This Future Challenges article summarizes views on future directions in immunological research presented at round-table discussions at the 4th Immunology workshop in the Lofoten Islands in Norway, held in August 2023, and subsequent responses to surveys sent to meeting participants. It also summarizes some of the conversations around the responsibility of scientists to communicate with the non-science community, and the approaches that we may use to meet this obligation.

In this article for the Highlights of 2023 Series, significant advancements in pediatric immunology are discussed, focusing on new diagnostic and therapeutic approaches. Key studies include the integration of genomic and proteomic profiling for better diagnosis of inborn errors of immunity, the impact of nongenetic factors such as autoantibodies on immune responses, the promising use of Janus kinase inhibitors and chimeric antigen receptor-T cell therapy for treating immune deficiencies and autoimmune diseases and the potential for a curative approach using prime editing. These developments mark a shift toward personalized and precision medicine in pediatric immunology.

In this article, I aim to give some pieces of career advise for young immunologists based on my own experiences.

UNASSIGNED: Autologous chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies achieves long-term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR-T manufacturing, the compromised fitness of patient-derived T cells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR-NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges.
UNASSIGNED: To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in-depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR-NK manufacturing pipeline.
UNASSIGNED: We identified adult peripheral blood-derived NK cells to be the superior source for generating a CAR-NK cell product because of a higher maximum yield of CAR-expressing NK cells combined with potent natural, as well as CAR-mediated anti-tumor effector functions.
UNASSIGNED: Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre- and post-transduction and high on-target cytotoxicity make peripheral blood-derived NK cells a feasible and attractive CAR-NK cell product for clinical utility.

UNASSIGNED: Colorectal cancer (CRC) is the third most common cancer worldwide in men and women. In the metastasized stage, treatment options and prognosis are limited. To address the high medical need of this patient population, we generated a CD276xCD3 bispecific antibody termed CC-3. CD276 is expressed on CRC cells and on tumor vessels, thereby allowing for a \"dual\" anticancer effect.
UNASSIGNED: This first-in-human clinical study is planned as a prospective multicenter trial, enrolling patients with metastatic CRC after three lines of therapy. During the dose-escalation part, initially, an accelerated titration design with single-patient cohorts is employed. Here, each patient will receive a fixed dose level (starting with 50 µg for the first patient); however, between patients, dose level may be increased by up to 100%, depending on the decision of a safety review committee. Upon occurrence of any adverse events (AEs) grade ≥2, dose-limiting toxicity (DLT), or reaching a dose level of ≥800 µg, the escalation will switch to a standard 3 + 3 dose design. After maximum tolerated dose (MTD) has been determined, defined as no more than one of the six patients experiencing DLT, an additional 14 patients receive CC-3 at the MTD level in the dose-expansion phase. Primary endpoints are incidence and severity of AEs, as well as the best objective response to the treatment according to response evaluation criteria in solid tumors (RECIST) 1.1. Secondary endpoints include overall safety, efficacy, survival, quality of life, and pharmacokinetic investigations.
UNASSIGNED: The CD276xCD3 study was approved by the Ethics Committee of the Medical Faculty of the Heinrich Heine University Düsseldorf and the Paul-Ehrlich-Institut (P00702). Clinical trial results will be published in peer-reviewed journals. Trial registration numbers: ClinicalTrials.cov Registry (NCT05999396) and EU ClinicalTrials Registry (EU trial number 2022-503084-15-00).

Associate Professor Victor Peperzak from the University Medical Center (UMC) Utrecht in the Netherlands discusses the opportunities and challenges of building a research group in the Netherlands. Victor highlights the scientific strengths of UMC Utrecht in juxtaposition with other universities and centers around the Netherlands, and highlights the collaborative nature of the Dutch research scene.

Katalin Karikó and Drew Weissman were given the 2023 Nobel Prize in Physiology or Medicine for their findings of nucleoside base modifications that lead to the development of effective mRNA vaccines against COVID-19. This was a remarkable achievement, given that their initial manuscript was rejected by Nature and Science in 2005. The development of mRNA vaccines lagged for more than a decade for several reasons, including the lack of funding, the perceived risks of the technology, and the scepticism of many scientists. Furthermore, Karikó and Weissman\'s study appeared to be technical and difficult to understand. The COVID-19 pandemic, on the other hand, has shown the importance of mRNA vaccine technology. COVID-19 mRNA vaccines have been highly effective in preventing serious illness, hospitalization, and death. The Nobel Prize for Karikó and Weissman highlights the importance of perseverance, diversity, and inclusion in translational immunology. We need to build a more inclusive scientific community, where scientists from all backgrounds are supported and their work is valued. This will result in more scientific breakthroughs and better healthcare for everyone.

UNASSIGNED: Thymus implantation is a recently FDA-approved therapy for congenital athymia. Patients receiving thymus implantation develop a functional but incomplete T cell compartment. Our objective was to develop a mouse model to study clinical thymus implantation in congenital athymia and to optimise implantation procedures to maximise T cell education and expansion of naïve T cells.
UNASSIGNED: Using Foxn1 nu athymic mice as recipients, we tested MHC-matched and -mismatched donor thymi that were implanted as fresh tissue or cultured to remove donor T cells. We first implanted thymus under the kidney capsule and then optimised intramuscular implantation. Using competitive adoptive transfer assays, we investigated whether the failure of newly developed T cells to expand into a complete T cell compartment was because of intrinsic deficits or whether there were deficits in engaging MHC molecules in the periphery. Finally, we tested whether recombinant IL-7 would promote the expansion of host naïve T cells educated by the implanted thymus.
UNASSIGNED: We determined that thymus implants in Foxn1 nu athymic mice mimic many aspects of clinical thymus implants in patients with congenital athymia. When we implanted cultured, MHC-mismatched donor thymus into Foxn1 nu athymic mice, mice developed a limited T cell compartment with notably underdeveloped naïve populations and overrepresented memory-like T cells. Newly generated T cells were predominantly educated by MHC molecules expressed by the donor thymus, thus potentially undergoing another round of selection once in the peripheral circulation. Using competitive adoptive transfer assays, we compared expansion rates of T cells educated on donor thymus versus T cells educated during typical thymopoiesis in MHC-matched and -mismatched environments. Once in the circulation, regardless of the MHC haplotypes, T cells educated on a donor thymus underwent abnormal expansion with initially more robust proliferation coupled with greater cell death, resembling IL-7 independent spontaneous expansion. Treating implanted mice with recombinant interleukin (IL-7) promoted homeostatic expansion that improved T cell development, expanded the T cell receptor repertoire, and normalised the naïve T cell compartment.
UNASSIGNED: We conclude that implanting cultured thymus into the muscle of Foxn1 nu athymic mice is an appropriate system to study thymus implantation for congenital athymia and immunodeficiencies. T cells are educated by the donor thymus, yet naïve T cells have deficits in expansion. IL-7 greatly improves T cell development after thymus implantation and may offer a novel strategy to improve outcomes of clinical thymus implantation.

Laboratory science sometimes looks like it\'s built exclusively for young people, but if you look closely, you\'ll find another group of scientists waiting to join your lab: those of us who didn\'t quite launch our careers on a normal trajectory. Welcoming a second-career scientist into your lab takes time and resources, but may just be well worth it. Here\'s what one second-career scientist wants you to know about supporting second careers in immunology.

Our work presents a high-throughput kinetic killing assay in the 3D matrix using high-content imaging that is a robust and powerful cytotoxicity assay for evaluating the killing efficiency of immune killer cells or conducting drug screening under physiologically and pathologically relevant scenarios, particularly in the context of solid tumors.