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BackgroundAn increasing body of evidence suggests that in addition to the type, density, and state of immune cells in the tumor microenvironment (TME), also their proximity to cancer cells influences immunotherapy outcome. For example, favorable responses to immune checkpoint inhibitors in melanoma are associated with higher densities of CD8+ tumor-infiltrating lymphocytes (TIL) within 20 μm distance of melanoma cells. This notion is in line with the understanding that upon specific antigen recognition, cytotoxic T cells physically engage with their target cells through their TCRs followed by immunological synapse formation. Indeed, structural and functional avidity of cytotoxic CD8+ T cells correlates strongly with their activity against cancer cells. Together, these observations point to the importance of direct interactions between cytotoxic T cells and tumor cells in the TME. This led us to investigate whether tumor-specific CD8+ T cells can be isolated from clinical cancer specimens as heterotypic clusters.Materials and MethodsWe employed a tumor cell-T cell co-culture in vitro model, patient samples and ex vivo assays. To evaluate functional interactions between human T cells and tumor cells, we made use of a system we engineered previously, comprising melanoma cells expressing both HLA-A*02:01 and the MART-1 tumor antigen. They were challenged with CD8+ T cells from PBMCs that were retrovirally transduced with a MART-1-specific TCR. To asses these interactions in patient material, upon surgical removal tissue was cut into small fragments, digested and analyzed by (image-based) flow cytometry. Interacting (cluster) and not-interacting (singlets) T cells were isolated and expanded in vitro. To characterize tumor cell:T cell interactions single cell TCR and RNA sequencing is used, as well as ex vivo co-cultures with autologous tumor cells.ResultsWe found that in defined co-cultures, tumor antigen-recognizing T cells were commonly enriched over non-specific T cells in heterotypic clusters with tumor cells, prompting us to investigate whether such specific clusters could be isolated also from cancer specimens. We observed that from 10/10 human melanoma metastases, we were able to isolate heterotypic clusters, comprising CD8+ T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs), which was validated by imaging flow cytometry. Upon expansion, CD8+ T cells from tumor cell clusters and APC clusters exerted on average 7.6-fold increased melanoma-killing activity over T cell singlets, which was associated with enhanced cytokine production. CD8+ T cells from clusters were enriched for tumor-reactive and exhausted gene signatures. Integration with T cell receptor (TCR)-sequencing showed increased clonality of clustered T cells, indicative of expansion upon antigen recognition.ConclusionsTogether, these results demonstrate that tumor-reactive CD8+ T cells are enriched in functional clusters with tumor cells and/or APCs, and that they can be isolated and expanded from clinical samples. Being often excluded in cell sorting procedures, these distinct heterotypic CD8+ T cell clusters serve as a valuable source amenable to deciphering functional tumor-immune cell interactions, while they may also be therapeutically explored. S. Ibáñez Molero: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL. J. Veldman: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL. J. J H Traets: None. A. George: None. K. Hoefakker: None. S. Pack: None. L. Tas: None. P. Alóndiga-Mérida: None. B. van den Broek: None. R. Harkes: None. M. Nieuwland: None. M. van Baalen: None. E. Mul: None. S. Tol: None. J.B.A.G. Haanen: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Amgen, Asher Bio, BioNTech, BMS, MSD, Novartis, Sastra Cell Therapy. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Neogene Tx. F. Consultant/Advisory Board; Modest; BMS, CureVac, GSK, Imcyse, Iovance Bio, Instil Bio, Immunocore, Ipsen, Merck Serono, MSD, Molecular Partners, Novartis, Pfizer, Roche/Genentech, Sanofi, Scenic, Third Rock Ventures, Achilles Tx, BioNTech US, Instil Bio, PokeAcell, T-Knife, Scenic, Neogene Therapeutics. W.J.V. Houdt: None. D.S. Peeper: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Oncode Institute, Dutch Cancer Society KWF. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL, Immagene. F. Consultant/Advisory Board; Modest; Immagene.

Adjuvant ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) both improve relapse-free survival of stage III melanoma patients 1 , 2 . In stage IV disease, the combination of ipilimumab + nivolumab is superior to ipilimumab alone and also appears to be more effective than nivolumab monotherapy 3 . Preclinical work suggests that neoadjuvant application of checkpoint inhibitors may be superior to adjuvant therapy 4 . To address this question and to test feasibility, 20 patients with palpable stage III melanoma were 1:1 randomized to receive ipilimumab 3 mg kg −1 and nivolumab 1 mg kg −1 , as either four courses after surgery (adjuvant arm) or two courses before surgery and two courses postsurgery (neoadjuvant arm). Neoadjuvant therapy was feasible, with all patients undergoing surgery at the preplanned time point. However in both arms, 9/10 patients experienced one or more grade 3/4 adverse events. Pathological responses were achieved in 7/9 (78%) patients treated in the neoadjuvant arm. None of these patients have relapsed so far (median follow-up, 25.6 months). We found that neoadjuvant ipilimumab + nivolumab expand more tumor-resident T cell clones than adjuvant application. While neoadjuvant therapy appears promising, with the current regimen it induced high toxicity rates; therefore, it needs further investigation to preserve efficacy but reduce toxicity. Neoadjuvant combination immunotherapy in patients with advanced melanoma shows favorable activity over adjuvant treatment and warrants future evaluation with modified dosing schedules to reduce treatment-related adverse events.

Background The gut microbiome plays an important role in immune modulation. Specifically, presence or absence of certain gut bacterial taxa has been associated with better antitumor immune responses. Furthermore, in trials using fecal microbiota transplantation (FMT) to treat melanoma patients unresponsive to immune checkpoint inhibitors (ICI), complete responses (CR), partial responses (PR), and durable stable disease (SD) have been observed. However, the underlying mechanism determining which patients will or will not respond and what the optimal FMT composition is, has not been fully elucidated, and a discrepancy in microbial taxa associated with clinical response has been observed between studies. Furthermore, it is unknown whether a change in the microbiome itself, irrespective of its origin, or FMT from ICI responding donors, is required for reversion of ICI-unresponsiveness. To address this, we will transfer microbiota of either ICI responder or nonresponder metastatic melanoma patients via FMT. Methods In this randomized, double-blinded phase Ib/IIa trial, 24 anti-PD1-refractory patients with advanced stage cutaneous melanoma will receive an FMT from either an ICI responding or nonresponding donor, while continuing anti-PD-1 treatment. Donors will be selected from patients with metastatic melanoma treated with anti-PD-1 therapy. Two patients with a good response (≥ 30% decrease according to RECIST 1.1 within the past 24 months) and two patients with progression (≥ 20% increase according to RECIST 1.1 within the past 3 months) will be selected as ICI responding or nonresponding donors, respectively. The primary endpoint is clinical benefit (SD, PR or CR) at 12 weeks, confirmed on a CT scan at 16 weeks. The secondary endpoint is safety, defined as the occurrence of grade ≥ 3 toxicity. Exploratory endpoints are progression-free survival and changes in the gut microbiome, metabolome, and immune cells. Discussion Transplanting fecal microbiota to restore the patients’ perturbed microbiome has proven successful in several indications. However, less is known about the potential role of FMT to improve antitumor immune response. In this trial, we aim to investigate whether administration of FMT can reverse resistance to anti-PD-1 treatment in patients with advanced stage melanoma, and whether the ICI-responsiveness of the feces donor is associated with its effectiveness. Trial registration ClinicalTrials.gov: NCT05251389 (registered 22-Feb-2022). Protocol V4.0 (08–02-2022).

Background: Because sunitinib can induce extensive necrosis in metastatic renal cell cancer (mRCC), we examined whether criteria defined by Choi might be valuable to predict early sunitinib efficacy. Methods: Computed tomography was used for measurement of tumour lesions in mm and lesion attenuation in Hounsfield units (HUs). According to Choi criteria partial response (PR) was defined as ⩾10% decrease in size or ⩾15% decrease in attenuation. Results: A total of 55 mRCC patients treated with sunitinib were included. At first evaluation, according to the Response Evaluation Criteria in Solid Tumours (RECIST) 7 patients had PR, 38 stable disease (SD), and 10 progressive disease (PD), whereas according to Choi criteria 36 patients had PR, 6 SD and 13 PD. Median tumour attenuation decreased from 66 to 47 HUs ( P ⩽0.001). In patients with PR, Choi criteria had a significantly better predictive value for progression-free survival and overall survival (both P s<0.001) than RECIST ( P =0.685 and 0.191 respectively). The predictive value for RECIST increased ( P =0.001 and <0.001 respectively), when best response during treatment was taken into account. Conclusion: Choi criteria could be helpful to define early mRCC patients who benefit from sunitinib, but the use of these criteria will not change the management of these patients.

Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. 'Empty MHC molecules' generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a high-throughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome.

Sunitinib has been registered for the treatment of advanced renal cell cancer (RCC). As patient inclusion was highly selective in previous studies, experience with sunitinib in general oncological practice remains to be reported. We determined the efficacy and safety of sunitinib in patients with advanced RCC included in an expanded access programme. ECOG performance status >1, histology other than clear cell and presence of brain metastases were no exclusion criteria. Eighty-two patients were treated: 23% reached a partial response, 50% had stable disease, 20% progressed and six patients were not evaluable. Median progression-free survival (PFS) was 9 months and median overall survival (OS) was 15 months. Importantly, 47 patients (57%) needed a dose reduction, 35 (43%) because of treatment-related adverse events, 10 (12%) because of continuous dosing, and two because of both. Stomatitis, fatigue, hand–foot syndrome and a combination of grade 1–2 adverse events were the most frequent reasons for dose reduction. In 40 patients (49%), there was severe toxicity, defined as dose reduction or permanent discontinuation, which was highly correlated with low body surface area, high age and female gender. On the basis of age and gender, a model was developed that could predict the probability of severe toxicity.

Background Usual type vulvar intraepithelial neoplasia (uVIN) is caused by HPV, predominantly type 16. Several forms of HPV immunotherapy have been studied, however, clinical results could be improved. A novel intradermal administration route, termed DNA tattooing, is superior in animal models, and was tested for the first time in humans with a HPV16 E7 DNA vaccine (TTFC-E7SH). Methods The trial was designed to test safety, immunogenicity, and clinical response of TTFC-E7SH in twelve HPV16 + uVIN patients. Patients received six vaccinations via DNA tattooing. The first six patients received 0.2 mg TTFC-E7SH and the next six 2 mg TTFC-E7SH. Vaccine-specific T-cell immunity was evaluated by IFNγ-ELISPOT and multiparametric flow cytometry. Results Only grade I-II adverse events were observed upon TTFC-E7SH vaccination. The ELISPOT analysis showed in 4/12 patients a response to the peptide pool containing shuffled E7 peptides. Multiparametric flow cytometry showed low CD4 + and/or CD8 + T-cell responses as measured by increased expression of PD-1 (4/12 in both), CTLA-4 (2/12 and 3/12), CD107a (5/12 and 4/12), or the production of IFNγ (2/12 and 1/12), IL-2 (3/12 and 4/12), TNFα (2/12 and 1/12), and MIP1β (3/12 and 6/12). At 3 months follow-up, no clinical response was observed in any of the twelve vaccinated patients. Conclusion DNA tattoo vaccination was shown to be safe. A low vaccine-induced immune response and no clinical response were observed in uVIN patients after TTFC-E7SH DNA tattoo vaccination. Therefore, a new phase I/II trial with an improved DNA vaccine format is currently in development for patients with uVIN.

Induction of immunity after DNA vaccination is generally considered a slow process. Here we show that DNA delivery to the skin results in a highly transient pulse of antigen expression. Based on this information, we developed a new rapid and potent intradermal DNA vaccination method. By short-interval intradermal DNA delivery, robust T-cell responses, of a magnitude sufficient to reject established subcutaneous tumors, are generated within 12 d. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations in which immunity is required in the shortest possible time.

Minor histocompatibility antigens (mHags) are immunogenic peptides from polymorphic cellular proteins that induce strong T-cell responses after human leukocyte antigen (HLA)-matched, mHag-mismatched stem-cell transplantation 1 , 2 . mHags with broad or limited tissue expression are target antigens for graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactivities 1 . Separation of these activities is crucial for adoptive immunotherapy of leukemia without GvH disease. Therefore, using a skin-explant assay we investigated the in situ activities of cytotoxic T lymphocytes (CTLs) specific for the ubiquitously expressed mHag H-Y and for the hematopoietic-restricted mHags HA-1 and HA-2. H-Y-specific CTLs, visualized by tetrameric HLA–mHag peptide complexes 3 , infiltrated male skin sections within 24 hours, induced severe GvH reactions of grade III–IV and produced high levels of IFN-γ. In contrast, CTLs specific for the hematopoietic system–specific mHags HA-1 and HA-2 induced no or low GvH reactions above background and produced little or no interferon-γ, unless the skin sections were preincubated with HA-1/HA-2 synthetic peptides. These results provide the first in situ dissection of GvH effects by mHag-specific CTLs and show that ubiquitously expressed mHags are the prime targets of GvH disease.