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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. Pooled CRISPR perturbation screens with multimodal RNA and protein single-cell profiling readout (Perturb-CITE-seq) applied to patient-derived melanoma and tumor-infiltrating lymphocyte co-cultures identifies new tumor immune evasion mechanisms.

The glycoprotein CD58, also known as lymphocyte-function antigen 3 (LFA-3), is a costimulatory receptor distributed on a broad range of human tissue cells. Its natural ligand CD2 is primarily expressed on the surface of T/NK cells. The CD2-CD58 interaction is an important component of the immunological synapse (IS) that induces activation and proliferation of T/NK cells and triggers a series of intracellular signaling in T/NK cells and target cells, respectively, in addition to promoting cell adhesion and recognition. Furthermore, a soluble form of CD58 (sCD58) is also present in cellular supernatant in vitro and in local tissues in vivo . The sCD58 is involved in T/NK cell-mediated immune responses as an immunosuppressive factor by affecting CD2-CD58 interaction. Altered accumulation of sCD58 may lead to immunosuppression of T/NK cells in the tumor microenvironment, allowing sCD58 as a novel immunotherapeutic target. Recently, the crucial roles of costimulatory molecule CD58 in immunomodulation seem to be reattracting the interests of investigators. In particular, the CD2-CD58 interaction is involved in the regulation of antiviral responses, inflammatory responses in autoimmune diseases, immune rejection of transplantation, and immune evasion of tumor cells. In this review, we provide a comprehensive summary of CD58 immunobiology.

The dissemination of HIV from an initial site of infection is facilitated by motile HIV-infected CD4(+) T-cells. However, the impact of infected target cell migration on antigen recognition by HIV-specific CD8(+) T-cells is unclear. Using a 3D in vitro model of tissue, we visualized dynamic interactions between HIV-infected or peptide-pulsed CD4(+) T-cells and HIV-specific CD8(+) T-cells. CTLs engaged motile HIV-infected targets, but ∼ 50% of targets broke contact and escaped. In contrast, immobilized target cells were readily killed, indicating target motility directly inhibits CD8(+) T-cell function. Strong calcium signals occurred in CTLs killing a motile target but calcium signaling was weak or absent in CTLs which permitted target escape. Neutralization of adhesion receptors LFA-1 and CD58 inhibited CD8(+) T-cell function within the 3D matrix, demonstrating that efficient motile target lysis as dependent on adhesive engagement of targets. Antigen sensitivity (a convolution of antigen density, TCR avidity and CD8 coreceptor binding) is also critical for target recognition. We modulated this parameter (known as functional avidity but referred to here as \"avidity\" for the sake of simplicity) by exploiting common HIV escape mutations and measured their impact on CTL function at the single-cell level. Targets pulsed with low avidity mutant antigens frequently escaped while CTLs killed targets bearing high avidity antigen with near-perfect efficiency. CTLs engaged, arrested, and killed an initial target bearing high avidity antigen within minutes, but serial killing was surprisingly rare. CD8 cells remained committed to their initial dead target for hours, accumulating TCR signals that sustained secretion of soluble antiviral factors. These data indicate that high-avidity CD8(+) T-cells execute an antiviral program in the precise location where antigen has been sensed: CTL effector functions are spatiotemporally coordinated with an early lytic phase followed by a sustained stationary secretory phase to control local viral infection.

BackgroundBispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been demonstrated, efficacy of T-cell engagers (TCEs) in solid tumors settings, molecular determinants of response, and underlying mechanisms of resistance to BiTE therapy require more investigation.MethodsTo uncover cancer cell-intrinsic genetic modifiers of TCE-mediated cytotoxicity, we performed genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loss-of-function and CRISPRa (CRISPR activation) gain-of-function screens using TCEs against two distinct tumor-associated antigens (TAAs). By using in vitro T-cell cytotoxicity assays and in vivo efficacy studies, we validated the roles of two common pathways identified in our screen, T-cell costimulation pathway and apoptosis pathway, as key modifiers of BiTE activity.ResultsOur genetic screens uncovered TAAs-independent cancer cell-intrinsic genes with functions in autophagy, T-cell costimulation, the apoptosis pathway, chromatin remodeling, and cytokine signaling that altered responsiveness to BiTE-mediated killing. Notably, loss of CD58 (the ligand of the CD2 T-cell costimulatory receptor), a gene frequently altered in cancer, led to decreased TCE-mediated cytotoxicity, T-cell activation and antitumor efficacy in vitro and in vivo. Moreover, the effects of CD58 loss were synergistically compounded by concurrent loss of CD80/CD86 (ligands for the CD28 T-cell costimulatory receptor), whereas joint CD2 and CD28 costimulation additively enhanced TCE-mediated killing, indicating non-redundant costimulatory mechanisms between the two pathways. Additionally, loss of CFLAR (Caspase-8 and FADD Like Apoptosis Regulator), BCL2L1, and BID (BH3 Interacting Domain Death Agonist) induced profound changes in sensitivity to TCEs, indicating that key regulators of apoptosis, which are frequently altered in cancer, impact tumor responsiveness to BiTE therapy.ConclusionsThis study demonstrates that genetic alterations central to carcinogenesis and commonly detected in cancer samples lead to significant modulation of BiTE antitumor activity in vitro and in vivo, findings with relevance for a better understanding of patient responses to BiTE therapy and novel combinations that enhance TCE efficacy.

Membrane nanotubes are thin membranous projections that physically connect two cells. While nanotubes have been studied in human natural killer (NK) cells and are implicated in aiding NK cell cytotoxic function, requirements for their formation to susceptible target cells remain incompletely understood. Here we demonstrate that the CD2-CD58/48 receptor-ligand interaction promotes and is required for nanotube formation in human NK cells. In the CD2(-) NK cell line YTS, a stable CD2 expression variant enabled effective nanotube formation, and was associated with better cytotoxic function. Importantly, only interactions between an NK cell and a susceptible target cell were associated with multiple nanotubes and the number of nanotubes was inversely correlated with their length. Quantitative live cell fluorescence microscopy of CD2 nanotubes revealed time-dependent enrichment and localization of CD2 to the nanotube tip, and blocking CD2 receptor-ligand interactions prevented nanotube formation. Increased nanotube formation was not simply a feature of receptor-ligand pairing, as a KIR-MHC interaction in the same cell line system failed to promote nanotube formation. Additionally, blocking LFA-1-ICAM and 2B4-CD48 receptor-ligand interactions failed to inhibit nanotube formation. Thus only specific receptor-ligand pairs promote nanotubes. CD2 also promoted nanotube formation in ex vivo NK cells suggesting that CD2 plays a crucial role in the generation of nanotubes between an NK cell and its target.

T-cell-mediated immune responses are typically low in conditions of malignant glioma which has been known to cause marked immunesuppression and dysregulate major T-cell signaling molecules. Thus, T-cell-based immunotherapies are currently in vogue in the treatment of malignant glioma. The novel glycopeptide, T11TS/S-LFA-3/S-CD58 has previously been shown by our group to be highly efficacious in glioma abrogation in in vivo and in vitro conditions. This glycopeptide ligands to the costimulatory CD2 molecule on T-cells, causing profound immune stimulation leading to glioma abrogation, suggesting probable involvement of T11TS in modulation of the T-cell signaling pathway. The present study offers a multi-targeted approach towards repair of some of the key components of the immunological synapse at the T-cell-APC interface and is therefore the first of its kind to offer a holistic model of restoration of immunological synapse components so as to trigger T-cells towards activation against glioma. The study thus indicates that the totally dysregulated molecular events at the immunological synapse in glioma are restored back to normal levels with the administration of T11TS, which finally culminates in glioma abrogation. The present study thus delineates an important T-cell signaling approach whereby T11TS acts as an anti-neoplastic agent, thus helping to chart out newer avenues in the fight against gliomas.

Early clearance of leukemic cells during induction therapy of childhood acute lymphoblastic leukemia (ALL) is a basis for treatment optimization. Currently, the most widely used methods for the detection of minute residual malignant cells in the bone marrow and/or peripheral blood, minimal residual disease (MRD), are PCR and flow cytometry (FCM). Immunophenotypic modulation (IM) is a well known factor that can hamper the accurate FCM analysis. To report the IM detected by 8-color FCM during the BFM-type remission induction in 24 consecutive MRD-positive samples of children with B-cell precursor ALL and the possible implications for MRD detection. Between 2010 and 2012 we prospectively followed up the MRD on days 15 and 33 of induction treatment in bone marrow (BM) samples and on day 8 in peripheral blood (PB). The IM was assessed by comparative analyses of the changes in the mean fluorescence intensity of 7 highly relevant antigens expressed by the leukemic cells and normal B-lymphocytes. IM occurred, to different extents, in all analyzed day 15 BM and in most day 33 BM samples. Statistically significant changes in the MFI-levels of four CDs expressed by the leukemic blasts were observed: downmodulation of CD10, CD19 and CD34 and upmodulation of CD20. No changes in the expression of CD38, CD58 and CD45 were noticed. Measuring the MRD by standardized 8-color flow cytometry helps improve the monitoring of the disease, leading to better therapeutic results. However, the IM of the different antigens expressed by the leukemic blasts should be taken into consideration and cautiously analyzed.

We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor Ags and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8⁺ T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8⁺ T cells is still unclear. The study reported here was an in vitro investigation of the proliferation and function of CEA-specific human memory CD8⁺ T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8⁺ T cells. The lytic capacity of memory CTLs toward CEA⁺ tumors was also significantly enhanced. IL-2Rα (CD25) was upregulated dramatically following APC-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8⁺ T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8⁺ T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8⁺ T cells.

The effect of infection with Chlamydia pneumoniae on host messenger RNA expression in human monocytic cells with complement DNA microarrays was studied. The data chronicle a cascade of transcriptional events affecting 128 genes, many of which have not previously been reported to be affected by C. pneumoniae infection. Down-regulated genes are primarily associated with RNA and DNA metabolism, chromosomal stability, and cell-cycle regulation. Up-regulated messages include those for a variety of genes with important proinflammatory functions. Many of the up-regulated genes—including the hyaluron receptor CD44, vasoconstrictor endothelin-1, smooth muscle growth factor heparin-binding EGF-like growth factor, and fatty acid binding protein-4-had been previously described as linked to the development of atherosclerosis and other chronic inflammatory diseases. C. pneumoniae-infected monocytes can contribute to the development and progression of diseases for which acute or chronic inflammation has been shown to be important, such as atherosclerosis.