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-T cell-mediated antitumor activity.

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR targeting a tumor epitope, is a form of adoptive cell therapy (ACT) that has exhibited promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, making them attractive targets for TCR-T cell therapy. However, 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 an epitope encompassing such mutations can bind HLA-A2 and induce T cell responses. We report post-translational modifications (PTMs) 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 the unmodified epitope, may be presented in HLA-A2 by tumor cells and impact TCR recognition. Using a novel computationally guided approach, 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 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/demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these new TCRs. Our findings highlight the role of PTMs in immune evasion and suggest identifying and targeting such modifications should make effective ACTs available for a substantially greater range of tumors than the current therapeutic landscape. One-sentence summary: Tumor cell methylation of KRAS G12V epitope in HLA-A2 permits immune evasion, and new TCRs were generated to overcome this with engineered cell therapy.

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.

Large libraries of single-chain trimer peptide-MHCs enable antigen-specific CD8+ T cell discovery and analysis.

The discovery and characterization of antigen-specific CD8+ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8+ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.

The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor.

Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.

Perceptions of ecosystem services: Comparing socio-cultural and environmental influences.

Ecosystem services such as food provisioning, climate regulation, nutrient cycling, or recreation in open landscapes underpin human wellbeing. They are highly dependent on land use, land cover and utilization pattern as well as environmental factors like climate, topography and soil. In consequence, ecosystem services supply shows a high spatial variability. However, it is less clear if the perception of the importance of ecosystem services is similarly heterogeneous in space and amongst societal actors. The aim of this large-scale study was to explore whether land cover and climate gradients as well as socio-cultural factors influence the perceptions of ecosystem services of four groups of societal actors: citizens, farmers, foresters and nature managers. Spatially explicit survey data of 3018 respondents allowed to gain insight into the distribution of perceived importance of 21 ecosystem services in the federal state of Bavaria, Germany together with the respondents' socio-cultural characterisation (e.g. gender, education and hobbies in nature). Responses were analysed through descriptive statistics, redundancy analysis, and Generalized Linear Models. Results reveal that the perceived importance of many ecosystem services was consistently high across groups, although perception differed for some ecosystem services (e.g. production of energy plants and timber as well as recreation in urban green space). Compared to other actor groups, farmers attributed slightly lower importance to all ES except provisioning services. Socio-cultural factors better explained variability in perceived importance of ecosystem services than land cover and climate gradients. This might be either explained by the fact that the environmental gradients vary not strong enough in our case study or that they do not shape the perceptions of respondents. A limitation of the study is that the sample of respondents obtained is not representative for the population, but biased towards persons interested in the topics of the survey. Still the consensus indicated by the overall positive perception of ecosystem services among respondents highlights the integrative potential of ecosystem services when included in decision-making.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions.

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25- (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor-ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

Adoptive immunotherapy of disseminated leukemia with TCR-transduced, CD8+ T cells expressing a known endogenous TCR.

Adoptive T-cell immunotherapy has shown promise in the treatment of human malignancies, but the challenge of isolating T cells with high avidity for tumor antigens in each patient has limited application of this approach. The transfer into T cells of T-cell receptor (TCR) genes encoding high-affinity TCRs recognizing defined tumor-associated antigens can potentially circumvent this obstacle. Using a well-characterized murine model of adoptive T-cell immunotherapy for widely disseminated leukemia, we demonstrate that TCR gene-modified T cells can cure mice of disseminated tumor. One goal of such adoptive therapy is to establish a persistent memory response to prevent recurrence; however, long-term function of transferred TCR-transduced T cells is limited due to reduced expression of the introduced TCR in vivo in quiescent resting T cells. However, by introducing the TCR into a cell with a known endogenous specificity, activation of these T cells by stimulation through the endogenous TCR can be used to increase expression of the introduced TCR, potentially providing a strategy to increase the total number of tumor-reactive T cells in the host and restore more potent antitumor activity.

T cell receptor gene therapy targeting WT1 prevents acute myeloid leukemia relapse post-transplant.

Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features1-3. When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells4. As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease5. Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens6. We therefore isolated a high-affinity Wilms' Tumor Antigen 1-specific TCR (TCRC4) from HLA-A2+ normal donor repertoires, inserted TCRC4 into Epstein-Bar virus-specific donor CD8+ T cells (TTCR-C4) to minimize graft-versus-host disease risk and enhance transferred T cell survival7,8, and infused these cells prophylactically post-HCT into 12 patients ( NCT01640301 ). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). TTCR-C4 maintained TCRC4 expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse.

Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3ß. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCRß chain libraries pre-enriched for target antigen specificity. Several TCRß chains paired with a transgenic TCRα chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.

The miR-106a~363Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27Kip1 cell cycle inhibitor.

The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363 Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363 Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27 Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.

Tracking the Fate and Origin of Clinically Relevant Adoptively Transferred CD8+ T Cells In Vivo.

Adoptively transferred tumor-specific cells can mediate tumor regression in cancers refractory to conventional therapy. Autologous polyclonal tumor-specific cytotoxic T cells (CTL) generated from peripheral blood and infused into patients with metastatic melanoma show enhanced persistence, compared to equivalent numbers of more extensively expanded monoclonal CTL, and are associated with complete remissions (CR) in select patients. We applied high-throughput T cell receptor Vß sequencing (HTTCS) to identify individual clonotypes within CTL products, track them in vivo post-infusion and then deduce the pre-adoptive transfer (endogenous) frequencies of cells ultimately responsible for tumor regression. The summed in vivo post-transfer frequencies of the top 25 HTTCS-defined clonotypes originally detected in the infused CTL population were comparable to enumeration by binding of antigen peptide-HLA multimers, revealing quantitative HTTCS is a reliable, multimer-independent alternative. Surprisingly, the polyclonal CTL products were composed predominantly of clonotypes that were of very low frequency (VLF) in the endogenous samples, often below the limit of HTTCS detection (0.001%). In patients who achieved durable CRs, the composition of transferred CTLs was dominated (57-90%) by cells derived from a single VLF clonotype. Thus, HTTCS now reveals that tumor-specific CTL enabling long-term tumor control originate from endogenous VLF populations that exhibit proliferative/survival advantages. Along with results indicating that naïve cell populations are most likely to contain cells that exist at VLF within the repertoire, our results provide a strong rationale for favoring T cells arising from VLF populations and with early-differentiation phenotypes when selecting subset populations for adoptive transfer.