Frequencies of 4 tumor-infiltrating lymphocytes potently predict survival in glioblastoma, an immune desert.

BACKGROUND: GBM is an aggressive grade 4 primary brain tumor (BT), with a 5%-13% 5-year survival. Most human GBMs manifest as immunologically "cold" tumors or "immune deserts," yet the promoting or suppressive roles of specific lymphocytes within the GBM tumor microenvironment (TME) is of considerable debate. METHODS: We used meticulous multiparametric flow cytometry (FC) to determine the lymphocytic frequencies in 102 GBMs, lower-grade gliomas, brain metastases, and nontumorous brain specimen. FC-attained frequencies were compared with frequencies estimated by "digital cytometry." The FC-derived data were combined with the patients' demographic, clinical, molecular, histopathological, radiological, and survival data. RESULTS: Comparison of FC-derived data to CIBERSORT-estimated data revealed the poor capacity of digital cytometry to estimate cell frequencies below 0.2%, the frequency range of most immune cells in BTs. Isocitrate dehydrogenase (IDH) mutation status was found to affect TME composition more than the gliomas' pathological grade. Combining FC and survival data disclosed that unlike other cancer types, the frequency of helper T cells (Th) and cytotoxic T lymphocytes (CTL) correlated negatively with glioma survival. In contrast, the frequencies of γδ-T cells and CD56bright natural killer cells correlated positively with survival. A composite parameter combining the frequencies of these 4 tumoral lymphocytes separated the survival curves of GBM patients with a median difference of 10 months (FC-derived data; P < .0001, discovery cohort), or 4.1 months (CIBERSORT-estimated data; P = .01, validation cohort). CONCLUSIONS: The frequencies of 4 TME lymphocytes strongly correlate with the survival of patients with GBM, a tumor considered an immune desert.

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools.

Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05-0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

CTGF/CCN2 has a possible detrimental role in the inflammation and the remyelination failure in the early stages of multiple sclerosis.

Connective tissue growth factor (CTGF/CCN2) is a proinflammatory and an oligodendrocyte-differentiating blocking agent. It is found in MS lesions, which raises the possibility of involvement in MS pathogenesis. We found that its CSF and serum levels were higher in RR-MS patients than in controls and for serum compared to PP and SP-MS. Immune cells of both RR-MS and controls secreted CTGF/CCN2, which was enhanced by CD3/CD28 stimulation or by LPS. Anti-CTGF treatment of mice with experimental autoimmune encephalitis ameliorated its clinical severity. CTGF/CCN2 may play a role in the immune pathogenesis of MS and in remyelination failure in early stages of MS.

T Cells Retain Pivotal Antitumoral Functions under Tumor-Treating Electric Fields.

Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields' locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)-T-based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag-specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients' glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.

Rh2-enriched Korean ginseng (Ginseng Rh2+) inhibits tumor growth and development of metastasis of non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: While there are multiple studies on the anti-tumoral effects of Panax ginseng as active ingredients (one or more ginsenosides derived from the extract) or as a whole plant extract, there is a lack of studies to assess the effects Panax ginseng's of active ingredients combined with the whole plant extract. Our aim was to study the effect of whole ginseng, enriched in the anti-tumoral Rh2 component and other ginsenosides (Ginseng Rh2+), on the metastatic capacity of non-small cell lung cancer (NSCLC). METHODS: We evaluated the effects of Ginseng Rh2+ on survival, migration and motility, induction of apoptosis, and expression of its apoptosis-related proteins in non-small cell lung cancer (NSCLC) cells in vitro and on primary tumor growth and metastatic capacity in a syngeneic mouse lung cancer model in vivo. The effects of Ginseng Rh2+ on NSCLC cells were studied in vitro using: a colorimetric tetrazolium salt (XTT) assay, annexin V-FITC/PI, western blotting, wound healing motility assay, Transwell migration and cell adhesion assays. In vivo, mice were inoculated with Lewis mouse lung carcinoma cells subcutaneously to evaluate local tumor growth, or intravenously to evaluate the effects of Ginseng Rh2+ on development of experimental metastases. Mice were treated by intraperitoneal administration of Ginseng Rh2+ (0.005-0.5 g kg-1) on days 6, 10, and 14 after tumor injection. RESULTS: We found that Ginseng Rh2+ increased the apoptosis of NSCLC cells in vitro, demonstrating dose dependent down-regulation of the Bcl-2 anti-apoptotic gene and concurrent up-regulation of the Bax pro-apoptotic gene. Ginseng Rh2+ inhibited the tumor cells' capacity to attach to the ECM-related matrix and reduced cell migration. In vivo, Ginseng Rh2+ inhibited local tumor growth and reduced the development of experimental lung metastases. CONCLUSION: Our study suggests that Ginseng Rh2+ may potentially be used as a therapeutic agent for treatment of NSCLC.

Immune Responses to SARS-CoV2 Mirror Societal Responses to COVID-19: Identifying Factors Underlying a Successful Viral Response.

The adaptive immune system was sculpted to protect individuals, societies, and species since its inception, developing effective strategies to cope with emerging pathogens. Here, we show that similar successful or failed dynamics govern personal and societal responses to a pathogen as SARS-CoV2. Understanding the self-similarity between the health-protective measures taken to protect the individual or the society, help identify critical factors underlying the effectiveness of societal response to a pathogenic challenge. These include (1) the quick employment of adaptive-like, pathogen-specific strategies to cope with the threat including the development of "memory-like responses"; (2) enabling productive coaction and interaction within the society by employing effective decision-making processes; and (3) the quick inhibition of positive feedback loops generated by hazardous or false information. Learning from adaptive anti-pathogen immune responses, policymakers and scientists could reduce the direct damages associated with COVID-19 and avert an avoidable "social cytokine storm" with its ensuing socioeconomic damage.

Evaluation of the Compatibility of Electric Tumor Treating Fields with Key Anti-tumoral T-Cell Functions.

BACKGROUND: Tumor treating fields (TTFields) are low-intensity, intermediate frequency electric fields that affect proliferating cells. TTFields are FDA approved for treatment of newly diagnosed and recurrent glioblastoma. Combining TTFields with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to the ability of TTFields to induce immunogenic cell death. Conversely, TTFields may interfere with immune functions critical for effective T-cell responses. OBJECTIVES: To evaluate the effects of TTFields on pivotal antitumoral T-cell functions. METHODS: T-cells from healthy donor peripheral blood (PB) or from viably dissociated human glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen stimulation. Multiparametric flow cytometry (8-color) was used to assess T-cell responses by monitoring select pivotal functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), and activation/exhaustion (PD-1). Cellular viability was assessed in a dedicated assay. A chimeric antigen receptor (CAR) T-cell-based assay directly evaluated cellular cytotoxicity. RESULTS: Activated PB T-cells and tumor-infiltrating T-cells (TILs) preserved all monitored anti- tumoral functions under TTFields, apart from proliferation. This finding also applied specifically to PD-1 + TILs, comprised predominantly of tumor antigen-specific cells. Activated T-cells that attempted to proliferate under TTFields demonstrated decreased viability, in line with TTField MOA. Small or no reduction in viability was found in T-cells that did not attempt to proliferate, whether activated or resting. CONCLUSIONS: All monitored anti-tumoral T cell functions, except for proliferation, were unhindered by TTFields. Our results support further investigation into combinations of TTFields with T-cell based immunotherapeutic approaches.

Cryopreservation of Stromal Vascular Fraction Cells Reduces Their Counts but Not Their Stem Cell Potency.

Adipose-derived stem cells are derived from the nonfat component of adipose tissue termed the stromal vascular fraction (SVF). The use of freshly isolated autologous SVF cells as an alternative to adult stem cells is becoming more common. Repeated SVF administration for improved clinical outcomes is complicated by the need for repeated liposuction. This can be overcome by cryopreservation of SVF cells. The current study aimed to assess whether SVF cells retain their stem cell potency during cryopreservation. METHODS: SVF cells isolated from lipoaspirates (donor age: 46.1 ± 11.7 y; body mass index: 29.3 ± 4.8 kg/m2) were analyzed either immediately after isolation or following cryopreservation at -196°C. Analyses included assessment of nucleated cell counts by methylene blue staining, colony-forming unit fibroblast counts, surface marker expression using a flow cytometric panel (CD45, CD34, CD31, CD73, CD29, and CD105), expansion in culture, and differentiation to fat and bone. RESULTS: While cryopreservation reduced the number of viable SVF cells, stem cell potency was preserved, as demonstrated by no significant difference in the proliferation, surface marker expression in culture, bone and fat differentiation capacity, and the number of colony-forming unit fibroblasts in culture, in cryopreserved versus fresh SVF cells. Importantly, reduced cell counts of cryopreserved cells were due, mainly, to a reduction in hematopoietic CD45+ cells, which was accompanied by increased proportions of CD45-CD34+CD31- stem cell progenitor cells compared to fresh SVF cells. CONCLUSIONS: Cryopreservation of SVF cells did not affect their in vitro stem cell potency and may therefore enable repeated SVF cell administrations, without the need for repeated liposuction.

Fas-L promotes the stem cell potency of adipose-derived mesenchymal cells.

Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.

A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

BACKGROUND: Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. RESULTS: Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. CONCLUSIONS: The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.

Dendritic Cells in the Context of Human Tumors: Biology and Experimental Tools.

Dendritic cells (DC) are the most potent and versatile antigen-presenting cells (APC) in the immune system. DC have an exceptional ability to comprehend the immune context of a captured antigen based on molecular signals identified from its vicinity. The analyzed information is then conveyed to other immune effector cells. Such capability enables DC to play a pivotal role in mediating either an immunogenic response or immune tolerance towards an acquired antigen. This review summarizes current knowledge on DC in the context of human tumors. It covers the basics of human DC biology, elaborating on the different markers, morphology and function of the different subsets of human DC. Human blood-borne DC are comprised of at least three subsets consisting of one plasmacytoid DC (pDC) and two to three myeloid DC (mDC) subsets. Some tissues have unique DC. Each subset has a different phenotype and function and may induce pro-tumoral or anti-tumoral effects. The review also discusses two methods fundamental to the research of DC on the single-cell level: multicolor flow cytometry (FCM) and image-based cytometry (IC). These methods, along with new genomics and proteomics tools, can provide high-resolution information on specific DC subsets and on immune and tumor cells with which they interact. The different layers of collected biological data may then be integrated using Immune-Cytomics modeling approaches. Such novel integrated approaches may help unravel the complex network of cellular interactions that DC carry out within tumors, and may help harness this complex immunological information into the development of more effective treatments for cancer.

Split immunity: immune inhibition of rat gliomas by subcutaneous exposure to unmodified live tumor cells.

Gliomas that grow uninhibited in the brain almost never metastasize outside the CNS. The rare occurrences of extracranial metastasis are usually associated with a suppressed immune system. This observation raises the possibility that some gliomas might not grow outside the CNS due to an inherent immune response, We report in this study that the highly malignant F98 Fischer rat undifferentiated glioma, which grows aggressively in the brain, spontaneously regresses when injected live s.c. We found that this regression is immune-mediated and that it markedly enhances the survival or cures rats challenged with the same tumor intracranially either before or after the s.c. live-cell treatment. Adoptive transfer experiments showed the effect was immune-mediated and that the CD8 T cell fraction, which exhibited direct tumor cytotoxicity, was more effective than the CD4 T cell fraction in mediating resistance to intracranial challenge of naive rats. Brain tumors from treated rats exhibited enhanced CD3(+)CD8(+)CD4(-) and CD3(+)CD4(+)CD8(-) T cell infiltration and IFN-γ secretion. The results in the F98 glioma were corroborated in the Lewis rat CNS-1 astrocytoma. In both tumor models, s.c. treatment with live cells was significantly better than immunization with irradiated cells. We propose in this study a location-based immunotherapeutic phenomenon we term "split immunity": a tumor that thrives in an immune-privileged site may be inhibited by injecting live, unmodified tumor cells into a site that is not privileged, generating protective immunity that spreads back to the privileged site. Split immunity could explain several long-standing paradoxes regarding the lack of overt extracranial metastasis in patients with primary brain tumors.

ImMucin: a novel therapeutic vaccine with promiscuous MHC binding for the treatment of MUC1-expressing tumors.

An optimal cancer vaccine should be able to induce highly potent, long-lasting, tumor-specific responses in the majority of the cancer patient population. One approach for achieving this is to use synthetic peptide vaccines derived from widely expressed tumor-associated antigens, that promiscuously bind multiple MHC class I and class II alleles. MUC1-SP-L (ImMucin, VXL100) is a 21mer peptide encoding the complete signal peptide domain of MUC1, a tumor-associated antigen expressed by over 90% of solid and non-solid tumors. MUC1-SP-L was predicted in silico to bind various MHC class I and MHC class II alleles, covering the majority of the Caucasian population. PBLs obtained from 13 naïve donors all proliferated, with a Stimulation Index (SI≥2), to the MUC1-SP-L peptide, producing mixed CD4+ and CD8+ responses. Similar results were manifested by MUC1-SP-L in PBLs derived from 9 of 10 cancer patients with MUC1 positive tumors. CD4+ and CD8+ T cell populations exhibited CD45RO memory markers and secreted IFN-gamma and IL-2 following stimulation with MUC1-SP-L. These T cells also exhibited proliferation to the MUC1-SP-L inner 9mer epitopes and cytotoxicity against tumor cell lines expressing MUC1 and a concordant MHC class I allele. Cytotoxicity to MUC1-expressing human and murine tumors was shown also in T cells obtained from HLA-A2 transgenic mice and BALB/c syngeneic mice immunized with the MUC1-SP-L and GM-CSF. In an immunotherapy model, BALB/c mice inoculated with metastatic MUC1 transfected murine DA3 mammary tumor cells, exhibited significantly prolonged survival following vaccination with MUC1-SP-L. Our results indicate superior immunological and anti-tumor properties of MUC1-SP-L compared to previously published MUC1-derived epitopes.

Signal peptides and trans-membrane regions are broadly immunogenic and have high CD8+ T cell epitope densities: Implications for vaccine development.

Cell mediated immune response has a major role in controlling the elimination of infectious agents. The rational design of sub-unit peptide vaccines against intracellular pathogens or cancer requires the use of antigenic sequence/s that can induce highly potent, long lasting and antigen-specific responses in the majority of the population. A promising peptide selection strategy is the detection of multi-epitope peptide sequences with an ability to bind multiple MHC alleles. While past research sought the best epitopes based on their specific antigenicity, we ask whether specific defined domains have high epitope densities. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The improved MHC binding of these domains relies on their hydrophobic nature and, in signal peptides, also on their specific sequence. The high epitope density of SP was computed using in-silico methods and corroborated by the high percentage of identified SP epitope in the IEDB (immune epitope database). The enhanced immunogenicity of SP was then experimentally confirmed using a panel of nine peptides derived from Mycobacterium tuberculosis (MTb) proteins used in human PBMC proliferation assays and T cell lines functional assays. Our results show the exceptionally high antigen specific response rates and population coverage to SP sequences compared with non-SP peptide antigens derived from the same proteins. The results suggest a novel scheme for the rational design of T cell vaccines using a domain based rather than an epitope based approach.

T cell vaccination induces the elimination of EAE effector T cells: analysis using GFP-transduced, encephalitogenic T cells.

T cell vaccination (TCV) with irradiated encephalitogenic T cells induces resistance to EAE. However, the fate of the encephalitogenic T cells in vivo following TCV has yet to be studied. Here we used anti-MBP encephalitogenic T cells that were transduced to express GFP to study the effects of TCV on these cells. In naïve rats or in control-vaccinated (Ova-GFP) rats injected i.v. with GFP-labeled effector cells, high numbers of effector T cells were found along with macrophages, CD8 T cells and Non-GFP CD4 cells in the spleens, parathymic lymph nodes (PTLN) and spinal cords. In contrast, the recipients that had been treated with TCV (anti-MBP T-cell lines) showed few if any GFP-labeled effector T cells throughout the disease (day 1-8) and their spinal cords were almost clear of macrophages, CD4 and CD8 cells. Splenocytes in the control groups secreted IFNgamma in response to MBP and showed high numbers of IFNgamma secreting CD4 and CD8 cells in their spinal cords at the disease peak. In the TCV-protected groups, splenocytes showed no reactivity to MBP but secreted IFNgamma in response to irradiated encephalitogenic T cells--an anti-idiotypic response. Thus, TCV leads to a marked decrease in the numbers of effector T cells in the CNS and lymphoid organs, to a marked reduction in the Th1 cytokine producing cells in the CNS, and to the appearance of T cells responsive to the anti-MBP effector T cells.

T-cell seeding: neonatal transfer of anti-myelin basic protein T-cell lines renders Fischer rats susceptible later in life to the active induction of experimental autoimmune encephalitis.

Fischer strain rats resist active induction of experimental autoimmune encephalomyelitis (EAE) following immunization with guinea-pig myelin basic protein (MBP) in complete Freund's adjuvant (CFA). Nevertheless, we now report that an encephalitogenic CD4(+) anti-MBP T-cell line could be developed from actively immunized Fischer rats. Adoptive transfer of the activated line mediated acute EAE in adult Fischer rats, but not in 1-day-old rats. Moreover, we found that both resting and activated anti-MBP T cells injected 1 day post-natally rendered these rats susceptible later in life to the active induction of EAE by immunization with MBP/CFA. The actively induced EAE manifested the accelerated onset of a secondary, memory-type response. Resting anti-MBP T cells injected even up to 2 weeks post-natally produced no clinical signs but seeded 50-100% of the recipients for an active encephalitogenic immune response to MBP. An earlier T-cell injection (1-2 days) produced a higher incidence and stronger response. The transferred resting T cells entered the neonatal spleen and thymus and proliferated there but did not change the total anti-MBP precursor number in adults. Splenocytes harvested from rats that were injected neonatally but not exposed to MBP in vivo proliferated strongly and produced significant amounts of interferon-gamma to MBP in vitro. Similar results were observed in rats injected with resting T-cell lines reactive to ovalbumin, suggesting that the neonatal injection of resting T cells specific for a self or for a foreign antigen can seed the immune system with the potential for an enhanced effector response to that antigen later in life.

Exuberated numbers of tumor-specific T cells result in tumor escape.

Cytotoxic T cells (CTL) play a major role in tumor rejection. Expansion of CTLs, either by immunization or adoptive transfer, is a prominent goal in current immunotherapy. The antigen-specific nature of these expansion processes inevitably initiates a clonotypic attack on the tumor. By injecting an Ovalbumin-expressing melanoma into OT-I mice, in which >90% of CTLs recognize an Ovalbumin peptide, we show that an increased number of tumor-specific CTLs causes emergence of escape variants. We show that these escape variants are a result of antigen silencing via a yet undetermined epigenetic mechanism, which occurs frequently and is spontaneously reversible. We further show that an increase in the time of tumor onset in OT-I compared with C57BL/6J is a result of immune selection.

Preventive and therapeutic vaccination with PAP-3, a novel human prostate cancer peptide, inhibits carcinoma development in HLA transgenic mice.

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201-PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.

Human CTL epitopes prostatic acid phosphatase-3 and six-transmembrane epithelial antigen of prostate-3 as candidates for prostate cancer immunotherapy.

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancer-associated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and six-transmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1+ LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1+ LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti-prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials.

Combined dendritic cell cryotherapy of tumor induces systemic antimetastatic immunity.

PURPOSE: Cryotherapy of localized prostate, renal, and hepatic primary tumors and metastases is considered a minimally invasive treatment demonstrating a low complication rate in comparison with conventional surgery. The main drawback of cryotherapy is that it has no systemic effect on distant metastases. We investigated whether intratumoral injections of dendritic cells following cryotherapy of local tumors (cryoimmunotherapy) provides an improved approach to cancer treatment, combining local tumor destruction and systemic anticancer immunity. EXPERIMENTAL DESIGNS: The 3LL murine Lewis lung carcinoma clone D122 and the ovalbumin-transfected B16 melanoma clone MO5 served as models for spontaneous metastasis. The antimetastatic effect of cryoimmunotherapy was assessed in the lung carcinoma model by monitoring mouse survival, lung weight, and induction of tumor-specific CTLs. The mechanism of cryoimmunotherapy was elucidated in the melanoma model using adoptive transfer of T cell receptor transgenic OT-I CTLs into the tumor-bearing mice, and analysis of Th1/Th2 responses by intracellular cytokine staining in CD4 and CD8 cells. RESULTS: Cryoimmunotherapy caused robust and tumor-specific CTL responses, increased Th1 responses, significantly prolonged survival and dramatically reduced lung metastasis. Although intratumor administration of dendritic cells alone increased the proliferation rate of CD8 cells, only cryoimmunotherapy resulted in the generation of effector memory cells. Furthermore, cryoimmunotherapyprotected mice that had survived primary MO5 tumors from rechallenge with parental tumors. CONCLUSIONS: These results present cryoimmunotherapy as a novel approach for systemic treatment of cancer. We envisage that cryotherapy of tumors combined with subsequent in situ immunotherapy by autologous unmodified immature dendritic cells can be applied in practice.