Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8+ and CD4+ T-cell responses with multiple specificities including a novel DR7-restricted epitope.

Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8+ and CD4+ T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8+ and NY-ESO-183-99 for CD4+ T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7+ patients generated strong CD4+ T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8+ and CD4+ T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach.

Magnetic nanoparticle-induced hyperthermia with appropriate payloads: Paul Ehrlich's "magic (nano)bullet" for cancer theranostics?

Effective multimodal cancer management requires the optimal integration of diagnostic and therapeutic modalities. Radiation therapy, chemotherapy and immunotherapy, alone or in combination, are integral parts of various cancer treatment protocols. Hyperthermia at 39-45°C is a potent radiosensitiser and has been shown to improve therapeutic outcomes in various tumours through its synergy with chemotherapy. Gene silencing approaches, using small interfering RNAs and microRNAs, are also being explored in clinical trials in oncology. The rapid developments in multifunctional nanoparticles provide ample opportunities to integrate both diagnostic and therapeutic modalities into a single effective cancer "theranostic" vector. Nanoparticles could extravasate passively into the tumour tissues in preference to the adjacent normal tissues by capitalizing on the enhanced permeability and retention effect. Tumour targeting might be further augmented by conjugating tumour-specific peptides and antibodies onto the surface of these nanoparticles or by activation through electromagnetic radiations, laser or ultrasound. Magnetic nanoparticles can induce hyperthermia in the presence of an alternating magnetic field, thereby multifunctionally with tumour-specific payloads empowering tumour specific radiotheranostics (for both imaging and radiotherapy), chemotherapy drug delivery, immunotherapy and gene silencing therapy. Such a (nano)bullet could realise the "magic bullet" conceived by Paul Ehrlich more than a century ago. This article discusses the various aspects of this "magic (nano)bullet" and the challenges that need to be addressed to usher in this new paradigm in modern cancer diagnostics and therapeutics.

Identification of melanoma cells and lymphocyte subpopulations in lymph node metastases by FTIR imaging histopathology.

While early stages of melanoma are usually cured by surgery, metastatic melanomas are difficult to treat because the widely available options have low response rates. Careful and precise diagnosis and staging are essential to determine patient's risk and to select appropriate treatments. Fortunately, the recent progress in immunotherapy is very encouraging. In this context, it is important to characterize the intratumoral infiltration of immune cells in each patient, which is however not done routinely due to the lack of standardized methods. In this study, we used Fourier transform infrared (FTIR) imaging combined with multivariate statistical analyses to investigate non-metastatic and metastatic lymph nodes from melanoma patients. Our results show that the different cell types have different infrared spectral features allowing automated identification of these cell types. High recognition rates were obtained using a supervised partial least square discriminant analysis (PLS-DA) model. Melanoma cells were recognized with 87.1% sensitivity and 85.7% specificity, showing that FTIR spectroscopy has similar detection power as immunohistochemistry. Besides, FTIR imaging could also distinguish lymphocyte subpopulations (B and T cells). Finally, we investigated the changes in lymphocytes due to the presence of metastases. Interestingly, specific features of spectra of lymphocytes present in metastatic or tumor-free lymph nodes could be evidenced by PCA. A PLS-DA model was capable of predicting whether lymphocytes originated from invaded or non-invaded lymph nodes. These data demonstrate that FTIR imaging is capable to distinguish known and also novel biological features in human tissues, with potential practical relevance for histopathological diagnosis and biomarker assessment.

An infrared spectral signature of human lymphocyte subpopulations from peripheral blood.

Metastatic melanomas are frequently refractory to most adjuvant therapies such as chemotherapies and radiotherapies. Recently, immunotherapies have shown good results in the treatment of some metastatic melanomas. Immune cell infiltration in the tumor has been associated with successful immunotherapy. More generally, tumor infiltrating lymphocytes (TILs) in the primary tumor and in metastases of melanoma patients have been demonstrated to correlate positively with favorable clinical outcomes. Altogether, these findings suggest the importance of being able to identify, quantify and characterize immune infiltration at the tumor site for a better diagnostic and treatment choice. In this paper, we used Fourier Transform Infrared (FTIR) imaging to identify and quantify different subpopulations of T cells: the cytotoxic T cells (CD8+), the helper T cells (CD4+) and the regulatory T cells (T reg). As a proof of concept, we investigated pure populations isolated from human peripheral blood from 6 healthy donors. These subpopulations were isolated from blood samples by magnetic labeling and purities were assessed by Fluorescence Activated Cell Sorting (FACS). The results presented here show that Fourier Transform Infrared (FTIR) imaging followed by supervised Partial Least Square Discriminant Analysis (PLS-DA) allows an accurate identification of CD4+ T cells and CD8+ T cells (>86%). We then developed a PLS regression allowing the quantification of T reg in a different mix of immune cells (e.g. Peripheral Blood Mononuclear Cells (PBMCs)). Altogether, these results demonstrate the sensitivity of infrared imaging to detect the low biological variability observed in T cell subpopulations.

IRF4 and BATF are critical for CD8⁺ T-cell function following infection with LCMV.

CD8(+) T-cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses, beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T-cell function, which are still incompletely understood. Here, we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B-cell-activating transcription factor (BATF) are necessary for sustained CD8(+) T-cell effector function. Although Irf4(-/-) CD8(+) T cells were initially capable of proliferation, IRF4 deficiency resulted in limited CD8(+) T-cell responses after infection with the lymphocytic choriomeningitis virus. Consequently, Irf4(-/-) mice established chronic infections, but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T-cell function, limited immunopathology, and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T-cell immunity.

Breast cancer and melanoma cell line identification by FTIR imaging after formalin-fixation and paraffin-embedding.

Over the past few decades, Fourier transform infrared (FTIR) spectroscopy coupled to microscopy has been recognized as an emerging and potentially powerful tool in cancer research and diagnosis. For this purpose, histological analyses performed by pathologists are mostly carried out on biopsied tissue that undergoes the formalin-fixation and paraffin-embedding (FFPE) procedure. This processing method ensures an optimal and permanent preservation of the samples, making FFPE-archived tissue an extremely valuable source for retrospective studies. Nevertheless, as highlighted by previous studies, this fixation procedure significantly changes the principal constituents of cells, resulting in important effects on their infrared (IR) spectrum. Despite the chemical and spectral influence of FFPE processing, some studies demonstrate that FTIR imaging allows precise identification of the different cell types present in biopsied tissue, indicating that the FFPE process preserves spectral differences between distinct cell types. In this study, we investigated whether this is also the case for closely related cell lines. We analyzed spectra from 8 cancerous epithelial cell lines: 4 breast cancer cell lines and 4 melanoma cell lines. For each cell line, we harvested cells at subconfluence and divided them into two sets. We first tested the "original" capability of FTIR imaging to identify these closely related cell lines on cells just dried on BaF2 slides. We then repeated the test after submitting the cells to the FFPE procedure. Our results show that the IR spectra of FFPE processed cancerous cell lines undergo small but significant changes due to the treatment. The spectral modifications were interpreted as a potential decrease in the phospholipid content and protein denaturation, in line with the scientific literature on the topic. Nevertheless, unsupervised analyses showed that spectral proximities and distances between closely related cell lines were mostly, but not entirely, conserved after FFPE processing. Finally, PLS-DA statistical analyses highlighted that closely related cell lines are still successfully identified and efficiently distinguished by FTIR spectroscopy after FFPE treatment. This last result paves the way towards identification and characterization of cellular subtypes on FFPE tissue sections by FTIR imaging, indicating that this analysis technique could become a potential useful tool in cancer research.

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

The activatory receptor 2B4 is expressed in vivo by human CD8+ effector alpha beta T cells.

The membrane receptor 2B4 is a CD2 family member that is involved in lymphocyte activation. A fraction of human CD8+ alphabeta T cells up-regulate 2B4 in vivo, and here we demonstrate that this correlates with the acquisition of effector cell properties such as granzyme B and perforin expression, rapid IFN-gamma production, and down-regulation of the lymph node homing chemokine receptor CCR7. In PBLs from healthy donors, cytomegalovirus-specific effector T cells were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag recognized by T cells-1)-specific T cells were 2B4 negative. In melanoma patients, Melan-A-specific T cells up-regulated 2B4 in parallel with in vivo differentiation. This occurred in PBLs after vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes, likely through disease-associated activation of Melan-A-specific T cells. Thus, 2B4 expression correlates with CD8+ T cell differentiation in vivo.

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

Ex vivo analysis of tumor antigen specific CD8+ T cell responses using MHC/peptide tetramers in cancer patients.

The development of soluble tetrameric MHC/peptide complexes has opened the possibility to directly identify and monitor antigen-specific CD8+ T cells in different clinical situations. This represents a technological breakthrough for the field of cell-mediated immunity. For example, the direct identification and enumeration of tumor-specific CD8+ T cells at the tumor site and in blood has recently provided compelling evidence that strong anti-tumoral responses naturally occur in some cancer patients. Moreover, the use of tetramers plays an essential role in the design of vaccination protocols aimed at inducing a strong and protective CD8+ T cell-mediated anti-tumoral response in cancer patients. The monitoring of antigen-specific T cell responses elicited by various peptide-based vaccines tested in phase I clinical trials clearly indicates that tumor-specific CD8+ T cells can be activated effectively at least in some cancer patients. Thus, multiparameter monitoring of antigen-specific T cell responses that combines ex vivo tetramer staining with various phenotyping and functional assays provides a novel approach to assess the functional potential of tumor-specific T lymphocytes and may also facilitate the optimization of vaccination protocols.

Ex vivo IFN-gamma secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases.

To elucidate the functional heterogeneity of Ag-specific T lymphocyte populations, we combined labeling of lymphocytes with MHC/peptide tetramers and a cell surface affinity matrix for IFN-gamma. Magnetic cell sorting of IFN-gamma-positive lymphocytes allowed the selective enrichment and identification of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, memory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells secreted IFN-gamma upon challenge with cognate peptide, consistent with an effector/memory status. The sensitivity of the approach allowed the detection of significant numbers of CMV-specific IFN-gamma-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-gamma staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-gamma upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RA(low) Melan-A/MART-1 tumor-specific cells from three of three melanoma patients presented levels of activity similar to those found for influenza- or CMV virus-specific lymphocytes, compatible with a functional differentiation into competent effector/memory T lymphocytes in vivo. Notably, a sizable fraction of Melan-A/MART-1-specific cells from a patient secreted IFN-gamma ex vivo following peptide-based vaccination. Thus, the high sensitivity of the assay provides a valuable tool to monitor effector T cell responses in different clinical situations.

Expansion and functional maturation of human tumor antigen-specific CD8+ T cells after vaccination with antigenic peptide.

Peptide-based vaccines are currently being tested for their ability to induce or augment tumor antigen (Ag)-specific CD8+ T-cell responses in cancer patients. Here we report that the frequency of circulating CD8+ T cells directed against the Melan-A/MART-1 Ag increased >20-fold in an HLA-A2 melanoma patient immunized repeatedly with the corresponding antigenic peptide, as assessed by staining with HLA-A2/peptide tetramers. Multiparameter flow cytometric analysis demonstrated that the increase in total Melan-A-specific cell number was accompanied by a marked increase in the proportion of the cells that expressed an activated/memory surface phenotype. As assessed by ELISPOT assays and intracellular staining, the absolute number of Melan-A-specific cells able to secrete IFN-gamma increased >50-fold upon vaccination. When tested directly after cell sorting on the basis of tetramer staining, Melan-A-specific cells were weakly cytolytic but became highly active after in vitro restimulation. Altogether, these results indicate that large numbers of functionally active tumor Ag-specific CD8+ T cells can be obtained and maintained at high levels after in vivo activation by repeated peptide-based vaccination.

Human CD8(+) T cells expressing HLA-DR and CD28 show telomerase activity and are distinct from cytolytic effector T cells.

Cycling lymphocytes may express the enzyme telomerase which is involved in maintenance of telomere length and cell proliferation potential. In CD8(+) T cells freshly isolated from peripheral blood, we found that in vivo cycling cells expressed HLA-DR. Furthermore, CD28-positive cells are known to have longer telomeres than CD28-negative T cells. Therefore we used HLA-DR- and CD28-specific antibodies to sort CD8(+) T cells and measure telomerase activity ex vivo. Relatively high levels of telomerase activity were found in HLA-DR/CD28 double-positive cells. In contrast, HLA-DR-negative and CD28-negative cells had almost no telomerase activity. In summary, HLA-DR expression correlates with proliferation, and CD28 expression with proliferative potential. We have previously identified that ex vivo cytolytic CD8(+) T cells are CD56 (NCAM) positive. Here we show that HLA-DR(+) cells were rarely CD56(+) and vice versa. This demonstrates that telomerase-expressing and cytolytic CD8(+) T cells can be separated on the basis of the cell surface markers HLA-DR and CD56. Thus, activated CD8(+) T cells specialize and exert distinct functions correlating with surface molecule expression.

TNF receptor 1 (TNFR1) and CD95 are not required for T cell deletion after virus infection but contribute to peptide-induced deletion under limited conditions.

Deletion of mature T cells maintains cellular homeostasis and is involved in the maintenance of self tolerance to some peripheral self antigens. Previous studies have presented conflicting evidence for a role of the tumor necrosis factor receptor (TNFR) family member CD95 (Fas) in peripheral T cell deletion using CD95-deficient mice. To evaluate cooperation between CD95 and another TNFR family molecule, TNFR1, we generated mice deficient for both CD95 and TNFR1. We showed that TNFR1 and CD95 do not contribute to the decline of antigen-specific cytotoxic T lymphocytes after virus infection. Using TNFR1 / CD95-deficient mice expressing the P14 TCR specific for a lymphocytic choriomeningitis virus-derived peptide (p33) we showed that deletion of p33-specific CD8(+) T cells following high dose p33 administration is also normal. However, in non-TCR-transgenic TNFR1 / CD95-deficient mice treated with the same p33 regimen, tolerance induction was defective. These data indicate that TNFR1 and CD95 are dispensable for deletion of antigen-specific T cells after viral infection. However, under certain conditions, both TNFR1 and CD95 appear to cooperate in CD8(+) T cell deletion.

Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression.

Recent data suggest that human effector CD8+ T cells express a distinct CD27-CD45RAhigh (CD57+CD28-CD11ahigh) phenotype. Here, we propose that CTL effector function correlates better with CD56 (neuronal cell adhesion molecule (NCAM)) surface expression. CD56 was absent on cord blood CD8+ T cells, but was expressed by 4-30% of freshly isolated circulating CD8+ T cells from 15 adults. Dramatic oligoclonal expansions in 3/3 individuals were confined to the CD56+ subset of CD8+ T cells. The CD56+ subset generally contained high amounts of intracellular perforin and granzyme B. Finally, direct cytolytic capacity was closely restricted to the CD56+(CD45RAhigh) cells, better than to CD27-CD45RAhigh cells in 5/5 individuals analyzed. Thus, the phenotype corresponding to the circulating effector CD8+ T cell pool may be simplified and more precisely defined by the use of just two surface markers: CD8 and CD56.

Optimal activation of tumor-reactive T cells by selected antigenic peptide analogues.

Many mechanisms have been proposed to explain why immune responses against human tumor antigens are generally ineffective. For example, tumor cells have been shown to develop active immune evasion mechanisms. Another possibility is that tumor antigens are unable to optimally stimulate tumor-specific T cells. In this study we have used HLA-A2/Melan-A peptide tetramers to directly isolate antigen-specific CD8(+) T cells from tumor-infiltrated lymph nodes. This allowed us to quantify the activation requirements of a representative polyclonal yet monospecific tumor-reactive T cell population. The results obtained from quantitative assays of intracellular Ca(2+) mobilization, TCR down-regulation, cytokine production and induction of effector cell differentiation indicate that the naturally produced Melan-A peptides are weak agonists and are clearly suboptimal for T cell activation. In contrast, optimal T cell activation was obtained by stimulation with recently defined peptide analogues. These findings provide a molecular basis for the low immunogenicity of tumor cells and suggest that patient immunization with full agonist peptide analogues may be essential for stimulation and maintenance of anti-tumor T cell responses in vivo.

TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) can interact with various members of the TNF receptor family. Previously, we reported that TRAF2-deficient mice die prematurely and have elevated serum TNF levels. In this study, we demonstrate that TRAF2-deficient macrophages produce increased amounts of nitric oxide (NO) and TNF in response to TNF stimulation. Furthermore, we could enhance the survival of TRAF2-deficient mice by eliminating either TNF or TNFR1. Using these double-knockout mice, we show that in the absence of TRAF2, the T helper-dependent antibody response, CD40-mediated proliferation, and NF-kappaB activation are defective. These data demonstrate two important roles of TRAF2, one as a negative regulator of certain TNFR1 signals and the other as a positive mediator of CD40 signaling.

High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.