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Heteroclitic XBP1 peptides evoke tumor-specific memory cytotoxic T lymphocytes against breast cancer, colon cancer, and pancreatic cancer cells

XBP1 is a critical transcriptional activator of the unfolded protein response (UPR), which increases tumor cell survival under prolonged endoplasmic reticulum (ER) stress and hypoxic conditions.This study was designed to evaluate the immunogenicity of heteroclitic XBP1 unspliced (US)184–192 (YISPWILAV) and heteroclictic XBP1 spliced (SP)367–375 (YLFPQLISV) HLA-A2 peptides, and to characterize the specific activities of XBP1 peptides-specific cytotoxic T lymphocytes (XBP1-CTL) against breast cancer, colon cancer, and pancreatic cancer cells.The XBP1-CTL had upregulated expression of critical T cell markers and displayed HLA-A2-restricted and antigen-specific activities against breast cancer, colon cancer and pancreatic cancer cells. XBP1-CTL were enriched withCD45RO+ memory CTL, which showed high expression of critical T cell markers (CD28, ICOS, CD69, CD40L), cell proliferation and antitumor activities as compared to CD45RO− non-memory CTL. The effector memory (EM: CD45RO+CCR7−) subset had the highest level of cell proliferation while the central memory (CM: CD45RO+CCR7+) subset demonstrated enhanced functional activities (CD107a degranulation, IFNγ/IL-2 production) upon recognition of the respective tumor cells. Furthermore, both the EM and CM XBP1-CTL subsets expressed high levels of Th1 transcription regulators Tbet and Eomes. The highest frequencies of IFNγ or granzyme B producing cells were detected within CM XBP1-CTL subset that were either Tbet+ or Eomes+ in responding to the tumor cells.These results demonstrate the immunotherapeutic potential of a cocktail of immunogenic HLA-A2 specific heteroclitic XBP1 US184–192 and heteroclictic XBP1 SP367–375 peptides to induce CD3+CD8+ CTL enriched for CM and EM cells with specific antitumor activities against a variety of solid tumors.


Introduction
High levels of proliferation characteristic of cancer cells lead to nutrient deprivation, hypoxia, and accumulation of unfolded/ misfolded proteins, which in turn leads to physiologically important ER stress responses, such as the UPR. UPR is a cellular adaptive mechanism that occurs in a majority of solid tumors, which allows for tumor cell survival under stress conditions by increasing its protein folding capacity. 1,2 X-box binding protein 1 (XBP1), a critical transcription activator in the UPR, regulates a subset of ER-resident chaperone genes essential for protein folding and maturation. 3,4 A recent report shows that XBP1 splicing plays an important role in the regulation of glucose homeostasis, which is independent of ER stress. 5 XBP1 exists as unspliced (XBP1u), a functional protein encoded by unspliced XBP1 mRNA, and spliced (XBP1s) forms via inositol requiring enzyme 1 a (IRE1 a)-mediated unconventional splicing, in which XBP1u facilitates the recruitment of XBP1 mRNA cotranslationally. 6 Genome-wide profiling, along with association studies and immunohistochemistry, demonstrated that XBP1 expression was induced in a variety of primary cancers or cancer cell lines including hematological malignancies such as multiple myeloma and acute lymphoblastic leukemias, [7][8][9] as well as breast cancers, 10,11 hepatocellular carcinoma, 12,13 pancreatic adenocarcinomas, 14,15 and colon cancer. 16,17 It has been demonstrated that XBP1 is activated in primary mammary tumors, and that its expression correlates with enhanced tumor growth. 18 In support of a direct role for XBP1 in tumorigenesis, the loss of XBP1 was shown to severely inhibit tumor growth. In addition, transformed cells with XBP1 deficiency were sensitized to hypoxia and underwent apoptosis, implicating XBP1 as a survival factor. 19 Thus, disrupting or targeting the XBP1 pathway is a rational approach for selective cancer cell killing, providing the basis for therapeutic strategies against multiple solid tumors.
The T-box transcription factors, T-bet and eomesodermin (Eomes), expressed in T cells have been implicated as master regulators of CD8 C T cell differentiation into Th1 type cells and the critical formation of effector and memory T cell subsets. [20][21][22] In the context of CD8 C memory T cells, T-bet and Eomes sustain memory phenotypes by stabilizing the expression of IL-2Rb, thus promoting IL-15 signaling, which is critical for continued proliferation of memory cells. 23,24 In addition, both T-box transcription factors cooperate to promote cytotoxic T lymphocyte (CTL) formation by inducing the expression of perforin and granzyme B during early stages of CD8 C T cell activation and promote migration to inflamed tissues by inducing chemokine receptors. [25][26][27] Importantly, sufficient clinical evidence demonstrates a correlation between longer survival of cancer patients and increased expression of genes representing type 1 effector T cells, in particular T-bet and Eomes. [28][29][30] Therefore, T-bet and Eomes are critical for both function and homeostasis of effector and memory T cells. However, their roles in the setting of memory T cell responses in response to tumor, and their expression and function in antigen-specific CTL are not well characterized.
Our group is interested in developing a peptide-based cancer vaccine against the XBP1 antigen using engineered heteroclitic XBP1 unspliced (US) [184][185][186][187][188][189][190][191][192] (YISPWILAV) and heteroclitic XBP1 spliced (SP) 367-375 (YLFPQLISV) HLA-A2 specific peptides. 31 Each of these selected peptides has been demonstrated to be highly immunogenic, inducing XBP1 antigen-specific CTL, which specifically target HLA-A2 C multiple myeloma (MM) cells. 31,32 In these studies, we further evaluated the immunogenicity of these heteroclitic XBP1 peptides, and characterized the resulting XBP1 peptides-specific CTL against a variety of solid tumor cancer cell lines, which overexpress the unspliced and spliced XBP1 antigens. Our results characterized distinct phenotypic profiles for XBP1-CTL and their specific antitumor activities against HLA-A2 C breast cancer, colon cancer and pancreatic cancer cells. The immunologic antitumor activities of the CM (CD45RO C CCR C ) and EM (CD45RO C CCR7 ¡ ) CD3 C CD8 C cells of XBP1-CTL were shown to be driven by T-bet and Eomes transcription regulator expression within the memory subsets. These results provide the rationale for designing an immunotherapeutic approach comprised of heteroclitic XBP1 US 184-192 and XBP1 SP 367-375 HLA-A2 peptides as a vaccine to induce distinct XBP1-CTL memory subsets expressing critical T cell markers and transcription regulators that result in specific antitumor activities against solid tumors including breast, colon and pancreatic cancers.  (YLFPQLISV) peptides. XBP1-CTL were generated from HLA-A2 C normal donors' CD3 C T cells by repeated stimulation with APC pulsed with a cocktail of heteroclitic XBP1 peptides. Compared to unstimulated control T cells, the XBP1-CTL (n D 3; donors A, B, C) showed enrichment of total CD3 C CD8 C T cells. A continuous increase in the frequency of CD3 C CD8 C T cells was observed from baseline (no stimulation) through four cycles of peptides stimulation (A). One week after four rounds of peptides stimulation, XBP1-CTL showed increases in both the frequency (% positive cells; B) and expression levels (MFI; C) of critical T cell markers.
Increased level of XBP1 gene expression in primary cancer cells from normal cells A significantly higher level of XBP1 gene expression was found in primary cancer cells from breast cancer patients or colon cancer patients as compared to cells from healthy donors through searching of the public databases canEvolve and Oncomine ( Table 2). A significant difference (p < 0.05) was detected in XBP1 gene expression using canEvolve in a series of "TCGA-colon" from colon cancer patients (n D 155) with normal donors (n D 24), along with a series of "TCGA-BRCA" cells from breast cancer patients (n D 536) to normal donors (n D 63). In addition, Oncomine database search showed significant differences in XBP1 gene expression between cells from normal donors and different types of colon cancer patients (n D 161) or breast cancer patients (n D 593). Pancreatic cancer patient samples were not available for the analyses.
Next, we investigated the expression levels of critical antigenspecific T cell markers on XBP1-CTL. Higher expression levels (MFI) were observed for CD28, ICOS (Inducible T-cell costimulator; CD278), CD69 and CD40L within the CD45RO C memory as compared to CD45RO ¡ non-memory CD3 C CD8 C XBP1-CTL (n D 3) (Fig. 4D). Further analyses also demonstrated increased frequencies of CD28, ICOS, CD69, and CD40L expression within the specific CM and EM subsets as compared to the na€ ıve and terminal effector XBP1-CTL subsets (Fig. 4E). In addition, the XBP1-CTL CM cells had higher levels of CD28, ICOS, CD69, and CD40L expression as compared to the EM subset, while not much difference was detected between the na€ ıve and terminal effector cells of XBP1-CTL subsets.
A high level of cell proliferation was included by memory subsets of XBP-CTL in response to breast, colon or pancreatic cancer cells in an antigen-specific and HLA-A2-restricted manner The antitumor activities of the XBP1-CTL were further analyzed using a 6 d CFSE-proliferation assay. The proliferation of XBP1-CTL was evidenced by a decrease in the fluorescence intensity of CFSE-labeled cells following stimulation with irradiated tumor cells. Flow cytometric analyses showed a high level of XBP1-CTL proliferation in response to HLA-A2 C breast cancer 70.3%, Panc1; 68.5%) (Fig. 5B), and HLA-A2 C colon cancer cells (LS180; 55.4%, SW480; 51.2%) (Fig. 5B).
The central memory subset of XBP-CTL possesses a high level of antitumor activities against breast, colon or pancreatic cancer cells Finally, we investigated the functional antitumor activities within the specific CM (CCR7 C CD45RO C /CD3 C CD8 C ) and EM (CCR7 ¡ CD45RO C /CD3 C CD8 C ) subsets from the XBP1-CTL. In contrast to the proliferation results, the CM subset of XBP1-CTL had greater cytokine production and CD107a degranulation as compared to the EM subset in response to the respective HLA-A2 C /XBP1 C solid tumor cell lines. Specifically, IFNg production was higher by the CM CTL population in response to breast cancer (MDA-MB-231: 76.8% CM vs. 17  EM; PL45: 52.0% CM vs. 4% EM) cells (Fig. 6C). XBP1-CTL generated from two different individuals showed the same pattern of antitumor responses, higher IFNg production (Fig. 6D), IL-2 production (Fig. 6E), and CD107a degranulation (Fig. 6F), by the CM subset as compared to EM cell population. Therefore, these results demonstrate that the CM subset of XBP1-CTL have superior cytokine production and cytolytic activity (CD107a degranulation) as compared to EM cells against breast cancer, colon cancer and pancreatic cancer cells.
T-bet C CM and Eomes C CM subsets of XBP1-CTL are enhanced for IFNg production in response to breast cancer, colon cancer or pancreatic cancer cells Next, we evaluated T-bet expression and IFNg production within the specific memory:na€ ıve subsets of XBP1-CTL (n D 3). The highest cell frequency of T-bet expression was detected within the EM subset (39-47%) followed by the CM (10-26%), effector (2-22%), and na€ ıve (< 1%) CD3 C CD8 C T cells (Fig. 8A). However, the highest frequency of T-bet C /IFNg C cells was detected within the CM subset of XBP1-CTL, consistently in response to the respective tumor cells including breast cancer (Fig. 8B), pancreatic cancer (Fig. 8C) or colon cancer (Fig. 8D)   Eomes expression (Fig. 9A). Additionally, IFNg C production was detected exclusively in Eomes C fraction of memory CD3 C CD8 C T cells of XBP1-CTL, in response to the various HLA-A2 C tumor cells. The highest level of IFNg C production was observed within the CM subset expressing Eomes in response to breast cancer (MB231, 12-19%; Fig. 9B), pancreatic cancer (Panc1, 25-34%; Fig. 9C) or colon cancer cells (SW480, 10-16%; Fig. 9D). Overall, IFNg production was higher in CD3 C CD8 C T cells expressing Tbet than Eomes in response to the various solid tumor cells. Therefore, these results provide additional evidence that heteroclitic XBP1 peptides induce different subsets of memory CD3 C CD8 C T cells, which express the critical transcription factors which regulate memory cell development and Th1 development.

Discussion
Peptide-based vaccines have distinct advantages over individualized vaccines in regards to cost of production, manufacture of vaccine products, and straightforward measurements to detect antigenspecific immune responses in patients. They have been shown to mount a safe and effective immune response to treat different types of cancer; for instance, in a randomized phase 3 trial in patients with advanced melanoma, patients receiving a vaccine consisting of the HLA-A2restricted gp100 peptide plus incomplete Freund's adjuvant, followed by IL-2 administration, showed a higher response rate and longer progression-free survival than the patient cohort who received IL-2 alone. 33 In addition, a vaccine for renal cell cancer patients consisting of multiple tumor-associated HLA-A2-restricted peptides demonstrated improved disease control in early stage clinical studies, evidenced by prolonged survival in patients who mounted a specific immune response to the vaccine combined with a single-dose cyclophosphamide. 34 Thus, application of promising immunogenic HLA-A2-specific peptides targeting tumor-associated antigens can offer new vaccine strategies to enhance antitumor efficacy under optimal conditions. During tumorigenesis, the high proliferation rate of cancer cells requires increased activities within the ER machinery to facilitate protein folding, assembly, and transport. This high demand leads to ER stress and accumulation of unfolded/misfolded proteins, ER calcium depletion, altered glycosylation, nutrient deprivation, oxidative stress, DNA damage, and/or energy perturbation/fluctuations. In addition, the high level of ER involvement triggers a group of signal transduction pathways, collectively termed as UPR, to modify transcriptional and translational programs and maintain ER homeostasis. [35][36][37] The UPR pathways are activated in a great variety of tumor types, and protect tumor cells from stressful conditions as well as immune surveillance. [38][39][40] Based on these observations, extensive research and clinical trials have been focused on targeting UPR pathways to treat different types of cancer patients including those with MM, mantle-cell or non-Hodgkin's lymphoma, acute myeloma leukemia, breast cancer, colorectal cancer, ovarian cancer, lung cancer, sarcoma, renal cancer or melanoma. [41][42][43][44][45][46][47] A few examples of UPR-targeted cancer drugs in development and their mechanisms include Bortezomib (proteasome inhibitor), Tanespimycin (HSP90 inhibitor), Retaspimycin (HSP90 inhibitor), Versipelostatin (GRP78 inhibitor), and Irestatins (IRE1a inhibitor). [48][49][50] The purpose of these studies was to evaluate an immunotherapeutic approach for targeting the XBP1, an important transcription activator of UPR, in a variety of solid tumor. Previously, we identified highly immunogenic heteroclitic XBP1 US [184][185][186][187][188][189][190][191][192] (YISPWILAV) and XBP1 SP 367-375 (YLFPQLISV) peptides with higher HLA-A2 binding affinities and stabilities than their native counterparts. 31 In these studies, XBP1-CTL were generated using a cocktail of HLA-A2 unspliced and spliced heteroclitic XBP1 peptides and were analyzed for their functional immune activities against solid tumors including breast, colon, and pancreatic cancer cells that highly overexpress XBP1 unspliced and spliced proteins. Our results confirmed our previous findings on the immunogenicity of the heteroclitic XBP1 peptides to induce CTL with distinct phenotypic profiles including highly activated memory CD8 C T cells. 31 In addition, these studies demonstrate distinctive upregulation of critical molecules on the expanded CD3 C CD8 C T cells including CD38 (cell activation marker), CD154 (CD40L; a co-stimulatory molecule), CD278 (ICOS; an inducible T cell co-stimulatory molecule) and CD137 (41BB; TNF family member critical for the survival of activated and memory CD8 C T cells) as well as the full range of memory CTL subsets. These studies also showed that the modifications of XBP1-CTL had poly-functional antitumor activities in response to various solid tumor cells beyond our previously tested MM cells.   CM (CD45RO C CCR7 C ) CD3 C CD8 C T cells retain a greater capacity for secondary re-expansion and long-term persistence and are enabled by efficient homeostatic self-renewal, whereas EM (CD45RO C CCR7 ¡ ) CD3 C CD8 C T cells rapidly exert effector functions upon antigen encounter. [51][52][53] However, there remain controversies in defining the immune response of the specific memory T subsets within antigen-specific CTL against tumor cells. 54,55 In these studies, we characterized the antitumor activities of the two different memory cell subsets, CM and EM, from XBP1 antigen-specific CTL against a variety of solid tumor cells. We chose to evaluate the XBP1-CTL after the fourth round of peptide stimulation, due to high levels of antigen-specific cells within both memory cell subsets of the CD3 C CD8 C T cells population. Our results demonstrate a higher level of XBP1-CTL proliferation within the EM as compared to the CM population in response to HLA-A2 C /XBP1 C breast, colon or pancreatic cancer cells. In contrast, XBP1-CTL functional antitumor activities, IFNg/IL-2 production and CD107a degranulation (Fig. 6) as well as granzyme B upregulation (data not shown), were the higher in CM cells as compared to the EM population. These outcomes were observed consistently in the XBP1-CTL generated from all individuals and against all solid tumor cell lines tested. Therefore, our results provide additional evidence that the heteroclitic XBP1 peptides are immunogenic and consistently induce a high proportion of memory CTL with a CM phenotype which have superior ability to mediate tumor regression as compared to the EM subset and supports the findings from other investigators' of the potential of CM in clinical benefit. [56][57][58][59][60] In future studies, we will further investigate the mechanism for arresting differentiation of antigen-specific memory CTL to potentially induce a greater frequency of antigen-specific CM CTL subset in order to better develop immunotherapeutic strategies.
Various transcription factors, repressors, and other regulators are known to be critical for generation of specific T cell subsets. 61,62 Among them, T-bet and Eomes have been demonstrated as important transcription regulators involved in the differentiation of CD8 C T cells into Th1 type cells. Both T- Fig. 5. Antigen-specific and HLA-A2-restricted cell proliferation of heteroclictic XBP1-CTL to solid tumor cells with higher response levels by memory CTL subsets. XBP1-CTL generated with a cocktail of heteroclitic XBP1 peptides were evaluated for their proliferative response to a variety of solid tumor cells using CFSE assays. Cell proliferation was measured by flow cytometry as the percent decrease in CFSE expression (CFSE low) on day 6 of culture after gating on the CD3 C CD8 C T cell population. HLA-A2-restricted and antigen-specific XBP1-CTL proliferation in response to HLA-A2 C /XBP1 C MDA-MB-231 breast cancer cells, but not HLA-A2 ¡ /XBP1 C BT-434 breast cancer cells, HLA-A2 C /XBP1 ¡ LNCaP, nor HLA-A2 ¡ /XBP1 ¡ VCaP prostate cancer cells (A). In addition, XBP1-CTL proliferated in response to HLA-A2 C /XBP1 C pancreatic cancer cells (PATU8902, Panc1) and colon cancer cells (LS180, SW480), but not to HLA-A2 mismatched pancreatic cancer (MiaPaCa-2), nor colon cancer cells (WiDr) (B). Specific XBP1-CTL proliferation was detected within the memory subsets (EM > CM) in response to HLA-A2 C /XBP1 C MDA-MB-231 breast cancer (C), LS180 colon cancer (D) or Panc1 pancreatic cancer cells (E). XBP1-CTL in media alone was used to determine background cell proliferation. bet and Eomes have a critical role in the generation and promotion of central and EM CD8 C T cells and in elimination of established tumors by the T cells. However, the role of these transcription factors in shaping the memory T cell pool and eliciting tumor-specific memory T cell subsets needs to be further elucidated. In current studies, we describe the association of T-bet and Eomes in antitumor activities within the memory T cell subsets of antigen-specific CTL in response to a variety of solid tumor cancers. Of particular interests were to define and characterize the expression patterns of T-bet and Eomes within the XBP1 antigen-specific effector or memory CD3 C CD8 C T cell subsets, which demonstrated specific functional activities against breast cancer, colon cancer, and pancreatic cancer. These studies showed that the XBP1-specific CTL express higher frequencies of T-bet C and Eomes C within the memory populations as compared to their non-memory counterparts. Specifically, XBP1-CTL EM subset had the highest frequency of T-bet C and Eomes C CD8 C T cells followed by the CM CTL subset. However, upon recognition of HLA-A2 C XBP1 antigen expressing cancer cells, the CM subset of XBP1-CTL expressing T-bet or Eomes had the greatest functional antitumor activities including IFNg production IL-2 production, granzyme B expression, and CD107a degranulation. Thus, our in vitro analyses provide the evidence of the critical role of T-bet and Eomes transcription factors regulating the antitumor activities within antigen-specific CTL and support results from other studies demonstrating that both transcription regulators cooperate to promote CTL formation during CD8 C T cell activation. [25][26][27] Recent studies have indicated that the administration of long peptides encompassing Th1 and CTL epitopes has the potential to elicit both CD4 C and CD8 C T-cell responses through crosspresentation, and more favorable clinical outcomes are associated with immune responses toward many MHC class I and class II epitopes derived from multiple tumor antigens. 63,64 Therefore, both CD4 C and CD8 C T cells play an important role in inducing and maintaining antitumor immunity, and we are interested in identifying long peptides specific to XBP1, CD138 and CS1 antigens and developing cancer vaccines based on respective single polypeptide encompassing epitopes, which can elicit both Th1 and CTL responses.
In summary, we demonstrate ex vivo that a cocktail of highly immunogenic HLA-A2 specific peptides, heteroclitic XBP1 US [184][185][186][187][188][189][190][191][192] and heteroclitic XBP1 SP 367-375 , can evoke XBP1-specific CTL with antitumor activities against a variety of solid tumor cancers that express high levels of unspliced and spliced XBP1 proteins. These data provide evidence for induction of a complete repertoire of XBP1-specific CM and EM CD3 C CD8 C T cells, which have a high proliferative capacity and strong antitumor activity against breast, colon and, pancreatic cancer cells. Therefore, we provide the framework for a cancer vaccine trial with the cocktail of heteroclitic XBP1 peptides to evoke CTL targeting both the unspliced and spliced XBP1 protein in order to delay or prevent disease progression in a variety of suitable cancer patient populations.  . High level of IFNg production, IL-2 production and CD107a degranulation by CM XBP1-CTL subset to various solid tumor cells. XBP1-CTL memory cell subsets were analyzed for their antitumor activity in response to a variety of solid tumor cells. Representative flow cytometric analyses showed higher levels of IFNg production (A), IL-2 production (B) and CD107a degranulation (C) within the CM subset of XBP1-CTL. Specific IFNg production (D), IL-2 production (E) and CD107a degranulation (F) were consistently higher against the respective tumor cells within the CM as compared to the EM subset of XBP1-CTL (n D 2). Results of the antitumor response are shown as a percentage of the parent CM or EM CD3 C CD8 C CTL population.
Analyses of XBP1 gene expression in cancer patients Expression analysis of the XBP1 gene was performed using canEvolve (www.canevolve.org) 65 and Oncomine (www.onco mine.org) 66 databases. The Oncomine database covers two subtypes for colon adenocarcinoma and six subtypes of breast cancer, as well as the appropriate control sample data. A differential analysis in Oncomine was used to compare expression in cancer versus healthy donors, with the student's t-test to calculate p values. 65 The canEvolve database uses linear models to calculate differential gene expression between groups and calculates the p values for given dataset. . Enhanced levels of T-bet, Eomes and granzyme B expression in memory CD3 C CD8 C T cell subsets of heteroclictic XBP1-CTL. The expression level of T-bet, Eomes and granzyme B was analyzed within the CD45RO C memory and CD45RO ¡ non-memory subsets of XBP1-CTL (n D 3). A higher frequency of cells expressing T-bet (A) and Eomes (B) was detected within the CD45RO C memory cells as compared to CD45RO ¡ non-memory CD3 C CD8 C T cells. In addition, the frequency of granzyme B C cells was increased in CD45RO C memory cells as compared to CD45RO ¡ non-memory CD3 C CD8 C T cells in response to the respective tumor cells (C).

Evaluation of XBP1 expression in tumor cell lines
The cancer cell lines were evaluated for intracellular expression of XBP1 unspliced or XBP1 spliced protein by flow cytometry. In brief, cells were permeabilized using the Cytofix/Cytoperm solution (BD) and stained with rabbit anti-human XBP1 unspliced isoform or mouse anti-human XBP1 spliced isoform mAbs for 30 min at room temperature, washed with Perm/Wash solution (BD) and stained with donkey anti-rabbit IgG-PE or goat antimouse IgG-PE, respectively, for 30 min at 4 C. The cells were washed with Perm/Wash solution and fixed in 2% formaldehyde-PBS. The cells were analyzed using a FACS Canto II TM flow cytometer and DIVA TM v6.1 software (Becton Dickinson) after gating on the tumor cells population.
Phenotypic analysis of XBP1-CTL and evaluation of critical T cell markers XBP1-CTL were evaluated for the frequency of CD3 C CD8 C T cells and expression levels (% positive cells, mean fluorescence intensity (MFI)) of critical T cell surface markers including CD45RO, CCR7, CD38, CD40L, CD69, 41BB, ICOS, and TCRab. After staining with each specific antibody, the cells were washed and fixed in 2% paraformaldehyde. The cells were  analyzed using a LSRII Fortessa TM flow cytometer and DIVA TM v7.1 software (BD) after gating on non-memory or memory CD3 C CD8 C T cell populations.
IFNg/IL-2 production, CD107a degranulation, granzyme B, and T-bet/Eomes expression in XBP1-CTL The functional antitumor activities of XBP1-CTL were examined against a variety of solid tumor cell lines for IFNg/IL-2 production, CD107a degranulation, granzyme B, and T-bet/ Eomes expression. XBP1-CTL were co-incubated with various stimulator cells in the presence of CD107a-FITC mAb, and Brefeldin A and Monensin (BD) were added after 1 h incubation. After an additional 5 h incubation, cells were stained with mAbs specific to surface antigens including CD3, CD8 C , CD45RO, CCR7, and/or CD69. The cells were fixed/permeabilized, and stained with mAb specific to IFNg, IL-2, granzyme B, T-bet or Eomes. The cells were analyzed using a LSRII Fortessa TM flow cytometer and DIVA TM v7.1 software after gating on non-memory or memory CD3 C CD8 C T cell populations.

XBP1-CTL proliferation in response to tumor cell lines
To evaluate tumor-specific cell proliferation, CFSE (Molecular Probes) labeled XBP1-CTL were co-incubated with irradiated (20 Gy) HLA-A2 C or HLA-A2 ¡ cancer cell lines. On day 6, the cultures were harvested, stained with anti-CD3, CD8 C , CD45RO and CCR7 mAbs, and analyzed by flow cytometry to determine their specific cell proliferation.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Figure 9. Eomes expression and IFNg production by memory XBP1-CTL subsets in response to solid tumor cells. The memory:na€ ıve cell subsets of XBP1-CTL (n D 3) were evaluated for Eomes expression by flow cytometry. A higher frequency of Eomes expressing cells was observed within the EM subset of XBP1-CTL as compared to the CM and TE subsets (A). However, the highest level of Eomes C /IFNg C cells was observed within the CM subset of XBP1-CTL against HLA-A2 C MDA-MB231 breast cancer (B), HLA-A2 C Panc1 pancreatic cancer (C) or HLA-A2 C SW480 colon cancer cells (D).