Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

An engineered IL-2 partial agonist promotes CD8+ T cell stemness

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients(1). Both the number of transferred T cells and their differentiation state are critical determinants of effective responses(2,3). T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells(4,5) and lower therapeutic efficacy(6), whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial(7). Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8(+) T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8(+) T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential

Serial magnetic cell enrichment of naturally occurring regulatory T cells.

<p>(a) Serial positive magnetic enrichment of CD4⁺CD25⁺CD45RA⁺ regulatory T cells (nTregs) from PBMCs. For pre-selection of CD4⁺ cells, PBMCs were first incubated with anti-CD4 Fab-multimers conjugated with <i>Strep</i>-Tactin-functionalized magnetic beads. The resulting positive fraction was then liberated from surface-bound label by D-biotin treatment and washed to remove anti-CD4 reagents. The second purification step comprised the selection for CD25 positive cells from the pre-selected CD4⁺ cell pool via specific anti-CD25 Fab bound to <i>Strep</i>-Tactin coated magnetic beads. Cell bound reagents were again removed from the resulting positive fraction by addition of D-biotin. In a third purification step, CD45RA⁺ cells were isolated from the enriched CD4⁺CD25⁺ cell population by using CD45RA-specific Fab-multimers conjugated to <i>Strep</i>-Tactin-coated magnetic beads. Living lymphocytes in the respective fractions of each selection step are shown. One representative experiment from five independent blood donors is shown. (b) Intracellular FoxP3 staining of triple positive enriched CD4⁺CD25⁺CD45RA⁺ regulatory T cells. (c) Overlay of the enriched CD4⁺CD25⁺CD45RA⁺ cell population (black dots) derived from serial magnetic selection as shown in (a) and the corresponding starting population (underlying grey dots). (d) Summary of cell purities obtained within each purification step of multiparameter magnetic bead-based nTregs purifications as performed in (a) with PBMCs derived from 5 different blood donors (left graph, mean values are indicated). In the right graph, yields (in %) of the target nTregs are shown; mean value is indicated. For all samples analyzed by flow cytometry, at least 50.000 events have been acquired.</p

Serial magnetic cell enrichment of central memory T cells.

<p>(a) Serial magnetic enrichment of CD8⁺CD62L⁺CD45RA^neg central memory T cells from fresh PBMCs. Cells were first incubated with anti-CD8 Fab-multimers conjugated with <i>Strep</i>-Tactin-functionalized magnetic beads in order to pre-select CD8⁺ cells. The resulting positive fraction was then treated with D-biotin and washed to remove all anti-CD8 reagents. In a second step, CD62L positive T cells were enriched from the pre-selected CD8⁺ T cell pool via specific anti-CD62L Fab bound to <i>Strep</i>-Tactin coated magnetic beads and subsequently liberated from the selection reagents as described above. In a final step CD45RA⁺ cells were depleted from the pre-enriched CD8⁺CD62L⁺ cell population using CD45RA specific Fab-multimers conjugated to <i>Strep</i>-Tactin-coated beads. Living lymphocytes in the respective fractions of each selection step are shown. One representative experiment from five independent blood donors is shown. (b) Overlay of the enriched CD8⁺CD62L⁺CD45RA^neg cell population (black dots) derived from serial magnetic selection as shown in (a) and the corresponding starting population (underlying grey dots). (c) Summary of cell purities obtained within each purification step of multiparameter magnetic bead-based T_CM purifications as performed in (a) with PBMCs derived from 5 different blood donors (left graph, mean values are indicated). In the right graph, yields (in %) of the target T_CMs are shown; mean value is indicated. For all samples analyzed by flow cytometry, at least 50.000 events have been acquired.</p

Identification of essential genes for cancer immunotherapy

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR–Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8⁺ T cells with the resistance or non-responsiveness of cancer to immunotherapies