Modulation of cell cycle progression by CTLA4-CD80/CD86 interactions on CD4+TCD4^+T cells depends on strength of the CD3 signal: critical role for IL-2

Cytotoxic T-lymphocyte antigen 4 (CTLA4) is a well-studied T cell costimulatory receptor that is known to inhibit T cell activation. In this study, the relationship between strength of the first signal and costimulatory interactions on primary mouse $CD4^+$ T cells was investigated. CTLA4-CD80/CD86 interactions differentially modulate T cell cycling based on the mode of CD3 signal: Activation with plate-bound (pb) anti-CD3 generates a strong signal compared with a weak signal with soluble (sol) anti-CD3, resulting in approximately sevenfold higher amounts of interleukin (IL)-2 and an increase in cell cycling. Activation of T cells with sol anti-CD3 (weak signal) together with CTLA4-CD80/CD86 blockade lowers IL-2 production and cell cycling, demonstrating an enhancing role for these interactions. Conversely, blockade of CTLA4-CD80/CD86 interactions on T cells activated with pb anti-CD3 (strong signal) increases proliferation, which is consistent with CTLA4 as a negative regulator. Also, coculture of T cells with Chinese hamster ovary cells expressing CD80 or CD86 demonstrates that the strength of the primary signal plays an important role. It is important that modulation of IL-2 amounts leads to distinct alterations in the functional effects of CTLA4-CD80/CD86 interactions. On increasing IL-2 amounts, activation of T cells stimulated with sol anti-CD3 (weak signal) and CTLA4-CD80/CD86 blockade is greater compared with control. Concurrently, neutralization of IL-2 greatly reduces activation of T cells stimulated with pb anti-CD3 (strong signal) and CTLA4-CD80/CD86 blockade compared with control. These results underscore the importance of strength of first signal, CTLA4-CD80/CD86 interactions, and IL-2 amounts in modulating primary $CD4^+$ T cell responses

Structural Basis of DNA Ligase IV-Artemis Interaction in Nonhomologous End-Joining

DNA ligase IV (LigIV) and Artemis are central components of the nonhomologous end-joining (NHEJ) machinery that is required for V(D)J recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD) in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ

IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice

Interferon-gamma (IFN-gamma) is an important immunomodulatory and pleiotropic cytokine produced, primarily, by activated T lymphocytes and natural killer (NK) cells. We have devised a nitric -oxide (NO)-based bioassay for mouse IFN-gamma using resident peritoneal exudate cells (PECs) from C57BL/6 mice. Comparison with three existing bioassays demonstrated that this assay was very sensitive and detected IFN-gamma in the linear range of similar to 0.03 -0.25 U/ml. Other cytokines, e.g. interleukin (IL)-2, IL-4, IL-6, IFN-alpha/beta and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination with IFN-gamma, did not greatly modulate NO levels produced by resident peritoneal exudate cells. The presence of exogenous NO3- and H2O2 did not interfere with the IFN-gamma induced NO production and detection. We also showed that the effect of lipopolysaccharide (LPS), which may be present in samples, could be suppressed by the use of Polymyxin B in the bioassay. The high sensitivity of the bioassay permitted the detection of low amounts of IFN-gamma in 1% mouse serum. In addition, this assay reproducibly detected bioactive IFN-gamma amounts in supernatants of activated T cells. The increase in IFN-gamma production by activated T cells in response to CD28 costimulation was similar to3-fold by this bioassay and similar to5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-gamma

Analysis of the Intratumoral Adaptive Immune Response in Well Differentiated and Dedifferentiated Retroperitoneal Liposarcoma

Treatment options are limited in well differentiated (WD) and dedifferentiated (DD) retroperitoneal liposarcoma. We sought to study the intratumoral adaptive immune response and explore the potential feasibility of immunotherapy in this disease. Tumor-infiltrating lymphocytes (TILs) were isolated from fresh surgical specimens and analyzed by flow cytometry for surface marker expression. Previously reported immune cell aggregates known as tertiary lymphoid structures (TLS) were further characterized by immunohistochemistry. In all fresh tumors, TILs were found. The majority of TILs were CD4 T cells; however cytotoxic CD8 T cells were also seen (average: 20% of CD3 T cells). Among CD8 T cells, 65% expressed the immune checkpoint molecule PD-1. Intratumoral TLS may be sites of antigen presentation as DC-LAMP positive, mature dendritic cells were found juxtaposed next to CD4 T cells. Clinicopathologic correlation, however, demonstrated that presence of TLS was associated with worse recurrence-free survival in WD disease and worse overall survival in DD disease. Our data suggest that an adaptive immune response is present in WD/DD retroperitoneal liposarcoma but may be hindered by TLS, among other possible microenvironmental factors; further investigation is needed. Immunotherapy, including immune checkpoint blockade, should be evaluated as a treatment option in this disease

Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion

© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc. Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)–JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion