Structural basis of T cell receptor specificity and cross-reactivity of two HLA-DQ2.5-restricted gluten epitopes in celiac disease.

Celiac disease is a T cell-mediated chronic inflammatory condition often characterized by human leukocyte antigen (HLA)-DQ2.5 molecules presenting gluten epitopes derived from wheat, barley, and rye. Although some T cells exhibit cross-reactivity toward distinct gluten epitopes, the structural basis underpinning such cross-reactivity is unclear. Here, we investigated the T-cell receptor specificity and cross-reactivity of two immunodominant wheat gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). We show by surface plasmon resonance that a T-cell receptor alpha variable (TRAV) 4+-T-cell receptor beta variable (TRBV) 29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 with similar affinity, whereas a TRAV4- (TRAV9-2+) TCR recognized HLA-DQ2.5-glia-ω1 only. We further determined the crystal structures of the TRAV4+-TRBV29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1, as well as the structure of an epitope-specific TRAV9-2+-TRBV7-3+ TCR-HLA-DQ2.5-glia-ω1 complex. We found that position 7 (p7) of the DQ2.5-glia-α1a and DQ2.5-glia-ω1 epitopes made very limited contacts with the TRAV4+ TCR, thereby explaining the TCR cross-reactivity across these two epitopes. In contrast, within the TRAV9-2+ TCR-HLA-DQ2.5-glia-ω1 ternary complex, the p7-Gln was situated in an electrostatic pocket formed by the hypervariable CDR3ß loop of the TCR and Arg70ß from HLA-DQ2.5, a polar network which would not be supported by the p7-Leu residue of DQ2.5-glia-α1a. In conclusion, we provide additional insights into the molecular determinants of TCR specificity and cross-reactivity to two closely-related epitopes in celiac disease.

T-Cell Specificity Influences Disease Heterogeneity in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Encouraged by the enormous progress that the identification of specific autoantigens added to the understanding of neurologic autoimmune diseases, we undertook here an in-depth study of T-cell specificities in the autoimmune disease multiple sclerosis (MS), for which the spectrum of responsible autoantigens is not fully defined yet. The identification of target antigens in MS is crucial for therapeutic strategies aimed to induce antigen-specific tolerance. In addition, knowledge of relevant T-cell targets can improve our understanding of disease heterogeneity, a hallmark of MS that complicates clinical management. METHODS: The proliferative response and interferon gamma (IFN-γ) release of CSF-infiltrating CD4+ T cells from patients with MS against several autoantigens was used to identify patients with different intrathecal T-cell specificities. Fresh CSF-infiltrating and paired circulating lymphocytes in these patients were characterized in depth by ex vivo immunophenotyping and transcriptome analysis of relevant T-cell subsets. Further examination of these patients included CSF markers of inflammation and neurodegeneration and a detailed characterization with respect to demographic, clinical, and MRI features. RESULTS: By testing CSF-infiltrating CD4+ T cells from 105 patients with MS against seven long-known myelin and five recently described GDP-l-fucose synthase peptides, we identified GDP-l-fucose synthase and myelin oligodendrocyte glycoprotein (35-55) responder patients. Immunophenotyping of CSF and paired blood samples in these patients revealed a significant expansion of an effector memory (CCR7- CD45RA-) CD27- Th1 CD4+ cell subset in GDP-l-fucose synthase responders. Subsequent transcriptome analysis of this subset demonstrated expression of Th1 and cytotoxicity-associated genes. Patients with different intrathecal T-cell specificities also differ regarding inflammation- and neurodegeneration-associated biomarkers, imaging findings, expression of HLA class II alleles, and seasonal distribution of the time of the lumbar puncture. DISCUSSION: Our observations reveal an association between autoantigen reactivity and features of disease heterogeneity that strongly supports an important role of T-cell specificity in MS pathogenesis. These data have the potential to improve patient classification in clinical practice and to guide the development of antigen-specific tolerization strategies.

Antigen-Specific Treg Therapy in Type 1 Diabetes - Challenges and Opportunities.

Regulatory T cells (Tregs) are key mediators of peripheral self-tolerance and alterations in their frequencies, stability, and function have been linked to autoimmunity. The antigen-specific induction of Tregs is a long-envisioned goal for the treatment of autoimmune diseases given reduced side effects compared to general immunosuppressive therapies. However, the translation of antigen-specific Treg inducing therapies for the treatment or prevention of autoimmune diseases into the clinic remains challenging. In this mini review, we will discuss promising results for antigen-specific Treg therapies in allergy and specific challenges for such therapies in autoimmune diseases, with a focus on type 1 diabetes (T1D). We will furthermore discuss opportunities for antigen-specific Treg therapies in T1D, including combinatorial strategies and tissue-specific Treg targeting. Specifically, we will highlight recent advances in miRNA-targeting as a means to foster Tregs in autoimmunity. Additionally, we will discuss advances and perspectives of computational strategies for the detailed analysis of tissue-specific Tregs on the single-cell level.

T Cell Specificity: A Great Challenge in Chagas Disease.

The CD4+ and CD8+ T cell immune response against T. cruzi, the parasite causing Chagas disease, are relevant for both parasite control and disease pathogenesis. Several studies have been focused on their phenotype and functionally, but only a few have drilled down to identify the parasite proteins that are processed and presented to these cells, especially to CD4+ T lymphocytes. Although approximately 10,000 proteins are encoded per haploid T. cruzi genome, fewer than 200 T cell epitopes from 49 T. cruzi proteins have been identified so far. In this context, a detailed knowledge of the specific targets of T cell memory response emerges as a prime tool for the conceptualization and development of prophylactic or therapeutic vaccines, an approach with great potential to prevent and treat this chronic disease. Here, we review the available information about this topic in a comprehensive manner and discuss the future challenges in the field.

Is It Feasible to Use CMV-Specific T-Cell Adoptive Transfer as Treatment Against Infection in SOT Recipients?

During the last decade, many studies have demonstrated the role of CMV specific T-cell immune response on controlling CMV replication and dissemination. In fact, it is well established that transplanted patients lacking CMV-specific T-cell immunity have an increased occurrence of CMV replication episodes and CMV-related complications. In this context, the use of adoptive transfer of CMV-specific T-cells has been widely investigated and applied to Hematopoietic Stem Cell Transplant patients and may be useful as a therapeutic alternative, to reconstitute the CMV specific T-cell response and to control CMV viremia in patients receiving a transplantation. However, only few authors have explored the use of T-cell adoptive transfer in SOT recipients. We propose a novel review in which we provide an overview of the impact of using CMV-specific T-cell adoptive transfer on the control of CMV infection in SOT recipients, the different approaches to stimulate, isolate and expand CMV-specific T-cells developed over the years and a discussion of the possible use of CMV adoptive cellular therapy in this SOT population. Given the timeliness and importance of this topic, we believe that such an analysis will provide important insights into CMV infection and its treatment/prevention.

Clonally Focused Public and Private T Cells in Resected Brain Tissue From Surgeries to Treat Children With Intractable Seizures.

Using a targeted transcriptomics approach, we have analyzed resected brain tissue from a cohort of 53 pediatric epilepsy surgery cases, and have found that there is a spectrum of involvement of both the innate and adaptive immune systems as evidenced by the differential expression of immune-specific genes in the affected brain tissue. The specimens with the highest expression of immune-specific genes were from two Rasmussen encephalitis cases, which is known to be a neuro-immunological disease, but also from tuberous sclerosis complex (TSC), focal cortical dysplasia, and hemimegalencephaly surgery cases. We obtained T cell receptor (TCR) Vß chain sequence data from brain tissue and blood from patients with the highest levels of T cell transcripts. The clonality indices and the frequency of the top 50 Vß clonotypes indicated that T cells in the brain were clonally restricted. The top 50 Vß clonotypes comprised both public and private (patient specific) clonotypes, and the TCR Vß chain third complementarity region (CDR3) of the most abundant public Vß clonotype in each brain sample was strikingly similar to a CDR3 that recognizes an immunodominant epitope in either human cytomegalovirus or Epstein Barr virus, or influenza virus A. We found that the frequency of 14 of the top 50 brain Vß clonotypes from a TSC surgery case had significantly increased in brain tissue removed to control recurrent seizures 11 months after the first surgery. Conversely, we found that the frequency in the blood of 18 of the top 50 brain clonotypes from a second TSC patient, who was seizure free, had significantly decreased 5 months after surgery indicating that T cell clones found in the brain had contracted in the periphery after removal of the brain area associated with seizure activity and inflammation. However, the frequency of a public and a private clonotype significantly increased in the brain after seizures recurred and the patient underwent a second surgery. Combined single cell gene expression and TCR sequencing of brain-infiltrating leukocytes from the second surgery showed that the two clones were CD8 effector T cells, indicating that they are likely to be pathologically relevant.

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL.

Combining synthetic biology with adoptive T-cell transfer has led to promising advances in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). Chimeric antigen receptors (CARs) are synthetic receptors that redirect T-cell specificity against cancer. CARs include "built-in" signaling domains that reprogram T-cell metabolism, enhance effector function, and support long-term persistence. Despite their success in blood-based malignancies, relapse can occur in CD19-redirected CAR T-cell therapies for several reasons, including poor engraftment, impaired in vivo proliferation, and T-cell senescence. Herein, we explain how subtle alterations in CAR design may overcome barriers to effective adoptive immunotherapy. We also discuss how the physiochemical properties of the single-chain variable fragment (scFv) affect differentiation and persistence. Moreover, we describe innovative advances in CAR engineering and provide insight into the development of humanized scFvs whose proposed benefits include increased persistence and improved clinical outcomes. Tumor cells can evade CAR T-cell-mediated detection and elimination due to the emergence or presence of CD19-negative leukemic cell subpopulations. We also discuss the opportunities and challenges in targeting other B-ALL-associated antigens. Identifying alternate targets is fundamentally necessary to restore the success of CAR T-cell therapies in CD19-negative patients with B-ALL.

Single-cell transcriptome analysis of the heterogeneous effects of differential expression of tumor PD-L1 on responding TCR-T cells.

Rationale: TCR-T cell therapy plays a critical role in the treatment of malignant cancers. However, it is unclear how TCR-T cells are affected by PD-L1 molecule in the tumor environment. We performed an in-depth evaluation on how differential expressions of tumor PD-L1 can affect the functionality of T cells. Methods: We used MART-1-specific TCR-T cells (TCR-TMART-1), stimulated with MART-127-35 peptide-loaded MEL-526 tumor cells, expressing different proportions of PD-L1, to perform cellular assays and high-throughput single-cell RNA sequencing. Results: Different clusters of activated or cytotoxic TCR-TMART-1 responded divergently when stimulated with tumor cells expressing different percentages of PD-L1 expression. Compared to control T cells, TCR-TMART-1 were more sensitive to exhaustion, and secreted not only pro-inflammatory cytokines but also anti-inflammatory cytokines with increasing proportions of PD-L1+ tumor cells. The gene profiles of chemokines were modified by increased expression of tumor PD-L1, which concurrently downregulated pro-inflammatory and anti-inflammatory transcription factors. Furthermore, increased expression of tumor PD-L1 showed distinct effects on different inhibitory checkpoint molecules (ICMs). In addition, there was a limited correlation between the enrichment of cell death signaling in tumor cells and T cells and increased tumor PD-L1 expression. Conclusion: Overall, though the effector functionality of TCR-T cells was suppressed by increased expression percentages of tumor PD-L1 in vitro, scRNA-seq profiles revealed that both the anti-inflammatory and pro-inflammatory responses were triggered by a higher expression of tumor PD-L1. This suggests that the sole blockade of tumor PD-L1 might inhibit not only the anti-inflammatory response but also the pro-inflammatory response in the complicated tumor microenvironment. Thus, the outcome of PD-L1 intervention may depend on the final balance among the highly dynamic and heterogeneous immune regulatory circuits.

HLA-E-restricted, Gag-specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities.

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.

Immune profiling of Mycobacterium tuberculosis-specific T cells in recent and remote infection.

BACKGROUND: Recent Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of progression to tuberculosis disease, compared to persistent infection after remote exposure. However, current immunodiagnostic tools fail to distinguish between recent and remote infection. We aimed to characterise the immunobiology associated with acquisition of M.tb infection and identify a biomarker that can distinguish recent from remote infection. METHODS: Healthy South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 months) and persistent (QuantiFERON-TB+ for >1.5 year) infection. We characterised M.tb-specific CD4 T cell functional (IFN-γ, TNF, IL-2, CD107, CD154), memory (CD45RA, CCR7, CD27, KLRG-1) and activation (HLA-DR) profiles by flow cytometry after CFP-10/ESAT-6 peptide pool or M.tb lysate stimulation. We then assessed the diagnostic performance of immune profiles that were differentially expressed between individuals with recent or persistent QuantiFERON-TB+. FINDINGS: CFP-10/ESAT-6-specific CD4 T cell activation but not functional or memory phenotypes distinguished between individuals with recent and persistent QuantiFERON-TB+. In response to M.tb lysate, recent QuantiFERON-TB+ individuals had lower proportions of highly differentiated IFN-γ+TNF+ CD4 T cells expressing a KLRG-1+ effector phenotype and higher proportions of early differentiated IFN-γ-TNF+IL-2+ and activated CD4 T cells compared to persistent QuantiFERON-TB+ individuals. Among all differentially expressed T cell features CFP-10/ESAT-6-specific CD4 T cell activation was the best performing diagnostic biomarker of recent infection. INTERPRETATION: Recent M.tb infection is associated with highly activated and moderately differentiated functional M.tb-specific T cell subsets, that can be used as biomarkers to distinguish between recent and remote infection. FUNDING: US National Institutes of Health (NIH), Bill and Melinda Gates Foundation, South African National Research Foundation, South African Medical Research Council, and Aeras.

Direct control of CAR T cells through small molecule-regulated antibodies.

Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.

Antigen specificities and functional properties of MR1-restricted T cells.

MR1 is an MHC class I-like molecule with unique structural and biological features that make it an important member among the molecules involved in antigen presentation to T cells. Distinctive features include ubiquitous expression of the MR1 gene and its monomorphism. Another relevant property is that the MR1 protein appears at very low levels on the plasma membrane and its surface expression is regulated by antigen binding. Finally, the nature of presented antigens differs from those that bind other presenting molecules and includes small metabolites of microbial and self-origin, small drugs and tumor-associated antigens. This opinion paper describes in detail some of those features and discusses recent literature in the field.

A novel chimeric antigen receptor redirecting T-cell specificity towards CD26+ cancer cells.

Chimeric antigen receptor (CAR) T-cell immunotherapy is rapidly emerging as a promising novel treatment for malignancies. To broaden the success of CAR T-cell treatment for chronic myeloid leukaemia (CML), we attempted to construct a CD26 CAR T-cell product to target tyrosine kinase inhibitor-insensitive leukaemia stem cells (LSCs), which have been a challenge to cure for several decades and can be discriminated from healthy stem cells by the robust biomarker CD26. Of additional interest is that CD26 has also been reported to be a multi-purpose therapeutic target for other malignancies. Here, we constructed CD26 CAR T cells utilizing lentiviral transduction methods and verified them by flow cytometry analysis and RNA-seq. We found that the initial expansion of CD26 CAR-transduced T cells was delayed due to transient fratricide, but subsequent expansion was accelerated. CD26 CAR T cells exhibited cytotoxicity against the CD26+ T-cell lymphoma cell line Karpas 299, CD26-overexpressing K562 cells and primary CML LSCs, activated multiple effector functions in co-culture assays, and limited tumour progression in a mouse model; but there was some off-tumour cytotoxicity towards activated lymphocytes. In conclusion, these results establish the feasibility of using CD26 as an antigen for CAR T cells targeting CD26+ tumour cells.

Tumor-Specific CD8+ T-Cell Responses Induced by DNA Vaccination.

DNA vaccines assisted by electroporation efficiently trigger antitumor cytotoxic CD8+ T cell responses in preclinical cancer models and hold potential for human use. They can be easily engineered to express either tumor-associated self-antigens, which are broadly expressed among tumor patients but also in healthy tissue, or tumor-specific neoantigens, which are uniquely expressed in tumors and differ among patients. Recently, it has been demonstrated that DNA vaccination generates both circulating and tissue-resident compartments of CD8+ T cells, which act concertedly against tumors. Here we describe the steps to obtain and test DNA vaccines against models of self-antigens and neoantigens in mice. It includes the evaluation of effector and memory CD8+ T cell responses, as well as assessing the antitumor potential in vivo using transplantable syngeneic tumor models.

Biased TCR gene usage in citrullinated Tenascin C specific T-cells in rheumatoid arthritis.

We aimed to search for common features in the autoreactive T cell receptor (TCR) repertoire in patients with rheumatoid arthritis (RA), focusing on the newly identified candidate antigen citrullinated Tenascin C (cit-TNC). Mononuclear cells from peripheral blood or synovial fluid of eight RA-patients positive for the RA-associated HLA-DRB1*04:01 allele were in-vitro cultured with recently identified citrullinated peptides from Tenascin C. Antigen-specific T cells were isolated using peptide-HLA tetramer staining and subsequently single-cell sequenced for paired alpha/beta TCR analyses by bioinformatic tools. TCRs were re-expressed for further studies of antigen-specificity and T cell responses. Autoreactive T cell lines could be grown out from both peripheral blood and synovial fluid. We demonstrate the feasibility of retrieving true autoreactive TCR sequences by validating antigen-specificity in T cell lines with re-expressed TCRs. One of the Tenascin C peptides, cit-TNC22, gave the most robust T cell responses including biased TCR gene usage patterns. The shared TCR-beta chain signature among the cit-TNC22-specific TCRs was evident in blood and synovial fluid of different patients. The identification of common elements in the autoreactive TCR repertoire gives promise to the possibility of both immune monitoring of the autoimmune components in RA and of future antigen- or TCR-targeted specific intervention in subsets of patients.

Robust Production of Merkel Cell Polyomavirus Oncogene Specific T Cells From Healthy Donors for Adoptive Transfer.

Virus positive Merkel cell carcinoma (VP-MCC) is an aggressive but immunogenic skin malignancy driven by Merkel cell polyomavirus (MCPyV) T antigen (TAg). Since adoptive T cell transfer (ACT) can be effective against virus-driven malignancies, we set out to develop a methodology for generating MCPyV TAg specific T cells. MCPyV is a common, asymptomatic infection and virus-exposed healthy donors represent a potential source of MCPyV TAg specific T cells for ACT. Virus specific T cells were generated using monocyte-derived dendritic cells (moDCs) pulsed with MCPyV TAg peptide libraries and co-cultured with autologous T cells in supplemented with pro-inflammatory and homeostatic cytokines for 14 days. Specific reactivity was observed predominantly within the CD4+ T cell compartment in the cultures generated from 21/46 random healthy donors. Notably, responses were more often seen in donors aged 50 years and older. TAg specific CD4+ T cells specifically secreted Th1 cytokines and upregulated CD137 upon challenge with MCPyV TAg peptide libraries and autologous transduced antigen presenting cells. Expanded T cells from healthy donors recognized epitopes of both TAg splice variants found in VP-MCC tumors, and minimally expressed exhaustion markers. Our data show that MCPyV specific T cells can be expanded from healthy donors using methods appropriate for the manufacture of clinical grade ACT products.

Specificity of the T Cell Response to Protein Biopharmaceuticals.

The anti-drug antibody (ADA) response is an undesired humoral response raised against protein biopharmaceuticals (BPs) which can dramatically disturb their therapeutic properties. One particularity of the ADA response resides in the nature of the immunogens, which are usually human(ized) proteins and are therefore expected to be tolerated. CD4 T cells initiate, maintain and regulate the ADA response and are therefore key players of this immune response. Over the last decade, advances have been made in characterizing the T cell responses developed by patients treated with BPs. Epitope specificity and phenotypes of BP-specific T cells have been reported and highlight the effector and regulatory roles of T cells in the ADA response. BP-specific T cell responses are assessed in healthy subjects to anticipate the immunogenicity of BP prior to their testing in clinical trials. Immunogenicity prediction, also called preclinical immunogenicity assessment, aims at identifying immunogenic BPs and immunogenic BP sequences before any BP injection in humans. All of the approaches that have been developed to date rely on the detection of BP-specific T cells in donors who have never been exposed to BPs. The number of BP-specific T cells circulating in the blood of these donors is therefore limited. T cell assays using cells collected from healthy donors might reveal the weak tolerance induced by BPs, whose endogenous form is expressed at a low level. These BPs have a complete human sequence, but the level of their endogenous form appears insufficient to promote the negative selection of autoreactive T cell clones. Multiple T cell epitopes have also been identified in therapeutic antibodies and some other BPs. The pattern of identified T cell epitopes differs across the antibodies, notwithstanding their humanized, human or chimeric nature. However, in all antibodies, the non-germline amino acid sequences mainly found in the CDRs appear to be the main driver of immunogenicity, provided they can be presented by HLA class II molecules. Considering the fact that the BP field is expanding to include new formats and gene and cell therapies, we face new challenges in understanding and mastering the immunogenicity of new biological products.

Novel MHC-Independent αßTCRs Specific for CD48, CD102, and CD155 Self-Proteins and Their Selection in the Thymus.

MHC-independent αßTCRs (TCRs) recognize conformational epitopes on native self-proteins and arise in mice lacking both MHC and CD4/CD8 coreceptor proteins. Although naturally generated in the thymus, these TCRs resemble re-engineered therapeutic chimeric antigen receptor (CAR) T cells in their specificity for MHC-independent ligands. Here we identify naturally arising MHC-independent TCRs reactive to three native self-proteins (CD48, CD102, and CD155) involved in cell adhesion. We report that naturally arising MHC-independent TCRs require high affinity TCR-ligand engagements in the thymus to signal positive selection and that high affinity positive selection generates a peripheral TCR repertoire with limited diversity and increased self-reactivity. We conclude that the affinity of TCR-ligand engagements required to signal positive selection in the thymus inversely determines the diversity and self-tolerance of the mature TCR repertoire that is selected.

Neoantigens in Hematologic Malignancies.

T cell cancer neoantigens are created from peptides derived from cancer-specific aberrant proteins, such as mutated and fusion proteins, presented in complex with human leukocyte antigens on the cancer cell surface. Because expression of the aberrant target protein is exclusive to malignant cells, immunotherapy directed against neoantigens should avoid "on-target, off-tumor" toxicity. The efficacy of neoantigen vaccines in melanoma and glioblastoma and of adoptive transfer of neoantigen-specific T cells in epithelial tumors indicates that neoantigens are valid therapeutic targets. Improvements in sequencing technology and innovations in antigen discovery approaches have facilitated the identification of neoantigens. In comparison to many solid tumors, hematologic malignancies have few mutations and thus fewer potential neoantigens. Despite this, neoantigens have been identified in a wide variety of hematologic malignancies. These include mutated nucleophosmin1 and PML-RARA in acute myeloid leukemia, ETV6-RUNX1 fusions and other mutated proteins in acute lymphoblastic leukemia, BCR-ABL1 fusions in chronic myeloid leukemia, driver mutations in myeloproliferative neoplasms, immunoglobulins in lymphomas, and proteins derived from patient-specific mutations in chronic lymphoid leukemias. We will review advances in the field of neoantigen discovery, describe the spectrum of identified neoantigens in hematologic malignancies, and discuss the potential of these neoantigens for clinical translation.

Detection of Tumor Antigen-Specific T-Cell Responses After Oncolytic Vaccination.

Oncolytic vaccines, which consist of recombinant oncolytic viruses (OV) encoding tumor-associated antigens (TAAs), have demonstrated potent antitumor efficacy in preclinical models and are currently evaluated in phase I/II clinical trials. On one hand, oncolysis of OV-infected malignant entities reinstates cancer immunosurveillance. On the other hand, overexpression of TAAs in infected cells further stimulates the adaptive arm of antitumor immunity. Particularly, the presence of tumor-specific CD8+ T lymphocytes within the tumor microenvironment, as well as in the periphery, has demonstrated prognostic value for cancer treatments. These effector CD8+ T cells can be detected through their production of the prototypical Tc1 cytokine: IFN-γ. The quantitative and qualitative assessment of this immune cell subset remains critical in the development process of efficient cancer vaccines, including oncolytic vaccines. The present chapter will describe a single-cell immunological assay, namely the intracellular cytokine staining (ICS), that allows the enumeration of IFN-γ-producing TAA-specific CD8+ T cells in various tissues (tumor, blood, lymphoid organs) following oncolytic vaccination.