Cytotoxic T Lymphocytes Recognition Research Articles

Targeted inhibition of glycogen synthase kinase-3 using 9-ING-41 (elraglusib) enhances CD8 T-cell-reactivity against neuroblastoma cells

The prognosis of patients with high-risk neuroblastoma remains poor, partly due to inadequate immune recognition of the tumor. Neuroblastomas display extremely low surface MHC-I, preventing recognition by cytotoxic T lymphocytes (CTLs) and contributing to an immunosuppressive tumor microenvironment. Glycogen synthase kinase-3 beta (GSK-3β) is involved in pathways that may affect the MHC-I antigen processing and presentation pathway. We proposed that therapeutic inhibition of GSK-3β might improve the surface display of MHC-I molecules on neuroblastoma cells, and therefore tested if targeting of GSK-3β using the inhibitor 9-ING-41 (Elraglusib) improves MHC-I-mediated CTL recognition. We analyzed mRNA expression data of neuroblastoma tumor datasets and found that non-MYCN-amplified neuroblastomas express higher GSK-3β levels than MYCN-amplified tumors. In non-MYCN-amplified cells SH-SY5Y, SK-N-AS and SK-N-SH 9-ING-41 treatment enhanced MHC-I surface display and the expression levels of a subset of genes involved in MHC-I antigen processing and presentation. Further, 9-ING-41 treatment triggered increased STAT1 pathway activation, upstream of antigen presentation pathways in two of the three non-MYCN-amplified cell lines. Finally, in co-culture experiments with CD8 + T cells, 9-ING-41 improved immune recognition of the neuroblastoma cells, as evidenced by augmented T-cell activation marker levels and T-cell proliferation, which was further enhanced by PD-1 immune checkpoint inhibition. Our preclinical study provides experimental support to further explore the GSK-3β inhibitor 9-ING-41 as an immunomodulatory agent to increase tumor immune recognition in neuroblastoma.

Abstract SY20-01: USP18 modulates the ISGylome immune signaling, and sensitizes tumor cells to irradiation and CTL recognition

Abstract Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target. Citation Format: Benedikt Kessler. USP18 modulates the ISGylome immune signaling, and sensitizes tumor cells to irradiation and CTL recognition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY20-01.

Abstract 1483: Examining the role of genome editing enzymes in anti tumor immunity

Abstract Background: Tumor antigens (TA) on cancer cells can trigger immune response TA are peptides that are either not present, or are present to a much lesser extent, on normal cells compared to tumor cells. CD8+ Cytotoxic T-cells (CTL) recognize TAs bound to class I major histocompatibility complex (MHC) molecules on the surface of tumor cells. We and others have shown that viruses subvert the mutational activity of the endogenous DNA-editing APOBEC enzymes in human host cells towards immune escape. Viruses do so by adapting their genomic DNA code to position APOBEC's favored mutational “hotspot” DNA sequences such that upon being mutated by the host APOBEC enzymes, the mutated viral protein is less immunogenic for CTL recognition. Like viruses, cancer cells have genomes that are highly plastic and adaptable through mutation; therefore, we hypothesize that the APOBEC DNA-editing enzymes are exploited by cancer cells for modulating TA immunogenicity. Aims of the Study: Aim1: To elucidate the impact of A3-mediated mutations on modulating TA immunogenicity for CTL recognition. Aim 2: To examine whether genomic sequences encoding TAs in the human genome have co-evolved to either attract or avoid APOBEC-driven mutations. Methods: Aim 1: Using the human reference proteome from EMBL/EBI, we located APOBEC-mutable hotspots in genomic DNA encoding the immunopeptidome. We simulated APOBEC-mediated mutations and translated to peptide TAs. We used NetMHCpan 4 to measure the impact of TA mutations on MHC class I binding affinity. In parallel, we are searching for already-identified APOBEC-mediated mutations in sequenced tumor genomes in cancer genome databases such as The Cancer Genome Atlas (TCGA), Catalogue of Observed Somatic Mutations in Cancer (COSMIC) and International Cancer Genome Consortium (ICGC). Aim 2: To investigate the enrichment of APOBEC-hotspots in genomic sequences encoding TAs restricted to each HLA, we are tabulating APOBEC hotspots in genomic DNA sequencing encoding TAs vs. non-TA-encoding sequences. At the sequence level, the ratio of hotspot frequency, in combination with scores of predicted impacts on immunogenicity as obtained in aim 1, will be used to glean whether and how the human genomic sequence has co-evolved with APOBECs from the standpoint of TAs. Results and Significance: Based on the preliminary data we observe that APOBEC-mediated mutations can diminish or enhance immunogenicity. Understanding the balance between these two effects is key in informing how APOBEC expression and activity ought to be regarded in the context of cancer immunotherapy. Citation Format: Faezeh Borzooee. Examining the role of genome editing enzymes in anti tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1483.

Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells.

Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.

Abstract LB-080: USP18 modulates the ISGylome, immune signaling and sensitizes tumor cells to irradiation and CTL recognition

Abstract The innate immune response plays a critical role in modulating the efficacy of and mechanisms conferring resistance to immune checkpoint blockade (ICB) therapies in humans. A major negative regulator of the Interferon (IFN) stimulated gene (ISG) pathway, is the ubiquitin-specific protease 18 (USP18). USP18 is the predominant human deubiquitylating enzyme that processes Interferon Stimulated Gene 15 ISG15, a ubiquitin-like protein that covalently modifies protein substrates, a tightly regulated process in the context of innate immunity. In this study, using advanced mass spectrometry and chemical biology tools, we defined the USP18 Interactome and ISG15ylome in chronic myeloid leukemia (CML)-derived cells (HAP1) treated with Interferon alpha (IFNα). Novel ISG15ylation targets were characterized that reduce the sensing of innate ligands and secretion of cytokines. In addition, we show that USP18 deletion leads to enhanced ISG15ylation profiles. Furthermore, we demonstrate that CML USP18-/- cells are more antigenic, leading to increased activation of cytotoxic T lymphocytes (CTLs), and are more susceptible to irradiation. Our results reinforce the role of USP18 as a key “brake” for inflammatory signals in tumor cells. As USP18 expression is upregulated in lung, breast and colon cancers, USP18 pharmacological inhibition may reflect a target opportunity in cancer immunotherapy. Citation Format: Adan Pinto-Fernandez, Helene Greenwood, Mariolina Salio, Jianzhou Chen, Thomas Partridge, George Vere, Hannah C. Scott, Andreas Damianou, Persephone Borrow, Ruth Muschel, Vincenzo Cerundolo, Benedikt M. Kessler. USP18 modulates the ISGylome, immune signaling and sensitizes tumor cells to irradiation and CTL recognition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-080.

Abstract 6616: Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden

Abstract Background: Cytotoxic T-lymphocytes (CTLs) infiltration in a bulk tumor is a positive prognostic factor in breast cancer. CTL recognition is believed to react to mutation-induced neoantigens, thus, higher tumor mutation burden (TMB) is considered as an important predictor of tumor immunogenicity and response to immunotherapy. Although majority of breast cancer have low tumor mutation burden (TMB), triple-negative breast cancer (TNBC) has higher TMB compared to other breast cancer subtypes. However, it is unclear if the prognostic value high CTL infiltration depends on the TMB as the primary driver of enhanced anti-cancer immunity or represents an independent prognostic marker which can be used as a complement to TMB to predict patients' outcomes. CTLs are identified by CD8 marker, is encoded by CD8A. Granzyme B (GZMB) is a serine protease that is secreted by functionally-active CTLs to induce apoptosis of the target cells. CXCL10 is a chemokine which selectively attracts activated CTLs into subsets of other cancers; but its role in breast cancer remains unknown. We investigated if TNBC infiltrated with high levels of functional CTLs, so called “hot tumors”, have improved survival independently of their TMB. Methods: Utilizing The Cancer Genome Atlas (TCGA) breast cancer cohort, we established Functional Hotness Score (FHS), based on the CD8A, GZMB and CXCL10 gene expression levels of bulk tumors. Total of 4149 breast cancer patients from publicly available multiple cohorts were analyzed to assess FHS and breast cancer patient prognosis as well as distinct immunity markers. Results: FHS predicted the breast cancer patient survival better than each consisting gene. The breast cancer patients with high FHS tumors showed significantly better survival in the testing cohort (TCGA), which was further confirmed in the validation cohort (METABRIC). FHS was higher in the primary breast cancer tissues with metastasis compared to that without metastasis in TCGA. Further, FHS was higher in the metastatic tumors compared to the primary tumors in GSE110590. Among breast cancer subtypes, TNBC showed highest FHS compared to other subtypes in testing TCGA, which was validated in the METABRIC cohort. The patients with high FHS tumor demonstrated significantly better long-term survival than that with low FHS only in the hormone receptor (HR)-negative breast cancers, but not in the HR-positive tumors. The high FHS TNBCs showed higher not only CD8-positive T cell infiltration, but also a broader type-1 anti-cancer immunity. To investigate if FHS predicts patient survival independent of TMB, the patients were divided into high and low TMB groups. Interestingly, the higher FHS patients showed better prognosis not only in the high TMB group but also in the low TMB among TNBC patients. The combination of high TMB with high FHS identified the unique subset of the patients who did not recur over time. Conclusion: In conclusion, TNBC with higher FHS based on the expression levels of CD8A, GZMB and CXCL10 showed improved prognosis with higher anti-cancer immunity regardless of TMB, and constituting an independent prognostic marker of survival, particularly when combined with TMB. Citation Format: Eriko Katsuta, Li Yan, Mateusz Opyrchal, Pawel Kalinski, Kazuaki Takabe. Functional hotness score generated by representative functional cytotoxic T-lymphocytes predicts long-term survival of triple-negative breast cancer independently to the tumor mutational burden [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6616.

Epigenetic modulation combined with PD-1/PD-L1 blockade enhances immunotherapy based on MAGE-A11 antigen-specific CD8+T cells against esophageal carcinoma.

Cancer testis antigens (CTAs) are promising targets for T cell-based immunotherapy and studies have shown that certain CT genes are epigenetically depressed in cancer cells through DNA demethylation. Melanoma-associated antigen A11 (MAGE-A11) is a CTA that is frequently expressed in esophageal cancer and is correlated with a poor esophageal cancer prognosis. Consequently, MAGE-A11 is a potential immunotherapy target. In this study, we evaluated MAGE-A11 expression in esophageal cancer cells and found that it was downregulated in several tumor cell lines, which restricted the effect of immunotherapy. Additionally, the specific recognition and lytic potential of cytotoxic T lymphocytes (CTLs) derived from the MAGE-A11 was determined. Specific CTLs could kill esophageal cancer cells expressing MAGE-A11 but rarely lysed MAGE-A11-negative tumor cells. Therefore, induction of MAGE-A11 expression is critical for CTLs recognition and lysis of esophageal cancer cells. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased MAGE-A11 expression in esophageal cancer cells and subsequently enhanced the cytotoxicity of MAGE-A11-specific CD8+T cells against cancer cell lines. Furthermore, we found that PD-L1 expression in esophageal cancer cells affected the antitumor function of CTLs. programmed death-1 (PD-1)/PD-L1 blockade could increase the specific CTL-induced lysis of HLA-A2+/MAGE-A11+ tumor cell lines treated with 5-aza-2'-deoxycytidine. These findings indicate that the treatment of tumor cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine augments MAGE-A11 expression in esophageal cancer cells. The combination of epigenetic modulation by 5-aza-2'-deoxycytidine and PD-1/PD-L1 blockade may be useful for T cell-based immunotherapy against esophageal cancer.

Human Leukocyte Antigen-Associated HIV-1 CRF02_AG gag and vif Polymorphisms in Ghana.

In human immunodeficiency virus type-1 (HIV-1) infections, cytotoxic T-lymphocyte (CTL) responses targeting human leukocyte antigen (HLA)-restricted viral epitopes exert strong suppressive pressure on viral replication and frequently select for mutations resulting in viral escape from CTL recognition. Numerous data on these HLA-associated mutations in HIV-1 subtypes B and C have been amassed with few reports described in other subtypes. In the present study, we investigated the HLA-associated mutations in HIV-1 subtype CRF02_AG prevailing in Ghana, Western Africa. We determined viral gag sequences in 246 out of 324 HIV-1-infected Ghanaians. Phylogeny analysis revealed that 200 (81.3%) individuals were infected with HIV-1 CRF02_AG. Full gag and vif sequences were obtained from 199 and 138, respectively, out of the 200 individuals infected with CRF02_AG and subjected to determination of HLA-associated mutations. The analysis found HLA-associated HIV-1 CRF02_AG non-synonymous polymorphisms at 19 sites; 13 in gag and six in vif, including those that were newly determined. Generation of this data is an important contribution to our understanding of HIV-1 CRF02_AG and host T cell interaction.

Effects of thymic selection on T cell recognition of foreign and tumor antigenic peptides

The advent of cancer immunotherapy has generated renewed hope for the treatment of many malignancies by introducing a number of novel strategies that exploit various properties of the immune system. These therapies are based on the idea that cytotoxic T lymphocytes (CTLs) directly recognize and respond to tumor-associated neoantigens (TANs) in much the same way as they would to foreign peptides presented on cell surfaces. To date, however, nearly all attempts to optimize immunotherapeutic strategies have been empirical. Here, we develop a model of T cell selection based on the assumption of random interaction strengths between a self-peptide and the various T cell receptors. The model enables the analytical study of the effects of selection on the CTL recognition of TANs and completely foreign peptides and can estimate the number of CTLs that can detect donor-matched transplants. We show that negative selection thresholds chosen to reflect experimentally observed thymic survival rates result in near-optimal production of T cells that are capable of surviving selection and recognizing foreign antigen. These analytical results are confirmed by simulation.

Long-term adaptation of the influenza A virus by escaping cytotoxic T-cell recognition.

The evolutionary adaptation of the influenza A virus (IAV) to human antibodies is well characterised. Much less is known about the long-term evolution of cytotoxic T lymphocyte (CTL) epitopes, which are important antigens for clearance of infection. We construct an antigenic map of IAVs of all human subtypes using a compendium of 142 confirmed CTL epitopes, and show that IAV evolved gradually in the period 1932–2015, with infrequent antigenic jumps in the H3N2 subtype. Intriguingly, the number of CTL epitopes per virus decreases with more than one epitope per three years in the H3N2 subtype (from 84 epitopes per virus in 1968 to 64 in 2015), mostly attributed to the loss of HLA-B epitopes. We confirm these observations with epitope predictions. Our findings indicate that selection pressures imposed by CTL immunity shape the long-term evolution of IAV.

Human cytomegalovirus miR-UL112-1 promotes the down-regulation of viral immediate early-gene expression during latency to prevent T-cell recognition of latently infected cells.

Human cytomegalovirus, a member of the herpesvirus family, can cause significant morbidity and mortality in immune compromised patients resulting from either primary lytic infection or reactivation from latency. Latent infection is associated with a restricted viral transcription programme compared to lytic infection which consists of defined protein coding RNAs but also includes a number of virally encoded microRNAs (miRNAs). One of these, miR-UL112-1, is known to target the major lytic IE72 transcript but, to date, a functional role for miR-UL112-1 during latent infection has not been shown. To address this, we have analysed latent infection in myeloid cells using a virus in which the target site for miR-UL112-1 in the 3' UTR of IE72 was removed such that any IE72 RNA present during latent infection would no longer be subject to regulation by miR-UL112-1 through the RNAi pathway. Our data show that removal of the miR-UL112-1 target site in IE72 results in increased levels of IE72 RNA in experimentally latent primary monocytes. Furthermore, this resulted in induction of immediate early (IE) gene expression that is detectable by IE-specific cytotoxic T-cells (CTLs); no such CTL recognition of monocytes latently infected with wild-type virus was observed. We also recapitulated these findings in the more tractable THP-1 cell line model of latency. These observations argue that an important role for miR-UL112-1 during latency is to ensure tight control of lytic viral immediate early (IE) gene expression thereby preventing recognition of latently infected cells by the host's potent pre-existing anti-viral CTL response.

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma.

IntroductionInterferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.ResultsWe could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.ConclusionWe suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.

Trouble at the core: BRAF(V600E) drives multiple modes of T-cell suppression in melanoma

ABSTRACTSeveral studies have demonstrated that oncogenic BRAF(V600E) promotes T-cell suppression in melanoma by upregulating the transcription of a multitude of immunomodulatory chemokine and cytokine genes. BRAF(V600E) has now been shown to act even more directly to evade cytotoxic T-cell recognition, by driving rapid internalization of human leukocyte antigen (HLA) class I from the tumor-cell surface and its intracellular sequestration.

Different Effects of Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutations on Cytotoxic T Lymphocyte Recognition between HIV-1 Subtype B and Subtype A/E Infections.

The effect of antiretroviral drug resistance mutations on cytotoxic T lymphocyte (CTL) recognition has been analyzed in HIV-1 subtype B infections, but it remains unclear in infections by other HIV-1 subtypes that are epidemic in countries where antiretroviral drugs are not effectively used. We investigated the effect of nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistance mutations (Y181C, Y181I, and Y181V) on epitope recognition by CTLs specific for 3 different HIV-1 epitopes (HLA-A*02:01-restricted IV10, HLA-B*35:01-restricted NY9, and HLA-C*12:02-restricted KY9) in subtype B and subtype A/E infections and the accumulation of these mutations in treatment-naive Japanese and Vietnamese. These NNRTI-resistance mutations critically affected NY9-specific and KY9-specific T cell responses in the subtype B infections, whereas they showed a different effect on IV10-specific T cell responses among the subtype B-infected individuals. These mutations affected IV10-specific T cell responses but weakly affected NY9-specific T cell responses in the subtype A/E infections. The substitution at position 3 of NY9 epitope which was found in the subtype A/E virus differently influenced the peptide binding to HLA-B*35:01, suggesting that the differences in peptide binding may result in the differences in T cell recognition between the subtype B virus and A/E virus infections. The Y181C mutation was found to be accumulating in treatment-naive Vietnamese infected with the subtype A/E virus. The present study demonstrated different effects of NNRTI-resistance RT181 mutations on CTL responses between the 2 subtype infections. The Y181C mutation may influence HIV-1 control by the CTLs in Vietnam, since this mutation has been accumulating in treatment-naive Vietnamese. Antiretroviral therapy leads to the emergence of drug-resistant HIV-1, resulting in virological and clinical failures. Though HIV-1-specific CTLs play a critical role in HIV-1 infection, some of drug resistance mutations located in CTL epitopes are known to affect HIV-1-specific CTL responses. Nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistance RT181 mutations are frequently observed in patients treated with NNRTIs. Such drug resistance mutations may have an influence on immune control by HIV-1-specific CTLs, especially in countries where antiretroviral drugs are not effectively used. We here investigated the effect of three NNRTI-resistance RT181 mutations on immune responses by HIV-1-specific CTLs and the recent accumulation of these mutations in treatment-naive Vietnamese infected with HIV-1 subtype A/E virus. RT181 mutations affected CTL recognition in both subtype A/E and B infections, while the RT Y181C mutation has been accumulating in treatment-naive Vietnamese. The results suggest that the Y181C mutation may influence HIV-1 control by CTLs in Vietnam.

Upregulation of class I MHC on neuroblastoma cells by NK cell exposure for enhanced CTL reactivity

Neuroblastoma is the most common solid tumor in pediatric patients. Survival rates are less than 20% for stage IV disease despite intensive treatment consisting of surgery, chemotherapy, radiotherapy and autologous hematopoietic stem cell transplantation. Hence there is clear unmet need for better treatment options for these patients. Immunotherapy is now considered a new promising treatment strategy, but neuroblastoma stage IV was shown to be low immunogenic. This project aims to improve immunogenicity of neuroblastoma for enhanced cytotoxic T lymphocyte (CTL)-mediated immune reactivity. Using five distinct neuroblastoma cell lines we found that the protein PRAME serves as an immunogenic tumor antigen for neuroblastoma, which can be presented in class I MHC/peptide complexes to reactive T cells. However, PRAME-expressing neuroblastoma cells were only targets for T cells when they were pre-treated with interferon-gamma to stimulate MHC class I upregulation. Interestingly however we observed that neuroblastoma cells also upregulated MHC class I by exposure to Natural Killer (NK) cells. After NK cell co-culture, neuroblastoma cells were exposed to PRAME-antigen-specific CTL to elucidate if improved immunogenicity can be corroborated by improved neuroblastoma antigen-specific CTL activation. We show that upon interaction with NK cells neuroblastoma cells can be recognized by PRAME-specific CTL. Our results demonstrate that neuroblastoma cells are transformed into CTL targets by interacting with NK cells. Neuroblastoma cells adjust their immunogenicity depending on signals derived from its direct environment, thereby evading anti-tumor immune responses. By making use of their plasticity neuroblastoma cells can be persuaded to increase their susceptibility for CTL recognition. This finding implicates that combined stimulation of class I MHC upregulation and CTL reactivity may be considered as an attractive multi-modal treatment option for neuroblastoma patients.

The B subunit of Escherichia coli enterotoxin helps control the in vivo growth of solid tumors expressing the Epstein-Barr virus latent membrane protein 2A.

Latent membrane protein 2A (LMP2A) is expressed on almost all Epstein–Barr virus (EBV)-associated tumors and is a potential target for immunotherapeutic intervention and vaccination. However, LMP2A is not efficiently processed and presented on major histocompatibility antigens class I molecules to generate potent cytotoxic T-lymphocytes (CTL) responses capable of killing these tumors. The B subunit of Escherichia coli enterotoxin (EtxB), causes rapid internalization and processing of membrane-bound LMP2A on EBV-infected B cells, and facilitates loading of processed-LMP2A peptides onto MHC class I. This re-directed trafficking/delivery of LMP2A to the MHC class I machinery enhances recognition and killing by LMP2A-specific CTL in vitro. To test the potential of EtxB to enhance immune targeting of LMP2A expressed in solid tumors, we generated a murine tumor model (Renca-LMP2A), in which LMP2A is expressed as a transgenic neoantigen on a renal carcinoma (Renca) cell line and forms solid tumors when injected subcutaneously into BALB/c mice. The data show that in BALB/c mice which have only low levels of peripheral Kd-LMP2A-specific CD8+ T cells, merely a transient inhibition of tumor growth is achieved compared with naïve mice; suggesting that there is suboptimal LMP2A-specifc CTL recognition and poorly targeted tumor killing. However, importantly, treatment of these mice with EtxB led to a significant delay in the onset of tumor growth and significantly lower tumor volumes compared with similar mice that did not receive EtxB. Moreover, this remarkable effect of EtxB was achieved despite progressive reduction in tumor expression of LMP2A and MHC class I molecules. These data clearly demonstrate the potential efficacy of EtxB as a novel therapeutic agent that could render EBV-associated tumors susceptible to immune control.

Upregulation of Class I MHC on Neuroblastoma Cells By NK Cell Exposure for Enhanced CTL Reactivity

Neuroblastoma is the most common solid tumor in pediatric patients, with a clear unmet need as survival rates are <20% for stage IV disease. As adjuvant immunotherapy is considered to have additional anti-tumor activity we aim to increase cytotoxic T lymphocyte (CTL)-mediated immune surveillance in neuroblastoma patients by administration of a dendritic cell-based vaccine. Neuroblastoma has developed mechanisms to circumvent CTL recognition; therefore it is our aim to find clinical strategies to make neuroblastoma susceptible for CTL killing

Cytotoxic T-Lymphocytes Recognize and Target Leukemic Stem Cells

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. Treatment for acute myeloid leukemia (AML) patients became more effective, yet relapse rates are still high. Immunotherapeutic approaches might overcome the immune escape mechanisms of LSC and might be a therapeutic option. Leukemia-associated antigens (LAA) represent targets that are recognized by cytotoxic T-lymphocytes (CTL) and might be useful for specific immunotherapy.We enriched the LSC containing fraction as CD34+CD38- cells (called LSC-EF) of 20 AML patients using FACS, analyzed expression patterns of LAA in LSC-EF and compared them to patterns in enriched HSC und bulk AML cells by microarray expression analysis and qRT-PCR. In immunoassays, we analysed the functional inhibition of LCS in colony forming assays (CFU) of AML patients by stimulated T cells.We detected a high expression of several LAA in LSC-EF. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC-EF compared to HSC. Survivin (p = 0.7), HAGE (p = 0.5), SSXIP2 (p = 0.4) and Aurorakinase A (p = 0.7) did not indicate a significant differential expression. BAGE does not seem to be an ideal candidate as it is significantly higher expressed on normal HSC (p = 0.0056). Further, we evaluated the expression differences of LSC-EF compared to leukemic bulk. PRAME, RHAMM and WT1 qualified as interesting potential targets as they are lower expressed on leukemic bulk than on LSC-EF. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk but lower in LSC-EF. qRT-PCR confirmed our array data.In immunoassays, T cells stimulated against LAA indicated a significant inhibition of CFUs in AML patient samples. PRAME, RHAMM and WT1 showed good immunogenic responses with CTL recognition rates in a range of 58-83%. PRAME hereby proved as most effective using two different patient samples, with a reduction of 83% colonies (p = 0.004). WT1 shows a significant reduction of 74% (p = 0.05) and RHAMM of 58% colonies (p = 0.2).Taken together, several LAA are significantly expressed on the LSC-EF, whereas PRAME, RHAMM and WT1 seemed most promising as these LAA are differentially expressed compared to normal stem cells and stimulation of cytotoxic T cells against these LAAs inhibit LSC in CFU assays. DisclosuresNo relevant conflicts of interest to declare.

HIV's ticket to ride: Cytotoxic T-lymphocyte-activated dendritic cells exploited for virus intercellular transfer.

For HIV-1 to effectively establish infection and persistence, it must circumvent host immune detection. The capacity of HIV-1 to efficiently mutate and generate multivariant strains in response to host immune selective pressure provides the virus with a particularly effective means of evading cytotoxic T-lymphocyte (CTL) recognition and elimination.1 However, a novel concept has emerged suggesting that HIV-1 can actually benefit from the strategic “baiting” and partial activation of HIV-1-specific CTL responders.2 Driven by immune pressure, CTL epitope divergence can result in the occurrence of surviving epitope variants capable of selectively activating the helper activity of preexisting cross-reactive CTL to create a safe haven for the virus within antigen-presenting dendritic cells (DC). Instead of dampening the immune response through CTL recognition and subsequent killing of these HIV-1 antigen-expressing DC targets, a dysfunctional CTL:DC cross-talk occurs. As a result, these DC become activated and programmed to differentiate into proinflammatory cells displaying unique features that contribute to viral dissemination and persistence.2 We have found that CTL-activated DC are capable of producing high levels of proinflammatory cytokines such as interleukin (IL)-12 and IL-6, but also simultaneously express some immunosuppressive factors such as IL-10 and PD-L1, which may contribute to their resistance to direct cellular attack. They are also programmed to produce a wide range of chemokines, including CCL4, CCL5, CXCL9, and CXCL10, that can preferentially attract activated CD4+ T helper (Th) cells, the main target of HIV-1 infection. Moreover, the production of CCL19 by these DC also promotes their interaction with CCR7+/CD45RA+ naive Th cells as well as CCR7+/CD45RO+ central memory Th cells, the major cellular reservoir of latent HIV-1.3 During this secondary interaction with Th cells, these CTL-programmed DC uniquely undergo fantastic morphological changes, characterized by their rapid development of numerous, far-reaching tunneling nanotube-like membrane protrusions. This intriguing phenomenon ultimately leads to the formation of complex nanotube networks, which facilitate the direct transfer of both cell surface and cytoplasmic components between interconnected neighboring and distant cells. Importantly, these membrane bridges can also serve as highways for HIV-1 to utilize for efficient cell-to-cell transfer (Fig. 1). FIG. 1. Live-cell differential interference contrast image of eukaryotic green fluorescent protein (EGFP)-expressing HIV-1-like particles trafficking via dendritic cell (DC) membrane bridges. Our findings add a new dimension to the proposed concept of “original antigenic sin”4 in which the promiscuous nature of preexisting CTL can be exploited by an evolving virus to modulate the functional and physical characteristics of the DC, and to create an inflammatory environment suitable for viral spread and persistence within the host. These findings also highlight the need to consider the detrimental potential of selectively activating dysfunctional memory CTL responses when implementing anti-HIV-1 vaccine strategies.

The route of HIV escape from immune response targeting multiple sites is determined by the cost-benefit tradeoff of escape mutations.

Cytotoxic T lymphocytes (CTL) are a major factor in the control of HIV replication. CTL arise in acute infection, causing escape mutations to spread rapidly through the population of infected cells. As a result, the virus develops partial resistance to the immune response. The factors controlling the order of mutating epitope sites are currently unknown and would provide a valuable tool for predicting conserved epitopes. In this work, we adapt a well-established mathematical model of HIV evolution under dynamical selection pressure from multiple CTL clones to include partial impairment of CTL recognition, , as well as cost to viral replication, . The process of escape is described in terms of the cost-benefit tradeoff of escape mutations and predicts a trajectory in the cost-benefit plane connecting sequentially escaped sites, which moves from high recognition loss/low fitness cost to low recognition loss/high fitness cost and has a larger slope for early escapes than for late escapes. The slope of the trajectory offers an interpretation of positive correlation between fitness costs and HLA binding impairment to HLA-A molecules and a protective subset of HLA-B molecules that was observed for clinically relevant escape mutations in the Pol gene. We estimate the value of from published experimental studies to be in the range (0.01–0.86) and show that the assumption of complete recognition loss () leads to an overestimate of mutation cost. Our analysis offers a consistent interpretation of the commonly observed pattern of escape, in which several escape mutations are observed transiently in an epitope. This non-nested pattern is a combined effect of temporal changes in selection pressure and partial recognition loss. We conclude that partial recognition loss is as important as fitness loss for predicting the order of escapes and, ultimately, for predicting conserved epitopes that can be targeted by vaccines.