Search

The therapeutic potential of recombinant cytokines has been limited by the severe side effects of systemic administration. We describe a strategy to reduce the dose-limiting toxicities of monomeric cytokines by designing two components that require colocalization for activity and that can be independently targeted to restrict activity to cells expressing two surface markers. We demonstrate the approach with a previously designed mimetic of cytokines interleukin-2 and interleukin-15—Neoleukin-2/15 (Neo-2/15)—both for trans-activating immune cells surrounding targeted tumor cells and for cis-activating directly targeted immune cells. In trans-activation mode, tumor antigen targeting of the two components enhanced antitumor activity and attenuated toxicity compared with systemic treatment in syngeneic mouse melanoma models. In cis-activation mode, immune cell targeting of the two components selectively expanded CD8+ T cells in a syngeneic mouse melanoma model and promoted chimeric antigen receptor T cell activation in a lymphoma xenograft model, enhancing antitumor efficacy in both cases.

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.

CDK4/6 inhibitors are approved to treat breast cancer and are in trials for other malignancies. We examined CDK4/6 inhibition in mouse and human CD8+ T cells during early stages of activation. Mice receiving tumor-specific CD8+ T cells treated with CDK4/6 inhibitors displayed increased T-cell persistence and immunologic memory. CDK4/6 inhibition upregulated MXD4, a negative regulator of MYC, in both mouse and human CD8+ T cells. Silencing of Mxd4 or Myc in mouse CD8+ T cells demonstrated the importance of this axis for memory formation. We used single-cell transcriptional profiling and T-cell receptor clonotype tracking to evaluate recently activated human CD8+ T cells in patients with breast cancer before and during treatment with either palbociclib or abemaciclib. CDK4/6 inhibitor therapy in humans increases the frequency of CD8+ memory precursors and downregulates their expression of MYC target genes, suggesting that CDK4/6 inhibitors in patients with cancer may augment long-term protective immunity. SIGNIFICANCE: CDK4/6 inhibition skews newly activated CD8+ T cells toward a memory phenotype in mice and humans with breast cancer. CDK4/6 inhibitors may have broad utility outside breast cancer, particularly in the neoadjuvant setting to augment CD8+ T-cell priming to tumor antigens prior to dosing with checkpoint blockade.This article is highlighted in the In This Issue feature, p. 2355.

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed “bystander” antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity. Keywords: tolerance; autoantibodies; antigen capture; antigen presentation; influenza

Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach diseaseassociated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4⁺ and CD8⁺ T cells) in an antigenspecific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes., United States. Defense Advanced Research Projects Agency (Contract HR0011-12-2-0015)

Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR., National Institutes of Health (U.S.) (RO1 Grant AI087879-01), Pancreatic Cancer Action Network

Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR., National Institutes of Health (U.S.) (Grant AI087879-01)

We generated a CD8 T-cell receptor (TCR) transnuclear (TN) mouse specific to the Ld -restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T-cell-mediated response against this pathogen. The TN T cells bound Ld -Gra6 tetramer and proliferated upon unspecific and peptide-specific stimulation. The TCR beta sequence of the Gra6-specific TN CD8 T cells is identical in its V- and J-region to the TCR-β harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma-infected mice carried Gra6 TN cells already at day 8 post-infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T-cell response to Toxoplasma in both the acute and the chronic phases of infection.

Resolving how the early signaling events initiated by cell–cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell–cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting single-cell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell., Singapore-MIT Alliance, American Association for Cancer Research. Pancreatic Cancer Action Network, Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science (Frank Quick Faculty Research Innovation Fellowship)

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers[superscript 1, 2, 3, 4, 5]. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition[superscript 6, 7, 8, 9, 10]. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for T, American Association for Cancer Research. Pancreatic Cancer Action Network

We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymatically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these experiments to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes., United States. Defense Advanced Research Projects Agency (Contract HR0011-12-2-0015)

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response., National Institutes of Health (U.S.)

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments., National Institutes of Health (U.S.) (Transformative Research Award 1R01CA173750), Dana-Farber/Harvard Cancer Center (Bridge Project), Lustgarten Foundation, Novartis Institutes of Biomedical Research, National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051), American Cancer Society (John W. Thatcher, Jr Postdoctoral Fellowship in Melanoma Research), Breast Cancer Research Foundation (Terri Brodeur Breast Cancer Foundation Postdoctoral Fellowship), National Institutes of Health (U.S.) (NIH T32 grant AI07386)

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments., National Institutes of Health (U.S.) (Transformative Research Award 1R01CA173750), Dana-Farber/Harvard Cancer Center (Bridge Project), Lustgarten Foundation, Novartis Institutes of Biomedical Research, National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051), American Cancer Society (John W. Thatcher, Jr Postdoctoral Fellowship in Melanoma Research), Breast Cancer Research Foundation (Terri Brodeur Breast Cancer Foundation Postdoctoral Fellowship), National Institutes of Health (U.S.) (NIH T32 grant AI07386)

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool., United States. Army Research Office. (Institute for Collaborative Biotechnologies Grant W911NF-09-0001)

Variability in cell-cell interactions is ubiquitous and particularly relevant for the immune system, where the reliability of cell-cell interactions is critical for the prevention of disease. This variability is poorly understood mainly due to the limitations of current methods. We have therefore designed a highly parallel microfluidic cell-pairing device and optimized its pairing efficiency using fluids modeling. The optimized device can hydrodynamically pair hundreds of primary mouse immune-cells at an efficiency of ~50%. We measured T cell activation dynamics of ~130 primary mouse T cells paired with B cells. Our findings represent the first time that variation has been observed in T cell activation dynamics., National Institutes of Health (U.S.) (NIH (EB008550)), Singapore-MIT Alliance

During eukaryotic development, the induction of a lineage-specific transcription factor typically drives differentiation of multipotent progenitor cells, while repressing that of alternative lineages. This process is often mediated by some extracellular signaling molecules, such as cytokines that can bind to cell surface receptors, leading to activation and/or repression of transcription factors. We explored the early differentiation of naive CD4 T helper (Th) cells into Th1 versus Th2 states by counting single transcripts and quantifying immunofluorescence in individual cells. Contrary to mutually exclusive expression of antagonistic transcription factors, we observed their ubiquitous co-expression in individual cells at high levels that are distinct from basal-level co-expression during lineage priming. We observed that cytokines are expressed only in a small subpopulation of cells, independent from the expression of transcription factors in these single cells. This cell-to-cell variation in the cytokine expression during the early phase of T helper cell differentiation is significantly larger than in the fully differentiated state. Upon inhibition of cytokine signaling, we observed the classic mutual exclusion of antagonistic transcription factors, thus revealing a weak intracellular network otherwise overruled by the strong signals that emanate from extracellular cytokines. These results suggest that during the early differentiation process CD4 T cells acquire a mixed Th1/Th2 state, instructed by extracellular cytokines. The interplay between extracellular and intracellular signaling components unveiled in Th1/Th2 differentiation may be a common strategy for mammalian cells to buffer against noisy cytokine expression., National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874), National Institutes of Health (U.S.) (Pioneer Award), National Institutes of Health (U.S.) (Grant R01-GM068957)

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool., United States. Army Research Office. (Institute for Collaborative Biotechnologies Grant W911NF-09-0001)

Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein–protein fusions, the method reported can be extended to connecting proteins with any entity of interest., Netherlands Organization for Scientific Research, Human Frontier Science Program (Strasbourg, France), National Institutes of Health (U.S.) (R01 Award AI033456), National Institutes of Health (U.S.) (R01 Award AI087879)

Protein quality control is a balance between chaperone-assisted folding and removal of misfolded proteins from the endoplasmic reticulum (ER). Cell-based assays have been used to identify key players of the dislocation machinery, including members of the Derlin family. We generated conditional knockout mice to examine the in vivo role of Derlin-2, a component that nucleates cellular dislocation machinery. In most Derlin-2-deficient tissues, we found constitutive upregulation of ER chaperones and IRE-1-mediated induction of the unfolded protein response. The IRE-1/XBP-1 pathway is required for development of highly secretory cells, particularly plasma cells and hepatocytes. However, B lymphocyte development and antibody secretion were normal in the absence of Derlin-2. Likewise, hepatocyte function was unaffected by liver-specific deletion of Derlin-2. Whole-body deletion of Derlin-2 results in perinatal death. The few mice that survived to adulthood all developed skeletal dysplasia, likely caused by defects in collagen matrix protein secretion by costal chondrocytes., National Institutes of Health (U.S.)

XBP-1, a transcription factor that drives the unfolded protein response (UPR), is activated in B cells when they differentiate to plasma cells. Here, we show that in the B cells, whose capacity to secrete IgM has been eliminated, XBP-1 is induced normally on induction of differentiation, suggesting that activation of XBP-1 in B cells is a differentiation-dependent event, but not the result of a UPR caused by the abundant synthesis of secreted IgM. Without XBP-1, B cells fail to signal effectively through the B-cell receptor. The signalling defects lead to aberrant expression of the plasma cell transcription factors IRF4 and Blimp-1, and altered levels of activation-induced cytidine deaminase and sphingosine-1-phosphate receptor. Using XBP-1-deficient/Blimp-1-GFP transgenic mice, we find that XBP-1-deficient B cells form antibody-secreting plasmablasts in response to initial immunization; however, these plasmablasts respond ineffectively to CXCL12. They fail to colonize the bone marrow and do not sustain antibody production. These findings define the role of XBP-1 in normal plasma cell development and have implications for management of B-cell malignancies., National Science Foundation (U.S.). Graduate Research Fellowship Program, Cancer Research Institute (New York, N.Y.) (Fellowship)

Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function., National Institutes of Health (U.S.)

Shiga-toxigenic Escherichia coli (STEC) use subtilase cytotoxin (SubAB) to interfere with adaptive immunity. Its inhibition of immunoglobulin secretion is both rapid and profound. SubAB favors cleavage of the newly synthesized immunoglobulin heavy chain–binding protein (BiP) to yield a C-terminal fragment that contains BiP’s substrate-binding domain. In the absence of its regulatory nucleotide-binding domain, the SubAB-cleaved C-terminal BiP fragment remains tightly bound to newly synthesized immunoglobulin light chains, resulting in retention of light chains in the endoplasmic reticulum (ER). Immunoglobulins are thus detained in the ER, making impossible the secretion of antibodies by SubAB-treated B cells. The inhibitory effect of SubAB is highly specific for antibody secretion, because other secretory proteins such as IL-6 are released normally from SubAB-treated B cells. Although SubAB also causes BiP cleavage in HepG2 hepatoma cells, (glyco)protein secretion continues unabated in SubAB-exposed HepG2 cells. This specific block in antibody secretion is a novel means of immune evasion for STEC. The differential cleavage of newly synthesized versus "aged" BiP by SubAB in the ER provides insight into the architecture of the ER compartments involved., National Institutes of Health (U.S.), Cancer Research Institute (New York, N.Y.)

Shiga-toxigenic Escherichia coli (STEC) use subtilase cytotoxin (SubAB) to interfere with adaptive immunity. Its inhibition of immunoglobulin secretion is both rapid and profound. SubAB favors cleavage of the newly synthesized immunoglobulin heavy chain–binding protein (BiP) to yield a C-terminal fragment that contains BiP’s substrate-binding domain. In the absence of its regulatory nucleotide-binding domain, the SubAB-cleaved C-terminal BiP fragment remains tightly bound to newly synthesized immunoglobulin light chains, resulting in retention of light chains in the endoplasmic reticulum (ER). Immunoglobulins are thus detained in the ER, making impossible the secretion of antibodies by SubAB-treated B cells. The inhibitory effect of SubAB is highly specific for antibody secretion, because other secretory proteins such as IL-6 are released normally from SubAB-treated B cells. Although SubAB also causes BiP cleavage in HepG2 hepatoma cells, (glyco)protein secretion continues unabated in SubAB-exposed HepG2 cells. This specific block in antibody secretion is a novel means of immune evasion for STEC. The differential cleavage of newly synthesized versus "aged" BiP by SubAB in the ER provides insight into the architecture of the ER compartments involved., National Institutes of Health (U.S.), Cancer Research Institute (New York, N.Y.)