Alloreactive T lymphocytes are the primary mediators of immune responses in transplantation, both in the graft-versus-host and host-versus-graft directions. While essentially all clones comprising the human T cell repertoire have been selected on self-peptide presented by self–human leukocyte antigens (self-HLAs), much remains to be understood about the nature of clones capable of responding to allo-HLA molecules. Quantitative tools to study these cells are critical to understand fundamental features of this important response; however, the large size and diversity of the alloreactive T cell repertoire in humans presents a great technical challenge. We have developed a high-throughput T cell receptor (TCR) sequencing approach to characterize the human alloresponse. We present a statistical method to model T cell clonal frequency distribution and quantify repertoire diversity. Using these approaches, we measured the diversity and frequency of distinct alloreactive CD4 ⁺ and CD8 ⁺ T cell populations in HLA-mismatched responder-stimulator pairs. Our findings indicate that the alloimmune repertoire is highly specific for a given pair of individuals, that most alloreactive clones circulate at low frequencies, and that a high proportion of TCRs is likely able to recognize alloantigens.

Background Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. Methods Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. Results Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. Conclusion These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.

In humans receiving intestinal transplantation (ITx), long-term multilineage blood chimerism often develops. Donor T cell macrochimerism (≥4%) frequently occurs without graft-versus-host disease (GVHD) and is associated with reduced rejection. Here we demonstrate that patients with macrochimerism had high graft-versus-host (GvH) to host-versus-graft (HvG) T cell clonal ratios in their allografts. These GvH clones entered the circulation, where their peak levels were associated with declines in HvG clones early after transplant, suggesting that GvH reactions may contribute to chimerism and control HvG responses without causing GVHD. Consistently, donor-derived T cells, including GvH clones, and CD34⁺ hematopoietic stem and progenitor cells (HSPCs) were simultaneously detected in the recipients’ BM more than 100 days after transplant. Individual GvH clones appeared in ileal mucosa or PBMCs before detection in recipient BM, consistent with an intestinal mucosal origin, where donor GvH-reactive T cells expanded early upon entry of recipient APCs into the graft. These results, combined with cytotoxic single-cell transcriptional profiles of donor T cells in recipient BM, suggest that tissue-resident GvH-reactive donor T cells migrated into the recipient circulation and BM, where they destroyed recipient hematopoietic cells through cytolytic effector functions and promoted engraftment of graft-derived HSPCs that maintain chimerism. These mechanisms suggest an approach to achieving intestinal allograft tolerance.

Introduction The prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) is dismal and conventional chemotherapy treatment delivers limited survival improvement. Immunotherapy may complement our current treatment strategies. We previously demonstrated that the combination of an allogeneic tumour-lysate dendritic cell (DC) vaccine with an anti-CD40 agonistic antibody resulted in robust antitumour responses with survival benefit in a murine PDAC model. In the Rotterdam PancrEAtic Cancer Vaccination-2 trial, we aim to translate our findings into patients. This study will determine the safety of DC/anti-CD40 agonistic antibody combination treatment, and treatment-induced tumour-specific immunological responses. Methods and analysis In this open-label, single-centre (Erasmus Univsersity Medical Center, Rotterdam, Netherlands), single-arm, phase I dose finding study, adult patients with metastatic pancreatic cancer with progressive disease after FOLFIRINOX chemotherapy will receive monocyte-derived DCs loaded with an allogeneic tumour lysate in conjunction with a CD40 agonistic antibody. This combination-immunotherapy regimen will be administered three times every 2 weeks, and booster treatments will be given after 3 and 6 months following the third injection. A minimum of 12 and a maximum of 18 patients will be included. The primary endpoint is safety and tolerability of the combination immunotherapy. To determine the maximum tolerated dose, DCs will be given at a fixed dosage and anti-CD40 agonist in a traditional 3+3 dose-escalation design. Secondary endpoints include radiographic response according to the RECIST (V.1.1) and iRECIST criteria, and the detection of antitumour specific immune responses. Ethics and dissemination The Central Committee on Research Involving Human Subjects (CCMO; NL76592.000.21) and the Medical Ethics Committee (METC; MEC-2021-0566) of the Erasmus M.C. University Medical Center Rotterdam approved the conduct of the trial. Written informed consent will be required for all participants. The results of the trial will be submitted for publication in a peer-reviewed scientific journal. Trial registration number NL9723.

The data described was acquired as part of a clinical study with the aim to investigate the potential of tumor-reactive T-cell response as response to vaccination of pancreatic cancer patients with an allogenic tumor cell lysate vaccine (Lau et al., 2022). Proteomics analysis was carried out to identify tumor antigens that are shared between the allogeneic tumor cell lysate used for the vaccine and pancreatic ductal adenocarcinoma (PDAC) tissue samples. To this objective, cell lysates of the vaccine and of nine tissue samples were enzymatically digested and isotopically labeled with tandem mass tags (TMT) in a so-called six-plex manner (Thermo Fisher Scientific). Three pools were prepared by mixing the samples according to their TMT-labels. Subsequently, the three sample pools were fractionated into 24 fractions with high-pH reversed phase chromatography. These fractions were first analyzed on a nano-liquid chromatography (LC) system online coupled to a high-resolution Eclipse Orbitrap mass spectrometer (MS) equipped with a high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) source using a data-dependent MS2 shotgun method. Overall, 126,618 unique peptide sequences, on basis of 768,638 peptide spectra matches and corresponding to 7,597 protein groups, were identified in the total sample set including 61 tumor antigens (Supplement Table S2 in Lau et al. 2022) that were prioritized by Cheever and co-workers as vaccine target antigens on basis of a series of objective criteria (Cheever et al., 2009). In the second phase of the experiment, this set of tumor antigens was targeted using a serial precursor selection (SPS) MS3 method. From this data, ion trap MS2 and Orbitrap MS3 fragment spectra were extracted for peptide identification (protein sequence database-dependent search) and relative quantification using the TMT labels, respectively. The dataset ultimately allowed the identification and quantification of 51 proteins and 163 related peptide precursors with the TMT labels (see Fig. 2B and Supplemental Fig. 8, Lau et al. 2022).