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The majority of proteins in mammalian cells are modified by covalent attachment of an acetyl-group to the N-terminus (Nt-acetylation). Paradoxically, Nt-acetylation has been suggested to inhibit as well as to promote substrate degradation. Contrasting these findings, proteome-wide stability measurements failed to detect any correlation between Nt-acetylation status and protein stability. Accordingly, by analysis of protein stability datasets, we found that predicted Nt-acetylation positively correlates with protein stability in case of GFP, but this correlation does not hold for the entire proteome. To further resolve this conundrum, we systematically changed the Nt-acetylation and ubiquitination status of model substrates and assessed their stability. For wild-type Bcl-B, which is heavily modified by proteasome-targeting lysine ubiquitination, Nt-acetylation did not correlate with protein stability. For a lysine-less Bcl-B mutant, however, Nt-acetylation correlated with increased protein stability, likely due to prohibition of ubiquitin conjugation to the acetylated N-terminus. In case of GFP, Nt-acetylation correlated with increased protein stability, as predicted, but our data suggest that Nt-acetylation does not affect GFP ubiquitination. Similarly, in case of the naturally lysine-less protein p16, Nt-acetylation correlated with protein stability, regardless of ubiquitination on its N-terminus or on an introduced lysine residue. A direct effect of Nt-acetylation on p16 stability was supported by studies in NatB-deficient cells. Together, our studies argue that Nt-acetylation can stabilize proteins in human cells in a substrate-specific manner, by competition with N-terminal ubiquitination, but also by other mechanisms that are independent of protein ubiquitination status.

Despite their low abundance in the tumor microenvironment (TME), classical type 1 dendritic cells (cDC1) play a pivotal role in anti-cancer immunity, and their abundance positively correlates with patient survival. However, their interaction with CD4+ T-cells to potentially enable the cytotoxic T lymphocyte (CTL) response has not been elucidated. Here we show that contact with activated CD4+ T-cells enables human ex vivo cDC1, but no other DC types, to induce a CTL response to cell-associated tumor antigens. Single cell transcriptomics reveals that CD4+ T-cell help uniquely optimizes cDC1 in many functions that support antigen cross-presentation and T-cell priming, while these changes don’t apply to other DC types. We robustly identify “helped” cDC1 in the TME of a multitude of human cancer types by the overlap in their transcriptomic signature with that of recently defined, tumor-infiltrating DC states that prove to be positively prognostic. As predicted from the functional effects of CD4+ T-cell help, the transcriptomic signature of “helped” cDC1 correlates with tumor infiltration by CTLs and Thelper(h)−1 cells, overall survival and response to PD-1-targeting immunotherapy. These findings reveal a critical role for CD4+ T-cell help in enabling cDC1 function in the TME and may establish the helped cDC1 transcriptomic signature as diagnostic marker in cancer.

Cancer immunotherapy aims to promote the activity of cytotoxic T lymphocytes (CTLs) within a tumour, assist the priming of tumour-specific CTLs in lymphoid organs and establish efficient and durable antitumour immunity. During priming, help signals are relayed from CD4+ T cells to CD8+ T cells by specific dendritic cells to optimize the magnitude and quality of the CTL response. In this Review, we highlight the cellular dynamics and membrane receptors that mediate CD4+ T cell help and the molecular mechanisms of the enhanced antitumour activity of CTLs. We outline how deficient CD4+ T cell help reduces the response of CTLs and how maximizing CD4+ T cell help can improve outcomes in cancer immunotherapy strategies. CD4+ T cells provide help to CD8+ T cells via lymph node-resident dendritic cells. In this Review, the authors discuss the molecular nature of help signals and how they can be harnessed to improve cancer immunotherapy.

BH3-only protein Bid is a key player in death receptor-induced apoptosis, because it provides the link with the mitochondrial route for caspase activation. In this pathway, Bid is activated upon cleavage by caspase-8. Its BH3 domain-containing carboxy-terminal fragment subsequently provokes mitochondrial outer membrane permeabilization by Bak/Bax activation. Bid has also been implicated in the apoptotic response to ionizing radiation (IR) and the topoisomerase inhibitor etoposide, anti-cancer regimens that cause double-strand (ds)DNA breaks. We confirm the existence of this pathway and show that it is p53-independent. However, the degree of Bid participation in the apoptotic response to dsDNA breaks depends on the nature of cell transformation. We used Bid-deficient mouse embryonic fibroblast (MEF) lines that were reconstituted with Bid to control the cellular background and demonstrated that the Bid-dependent apoptotic pathway induced by IR and etoposide operates in MEFs that are transformed by SV40, but is not evident in E1A/Ras-transformed MEFs. The Bid-dependent apoptotic response in p53-deficient SV40-transformed MEFs contributed to clonogenic execution of the cells, implying relevance for treatment outcome. In these cells, Bid acted in a conventional manner in that it required its BH3 domain to mediate apoptosis in response to IR and etoposide, and triggered apoptotic execution by indirect activation of Bak/Bax, mitochondrial permeabilization and caspase-9 activation. However, the mechanism of Bid activation was unconventional, because elimination of all known or suspected cleavage sites for caspases or other proteolytic enzymes and even complete elimination of its unstructured cleavage loop left Bid's pro-apoptotic role in the response to IR and etoposide unaffected.

In many tumor cell types, ionizing radiation or DNA-damaging anticancer drugs enhance sensitivity to death receptor-mediated apoptosis, which is of clinical interest. APO010, a form of CD95/Fas ligand is currently in a phase I trial in patients with solid tumors. To analyze the potential of combined modality treatment with APO010, we used p53-mutant Jurkat T leukemic cells, in which the mitochondrial pathway was blocked by Bcl-2 overexpression. These cells were strongly sensitized to APO010 by pretreatment with ionizing - or UV radiation, etoposide, histone deacetylase - or proteasome inhibitors. These stimuli alone did not induce apoptosis in J16-Bcl-2 cells. Sensitization could not be explained by the overruling of mitochondrial resistance imposed by Bcl-2, upregulation of CD95 membrane levels or modulation of inhibitor of apoptosis proteins. Rather, the stimuli commonly downregulated c-FLIP(L/S) protein levels, which was causally related to the sensitization: deliberate c-FLIP(L/S) downregulation by RNA interference largely overruled the capacity of the various stimuli to sensitize Jurkat-Bcl-2 cells to apoptotic execution by APO010. In p53-mutant, Bcl-2 overexpressing HCT-15 colon carcinoma cells, c-FLIP downregulation correlated with sensitization to APO010 for some, but not all stimuli. We conclude that c-FLIP downregulation represents a mechanism by which diverse anticancer regimens can facilitate tumor cell execution by CD95/Fas through the direct pathway of caspase activation.

Breast cancer cells often show increased activity of the mitogen-activated protein kinase (MAPK) pathway. We report here that this pathway reduces their sensitivity to death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and present the underlying mechanism. Activation of protein kinase C (PKC) inhibited TRAIL-induced apoptosis in a protein synthesis-independent manner. Deliberate activation of MAPK was also inhibitory. In digitonin-permeabilized cells, PKC activation interfered with the capacity of recombinant truncated (t)Bid to release cytochrome c from mitochondria. MAPK activation did not affect TRAIL or tumor necrosis factor (TNF)alpha-induced Bid cleavage. However, it did inhibit translocation of (t)Bid to mitochondria as determined both by subcellular fractionation analysis and confocal microscopy. Steady state tBid mitochondrial localization was prohibited by activation of the MAPK pathway, also when the Bcl-2 homology domain 3 (BH3) domain of tBid was disrupted. We conclude that the MAPK pathway inhibits TRAIL-induced apoptosis in MCF-7 cells by prohibiting anchoring of tBid to the mitochondrial membrane. This anchoring is independent of its interaction with resident Bcl-2 family members.

It has been proposed that HIV-1, in addition to directly infecting and killing CD4+ T cells, causes T cell dysfunction and T cell loss by chronic immune activation. We analyzed the effects of chronic immune activation in mice that constitutively expressed CD70, the ligand for the tumor necrosis factor receptor family member CD27, on B cells. CD70 transgenic (CD70 Tg) mice showed a progressive conversion of naive T cells into effector-memory cells, which culminated in the depletion of naive T cells from lymph nodes and spleen. T cell changes depended on continuous CD27-CD70 interactions and T cell antigen receptor stimulation. Despite this hyperactive immune system, CD70 Tg mice died aged 6-8 months from Pneumocystis carinii infection, a hallmark of T cell immunodeficiency. Thus, persistent delivery of costimulatory signals via CD27-CD70 interactions, as may occur during chronic active viral infections, can exhaust the T cell pool and is sufficient to induce lethal immunodeficiency.

Recently, a select number of viral and cellular proteins sharing a unique property have been identified, namely apoptin, HAMLET, TRAIL, MDA7, E4ORF4, NS1 and Brevinin-2R, where all have the remarkable ability to selectively kill transformed and/or tumour cells, while causing no or only minor cytotoxicity in normal and non-transformed cells. Research worldwide is now starting to unravel the molecular signalling pathways by which each of these proteins exerts its tumour-selective function, and the first (pre)clinical studies are very promising. The workshop ‘Proteins Killing Tumour Cells’ brought together, for the first time, researchers working on this novel class of intriguing proteins. Participants from 14 different countries shared their views on (possible common) mechanisms behind tumour-specific apoptosis induction and strategies to implement this knowledge in the clinic.

B-cell commitment is characterized by the expression of specific membrane proteins and the rearrangement and expression of immunoglobulin (Ig) heavy (H) and light (L) chain genes. At early stages of B-cell development, unrearranged Ig loci are transcribed, which correlates with these regions becoming accessible for Ig gene rearrangement. Some germline transcripts can be translated into protein and potentially play a role in cell signaling during B-cell development. In this report an early stage in human B-cell development is characterized using Epstein-Barr virus (EBV)-transformed cell lines from patients with a severe combined immunodeficiency (SCID). These lines were shown to produce germline constant (C) gene transcripts from the IGH and IGK loci. We demonstrate here that these cells are committed to the B-cell lineage as substantiated by expression of CD79a and CD79b. No surrogate light chain (SLC) gene transcription was detected, indicative of a very early differentiation stage. From these cell lines two types of germline IgV gene transcripts were isolated: a transcript containing the IGKV4-1 gene and a germline IGHV-1 transcript nearly identical to IGHV1/OR15-1 (HC15-1, DP-1), an orphon V H gene on chromosome 15. Germline V H transcripts originating from the V H locus on chromosome 14 could not be detected. It is of interest that, apart from Ig V and C genes (non-functional), V genes that reside outside the Ig locus are a target for the transcription factors that are postulated to initiate Ig gene rearrangement early in B-cell development.

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.

Dexamethasone treatment during the expansion phase sustains stemness of mesenchymal stem cells from human bone marrow

Classical T lymphocytes recognize foreign antigens in the context of self major histocompatibility complex (MHC) molecules by means of the T-cell receptor (TCR)alpha beta heterodimer. The genes for TCR beta-chains, like immunoglobulin genes, are subject to allelic exclusion. The introduction of a functional TCR-beta gene into the germline of mice prevents rearrangement of endogenous TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta gene with a deletion of the major part of the variable region (delta V-TCR-beta), also inhibits endogenous TCR-beta gene rearrangement. This inhibition is mediated via the encoded protein because impairment of endogenous TCR-beta gene rearrangement is not found if a frameshift mutation is introduced into the DJ region of the delta V-TCR-beta transgene. The delta V-TCR-beta transgene can lead to two phenotypes, in which lymphoid development is perturbed. Phenotype A is characterized by a severe impairment of both T and B cell development as reflected by the complete absence of certain lymphoid organs. In phenotype B, lymphoid organs are macroscopically normal, but T cell differentiation is impeded. Virtually all thymocytes lack membrane expression of TCR-alpha beta, but nevertheless carry the CD4 and CD8 antigens (CD4+CD8+ phenotype); they do not, however, mature further. The defect in mice of phenotype B but not of phenotype A can be corrected by the introduction of a functional TCR-beta gene.

Cloned blood lymphocytes that do not express the alpha- and beta-chains of the T-cell receptor show MHC-unrestricted cytotoxicity. These cells carry the gamma-protein, disulphide-linked either to another molecule or to itself, and associated with the CD3 complex. These observations may help to solve the mystery posed by the discovery of the gamma gene.

The T3/T-cell receptor complex on the surface of human thymus-derived lymphocytes consists of four glycoproteins: the alpha-chain of relative molecular mass (Mr) 40,000-50,000 (40-50K), the beta-chain (37-45K); the gamma-chain (25K) and the delta-chain (20K). The T3 alpha- and beta-chains have been identified as clonotypic T-cell receptors, but functionally the T3/T-cell receptor chains seem to form a single complex: monoclonal antibodies directed at the 20K T3 components are mitogenic for normal human T lymphocytes and, at higher concentrations, anti-clonotypic and anti-20K reagents block T-cell function. Recently, Zanders et al. showed that incubation of human T-helper clones with high concentrations of antigen abolishes antigen-specific proliferation and induces disappearance of T3 from the cell surface. Thus, the T3/T-cell receptor complex consists of two variable subunits, the T3 alpha- and beta-chains, which interact with antigen and the monomorphic 20K/25K T3 chains. Recently, the existence of a fifth polypeptide chain, the unglycosylated T3 epsilon-chain, has been postulated. Here we confirm that a 20K epsilon-chain does exist. The T3 epsilon-chain differs from the T3 delta-chain in primary structure as judged by N-terminal amino acid sequencing, peptide mapping and immunoblotting with anti-T3-delta and anti-T3-epsilon antibodies. Treatment with endoglycosidase F revealed two nonglycosylated T3 delta polypeptide backbone chains (16K and 14K) with identical amino termini. Together with previous pulse-chase experiments this observation suggests that the 14K T3 polypeptide is derived from the 16K T3 precursor by proteolytic processing near the C-terminus of the molecule.