
To transduce TCR-encoding lentivirus vector into CD8 + T cells, CD8 + T cells were stimulated with RetroNectin-and OKT-3-coated 48-well plate in the presence of IL-2 (40 IU/ml). On the next day, the stimulated CD8 + T cells were spin-infected with lentivirus vector in the presence of polybrene (10 μg/ml, Sigma-Aldrich, H9268) at 1000 g for 2 hours at 32° C. After 6-12 hours, the culture medium was changed into fresh medium supplemented with 10% heat inactivated human AB serum and IL-2 (100 IU/ml). To establish WT1 235specific TCR-transduced 2D3 cells, 2D3 cells were transfected with TCR-encoding lentivirus in the presence of polybrene. Venus-positive 2D3 cells were sorted and used for NFAT-GFP reporter assay as described below.

Proliferation assay
CD8 + T cells were transduced with WT1 235 -specific TCRs as described above, and the transduced CD8 + T cells (5 × 10 5 ) were stimulated by the co-culture with irradiated mWT1 235 peptide-pulsed autologous PBMCs. Seven days later, the expanded cells were harvested, counted by trypan blue, and measured for the frequency of WT1 235 -specific TCR-transduced CD8 + T cells using tetramer assay. Five hundred thousand cells out of the expanded cells were restimulated and re-expanded as described above. The series of experiments was three times repeated. The number of WT1 235 -specific TCR-transduced CD8 + T cells was calculated as the number of venus + WT1 235 tetramer + cells accumulated. 51 Cr release assay 51 Cr release assays were performed as previously described [19].

Statistical analysis
The statistical analysis and the calculation of EC50 values were performed with GraphPad Prism 7 (GraphPad Prism Software, San Diego, CA, USA).