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Cholangiocarcinomas are rare but aggressive liver tumors of high lethality with scarce treatment options. Here we report on the follow-up of a patient diagnosed with an intrahepatic cholangiocarcinoma who experienced repeated tumor recurrences including distant metastasis, therefore facing a dismal prognosis. At present, this patient is tumor-free for more than 8 years following repeated surgery and application of two successive personalized vaccines. In-depth functional immune cell analyses revealed a dominant CD4+ T-cell response against the vaccine antigens with infiltration of the tumor site and immune responses prevailing for years following the last vaccine administration. Additionally, spontaneous tumor neoantigen-specific CD4+ and CD8+ T-cell responses have been detected, which might have contributed to the outstanding outcome witnessed in this patient. This case report highlights vaccination strategies targeting non-mutated antigens as well as the increasingly recognized central role of antitumor CD4+ T cells.

Background: A previous phase I/II study demonstrated potent and long-term immune responses in men with prostate cancer following vaccination with a 20mer synthetic peptide (RV001) derived from the Ras homolog gene family member C protein (RhoC). Moreover, a fraction of patients experienced prostate-specific antigen (PSA) responses, which prompted the initiation of a phase II double-blind randomized trial (NCT04114825). The primary endpoint was to study whether vaccination could postpone PSA progression. Furthermore, the study included an evaluation of vaccination-induced immune responses, and in-depth in vitro studies of RhoC-specific CD4+ T cell responses. Methods: Men with non-metastatic biochemical recurrence after either radical prostatectomy or radiation therapy were eligible for the study. Participants were randomized 1:1 to either subcutaneous injections of 0.1 mg/mL RV001 emulsified in Montanide ISA 51, or a placebo. Vaccinations were administered every 2 weeks for the first six times, then five times every 4 weeks for a total treatment time of 30 weeks. Blood samples were collected from a subset of patients (n = 38) over the course of vaccination, and peripheral blood mononuclear cells (PBMCs) isolated for immunological assessment of vaccine-induced immune responses. Experiments using PBMCs from a healthy donor and a patient were performed to study the phenotype and function of RV001-specific CD4+ T cells. Results: A total of 192 men entered the study. There was no difference in time to PSA doubling, with 7.5 versus 9.3 months, or in time to initiating further therapies, 11.2 versus 17.6 months for treatment and control groups, respectively. At long-term follow-up, 12.9% of the patients in the vaccination arm had developed metastasis compared to 12% in the placebo arm. No serious treatment-related side effects were observed, and treatment-related adverse events did not differ between groups. Immunological examinations in a subset of patients demonstrated that the vaccination induced potent, long-lasting CD4+ T cell responses capable of proliferation and cytokine production. RV001-specific CD4+ T cells were shown to mediate cytotoxicity against a RhoC-expressing cancer cell line in an HLA-class II-dependent manner. Conclusions: Men randomized to active treatment with RV001V demonstrated the induction of potent, functionally capable, anti RhoC-CD4+ T cell responses. However, there was no benefit in time to biochemical progression, and no difference in time to the initiation of second-line therapies.

BackgroundPeptide-based vaccination is a rational option for immunotherapy of prostate cancer. In this first-in-man phase I/II study, we assessed the safety, tolerability and immunological impact of a synthetic long peptide vaccine targeting Ras homolog gene family member C (RhoC) in patients with prostate cancer. RhoC is a small GTPase overexpressed in advanced solid cancers, metastases and cancer stem cells.MethodsTwenty-two patients who had previously undergone radical prostatectomy received subcutaneous injections of 0.1 mg of a single RhoC-derived 20mer peptide emulsified in Montanide ISA-51 every 2 weeks for the first six times, then five times every 4 weeks for a total treatment time of 30 weeks. The drug safety and vaccine-specific immune responses were assessed during treatment and thereafter within a 13-month follow-up period. Serum level of prostate-specific antigen was measured up to 26 months postvaccination.ResultsMost patients (18 of 21 evaluable) developed a strong CD4 T cell response against the vaccine, which lasted at least 10 months following the last vaccination. Three promiscuouslypresented HLA-class II epitopes were identified. Vaccine-specific CD4 T cells were polyfunctional and effector memory T cells that stably expressed PD-1 (CD279) and OX-40 (CD134), but not LAG-3 (CD223). One CD8 T cell response was detected in addition. The vaccine was well tolerated and no treatment-related adverse events of grade ≥3 were observed.ConclusionTargeting of RhoC induced a potent and long-lasting T cell immunity in the majority of the patients. The study demonstrates an excellent safety and tolerability profile. Vaccination against RhoC could potentially delay or prevent tumor recurrence and metastasis formation.Trial registration number NCT03199872.

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8+ and CD4+ cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8+ T cells was dependent on the elongation site of the EP, whereas CD4+ T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8+ and CD4+ T cell reactivities.

BackgroundCD4+ T cells are conventionally thought of as mainly ‘help providers’ in anti-cancer immune responses, supporting the generation of robust CD8+ T cell responses. However, more recently CD4+ T cells and their role in anti-cancer responses have gained new interest based on data suggesting that CD4+ T cells are often induced in high frequency in therapeutic vaccination against cancer. Recently, vaccination with a RhoC derived long-synthetic peptide (RV001) induced a long-lasting CD4+ T cell response in prostate cancer patients.1 Nonetheless, it is not known whether endogenous RhoC protein is processed and presented as peptides in the context of HLA Class II molecules for recognition by CD4+ T cells. Similarly, it remains unclear whether the CD4+ T cells induced by the vaccine have the ability to directly kill cancer cells. To address these questions, we mimicked vaccine-induced RV001 specific CD4+ T cells in vitro and tested their capacity to kill cancer cells expressing their cognate antigen.MethodsT cells were isolated from the peripheral blood of a healthy donor and stimulated with peptide-pulsed autologous dendritic cells or PBMCs for five rounds. After each stimulation round, peptide reactivity was evaluated using EliSpot and intracellular cytokine staining (ICS). Peptide specific CD4+ T cells were sorted based on cytokine secretion and subsequently cloned by limiting dilution. Expanded clones were tested for cytotoxicity against FM3 cancer cells using xCELLigence Real-Time Cell Analysis and T cell receptor (TCR) assessment was performed using flow cytometry and polymerase chain reaction (PCR).ResultsAfter multiple rounds of stimulation, we successfully generated RV001-specific CD4+ T cells from a healthy donor. These reactive CD4+ T cells exhibited production of TNFα and IFNγ, along with the surface expression of the degranulation marker CD107a. Upon restimulation with the peptide, the generated CD4+ T cell clones demonstrated robust production of TNFα and IFNγ, as well as the synthesis of the cytotoxic molecules granzyme B and granulysin. Next, all clones showed cytotoxic activity upon peptide-pulsed FM3 cancer cells. For two of the clones, killing of unpulsed FM3, which express endogenous RhoC, was seen. Notably, the same TCR beta variable chain was found across the generated clones.ConclusionsWe showed that peptide-specific CD4+ T cells can produce cytotoxic molecules and mediate tumor cell killing. These findings offer a glimpse of the possible benefits of targeting CD4+ T responses in therapeutic cancer vaccination.ReferenceSchuhmacher J, Heidu S, Balchen T. Vaccination against RhoC induces long-lasting immune responses in patients with prostate cancer: results from a phase I/II clinical trial. J Immunother Cancer. 2020;8:e001157

We have previously shown that conformational change in the β 2 -integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8 + T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4 + T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the β 2 -integrin. The kinetics of β 2 -integrin activation was different on CD4 + and CD8 + T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4–6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4 + and CD8 + T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining β 2 -integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4 + T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4 + and CD8 + T cell reactivities, with versatile applicability in clinical and vaccination studies.

The B-cell maturation antigen (BCMA) is currently being evaluated as promising tumor-associated surface antigen for T-cell-based immunotherapy approaches, such as CAR T cells and bispecific antibodies, in multiple myeloma (MM). Cytotoxic T cells bearing BCMA-specific T-cell receptors might further allow targeting HLA-presented antigens derived from the intracellular domain of BCMA. By analyzing a mass spectrometry-acquired immunopeptidome dataset of primary MM samples and MM cell lines for BCMA-derived HLA ligands, we identified the naturally presented HLA-B*18-restricted ligand P(BCMA)B*18. Additionally, P(BCMA)B*18 was identified on primary CLL samples, thereby expanding the range for possible applications. P(BCMA)B*18 induced multifunctional BCMA-specific cells de novo from naïve CD8+ T cells of healthy volunteers. These T cells exhibited antigen-specific lysis of autologous peptide-loaded cells. Even in the immunosuppressive context of MM, we detected spontaneous memory T-cell responses against P(BCMA)B*18 in patients. By applying CTLA-4 and PD-1 inhibition in vitro we induced multifunctional P(BCMA)B*18-specific CD8+ T cells in MM patients lacking preexisting BCMA-directed immune responses. Finally, we could show antigen-specific lysis of autologous peptide-loaded target cells and even MM.1S cells naturally presenting P(BCMA)B*18 using patient-derived P(BCMA)B*18-specific T cells. Hence, this BCMA-derived T-cell epitope represents a promising target for T-cell-based immunotherapy and monitoring following immunotherapy in B-cell malignancy patients.

Tumour cells do not exist as isolated entities. Instead, they are surrounded by a variety of cells and extracellular matrix, which form the tumour microenvironment (TME). The interaction between cancer cells and their microenvironment is increasingly acknowledged as essential in dictating the outcome of the patients. The TME includes everything that surrounds tumour cells and is often highjacked by the latter to promote their growth, invasion, and immune escape. Immune cells and cancer-associated fibroblasts (CAFs) are essential components of the TME, and there is increasing evidence that their interaction constitutes a major player not only for tumour progression but also for therapy response.Recent work in the field of immuno-oncology resulted in the development of novel therapies that aim at activating immune cells against cancer cells to eliminate them. Despite their unprecedented success, the lack of response from a large portion of patients highlights the need for further progress and improvement. To achieve its ultimate goal, the interaction between cancer cells and the TME needs to be studied in-depth to allow the targeting of mechanisms that are involved in resistance or refractoriness to therapy. Moreover, predictive and prognostic biomarkers for patient stratification are still missing. In this review, we focus on and highlight the complexity of CAFs within the TME and how their interaction, particularly with immune cells, can contribute to treatment failure. We further discuss how this crosstalk can be further dissected and which strategies are currently used to target them.

Cancer immunotherapy activates the immune system to specifically target malignant cells. Research has often focused on CD8+ cytotoxic T cells, as those have the capacity to eliminate tumor cells after specific recognition upon TCR-MHC class I interaction. However, CD4+ T cells have gained attention in the field, as they are not only essential to promote help to CD8+ T cells, but are also able to kill tumor cells directly (via MHC-class II dependent recognition) or indirectly (e.g., via the activation of other immune cells like macrophages). Therefore, immunotherapy approaches have shifted from only stimulating CD8+ T cells to targeting and assessing both, CD4+ and CD8+ T cell subsets. Here, we discuss the various subsets of CD4+ T cells, their plasticity and functionality, their relevance in the antitumor immune response in patients affected by cancer, and their ever-growing role in therapeutic approaches for human cancer.

Monitoring T cells is of major importance for the development of immunotherapies. Recent sophisticated assays can address particular aspects of the anti-tumor T-cell repertoire or support very large-scale immune screening for biomarker discovery. Robust methods for the routine assessment of the quantity and quality of antigen-specific T cells remain, however, essential. This review discusses selected methods that are commonly used for T-cell monitoring and summarizes the advantages and limitations of these assays. We also present a new functional assay, which specifically detects activated β2 integrins within a very short time following CD8+ T-cell stimulation. Because of its unique and favorable characteristics, this assay could be useful for implementation into our T-cell monitoring toolbox.