Improving Agri-environmental Schemes: Suggestions from Farmers and Nature Managers in a Central European Region.

Agri-environmental schemes (AES) are important policy instruments within the Common Agricultural Policy (CAP) of the European Union for environmental protection. Due to the voluntary nature of AES, their attractiveness to farmers and stakeholders involved in nature management and protection (nature managers) is essential for high participation levels. This study aims to assess farmers' and nature managers' ideas to improve agri-environmental schemes. We analyzed suggestions of 825 farmers and 118 nature managers for improvements of AES collected in a large-scale survey in Bavaria, Germany. A content analysis was applied to categorize and compare suggestions by farmers (differentiated into two groups through a cluster analysis) and nature managers. The results reveal that stakeholders were highly willing to share ideas and made detailed suggestions for improvements and individual measures. They were aware of the importance of protecting nature and promoting biodiversity in agricultural landscapes and acknowledged the necessity of (financial) support programs. Farmers placed more emphasis on the practicability and profitability of measures on arable land, while nature managers tended to propose policy-related ideas focusing on nature protection, biodiversity, and specific species. Among farmers, suggestions differed with farm characteristics such as the operation mode (full-time, part-time). These findings can support the design of future AES, accounting for different background situations and thereby increasing acceptability. This includes considering perspectives from different stakeholder groups and creating regionally adapted programs with varying levels of flexibility and practicability.

Teaching the Modeling of Human-Environment Systems: Acknowledging Complexity with an Agent-Based Model.

Agent-based modeling is a promising tool for familiarizing students with complex systems as well as programming skills. Human-environment systems, for instance, entail complex interdependencies that need to be considered when modeling these systems. This complexity is often neglected in teaching modeling approaches. For a heterogeneous group of master's students at a German university, we pre-built an agent-based model. In class, this was used to teach modeling impacts of land use policies and markets on ecosystem services. As part of the course, the students had to perform small research projects with the model in groups of two. This study aims to evaluate how well students could deal with the complexity involved in the model based on their group work outcomes. Chosen indicators were, e.g., the appropriateness of their research goals, the suitability of the methods applied, and how well they acknowledged the limitations. Our study results revealed that teaching complex systems does not need to be done with too simplistic models. Most students, even with little background in modeling and programming, were able to deal with the complex model setup, conduct small research projects, and have a thoughtful discussion on the limitations involved. With adequate theoretical input during lectures, we recommend using models that do not hide the complexity of the systems but foster a realistic simplification of the interactions. Supplementary Information: The online version contains supplementary material available at 10.1007/s10956-022-10022-z.

Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains.

Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αß heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (KD values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at higher surface levels than TCRs, they were 10-100-fold less sensitive, even in the absence of the CD8 coreceptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high Ag density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high Ag density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for the next-generation design of receptors used in adoptive T cell therapies.

A CD200R-CD28 fusion protein appropriates an inhibitory signal to enhance T-cell function and therapy of murine leukemia.

Acute myeloid leukemia (AML), the most common adult acute leukemia in the United States, has the poorest survival rate, with 26% of patients surviving 5 years. Adoptive immunotherapy with T cells genetically modified to recognize tumors is a promising and evolving treatment option. However, antitumor activity, particularly in the context of progressive leukemia, can be dampened both by limited costimulation and triggering of immunoregulatory checkpoints that attenuate T-cell responses. Expression of CD200 (OX2), a negative regulator of T-cell function that binds CD200 receptor (CD200R), is commonly increased in leukemia and other malignancies and is associated with poor prognosis in leukemia patients. To appropriate and redirect the inhibitory effects of CD200R signaling on transferred CD8+ T cells, we engineered CD200R immunomodulatory fusion proteins (IFPs) with the cytoplasmic tail replaced by the signaling domain of the costimulatory receptor, CD28. An analysis of a panel of CD200R-CD28 IFP constructs revealed that the most effective costimulation was achieved in IFPs containing a dimerizing motif and a predicted tumor-T-cell distance that facilitates localization to the immunological synapse. T cells transduced with the optimized CD200R-CD28 IFPs exhibited enhanced proliferation and effector function in response to CD200+ leukemic cells in vitro. In adoptive therapy of disseminated leukemia, CD200R-CD28-transduced leukemia-specific CD8 T cells eradicated otherwise lethal disease more efficiently than wild-type cells and bypassed the requirement for interleukin-2 administration to sustain in vivo activity. The transduction of human primary T cells with the equivalent human IFPs increased proliferation and cytokine production in response to CD200+ leukemia cells, supporting clinical translation. This trial was registered at www.clinicaltrials.gov as #NCT01640301.

Re-adapting T cells for cancer therapy: from mouse models to clinical trials.

Adoptive T-cell therapy involves the isolation, expansion, and reinfusion of T lymphocytes with a defined specificity and function as a means to eradicate cancer. Our research has focused on specifying the requirements for tumor eradication with antigen-specific T cells and T cells transduced to express a defined T-cell receptor (TCR) in mouse models and then translating these strategies to clinical trials. Our design of T-cell-based therapy for cancer has reflected efforts to identify the obstacles that limit sustained effector T-cell activity in mice and humans, design approaches to enhance T-cell persistence, develop methods to increase TCR affinity/T-cell functional avidity, and pursue strategies to overcome tolerance and immunosuppression. With the advent of genetic engineering, a highly functional population of T cells can now be rapidly generated and tailored for the targeted malignancy. Preclinical studies in faithful and informative mouse models, in concert with knowledge gained from analyses of successes and limitations in clinical trials, are shaping how we continue to develop, refine, and broaden the applicability of this approach for cancer therapy.

Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection.

Many of the most promising tumor antigens for T-cell-based cancer immunotherapies are unmodified self-antigens. Unfortunately, the avidity of T cells specific for these antigens is limited by central tolerance during T-cell development in the thymus, resulting in decreased anti-tumor efficacy of these T cells. One approach to overcoming this obstacle is to mutate T-cell receptor (TCR) genes from naturally occurring T cells to enhance the affinity for the target antigen. These enhanced-affinity TCRs can then be developed for use in TCR gene therapy. Although TCRs with significantly enhanced affinity have been generated using this approach, it is not clear whether these TCRs, which bypass the affinity limits imposed by negative selection, remain unresponsive to the low levels of self-antigen generally expressed by some normal tissues. Here we show that 2 variants of a high-affinity WT1-specific TCR with enhanced affinity for WT1 are safe and do not mediate autoimmune tissue infiltration or damage when transduced into peripheral CD8 T cells and transferred in vivo. However, if expressed in developing T cells and subjected to thymic selection, the same enhanced-affinity TCRs signal tolerance mechanisms in the thymus, resulting in T cells with attenuated antigen sensitivity in the periphery.

Abrogation of SRC homology region 2 domain-containing phosphatase 1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo.

T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8(+) T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8(+) T cells alone or in the context of also providing supplemental IL-2. SHP-1(-/-) and SHP-1(+/+) effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1(-/-) effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1(-/-) effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1(-/-) effector CD8(+) T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1-specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.

Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity.

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR specific for a tumor epitope, is a form of adoptive cell therapy (ACT) that has demonstrated promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, but also attractive targets for TCR-T cell therapy. However, MHC class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence that an epitope encompassing the mutation can bind HLA-A2 and induce T cell responses. We report that post-translational modifications of the protein on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRAS G12V peptide, rather than unmodified epitope, may be presented in HLA-A2 by tumor cells and impact recognition by TCRs. Using a novel computationally guided approach to design TCRs, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from rare primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate or demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these TCRs. These findings highlight the role of post-translational modifications in immune evasion and suggest that identifying and targeting such modifications should facilitate development of more effective TCR-T cell therapies.

Tumor Regression Following Engineered Polyomavirus-Specific T Cell Therapy in Immune Checkpoint Inhibitor-Refractory Merkel Cell Carcinoma.

Although immune check-point inhibitors (CPIs) revolutionized treatment of Merkel cell carcinoma (MCC), patients with CPI-refractory MCC lack effective therapy. More than 80% of MCC express T-antigens encoded by Merkel cell polyomavirus, which is an ideal target for T-cell receptor (TCR)-based immunotherapy. However, MCC often repress HLA expression, requiring additional strategies to reverse the downregulation for allowing T cells to recognize their targets. We identified TCRMCC1 that recognizes a T-antigen epitope restricted to human leukocyte antigen (HLA)-A*02:01. Seven CPI-refractory metastatic MCC patients received CD4 and CD8 T cells transduced with TCRMCC1 (TTCR-MCC1) preceded either by lymphodepleting chemotherapy or an HLA-upregulating regimen (single-fraction radiation therapy (SFRT) or systemic interferon gamma (IFNγ)) with concurrent avelumab. Two patients who received preceding SFRT and IFNγ respectively experienced tumor regression. One experienced regression of 13/14 subcutaneous lesions with 1 'escape' lesion and the other had delayed tumor regression in all lesions after initial progression. Although TTCR-MCC1 cells with an activated phenotype infiltrated tumors including the 'escape' lesion, all progressing lesions transcriptionally lacked HLA expression. While SFRT/IFNγ did not immediately upregulate tumor HLA expression, a secondary endogenous antigen-specific T cell infiltrate was detected in one of the regressing tumors and associated with HLA upregulation, indicating in situ immune responses have the potential to reverse HLA downregulation. Indeed, supplying a strong co-stimulatory signal via a CD200R-CD28 switch receptor allows TTCR-MCC1 cells to control HLA-downregulated MCC cells in a xenograft mouse model, upregulating HLA expression. Our results demonstrate the potential of TCR gene therapy for metastatic MCC and propose a next strategy for overcoming epigenetic downregulation of HLA in MCC.

Opposite effects of endogenous peptide-MHC class I on T cell activity in the presence and absence of CD8.

Nonstimulatory or endogenous peptide-MHC (pepMHC) presented on the surfaces of APCs, either alone or alongside agonist pepMHC, plays various roles in T cell selection and activation. To examine these properties in more detail, we explored several model systems of TCR and pepMHC ligands with sufficient affinity to be activated in the absence of CD8. The TCRs had a range of affinities for agonist and nonstimulatory ligands and were restricted by MHC class I alleles with different properties. We observed CD8-independent antagonism from TCR-pepMHC interactions with very low affinities (e.g., K(D) = 300 µM). In addition, endogenous peptide-L(d) complexes on APCs antagonized activation of coreceptor (CD8)-negative 2C T cells even by the strong agonist QL9-L(d). In contrast, TCRs m33 and 3D-PYY, restricted by K(b) and D(b), respectively, did not show signs of antagonism by endogenous pepMHC in the absence of CD8. This did not appear to be an inherent difference in the ability of the TCRs to be antagonized, as altered peptide ligands could antagonize each TCR. In the presence of CD8, endogenous pepMHC ligands acted in some cases as coagonists. These results show that endogenous pepMHC molecules exhibit complex behavior in T cells, leading to either reduced activity (e.g., in cases of low coreceptor levels) or enhanced activity (e.g., in presence of coreceptor). The behavior may be influenced by the ability of different TCRs to recognize endogenous pepMHC but also perhaps by the inherent properties of the presenting MHC allele.