Transgene-enforced co-stimulation of CD4+ T cells leads to enhanced and sustained anti-tumor effector functioning

Background The role of co-stimulation in CD4+ T cell activation by professional APC is well established, while less is known of the role co-stimulation plays when CD4+ T cells interact directly with tumor cells. Methods Through genetic engineering of human CD4+ T cells, we tested the hypothesis that integration of co-stimulatory signaling domains within a tumor-targeting chimeric Ag receptor (CAR), the IL-13Ralpha2-specific IL-13-zetakine (IL13zeta), would enhance CD4+ T cell mediated responses against tumors that fail to express ligands for co-stimulatory receptors. Results Compared with CD3zeta-mediated activation alone, CD4+ effector T cells expressing the IL13-CD28-41BBzeta CAR exhibited augmented/sustained MAPK and AKT activity, up-regulated Th1 cytokine production, and enhanced cytolytic potency against tumor targets. Moreover, upon recursive stimulation with tumor, the IL13-CD28-41BBzeta+ cells retained/recycled their lytic function, whereas IL-13zeta+ CD4+ cells became anergic/exhausted. These in vitro observations correlated with enhanced in vivo control of established orthotopic CNS glioma xenografts in immunodeficient mice mediated by adoptively transferred ex vivo-expanded CD4+ T cells expressing the co-stimulatory CAR. Discussion Together these studies demonstrate the importance of integrating co-stimulation with CD3zeta signaling events to activate fully CD4+ anti-tumor effector cells for sustained function in the tumor microenvironment.     

Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB

The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8(+) T cells. The starting repertoire of CD8+ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8(+) T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy.     

Mechanism for down-regulation of CD28 by Nef

SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event.     

Inhibition of cell growth and induction of apoptotic cell death by the human tumor-associated antigen RCAS1.

Tumor-associated antigens that can be recognized by the immune system include the MAGE-family, p53, MUC-1, HER2/neu and p21ras. Despite their expression of these distinct antigens, tumor elimination by the immune system is often inefficient. Postulated mechanisms include insufficient expression of co-stimulatory or adhesion molecules by tumor cells, or defective processing and presentation of antigens on their cell surfaces. Tumor cells may also evade immune attack by expressing CD95 (APO-1/Fas) ligand or other molecules that induce apoptosis in activated T cells. Here we describe RCAS1 (receptor-binding cancer antigen expressed on SiSo cells), a membrane molecule expressed on human cancer cells. RCAS1 acts as a ligand for a putative receptor present on various human cell lines and normal peripheral lymphocytes such as T, B and NK cells. The receptor expression was enhanced by activation of the lymphocytes. RCAS1 inhibited the in vitro growth of receptor-expressing cells and induced apoptotic cell death. Given these results, tumor cells may evade immune surveillance by expression of RCAS1, which would suppress clonal expansion and induce apoptosis in RCAS1 receptor-positive immune cells.     

Anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells provokes potent T-lymphocyte activation and cytotoxicity.

The targeting of tumor cells by cytotoxic T lymphocytes is a promising strategy for biotherapy, but T cells require 2 signals via the T-cell receptor - CD3 complex and CD28 molecules for activation. To bridge the gap between cytotoxic T lymphocytes and tumor cells, our objective in this study was to describe the construction and the cell surface-anchored expression of a fusion protein, anti-CD3 scFv-B7.1, derived from inserting a fusion gene encoding anti-CD3 scFv and the extra-cellular domain of B7.1 fused by the splicing by overlap extension method into a mammalian expression vector, pDisplay. Transfection of the recombinant vector by electroporation into HeLa cells resulted in the production of protein migrating at approximately 57 kDa under reducing conditions. The expressed fusion protein could bind to T lymphocytes and induce strong T-cell activation. Meanwhile, a potent cytotoxicity was induced in the mixed culture of T-cell-modified tumor cells in a 96 h methyl-thiazolyl-diphenyl tetrazolium bromide assay. Our results indicate that this bifunctional protein, through activating T lymphocytes to lyse homologous human carcinomas, may be of potential value for T-cell-based immunotherapeutical treatment protocols in vivo.     

Comparative analysis of DC fused with allogeneic hepatocellular carcinoma cell line HepG2 and autologous tumor cells as potential cancer vaccines against hepatocellular carcinoma

Fusions of patient-derived dendritic cells (DCs) and autologous tumor cells induce T-cell responses against autologous tumors in animal models and human clinical trials. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here we fused autologous DCs from patients with hepatocellular carcinoma (HCC) to an allogeneic HCC cell line (HepG2). These fusion cells co-expressed tumor-associated antigens (TAAs) and DC-derived costimulatory and MHC molecules. Both CD4⁺ and CD8⁺ T cells were activated by the fusion cells. Cytotoxic T lymphocytes (CTLs) induced by the fusion cells were able to kill autologous HCC by HLA-A2- and/or HLA-A24-restricted mechanisms. CTL activity against shared TAAs indicates that the presence of alloantigens does not prevent the development of CTLs with activity against autologous HCC cells. These fusion cells may have applications in anti-tumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.     

A new recombinant single chain trispecific antibody recruits T lymphocytes to kill CEA (carcinoma embryonic antigen) positive tumor cells in vitro efficiently

Anti-tumor associated antigen (TAA).CD3.CD28 trispecific antibody(TsAb) is able to provide two signals for fully and continuously activation of T lymphocytes and recruit them around tumor cells, presenting an attractive concept in tumor immunotherapy. Here, a new single chain trispecific antibody (scTsAb), named CEA-scTsAb, was constructed by fusion of anti-CEA (Carcinoma Embryonic Antigen) single chain antibody (scFv), anti-CD3 scFv and anti-CD28 VH, spaced by polypeptide interlinkers taken from the fragment of constant region (FC) of human IgG and human serum albumin (HSA). It was expressed in Escherichia coli at low temperature (30 degrees C) with up to 50% of the antibody being present in soluble form. After one step of DEAE anion chromatography, the soluble product was sufficiently pure for further in vitro activity assays. First, it was proved that CEA-scTsAb could recognize three antigens (CEA, CD28 and Jurkat cell membrane antigen) specifically and could distinguish antigen positive cells from antigen negative cells in vitro. Then fresh PBMC (peripheral blood mononuclear cells), without being pre-treated by co-stimulatory reagents, such as IL-2 or CD28 mAb, were used as effector cells to test their ability to mediate tumor specific cytolysis of CEA-positive tumor cells, SW1116. It was found by photomicrography that T lymphocytes were attracted to SW1116 cells in the presence of CEA-scTsAb, which resulted in effective cytolysis of tumor cells. As shown by MTT assay, the efficacy of tumor specific cytolysis mediated by CEA-scTsAb related to both the quantity and activation of PBMC. At an effector cells/target cells ratio (E/T) of 5, it was proved by dual-color FACS with propidium iodide (PI) and FITC-annexin V that both necrosis and apoptosis of tumor cells were causes of tumor specific cytolysis. In summary, a new single chain trispecific (CEA x CD3 x CD28) antibody was constructed and characterized carefully in this paper and was found to possess functions: (i) to activate T lymphocytes independently of additional co-stimulatory signal, (ii) to attract activated T lymphocytes around CEA-positive tumor cells, (iii) to attack CEA-positive tumor cells with recruited T lymphocytes. Because it recognizes a widely distributed tumor antigen (CEA), with moderate molecular weight (about 75 kDa) and a simple production procedure, and is able to mediate a high level of tumor specific cytolysis without any additional co-stimulating reagents, CEA-scTsAb is very promising for the task of immunotherapy in future.     

Co-stimulatory members of the TNFR family: keys to effective T-cell immunity?

Interactions between co-stimulatory ligands and their receptors are crucial for the activation of T cells, the prevention of tolerance and the development of T-cell immunity. It is now evident that members of the immunoglobulin-like CD28-B7 co-stimulatory family cannot fully account for an effective long-lasting T-cell response or the generation of memory T cells. Several members of the tumour-necrosis factor receptor (TNFR) superfamily--OX40, 4-1BB, CD27, CD30 and HVEM (herpes-virus entry mediator)--are poised to deliver co-stimulatory signals both early and late after encounter with antigen. The roles of these molecules in initiating and sustaining the T-cell response and in promoting long-lived immunity are discussed.     

Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population

T-cell co-stimulation delivered by the molecules B7-1 or B7-2 through CD28 has a positive effect on T-cell activation, whereas engagement of cytotoxic T-lymphocyte antigen 4 (CTLA-4) by these molecules inhibits activation. In vivo administration to mice of blocking monoclonal antibodies or Fab fragments against CTLA-4 can augment antigen-specific T-cell responses and, thus, therapy with monoclonal antibody against CTLA-4 has potential applications for tumor therapy and enhancement of vaccine immunization. The effects of B7-1 and B7-2 co-stimulation through CD28 depend on the strength of the signal delivered through the T-cell receptor (TCR) and the activation state of T cells during activation. Thus, we sought to determine whether these factors similarly influence the effect of B7-mediated signals delivered through CTLA-4 during T-cell activation. Using freshly isolated human T cells and Fab fragments of a monoclonal antibody against CTLA-4, we demonstrate here that CTLA-4 blockade can enhance or inhibit the clonal expansion of different T cells that respond to the same antigen, depending on both the T-cell activation state and the strength of the T-cell receptor signal delivered during T-cell stimulation. Thus, for whole T-cell populations, blocking a negative signal may paradoxically inhibit immune responses. These results provide a theoretical framework for clinical trials in which co-stimulatory signals are manipulated in an attempt to modulate the immune response in human disease.     

Lymphocyte coreceptors

The activation of the immune system is tightly regulated by positive and negative receptors that allow the fine tuning of the immune cells. This regulation relies on receptors that were initially described in T lymphocytes, but have now been identified on cells from both innate and acquired immunity. The co-stimulatory receptors can allow cell activation or amplify it, regulate cell suvival and determine their effector functions. The co-inhibitory receptors can either prevent, decrease of inhibit the activation and differentiation process. The co-stimulatory and co-inhibitory molecules belong mainly to the so-called Ig superfamily and historically were called < CD28 and B7 family >. The members of the tumor necrosis factor receptor (TNFR) family devoid of intra-cytoplasmic death domain but binding TNF receptor associated factors (TRAF) are also important but are up to now mainly co-stimulatory. The prototypical co-stimulatory molecules belonging to CD28 family are CD28 and ICOS, whereas the co-inhibitory molecules identified so far are CTLA-4, PD-1 and BTLA. Their receptors belong in most instances to the B7 family. For instance, B7.1/CD80 and B7.2/CD86 interact both with CD28 and CTLA-4 ; PDL1 and PDL2 bind to PD-1. The exception being so far BTLA which interacts with the TNFR family member HVEM (Herpes virus entry mediator). Three other B7 family members B7-H3, B7-H4 and BT3.1 are orphan receptors until now. The basis of co-inhibition rely on distinct mechanisms, one that has been postulated being the ability of the intracytoplasmic domain of PD-1 and BTLA to bind to the protein tyrosine phosphatases SHP-1 and SHP-2. The pathways used by the co-stimulatory receptors are also not completely understood and rely for CD28 both on signal similar to the one elicited by TcR and consequently increasing the overall signal and other more specific, elicited by the activation of PI3-OH kinase, vav1 and rearrangement of cytoskeleton. Recently, reverse signaling has been described for B7 family members which further increases the spectrum of functions elicited by these families. Co-stimulation and co-inhibition are among the most promising molecules and pathways to be targeted by mAbs, recombinant proteins and drugs in auto-immune diseases, transplantation and cancer.     

Human immunodeficiency virus-specific circulating CD8 T lymphocytes have down-modulated CD3zeta and CD28, key signaling molecules for T-cell activation

Although human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3zeta, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3zeta down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3zeta-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3zeta(-). CD8 T cells with down-modulated CD3zeta also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR(+) CD62L(-)). After T-cell activation, CD3zeta-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor alpha-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3zeta is not reexpressed even after IL-2 exposure.     

Activation of c-jun N-terminal kinase by 4-1BB (CD137), a T cell co-stimulatory molecule

It has been widely accepted that T cell activation requires two signals; one from the binding of the antigen/major histocompatibility complex to the T-cell receptor (TCR)/CD3 complex and the other from the interaction between a surface molecule on antigen presenting cells and its receptor on T cells. The second signal is considered as co-stimulatory and the B7/CD28 pair has been well studied as a prototype. Recently 4-1BB (CD137) has been characterized as another co-stimulatory molecule for T cell activation. However, unlike the CD28/B7 pair, 4-1BB and its ligand 4-1BBL constitute a member of the tumor necrosis factor (TNF) receptor/TNF pair superfamily. The signaling mechanism of 4-1BB has not been revealed in detail. To investigate whether 4-1BB takes the signaling pathways analogous to those for TNF receptors, we generated polyclonal antibodies against human 4-1BB and 4-1BBL and established stable transfectants of the receptor and the ligand with a high level of cell surface expression. Over-expression of h4-1BB was found to result in the activation of c-Jun N-terminal kinase (JNK) in the human embryonic kidney cell line 293. In T cells, it has been previously demonstrated that JNK activation requires dual signals such as the ligation of TCR/CD3 complex plus CD28 co-stimulation or PMA plus ionomycin. The JNK activation by 4-1BB in Jurkat T cells was also found to require stimulation of the TCR/CD3 complex, consistent with the notion that 4-1BB functions as a co-stimulatory molecule for T cell activation.     

Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4.

Interaction of the B7 molecule on antigen-presenting cells with its receptors CD28 and CTLA-4 on T cells provides costimulatory signals for T cell activation. We have studied the effects of B7 on antitumor immunity to a murine melanoma that expresses a rejection antigen associated with the E7 gene product of human papillomavirus 16. While this E7+ tumor grows progressively in immunocompetent hosts, cotransfection of its cells with B7 led to tumor regression by a B7-dependent immune response mediated by CD8+ cytolytic T lymphocytes. The immune response induced by E7+B7+ tumor cells also caused regression of E7+B7- tumors at distant sites and was curative for established E7+B7- micrometastases. Our findings suggest that increasing T cell costimulation through the CD28 and CTLA-4 receptors may have therapeutic usefulness for generating immunity against tumors expressing viral antigens.     

CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.     

Expression of anti-HVEM single-chain antibody on tumor cells induces tumor-specific immunity with long-term memory

Genetic engineering of tumor cells to express immune-stimulatory molecules, including cytokines and co-stimulatory ligands, is a promising approach to generate highly efficient cancer vaccines. The co-signaling molecule, LIGHT, is particularly well suited for use in vaccine development as it delivers a potent co-stimulatory signal through the Herpes virus entry mediator (HVEM) receptor on T cells and facilitates tumor-specific T cell immunity. However, because LIGHT binds two additional receptors, lymphotoxin β receptor and Decoy receptor 3, there are significant concerns that tumor-associated LIGHT results in both unexpected adverse events and interference with the ability of the vaccine to enhance antitumor immunity. In order to overcome these problems, we generated tumor cells expressing the single-chain variable fragment (scFv) of anti-HVEM agonistic mAb on the cell surface. Tumor cells expressing anti-HVEM scFv induce a potent proliferation and cytokine production of co-cultured T cells. Inoculation of anti-HVEM scFv-expressing tumor results in a spontaneous tumor regression in CD4+ and CD8+ T cell-dependent fashion, associated with the induction of tumor-specific long-term memory. Stimulation of HVEM and 4-1BB co-stimulatory signals by anti-HVEM scFv-expressing tumor vaccine combined with anti-4-1BB mAb shows synergistic effects which achieve regression of pre-established tumor and T cell memory specific to parental tumor. Taken in concert, our data suggest that genetic engineering of tumor cells to selectively potentiate the HVEM signaling pathway is a promising antitumor vaccine therapy.     

Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma

Cytotoxic T lymphocytes (CTLs) directed to nonviral tumor-associated antigens do not survive long term and have limited antitumor activity in vivo, in part because such tumor cells typically lack the appropriate costimulatory molecules. We therefore engineered Epstein-Barr virus (EBV)-specific CTLs to express a chimeric antigen receptor directed to the diasialoganglioside GD2, a nonviral tumor-associated antigen expressed by human neuroblastoma cells. We reasoned that these genetically engineered lymphocytes would receive optimal costimulation after engagement of their native receptors, enhancing survival and antitumor activity mediated through their chimeric receptors. Here we show in individuals with neuroblastoma that EBV-specific CTLs expressing a chimeric GD2-specific receptor indeed survive longer than T cells activated by the CD3-specific antibody OKT3 and expressing the same chimeric receptor but lacking virus specificity. Infusion of these genetically modified cells seemed safe and was associated with tumor regression or necrosis in half of the subjects tested. Hence, virus-specific CTLs can be modified to function as tumor-directed effector cells.     

Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors     

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA⁺ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.