An arginase1- and PD-L1-derived peptide-based vaccine for myeloproliferative neoplasms: A first-in-man clinical trial.

Introduction: Arginase-1 (ARG1) and Programed death ligand-1 (PD-L1) play a vital role in immunosuppression in myeloproliferative neoplasms (MPNs) and directly inhibit T-cell activation and proliferation. We previously identified spontaneous T-cell responses towards PD-L1 and ARG1 derived peptide epitopes in patients with MPNs. In the present First-in-Man study we tested dual vaccinations of ARG1- derived and PD-L1-derived peptides, combined with Montanide ISA-51 as adjuvant, in patients with Janus Kinase 2 (JAK2) V617F-mutated MPN. Methods: Safety and efficacy of vaccination with ARG1- derived and PD-L1-derived peptides with montanide as an adjuvant was tested in 9 patients with MPN The primary end point was safety and toxicity evaluation. The secondary end point was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT04051307). Results: The study included 9 patients with JAK2-mutant MPN of which 8 received all 24 planned vaccines within a 9-month treatment period. Patients reported only grade 1 and 2 vaccine related adverse events. No alterations in peripheral blood counts were identified, and serial measurements of the JAK2V617F allelic burden showed that none of the patients achieved a molecular response during the treatment period. The vaccines induced strong immune responses against both ARG1 and PD-L1- derived epitopes in the peripheral blood of all patients, and vaccine-specific skin-infiltrating lymphocytes from 5/6 patients could be expanded in vitro after a delayed-type hypersensitivity test. In two patients we also detected both ARG1- and PD-L1-specific T cells in bone marrow samples at the end of trial. Intracellular cytokine staining revealed IFNγ and TNFγ producing CD4+- and CD8+- T cells specific against both vaccine epitopes. Throughout the study, the peripheral CD8/CD4 ratio increased significantly, and the CD8+ TEMRA subpopulation was enlarged. We also identified a significant decrease in PD-L1 mRNA expression in CD14+ myeloid cells in the peripheral blood in all treated patients and a decrease in ARG1 mRNA expression in bone marrow of 6 out of 7 evaluated patients. Conclusion: Overall, the ARG1- and PD-L1-derived vaccines were safe and tolerable and induced strong T-cell responses in all patients. These results warrant further studies of the vaccine in other settings or in combination with additional immune-activating treatments.

Pre-existing TGF-ß-specific T-cell immunity in patients with pancreatic cancer predicts survival after checkpoint inhibitors combined with radiotherapy.

BACKGROUND: Circulating transforming growth factor-ß (TGF-ß)-specific T cells that recognize TGF-ß-expressing immune regulatory cells have been described in patients with cancer. TGF-ß-derived peptide vaccination modulates the tumor microenvironment and has shown clinical effects in animal models of pancreatic cancer (PC). TGF-ß-expressing regulatory cells are especially elevated in PC and may prevent the clinical response to immune checkpoint inhibitors (ICIs). Thus, in the present study we investigated the significance of TGF-ß-specific T-cell immunity in patients with PC treated with ICI combined with radiotherapy in a randomized phase 2 study (CheckPAC). METHODS: Immune responses to a TGF-ß-derived epitope entitled TGF-ß-15 as well as epitopes from Clostridium tetani (tetanus) and influenza were measured in peripheral blood mononuclear cells (PBMCs) with interferon-É£ enzyme-linked immunospot assays. PBMCs were isolated before and after treatment. Correlations between immune response data and clinical data were evaluated with parametric and non-parametric statistical methods. Survival was analyzed with univariate and multivariate Cox-regression. TGF-ß-15 specific T cells were isolated and expanded and examined for recognition of autologous regulatory immune cells by flow cytometry. RESULTS: PBMCs from 32 patients were analyzed for immune responses to the TGF-ß-derived epitope entitled TGF-ß-15. Patients with a strong TGF-ß-specific immune response at treatment initiation had longer progression-free and overall survival, compared with patients with a weak or no TGF-ß-specific immune response. This remained significant in multivariate analysis. Patients with weak and strong TGF-ß-specific responses displayed similar responses towards viral antigens. Furthermore, we show that TGF-ß-specific T cells from a clinical responder specifically reacted to and lysed autologous, regulatory immune cells. Finally, mimicking a TGF-ß-15 vaccination, we showed that repeated stimulations with the TGF-ß-15 epitope in vitro enhanced the immune response to TGF-ß-15. CONCLUSION: A strong TGF-ß-15 specific immune response was associated with clinical benefit and improved survival after ICI/radiotherapy for patients with PC. Importantly, the lack of TGF-ß-specific T cells in some patients was not caused by a general immune dysfunction. TGF-ß-specific T cells recognized regulatory immune cells and could be introduced in vitro in patients without spontaneous responses. Taken together, our data suggest that combining TGF-ß-based vaccination with ICI/radiotherapy will be beneficial for patients with PC.

Reduced vitamin D-induced cathelicidin production and killing of Mycobacterium tuberculosis in macrophages from a patient with a non-functional vitamin D receptor: A case report.

Tuberculosis (TB) presents a serious health problem with approximately a quarter of the world's population infected with Mycobacterium tuberculosis (M. tuberculosis) in an asymptomatic latent state of which 5-10% develops active TB at some point in their lives. The antimicrobial protein cathelicidin has broad antimicrobial activity towards viruses and bacteria including M. tuberculosis. Vitamin D increases the expression of cathelicidin in many cell types including macrophages, and it has been suggested that the vitamin D-mediated antimicrobial activity against M. tuberculosis is dependent on the induction of cathelicidin. However, unraveling the immunoregulatory effects of vitamin D in humans is hampered by the lack of suitable experimental models. We have previously described a family in which members suffer from hereditary vitamin D-resistant rickets (HVDRR). The family carry a mutation in the DNA-binding domain of the vitamin D receptor (VDR). This mutation leads to a non-functional VDR, meaning that vitamin D cannot exert its effect in family members homozygous for the mutation. Studies of HVDRR patients open unique possibilities to gain insight in the immunoregulatory roles of vitamin D in humans. Here we describe the impaired ability of macrophages to produce cathelicidin in a HVDRR patient, who in her adolescence suffered from extrapulmonary TB. The present case is a rare experiment of nature, which illustrates the importance of vitamin D in the pathophysiology of combating M. tuberculosis.

Cytotoxic T cells isolated from healthy donors and cancer patients kill TGFß-expressing cancer cells in a TGFß-dependent manner.

Transforming growth factor-beta (TGFß) is a highly potent immunosuppressive cytokine. Although TGFß is a tumor suppressor in early/premalignant cancer lesions, the cytokine has several tumor-promoting effects in advanced cancer; abrogation of the antitumor immune response is one of the most important tumor-promoting effects. As several immunoregulatory mechanisms have recently been shown to be targets of specific T cells, we hypothesized that TGFß is targeted by naturally occurring specific T cells and thus could be a potential target for immunomodulatory cancer vaccination. Hence, we tested healthy donor and cancer patient T cells for spontaneous T-cell responses specifically targeting 38 20-mer epitopes derived from TGFß1. We identified numerous CD4+ and CD8+ T-cell responses against several epitopes in TGFß. Additionally, several ex vivo responses were identified. By enriching specific T cells from different donors, we produced highly specific cultures specific to several TGFß-derived epitopes. Cytotoxic CD8+ T-cell clones specific for both a 20-mer epitope and a 9-mer HLA-A2 restricted killed epitope peptide were pulsed in HLA-A2+ target cells and killed the HLA-A2+ cancer cell lines THP-1 and UKE-1. Additionally, stimulation of THP-1 cancer cells with cytokines that increased TGFß expression increased the fraction of killed cells. In conclusion, we have shown that healthy donors and cancer patients harbor CD4+ and CD8+ T cells specific for TGFß-derived epitopes and that cytotoxic T cells with specificity toward TGFß-derived epitopes are able to recognize and kill cancer cell lines in a TGFß-dependent manner.

Frequent adaptive immune responses against arginase-1.

The enzyme arginase-1 reduces the availability of arginine to tumor-infiltrating immune cells, thus reducing T-cell functionality in the tumor milieu. Arginase-1 is expressed by some cancer cells and by immune inhibitory cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), and its expression is associated with poor prognosis. In the present study, we divided the arginase-1 protein sequence into overlapping 20-amino-acid-long peptides, generating a library of 31 peptides covering the whole arginase-1 sequence. Reactivity towards this peptide library was examined in PBMCs from cancer patients and healthy individuals. IFNγ ELISPOT revealed frequent immune responses against multiple arginase-1-derived peptides. We further identified a hot-spot region within the arginase-1 protein sequence containing multiple epitopes recognized by T cells. Next, we examined in vitro-expanded tumor-infiltrating lymphocytes (TILs) isolated from melanoma patients, and detected arginase-1-specific T cells that reacted against epitopes from the hot-spot region. Arginase-1-specific CD4+T cells could be isolated and expanded from peripheral T cell pool of a patient with melanoma, and further demonstrated the specificity and reactivity of these T cells. Overall, we showed that arginase-1-specific T cells were capable of recognizing arginase-1-expressing cells. The activation of arginase-1-specific T cells by vaccination is an attractive approach to target arginase-1-expressing malignant cells and inhibitory immune cells. In the clinical setting, the induction of arginase-1-specific immune responses could induce or increase Th1 inflammation at the sites of tumors that are otherwise excluded due to infiltration with MDSCs and TAMs.

Hyperdimensional analysis of amino acid pair distributions in proteins.

Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis.