Compromising the Unfolded Protein Response Induces Autophagy-Mediated Cell Death in Multiple Myeloma Cells

OBJECTIVE: To determine whether the Unfolded Protein Response (UPR) sensors (PERK, ATF6 and IRE-1) can be targeted to promote death of Multiple Myeloma (MM) cells. METHODS: We have knocked-down separately each UPR stress sensor in human MM cell lines using RNA interference and followed MM cell death by monitoring the membrane, mitochondrial and nuclear alterations. Involvement of caspases in MM cell death consecutive to UPR sensor knock-down was analyzed by western blotting, measurement of their enzymatic activity using fluorigenic substrates and susceptibility to a pan-caspase inhibitor. Activation of the autophagic process was measured directly by detection of autophagosomes (electronic microscopy), monodansylcadaverine staining, production of the cleaved form of the microtubule-associated protein 1A/1B light chain 3 (LC3) and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as 3MA and bafilomycin A1. RESULTS: We show that extinction of a single UPR stress sensor (PERK) induces a non-apoptotic form of cell death in MM cells that requires autophagy for its execution. We also show that this cytotoxic autophagic process represses the apoptosis program by reducing the cytosolic release of the apoptogenic factors Smac/DIABLO and cytochrome c. INTERPRETATION: Altogether our findings suggest that autophagy can contribute to execution of death in mammalian cells that are exposed to mild ER stress. They also suggest that the autophagic process can regulate the intrinsic apoptotic pathway by inhibiting production of death effectors by the mitochondria, thus preventing formation of a functional apoptosome. Altogether these findings give credit to the idea that UPR sensors can be envisaged as therapeutic targets for the treatment of MM

Adjuvant-specific regulation of long-term antibody responses by ZBTB20

The duration of antibody production by long-lived plasma cells varies with the type of immunization, but the basis for these differences is unknown. We demonstrate that plasma cells formed in response to the same immunogen engage distinct survival programs depending on the adjuvant. After alum-adjuvanted immunization, antigen-specific bone marrow plasma cells deficient in the transcription factor ZBTB20 failed to accumulate over time, leading to a progressive loss of antibody production relative to wild-type controls. Fetal liver reconstitution experiments demonstrated that the requirement for ZBTB20 was B cell intrinsic. No defects were observed in germinal center numbers, affinity maturation, or plasma cell formation or proliferation in ZBTB20-deficient chimeras. However, ZBTB20-deficient plasma cells expressed reduced levels of MCL1 relative to wild-type controls, and transgenic expression of BCL2 increased serum antibody titers. These data indicate a role for ZBTB20 in promoting survival in plasma cells. Strikingly, adjuvants that activate TLR2 and TLR4 restored long-term antibody production in ZBTB20-deficient chimeras through the induction of compensatory survival programs in plasma cells. Thus, distinct lifespans are imprinted in plasma cells as they are formed, depending on the primary activation conditions. The durability of vaccines may accordingly be improved through the selection of appropriate adjuvants

B1b cells recognize protective antigens after natural infection and vaccination

There are multiple, distinct B-cell populations in human beings and other animals such as mice. In the latter species, there is a well-characterized subset of B-cells known as B1 cells, which are enriched in peripheral sites such as the peritoneal cavity but are rare in the blood. B1 cells can be further subdivided into B1a and B1b subsets. There may be additional B1 subsets, though it is unclear if these are distinct populations or stages in the developmental process to become mature B1a and B1b cells. A limitation in understanding B1 subsets is the relative paucity of specific surface markers. In contrast to mice, the existence of B1 cells in human beings is controversial and more studies are needed to investigate the nature of these enigmatic cells. Examples of B1b antigens include pneumococcal polysaccharide and the Vi antigen from Salmonella Typhi, both used routinely as vaccines in human beings and experimental antigens such as haptenated-Ficoll. In addition to inducing classical T-dependent responses some proteins are B1b antigens and can induce T-independent (TI) immunity, examples include factor H binding protein from Borrelia hermsii and porins from Salmonella. Therefore, B1b antigens can be proteinaceous or non-proteinaceous, induce TI responses, memory, and immunity, they exist in a diverse range of pathogenic bacteria, and a single species can contain multiple B1b antigens. An unexpected benefit to studying B1b cells is that they appear to have a propensity to recognize protective antigens in bacteria. This suggests that studying B1b cells may be rewarding for vaccine design as immunoprophylactic and immunotherapeutic interventions become more important due to the decreasing efficacy of small molecule antimicrobials

Outer Membrane Protein Complex of Meningococcus Enhances the Antipolysaccharide Antibody Response to Pneumococcal Polysaccharide–CRM197 Conjugate Vaccine ▿

Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM197 conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM197 conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands

The cell death promoted by the PERK siRNA is morphologically distinct from apoptosis.

<p>NCI-H929 cells were either transfected with the non-targeting (C) or with the PERK (D–H) siRNA, treated with staurosporine (B) or left untreated (A) for 24 h and then examined by electron microscopy. The two stages or the death process induced by PERK silencing are illustrated in panels D and E. F–H Visualization at higher magnification of the cytosolic electron-dense structures identified by the arrowheads in D. In F, a pre-autophagosomal-like structure, in G and H, autophagosomes with a double membrane sequestering cellular material.</p

Silencing UPR sensors induces death of human MM cell lines.

<p>A. NCI-H929 cells were transfected with the PERK, ATF6, IRE1 or non-targeting siRNAs. The levels of expression of the targeted transcripts were determined by real-time RT-PCR 24 h after transfection. B. Percentages of transcript extinction induced by the targeting siRNAs in U266 and NCI-H929 cells (mean ± SD values of three independent experiments). C. Whole cell lysates were prepared from NCI-H929 cells transfected with the targeting or non-targeting siRNAs and blotted with anti-PERK, anti-ATF6, anti IRE1 or anti-ß actin mAbs. Representative of two independent experiments. D. NCI-H929 cells were transfected with the PERK (siPERK) or control siRNA (siCtrl). Percentages of cells with Δm loss (TMRE^lo) or with membrane alterations (Annexin V+ or PI+) were evaluated by TMRE and annexin V or PI stainings, respectively. Results represent the mean+SD values of 2 experiments.</p

Extinction of the UPR sensors induces the autophagic cell death of MM cells.

<p>A. NCI-H929 cells were serum-starved or transfected with the non-targeting or PERK siRNAs for 24 h then stained with MDC and examined by fluorescence microscopy. B. NCI-H929 cells cultured as in A, in the presence or absence of 3-MA, were stained with MDC and analyzed by flow cytometry. C and D, NCI-H929 cells were serum-starved (C) or transfected either with the non-targeting or the PERK siRNAs (D). All cultures were conducted with or without bafilomycin A1. Accumulation of autophagosomes was visualized by the conversion of endogenous LC3-I (18 kDa) to LC3-II (16 kDa). E. Average proportion of MDC⁺ cells in NCI-H929 cells 24 h after transfection with the targeting or non-targeting siRNAs or 24 h after culture in serum-starved conditions. All cultures were conducted with or without 3-MA. Results are expressed as the mean ± SD percentages of positive cells as calculated from duplicate determinations. The data shown are representative of two independent experiments. F. NCI-H929 cells were cultured as in E. Membrane alterations were estimated by Annexin V staining after 24 h of culture. Results are expressed as the mean ± SD percentages of positive cells as calculated from duplicate determinations and are representative of three independent experiments. (* <i>p</i><0.05; ** <i>p</i><0.01; ***<i>p</i><0.005; <i>ns</i> = non significant).</p

PERK silencing represses apoptosis of MM cells.

<p>A. NCI-H929 cells were transfected with the non-targeting or PERK siRNA and treated or not with STS 8 h later. Control cultures in which untransfected cells were treated with STS were also conducted. DNA fragmentation (A) and PS exposure (B) were assessed 24 h after transfection. Results are expressed as the mean ± SD percentages of positive cells as calculated from duplicate determinations and are representative of two independent experiments. C. NCI-H929 cells were cultured as in B and C in the presence or absence of 3-MA. DNA fragmentation was assessed by the TUNEL assay. Mean ± SD values of two independent experiments are shown. (* <i>p</i><0.05).</p