Functional endogenous cytotoxic T lymphocytes are generated to multiple antigens co-expressed by progressing tumors; after intra-tumoral IL-2 therapy these effector cells eradicate established tumors

Tumors contain many antigens that may be recognized by the immune system. It is not known whether these antigens, and the epitopes within these antigens, can all be recognized by the anti-tumor immune response or if such responses are restricted to a few dominant epitopes. Effector function of endogenous cytotoxic T lymphocytes (CTL) generated during tumor progression has previously been assessed by indirect, ex vivo assays, which often focused on a single antigen. Therefore, we evaluated the endogenous in vivo CTL response to multiple neo tumor antigens using murine Lewis lung carcinoma tumor cells transfected with ovalbumin or a polyepitope construct. Both express multiple MHC class I-restricted epitopes. Ovalbumin contains a known hierarchy of epitopes for given MHC molecules, whilst the polyepitope expresses a number of dominant epitopes. We show that as tumors progress, potent effector CTL are generated in vivo that are restricted to dominant epitopes; we did not see the responses to subdominant or cryptic epitopes. Our data show that the CTL recognizing tumor antigens vary in their lytic capacity, as the CTL responding to two of the four epitopes were particularly potent killers. The presence of these effector CTLs did not prevent tumor growth. However, intra-tumoral IL-2 treatment altered the potency, but not the hierarchy, of these CTL such that they mediated tumor regression. These results have implications for immunotherapy protocols.     

Defining MHC class II T helper epitopes for WT1 tumor antigen

The product of Wilms‘ tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4⁺ helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1_124–138 and WT1_247–261, were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.     

An efficient and versatile mammalian viral vector system for major histocompatibility complex class I/peptide complexes

We report a Sendai virus (SeV) vector system for expression of major histocompatibility complex (MHC) class I/peptide complexes. We cloned the extracellular domain of a human MHC class I heavy chain, HLA-A*2402, and human beta-2 microglobulin (beta2m) fused with HLA-A*2402-restricted human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte (CTL) epitopes (e-beta2m) in separate SeV vectors. When we coinfected nonhuman mammalian cells with the SeVs, naturally folded human MHC class I/peptide complexes were secreted in the culture supernatants. Biotin binding peptide sequences on the C terminus of the heavy chain were used to tetramerize the complexes. These tetramers made in the SeV system recognized specific CD8-positive T cells in peripheral blood mononuclear cells of HIV-1-positive patients with a specificity and sensitivity similar to those of MHC class I tetramers made in an Escherichia coli system. Solo infection of e-beta2m/SeV produced soluble e-beta2m in the culture supernatant, and cells pulsed with the soluble protein were recognized by specific CTLs. Furthermore, when cells were infected with e-beta2m/SeV, these cells were recognized by the specific CTLs more efficiently than the protein pulse per se. SeV is nonpathogenic for humans, can transduce foreign genes into nondividing cells, and may be useful for immunotherapy to enhance antigen-specific immune responses. Our system can be used not only to detect but also to stimulate antigen-specific cellular immune responses.     

The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8⁺ or CD4⁺ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.     

Recognition of naturally processed and ovarian cancer reactive CD8<Superscript>+</Superscript> T cell epitopes within a promiscuous HLA class II T-helper region of NY-ESO-1

NY-ESO-1 is frequently expressed in epithelial ovarian cancer (EOC) and elicits spontaneous humoral and cellular immune responses in a proportion of EOC patients. The identification of NY-ESO-1 peptide epitopes with dual HLA-class I and class II specificities might be useful in vaccination strategies for generating cognate CD4+ T cell help to augment CD8+ T cell responses. Here, we describe two novel NY-ESO-1-derived MHC class I epitopes from EOC patients with spontaneous humoral immune response to NY-ESO-1. CD8+ T cells derived from NY-ESO-1 seropositive EOC patients were presensitized with a recombinant adenovirus encoding NY-ESO-1or pooled overlapping peptides. These epitopes, ESO127-136 presented by HLA-A68 molecule, and ESO127-135 restricted by HLA-Cw15 allele, are located within ESO119-143, a promiscuous HLA-class II region containing epitopes that bind to multiple HLA-DR alleles. The novel epitopes were naturally processed by APC or naturally presented by tumor cell lines. In addition, these epitopes induced NY-ESO-1-specific CTL in NY-ESO-1 seropositive EOC patients. Together, the results indicate that ESO119-143 epitope has dual HLA classes I and II specificities, and represents a potential vaccine candidate in a large number of cancer patients.     

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.     

Using the natural evolution of a rotavirus-specific human monoclonal antibody to predict the complex topography of a viral antigenic site

Background

Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8⁺ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL.

Results

Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-γ ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8⁺ T cell responses were observed during the protective immune response against sporozoite challenge.

Conclusion

The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

In vivo generation of cytotoxic T cells from epitopes displayed on peptide-based delivery vehicles

The development of nonviral, peptide-based constructs able to elicit protective in vivo CTL responses represents a major challenge in the design of future vaccines. We report the design of branched peptide delivery vehicles, termed loligomers, that facilitate the import, processing, and presentation of CTL epitopes onto nascent MHC class I molecules. These complexes are then effectively displayed on the surface of APCs. The intracellular delivery of CTL epitopes by loligomers prolonged the expression of Ag-MHC class I complexes on the surface of APCs in comparison with free CTL epitope alone. Furthermore, the injection of CTL epitope-containing loligomers into mice led to the generation of in vivo CTL responses and the induction of autoimmune disease in an animal model. Synthetic epitope-carrying, peptide-based delivery vehicles may represent useful components to be included in the formulation of future vaccines.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Tumor-specific cytolysis by lymphocytes infiltrating human melanomas

Tumor infiltrating lymphocytes (TIL) were grown in IL-2 from single cell tumor suspensions of 14 human melanomas resected from 12 patients. As a function of time in culture, 4 of 14 TIL cultures eventually expressed highly specific cytolytic activity against fresh autologous melanoma targets in short term chromium release assays, failing to lyse multiple allogeneic tumors or autologous normal cells. These highly specific TIL were identified as CTL by phenotype (CD3+/CD4-/CD8+/Leu7-) and by function (lysis inhibited by antibodies directed against CD3 and MHC class I molecules). Cell separation experiments using immunomagnetic beads identified a highly tumor-specific CTL subpopulation within a nonspecific TIL culture, suggesting that the lytic activity of tumor-specific CTL may be diluted by the nonspecific killer activity present in heterogeneous TIL cultures. These studies provide evidence for specific MHC-restricted human immune responses against autologous tumor in cancer-bearing patients, and may be of importance to ongoing clinical trials using TIL in the immunotherapy of advanced malignancies.     

Survivin--a universal tumor antigen

Tumor-associated antigens recognized by cellular effectors of the immune system are potential targets for antigen-specific cancer immunotherapy. These antigens are classified as tissue (melanocyte)-specific proteins, cancer-testis antigens (proteins expressed in normal testis and various cancers), tumor-specific peptides derived from mutations in tumor cells, and others. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific cytotoxic T lymphocytes (CTL) responses in vivo. However, most of the peptide epitopes used in these vaccination trials are melanocyte-specific, and these peptides cannot be applied for tumors of non-melanocyte origin. Furthermore, the expression of most tumor antigens is heterogeneous among tumors from different patients and can even vary among metastases obtained from one patient. Immune selection of antigen loss variants may prove to be an additional obstacle for the clinical applicability of most of the known CTL epitopes. Recently, a new tumor antigen, survivin, has been identified on the basis of spontaneous CTL responses in different cancer patients. Survivin is expressed in most human neoplasms, but not in normal, differentiated tissues. Importantly, downregulation or loss of survivin would severely inflict the growth potential of the tumor cell. Since survivin is expressed by a variety of different tumors MHC-restricted survivin epitopes may serve as important and widely applicable targets for anti-cancer immunotherapeutic strategies.     

In vivo cross-presentation of a soluble protein antigen: kinetics, distribution, and generation of effector CTL recognizing dominant and subdominant epitopes

Cross-presentation of exogenous Ags via the MHC class I pathway is now recognized for its role in self-tolerance, tumor immunity, and vaccine development. However, little is known about the in vivo distribution and kinetics of cross-presented protein Ags, nor the subsequent development of CTL effector responses to dominant or subdominant epitopes. We examined the location and duration of cross-presented Ag by using 5,6-carboxy-succinimidyl-fluorescein ester-labeled T cells from class I-restricted Ag-specific TCR mice. Comparisons of results from an in vitro (51)Cr release CTL assay with an in vivo CTL assay provided physiologically relevant insights into the functional capacities of CTL specific for epitopes with differing affinities. These data demonstrate that efficient cross-presentation of a dominant class I-restricted Ag is dose related and remains largely localized, but not limited to the draining lymph nodes for up to 3 wk following a single injection of soluble protein. Within this period, dominant peptide-specific CTL are fully functional in vivo throughout the secondary lymphoid system. However, no in vivo responses are seen to a subdominant or cryptic epitope. Prolonging Ag cross-presentation via use of IFA promoted persisting in vivo dominant epitope-specific CTL activity and revealed dose-responsive precursor CTL to the subdominant, but not to a cryptic epitope. Analysis of functional in vivo CTL responses demonstrated that, in the presence of strong ongoing responses to the dominant peptide, lytic activity of CTL directed at weaker epitopes is undetectable.     

Chemotherapy broadens the range of tumor antigens seen by cytotoxic CD8+ T cells in vivo

Cytotoxic chemotherapies may expose the immune system to high levels of tumor antigens and expand the CD8⁺ T-cell response to include weak or subdominant antigens. Here, we evaluated the in vivo CTL response to tumor antigens using a murine mesothelioma tumor cell line transfected with a neotumor antigen, ovalbumin, that contains a known hierarchy of epitopes for MHC class I molecules. We show that as tumors progress, effector CTLs are generated in vivo that focus on the dominant epitope SIINFEKL, although a weak response was seen to one (KVVRFDKL) subdominant epitope. These CTLs did not prevent tumor growth. Cisplatin treatment slowed tumor growth, slightly improved in vivo SIINFEKL presentation to T cells and reduced SIINFEKL-CTL activity. However, the CTL response to KVVRFDKL was amplified, and a response to another subdominant epitope, NAIVFKGL, was revealed. Similarly, gemcitabine cured most mice, slightly enhanced SIINFEKL presentation, reduced SIINFEKL-CTL activity yet drove a significant CTL response to NAIVFKGL, but not KVVRFDKL. These NAIVFKGL-specific CTLs secreted IFN?? and proliferated in response to in vitro NAIVFKGL stimulation. IL-2 treatment during chemotherapy refocused the response to SIINFEKL and simultaneously degraded the cisplatin-driven subdominant CTL response. These data show that chemotherapy reveals weaker tumor antigens to the immune system, a response that could be rationally targeted. Furthermore, while integrating IL-2 into the chemotherapy regimen interfered with the hierarchy of the response, IL-2 or other strategies that support CTL activity could be considered upon completion of chemotherapy.     

Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression

Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and c separately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRB loci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.     

The varicellovirus-encoded TAP inhibitor UL49.5 regulates the presentation of CTL epitopes by Qa-1b1

Impairment of MHC class I Ag processing is a commonly observed mechanism that allows viruses and tumors to escape immune destruction by CTL. The peptide transporter TAP that is responsible for the delivery of MHC class I-binding peptides into the endoplasmic reticulum is a pivotal target of viral-immune evasion molecules, and expression of this transporter is frequently lost in advanced cancers. We recently described a novel population of CTL that intriguingly exhibits reactivity against such tumor-immune escape variants and that recognizes self-peptides emerging at the cell surface due to defects in the processing machinery. Investigations of this new type of CTL epitopes are hampered by the lack of an efficient inhibitor for peptide transport in mouse cells. In this article, we demonstrate that the varicellovirus protein UL49.5, in contrast to ICP47 and US6, strongly impairs the activity of the mouse transporter and mediates degradation of mouse TAP1 and TAP2. Inhibition of TAP was witnessed by a strong reduction of surface MHC class I display and a decrease in recognition of conventional tumor-specific CTL. Analysis of CTL reactivity through the nonclassical molecule Qa-1(b) revealed that the presentation of the predominant leader peptide was inhibited. Interestingly, expression of UL49.5 in processing competent tumor cells induced the presentation of the new category of peptides. Our data show that the varicellovirus UL49.5 protein is a universal TAP inhibitor that can be exploited for preclinical studies on CTL-based immune intervention.     

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2⁺ tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369–377, 435–443 and 689–697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4⁺ T cells and antibodies are important components. S. Vertuani and C. Triulzi contributed equally to this work.     

Lactobacilli secreting a tumor antigen and IL15 activates neutrophils and dendritic cells and generates cytotoxic T lymphocytes against cancer cells

Lactobacillus rhamnosus GG (LGG) has been used to successfully induce tumor regression in an orthotopic model of bladder cancer. Increased infiltration of neutrophils and macrophages into the tumor mass was observed after therapy. This study evaluates the potential of LGG to induce a directed anti-tumor response. Lactobacilli were modified to secrete the prostate specific antigen (PSA) or IL15 and PSA (IL-15-PSA). Neutrophils and DC were exposed to LGG for 2 h as in clinical therapy for bladder cancer. Recombinant LGG activated neutrophils (elevated MHC class I expression) induced DC maturation (increased expression of CD86, CD80, CD40, MHC II and CD83), T cell proliferation and PSA specific cytotoxic T lymphocytes (CTL) activity. IL15 enhanced direct DC activation of CTL. Thus LGG secreting tumor antigens may activate antigen specific immune responses when instilled intravesically and IL15 could enhance this response.