Miniaturized CAR knocked onto CD3ε extends TCR function with CAR specificity under control of endogenous TCR signaling cascade.

Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.

Memory T cells effectively recognize the SARS-CoV-2 hypermutated BA.2.86 variant.

T cells are critical in mediating the early control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection. However, it remains unknown whether memory T cells can effectively cross-recognize new SARS-CoV-2 variants with a broad array of mutations, such as the emergent hypermutated BA.2.86 variant. Here, we report in two separate cohorts, including healthy controls and individuals with chronic lymphocytic leukemia, that SARS-CoV-2 spike-specific CD4+ and CD8+ T cells induced by prior infection or vaccination demonstrate resilient immune recognition of BA.2.86. In both cohorts, we found largely preserved SARS-CoV-2 spike-specific CD4+ and CD8+ T cell magnitudes against mutated spike epitopes of BA.2.86. Functional analysis confirmed that both cytokine expression and proliferative capacity of SARS-CoV-2 spike-specific T cells to BA.2.86-mutated spike epitopes are similarly sustained. In summary, our findings indicate that memory CD4+ and CD8+ T cells continue to provide cell-mediated immune recognition to highly mutated emerging variants such as BA.2.86.

Additive effects of booster mRNA vaccination and SARS-CoV-2 Omicron infection on T cell immunity across immunocompromised states.

Suboptimal immunity to SARS-CoV-2 mRNA vaccination has frequently been observed in individuals with various immunodeficiencies. Given the increased antibody evasion properties of emerging SARS-CoV-2 subvariants, it is necessary to assess whether other components of adaptive immunity generate resilient and protective responses against infection. We assessed T cell responses in 279 individuals, covering five different immunodeficiencies and healthy controls, before and after booster mRNA vaccination, as well as after Omicron infection in a subset of patients. We observed robust and persistent Omicron-reactive T cell responses that increased markedly upon booster vaccination and correlated directly with antibody titers across all patient groups. Poor vaccination responsiveness in immunocompromised or elderly individuals was effectively counteracted by the administration of additional vaccine doses. Functionally, Omicron-reactive T cell responses exhibited a pronounced cytotoxic profile and signs of longevity, characterized by CD45RA+ effector memory subpopulations with stem cell-like properties and increased proliferative capacity. Regardless of underlying immunodeficiency, booster-vaccinated and Omicron-infected individuals appeared protected against severe disease and exhibited enhanced and diversified T cell responses against conserved and Omicron-specific epitopes. Our findings indicate that T cells retain the ability to generate highly functional responses against newly emerging variants, even after repeated antigen exposure and a robust immunological imprint from ancestral SARS-CoV-2 mRNA vaccination.

[The Journal "Deine Gesundheit" - A Reflection of Social Psychiatry in the GDR?] / "Deine Gesundheit" ­ Spiegelbild der Sozialpsychiatrie in der DDR?

BACKGROUND: Articles on psychiatric care in the GDR published in the journal "Deine Gesundheit" are identified. This involved examining how psychiatry was presented to the public and the intentions of addressing a lay audience. METHODOLOGY: All booklets published between 1955 and 1989 were systematically reviewed, the role of the publishers examined, and an assessment made in the context of social psychiatry and sociopolitical conditions. RESULTS: There was a lively publication activity of psychiatric topics by mainly professional actors. The temporal accumulation in the context of psychiatric reform efforts is striking. CONCLUSIONS: Reform-oriented psychiatrists in particular used the popular science medium to reach a broad public and thus greater social acceptance of community psychiatric care concepts.

Immunodeficiency syndromes differentially impact the functional profile of SARS-CoV-2-specific T cells elicited by mRNA vaccination.

Many immunocompromised patients mount suboptimal humoral immunity after SARS-CoV-2 mRNA vaccination. Here, we assessed the single-cell profile of SARS-CoV-2-specific T cells post-mRNA vaccination in healthy individuals and patients with various forms of immunodeficiencies. Impaired vaccine-induced cell-mediated immunity was observed in many immunocompromised patients, particularly in solid-organ transplant and chronic lymphocytic leukemia patients. Notably, individuals with an inherited lack of mature B cells, i.e., X-linked agammaglobulinemia (XLA) displayed highly functional spike-specific T cell responses. Single-cell RNA-sequencing further revealed that mRNA vaccination induced a broad functional spectrum of spike-specific CD4+ and CD8+ T cells in healthy individuals and patients with XLA. These responses were founded on polyclonal repertoires of CD4+ T cells and robust expansions of oligoclonal effector-memory CD45RA+ CD8+ T cells with stem-like characteristics. Collectively, our data provide the functional continuum of SARS-CoV-2-specific T cell responses post-mRNA vaccination, highlighting that cell-mediated immunity is of variable functional quality across immunodeficiency syndromes.

Ancestral SARS-CoV-2-specific T cells cross-recognize the Omicron variant.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron (B.1.1.529) variant of concern (VOC) has destabilized global efforts to control the impact of coronavirus disease 2019 (COVID-19). Recent data have suggested that B.1.1.529 can readily infect people with naturally acquired or vaccine-induced immunity, facilitated in some cases by viral escape from antibodies that neutralize ancestral SARS-CoV-2. However, severe disease appears to be relatively uncommon in such individuals, highlighting a potential role for other components of the adaptive immune system. We report here that SARS-CoV-2 spike-specific CD4+ and CD8+ T cells induced by prior infection or BNT162b2 vaccination provide extensive immune coverage against B.1.1.529. The median relative frequencies of SARS-CoV-2 spike-specific CD4+ T cells that cross-recognized B.1.1.529 in previously infected or BNT162b2-vaccinated individuals were 84% and 91%, respectively, and the corresponding median relative frequencies for SARS-CoV-2 spike-specific CD8+ T cells were 70% and 92%, respectively. Pairwise comparisons across groups further revealed that SARS-CoV-2 spike-reactive CD4+ and CD8+ T cells were functionally and phenotypically similar in response to the ancestral strain or B.1.1.529. Collectively, our data indicate that established SARS-CoV-2 spike-specific CD4+ and CD8+ T cell responses, especially after BNT162b2 vaccination, remain largely intact against B.1.1.529.

Orthotopic T-cell receptor replacement in primary human T cells using CRISPR-Cas9-mediated homology-directed repair.

Adoptive T cell therapy using T-cell receptor (TCR)-engineered T cells allows to redirect T cell specificity and to target any antigen of interest. Here, we apply advanced genetic engineering using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) for simultaneous editing of TCR α- and ß-chains in primary human T cells. Together with non-virally delivered template DNA, this CRISPR-Cas9-system allows for elimination of the endogenous TCR and orthotopic placement of TCR α- and ß-chains. For complete details on the use and execution of this protocol, please refer to Schober et al. (2019) and Müller et al. (2021).

Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections.

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. T cell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive T cell transfer (ACT) approaches. T cell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific T cells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated ex vivo and preclinically engineered into the TCR locus of allogeneic third-party primary T cells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited T cells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, ex vivo isolated TCRs with clinical proven protection through ACT could be redirected into T cells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections.

Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.

A T-cell reporter platform for high-throughput and reliable investigation of TCR function and biology.

OBJECTIVE: Transgenic re-expression enables unbiased investigation of T-cell receptor (TCR)-intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαß constructs now facilitate large-scale transgenic TCR re-expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisation of re-expressed TCRs is still a complicated and laborious matter. Here, we propose a Jurkat-based triple parameter TCR signalling reporter endogenous TCR knockout cellular platform (TPRKO) that offers an unbiased, easy read-out of TCR functionality and facilitates high-throughput screening approaches. METHODS: As a proof-of-concept, we transgenically re-expressed 59 human cytomegalovirus-specific TCRs and systematically investigated and compared TCR function in TPRKO cells versus primary human T cells. RESULTS: We demonstrate that the TPRKO cell line facilitates antigen-HLA specificity screening via sensitive peptide-MHC-multimer staining, which was highly comparable to primary T cells. Also, TCR functional avidity in TPRKO cells was strongly correlating to primary T cells, especially in the absence of CD8αß co-receptor. CONCLUSION: Overall, our data show that the TPRKO cell lines can serve as a surrogate of primary human T cells for standardised and high-throughput investigation of TCR biology.

Orthotopic T-Cell Receptor Replacement-An "Enabler" for TCR-Based Therapies.

Natural adaptive immunity co-evolved with pathogens over millions of years, and adoptive transfer of non-engineered T cells to fight infections or cancer so far exhibits an exceptionally safe and functional therapeutic profile in clinical trials. However, the personalized nature of therapies using virus-specific T cells, donor lymphocyte infusion, or tumor-infiltrating lymphocytes makes implementation in routine clinical care difficult. In principle, genetic engineering can be used to make T-cell therapies more broadly applicable, but so far it significantly alters the physiology of cells. We recently demonstrated that orthotopic T-cell receptor (TCR) replacement (OTR) by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) can be used to generate engineered T cells with preservation of near-physiological function. In this review, we present the current status of OTR technology development and discuss its potential for TCR-based therapies. By providing the means to combine the therapeutic efficacy and safety profile of physiological T cells with the versatility of cell engineering, OTR can serve as an "enabler" for TCR-based therapies.

Reverse TCR repertoire evolution toward dominant low-affinity clones during chronic CMV infection.

Adaptive evolution is a key feature of T cell immunity. During acute immune responses, T cells harboring high-affinity T cell antigen receptors (TCRs) are preferentially expanded, but whether affinity maturation by clonal selection continues through the course of chronic infections remains unresolved. Here we investigated the evolution of the TCR repertoire and its affinity during the course of infection with cytomegalovirus, which elicits large T cell populations in humans and mice. Using single-cell and bulk TCR sequencing and structural affinity analyses of cytomegalovirus-specific T cells, and through the generation and in vivo monitoring of defined TCR repertoires, we found that the immunodominance of high-affinity T cell clones declined during the chronic infection phase, likely due to cellular senescence. These data showed that under conditions of chronic antigen exposure, low-affinity TCRs preferentially expanded within the TCR repertoire, with implications for immunotherapeutic strategies.

Orthotopic replacement of T-cell receptor α- and ß-chains with preservation of near-physiological T-cell function.

Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and ß-chains combined with orthotopic TCR placement leads to accurate αß-pairing and results in TCR regulation similar to that of physiological T cells.

FLEXamers: A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers.

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.

Efficient immunoaffinity chromatography of lymphocytes directly from whole blood.

We show that defined lymphocytes can be rapidly purified by immunoaffinity chromatography starting directly from whole blood. The method relies on low-affinity Fab-fragments attached to a column-matrix combined with the reversible Strep-tag technology. Compared to established cell enrichment protocols, the Strep-tag affinity chromatography of cells is independent of erythrocyte lysis or centrifugation steps, allowing for simple cell-enrichment with good yields, high purities, and excellent functionality of purified cells.

Single-agent pemetrexed or sequential pemetrexed/gemcitabine as front-line treatment of advanced non-small cell lung cancer in elderly patients or patients ineligible for platinum-based chemotherapy: a multicenter, randomized, phase II trial.

INTRODUCTION: This randomized phase II trial evaluated single-agent pemetrexed or sequential pemetrexed/gemcitabine in patients with non-small cell lung cancer (NSCLC) who were elderly (> or = 70 years) or younger than 70 years and ineligible for platinum-based chemotherapy. METHODS: Chemonaive patients with stage IIIB/IV NSCLC and an Eastern Cooperative Oncology Group performance status of 0 to 2 received either 500 mg/m2 of pemetrexed (day 1, every 3 weeks) for eight cycles, or the same dosage of pemetrexed for cycles 1 and 2 and then 1200 mg/m2 of gemcitabine (days 1 and 8, every 3 weeks) for cycles 3 and 4 (repeated once for a total of eight cycles). All patients were given vitamin B12 and folic acid supplementation. RESULTS: From July 2003 to July 2004, 87 patients (44 pemetrexed; 43 pemetrexed/gemcitabine) received treatment. The median time to progression was 4.5 (95% confidence interval: 3.0-9.3) and 4.1 months (95% confidence interval: 1.7-5.8) for the pemetrexed and pemetrexed/gemcitabine arms, respectively, and the median progression-free survival time was 3.3 months for both arms. Tumor response rates for the pemetrexed and pemetrexed/gemcitabine arms were 4.5% and 11.6%, respectively. The median overall survival time was 4.7 months for the pemetrexed arm and 5.4 months for the pemetrexed/gemcitabine arm, with respective 1-year survival rates of 28.5% and 28.1%. Grade 3/4 hematologic toxicity consisted of neutropenia (4.5% pemetrexed; 2.3% pemetrexed/gemcitabine), febrile neutropenia (4.5% pemetrexed; 4.7% pemetrexed/gemcitabine), thrombocytopenia (4.5% pemetrexed; 7.0% pemetrexed/gemcitabine), and anemia (6.8% pemetrexed; 4.7% pemetrexed/gemcitabine). No grade 3/4 nonhematologic toxicities exceeded 4.7% in either arm. CONCLUSIONS: Single-agent pemetrexed and sequential pemetrexed/gemcitabine have shown moderate activity and are well tolerated as first-line treatments for advanced NSCLC in elderly patients or patients unsuitable for platinum-based combination chemotherapy.