Cytoplasmic Domain Of CD4 Research Articles

TRAF6 and TRAF2/3 Binding Motifs in CD40 Differentially Regulate B Cell Function in T-Dependent Antibody Responses and Dendritic Cell Function in Experimental Autoimmune Encephalomyelitis.

Expression of the costimulatory molecule CD40 on both B cells and dendritic cells (DCs) is required for induction of experimental autoimmune encephalomyelitis (EAE), and cell-autonomous CD40 expression on B cells is required for primary T-dependent (TD) Ab responses. We now ask whether the function of CD40 expressed by different cell types in these responses is mediated by the same or different cytoplasmic domains. CD40 has been reported to possess multiple cytoplasmic domains, including distinct TRAF6 and TRAF2/3 binding motifs. To elucidate the invivo function of these motifs in B cells and DCs involved in EAE and TD germinal center responses, we have generated knock-in mice containing distinct CD40 cytoplasmic domain TRAF-binding site mutations and have used these animals, together with bone marrow chimeric mice, to assess the roles that these motifs play in CD40 function. We found that both TRAF2/3 and TRAF6 motifs of CD40 are critically involved in EAE induction and demonstrated that this is mediated by a role of both motifs for priming of pathogenic T cells by DCs. In contrast, the TRAF2/3 binding motif, but not the TRAF6 binding motif, is required for B cell CD40 function in TD high-affinity Ab responses. These data demonstrate that the requirements for expression of specific TRAF-binding CD40 motifs differ for B cells or DCs that function in specific immune responses and thus identify targets for intervention to modulate these responses.

CD19-CAR Cytokine Induced Killer Cells Armored with IL-18 Control Tumor Burden, Prolong Mouse Survival and Result in In Vivo Persistence of CAR-CIK Cells in a Model of B-Cell Acute Lymphoblastic Leukemia

Rationale: Cytokine induced killer cells (CIK) are a promising cancer immunotherapy. Recently CIK cells modified to express chimeric antigen receptors (CAR) target CD19 expressing hematological malignancies including B-cell acute lymphoblastic leukemia (B-ALL). Despite encouraging clinical trial results, insufficient CAR-CIK anti-tumor activity and persistence pose major obstacles towards improved efficacy in vivo. Therefore, we optimized our CAR-CIK platform by introducing two modifications to the CD19-CAR construct. The first modification “armored” CAR-CIK cells by inserting the IL-18 gene in a bicistronic DNA plasmid enabling simultaneous surface expression of the CAR protein and secretion of IL-18, which demonstrated improved CAR-T cell function. The second modification attenuated the CD28 cytoplasmic signaling domain of the CAR molecule to enhance anti-tumor activity and in vivo persistence since recent findings demonstrate that persistent or chronic CAR signaling can impair anti-tumor activity and in vivo persistence. Methods: CARCIK-1918 cells generated from donor-derived CIK cells are engineered to express CAR and IL-18 using the non-viral Sleeping Beauty (SB) transposon system. CIK cells were generated from donor PBMCs by the sequential addition of IFN-ϒ and stimulation with anti-CD3 antibody plus IL-2. After 2 days of stimulation CIK cells were co-electroporated with SB100X transposase mRNA and plasmid DNA containing a bicistronic transgene encoding both the anti-CD19 CAR and IL-18. This second-generation CAR contains a fusion of the CD28 transmembrane domain, intracellular CD28 and, TCR CD3ζ chain. In some instances, the cytoplasmic CD28 signaling domain is modified to attenuate CD28 signaling. This method of gene transfer results in stable surface expression of the CAR on CD3+ cells through a 17-day culture period. Day 17 cells are harvested, frozen and used for all subsequent studies. CARCIK-1918 cells are stimulated in vitro with CD19+ REH tumor cells and the phenotype of activated cells was characterized by multi-color flow cytometry. IL-18 secretion was determined by cytokine bead array analysis of supernatants collected post stimulation. CARCIK-1918 cells with or without the attenuated CD28 signaling domain were tested in vivo in a xenograft model of B-ALL, using NSG mice engrafted with CD19+ Raji tumor cells. Tumor burden was assessed weekly by bioluminescence imaging, the in vivo persistence of CARCIK-1918 cells was measured by flow cytometry, and survival was monitored throughout the study. Results: CARCIK-1918 cells express both IL-18 and CAR with or without attenuated CD28 signaling. Furthermore, stimulation of CARCIK-1918 cells with CD19+ REH tumor cells increases the frequency of CAR-CIK cells with a memory phenotype and concurrent upregulation of the high affinity IL-2 receptor, CD25, and the costimulatory receptor CD137. Expression of IL-18 leads to higher engraftment of CAR-CIK cells in tumor bearing NSG mice compared to unarmored controls. NSG mice engrafted with luciferase positive Raji cells demonstrated decreased bioluminescent signal following CARCIK-1918 treatment, consistent with CARCIK derived anti-leukemic activity. This anti-tumor activity corresponded to a statistically significant improvement in survival as mice treated with 2 nd generation CAR transgene CARCIK cells with or without attenuated CD28 signaling had a median survival of 37.5 (n=10; p=0.0001) and 38 (n=12; p =0.0001 days), respectively, compared to untreated mice that develop progressive highly disseminated tumor and succumb to disease by 3 weeks post tumor inoculation (n=11; median survival 18 days). Interestingly, mice treated with CARCIK-1918 cells with the CD28 attenuated signaling domain exhibited better tumor control and enhanced persistence in vivo, despite no significant improvement in survival compared to WT CD28 signaling CARCIK-1918 cells. Summary: This study demonstrates that administration of CAR-CIK cells armored with IL-18 results in a significant survival advantage for tumor challenged animals versus untreated animals. Furthermore, modifying the cytoplasmic CD28 domain to attenuate signaling, results in better control of tumor burden, and persistence of CARCIK-1918 cells in vivo. Our study is the first report of IL-18 armored CAR-CIK cells which may present a promising, novel strategy for the treatment of B-ALL.

Endoplasmic reticulum sequestration empowers phosphorylation profiling on the yeast surface

AbstractCells sense cues in their surrounding environment, turning extracellular stimuli into intracellular responses. Transient posttranslational modifications, such as phosphorylation, often guide these responses through tightly regulated signaling networks. There is great interest in understanding phosphorylation events that yield responses in cell signaling, but to date, methods to characterize the phosphoproteome rely on low‐throughput methods. To overcome this limitation, this study optimizes a yeast surface display system for high‐throughput screening of kinase–substrate interaction using endoplasmic reticulum (ER) sequestration. We demonstrate the ability of yeast to display phosphorylated full‐length receptor cytoplasmic domains and additionally optimized magnetic bead selections to efficiently recover yeast displaying phosphorylated domains. These lessons were applied to profiling the substrate specificity of the tyrosine kinase lymphocyte cell‐specific protein‐tyrosine kinase by mutating a phosphoacceptor motif within the CD28 cytoplasmic domain and to building multienzyme phosphorylation cascades in the yeast ER. Collectively, this study provides a facile approach for profiling kinase–substrate reactions in high throughput.

Abstract 5560: KIR-based CAR design showed durable antitumor efficacy by altered internalization kinetics following persistent antigenic stimulation

Abstract Poor persistence of functional chimeric antigen receptor (CAR) T cells in the tumor microenvironment can limit the effectiveness of the antitumor immune response. We previously showed that T cells expressing a multichain CAR design that uses an activating killer immunoglobulin-like receptor (KIR) transmembrane (TM) and cytoplasmic domain co-expressed with the ITAM-containing adapter, DAP12 (KIR-CAR) are able to control large, pre-established tumors that cannot be controlled by 2nd generation CD3ζ-based CAR T cells. This improved function is associated with maintenance of KIR-CAR expression on the tumor infiltrating lymphocytes compared with CD3ζ-based CARs. In the present study, we explored the mechanism for the improved KIR-CAR expression, which we hypothesized was related to reduced receptor internalization following receptor activation compared with standard CD3ζ-based CARs. To monitor receptor dynamics at the T cell surface, we took advantage of a surface protein tagging method that employs the SpyCatcher-SpyTag system to construct a KIR-based or CD3ζ-based CAR with the ability to pulse label the CAR at the cell surface with a fluorescent molecule. In this approach, a CAR ectodomain comprised of a SpyCatcher domain is linked to either the KIR TM and cytoplasmic domain (SpyKIR-CAR) or CD28 TM and cytoplasmic domain with CD3ζ (SpyCAR). The antigenic specificity of the CAR is conferred by addition of a fluorescently-labeled binder comprised of an antibody-like Designed Ankyrin Repeat Protein (DARPin) fused to a SpyTag sequence (SpyDARPin) that specifically binds the spyCatcher domain covalently via its spyTag and recognizes the EGFR antigen. The SpyKIR-CAR or Spy-CAR expression is confirmed by flow cytometry using an antibody to a myc-tag within the DARPin sequence. Uniform fluorescence labeling at the plasma membrane is observed following addition of the SpyDARPin as assessed by image cytometry and confocal microscopy. The kinetics of SpyCAR or SpyKIR-CAR fluorescence loss were similar in the absence of EGFR ligand with a half-life of 2 days consistent with normal membrane turnover by pinocytosis. Upon ligand-induced CAR activation, the SpyCAR fluorescence rapidly reduced beginning at 30 min post-activation to background fluorescence levels of unlabeled SpyCAR cells within 8 hours of activation. In contrast, the mean SpyKIR-CAR fluorescence showed no change in fluorescence at 30 min, while exhibiting a reduction to 80% of the mean baseline fluorescence at 8 hours and 4-fold above the background fluorescence. Confocal imaging confirmed the retention of labeled KIR-CAR at the plasma membrane over the time frame in which the labeled SpyCAR is lost. Our data support differences in CAR internalization upon activation as an important mechanism underlying the enhanced function of KIR-CAR T cells in solid tumors compared with traditional CD3ζ-based CAR designs. Citation Format: Qian Zhang, Eric Nigel, Alvin Yu, Morgan Hresko, Nicholas Minutolo, Daniel Powell, Michael C. Milone. KIR-based CAR design showed durable antitumor efficacy by altered internalization kinetics following persistent antigenic stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5560.

B7-CD28 co-stimulation enforces central tolerance by thymic clonal deletion and Treg cell generation through distinct mechanisms

Abstract To establish central T cell tolerance in the thymus, thymocytes expressing TCR strongly reactive to self-Ag-MHC complex are eliminated from the conventional T cell (Tconv) repertoire by two distinct processes, clonal deletion or clonal diversion to non-conventional T cell lineages such as Foxp3+ regulatory T (Treg) cells. We analyzed the requirements for B7-CD28 co-stimulation for thymic clonal deletion and Treg cell generation of endogenous tissue-restricted-antigen (TRA)-specific thymocytes. We found that: 1) both clonal deletion and Treg cell generation of TRA-specific thymocytes were B7-CD28-dependent, and that clonal deletion had a greater effect than Treg diversion in eliminating TRA-specific T cells from the Tconv repertoire; 2) B7-expressing APC requirements differed for clonal deletion and Treg generation; and 3) although B7-CD28 mediated both clonal deletion and Treg cell generation, CD28 cytoplasmic domain requirements for these two processes were different. These results indicate that qualitatively distinct CD28 signaling determines fates of strongly self-reactive thymocytes. To further characterize the CD28 signaling pathways involved in clonal deletion and Treg cell generation, single cell transcriptomic analyses of TRA-specific thymocyte are underway.

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling.

Affinity purification coupled to mass spectrometry (AP-MS) is a popular approach for deciphering the architecture of protein interaction networks. Protein lysates (100 μg) are typically required for multistep sample processing in large volumes, which often causes sample loss and reduces the MS analysis sensitivity. Herein, we reported a fully integrated spintip-based AP-MS technology, termed FISAP, for multiplexed and sensitive interactome profiling. The FISAP device can be easily employed for routine use by introducing AP beads into a C18 StageTip. Taking advantage of the switchable functionalization of the C18 matrix by sodium dodecyl sulfate, all the sample preparation steps encompassing peptide or antibody-based AP, reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting can be performed in a single tip with a benchtop centrifuge in 4 h. Using a biotinylated tyrosine phosphorylated (pTyr) peptide as an affinity ligand, we mapped the pTyr-dependent interactome of the pY191 motif on the immune receptor CD28 cytoplasmic domain. When processing 50 μg of protein lysates, FISAP showed a comparable interactome identification performance but better quantification performance and lower background interference compared to the traditional tube-based method. Furthermore, a cost-effective on-column TMT labeling protocol was established and integrated into the FISAP pipeline with increased sensitivity. Compared to the tube-based method, the usage of a synthetic peptide probe and a TMT reagent was both reduced by 20 times. As low as 1 μg of protein lysates could be applied for interactome profiling. Finally, we expanded the applicability of the FISAP technology to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex using 100 times less protein lysate than a previous report. Collectively, FISAP is an easy-to-use and sensitive technology for quantitatively profiling protein complexes when the starting material and affinity reagent are the limitation, especially for applications in biomedical research and chemical biology.

Evaluating nuclear translocation of surface receptors: recommendations arising from analysis of CD44.

CD44 is a transmembrane receptor that acts as adhesion protein, fundamentally recognizing hyaluronan, an essential component of the extracellular matrix. It has a well-established functional association with cancer metastasis, particularly the CD44 variant forms which are considered essential markers of cancer stem cells. CD44 itself lacks intrinsic kinase activity but rather engages in signalling through specific interactions with kinases and other signalling components. Proteolysis within its transmembrane region also leads to release of the CD44 cytoplasmic domain, which can translocate to the nucleus and regulate transcription. A third signalling modality has been reported where the intact CD44 receptor translocates to the nucleus. Here, we investigated the latter using imaging techniques together with biochemical analyses. Our findings support observations where CD44 is cleaved prior to nuclear translocation and challenges the evidence for the presence of intact CD44 receptors in the cell nucleus. Conclusions regarding the presence of intact CD44 in the cell nucleus as a signalling modality, therefore, require re-evaluation. We highlight artefacts and common technical issues associated with these experiments that can lead to misinterpretation.

Abstract 3583: NKG2D as a chimeric antigen receptor - DAP 10 provides optimal co stimulation for NKG2D based CARs

Abstract Chimeric antigen receptor (CAR) protein are artificial protein created by the fusion of an extracellular domain targeting one or several cell surface antigens, a transmembrane domain and an intracellular domain responsible for signal initiation and transmission. First-generation CARs consist of an intracellular signaling domain, often a cytoplasmic tail of the CD3ζ chain. The second and third generation include the addition of one or two co-stimulation domains (e.g. cytoplasmic domain of CD28 and/or 4-1BB). NKG2D is a receptor present on the cell surface of NK cells, NK T cells and subgroups of CD8+ T cells. The receptor exists as a dimer that interacts with a co-adaptor protein called DAP10. Upon interaction of NKG2D with one of its ligand (MICA, MICB, and ULBP1-ULBP6), the DAP10 cytoplasmic tail will induce downstream signaling. DAP10 has a similar signaling motif to CD28 and similarly gets phosphorylated and recruits kinases. NKG2D ligands are expressed on a vast majority of solid and liquid tumors and are thus a very attractive target for CAR T cells. In this study, multiple NKG2D constructs were created and compared to one another. The first CAR construct tested (chNKG2D.1) consists of a fusion of full-length wild-type NKG2D and the cytoplasmic domain of CD3ζ. This construct is currently tested in clinical settings. This CAR benefits from the co-stimulatory signal provided by DAP10 and can therefore be considered as a second generation CAR. Next to this, a new chNKG2D was constructed by combining DAP10 overexpression (chNKG2D.1.DAP10). Secondly classic second-generation CARs were created by adding CD28 (chNKG2D.2.1) or 4-1BB (chNKG2D.2.2) co-stimulatory domains to the chNKG2D. Co-expression of DAP10 increased chNKG2D expression at the cell surface of both CD4 and CD8 T cells (although the increase was more pronounced in CD4 T cells). Furthermore, co-cultures of T cells expressing this construct with K562 cells led to similar IFN-γ levels irrelevant of the CD4/CD8 ratio. Other CD4 specific cytokines were highly increased when the ratio was in favor of CD4 positive T cells. Interestingly, the addition of the co-stimulatory domains to the construct (chNKG2D.2.1 and .2.2) did not lead to higher levels of IFN-γ or any of the CD4 cytokines. Next to cytokine release, cytolytic activity was also assessed which interestingly showed no difference in any of the conditions even when the CD4/CD8 cell ratio was different. In conclusion NKG2D CAR T cells only showed increased cytokine release when DAP10 was overexpressed, all other conditions did not lead to any changes when compared to the ‘NKG2D CAR T cells currently tested in the clinic. This implies that NKG2D is optimally costimulated through the DAP 10 co-signaling, and that this co-signaling is a least as potent as traditional CD28 or 4-1BB based co stimulation. Citation Format: Jennifer BOLSEE, Eytan BREMAN, Fanny HUBERTY, Benjamin VIOLLE, Jerome MARIJSSE, Céline JACQUES-HESPEL, Celine MARCHAND, Nancy RAMELOT, Thuy NGUYEN, Julien HOUSSA, David E. GILHAM, Sophie AGAUGUE. NKG2D as a chimeric antigen receptor - DAP 10 provides optimal co stimulation for NKG2D based CARs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3583.

Abstract POSTER-THER-1433: Overcoming resistance to antibody targeted therapy in ovarian cancer

Abstract Targeted therapies utilizing monoclonal antibodies (mAbs) are now the major class of successful therapeutics for treating a variety of malignancies. Mechanistically, mAbs can have direct antitumoral activity but often their effectiveness relies upon antibody-dependent cellular cytotoxicity (ADCC). For instance, Trastuzumab is a therapeutic monoclonal antibody directed against HER2 that is well-tolerated and useful in the treatment of HER2-positive breast cancer, where efficacy is mediated in part by ADCC. In contrast, Trastuzumab is relatively ineffective in the treatment of epithelial ovarian cancer (EOC) where virtually all cancer cells express detectable levels of HER2. Poor response to mAb therapy in EOC and other types of cancers may be explained in part by diminished numbers and cytotoxic potential of NK cells in cancer patients, compared to healthy individuals. Therefore, reports of poor antitumor effect of Ab-therapy in cancer led us to hypothesize that the development of potent effector cells with the capacity to bind tumor-bound mAb and mediate strong antibody-directed cellular cytotoxicity would markedly improve the efficacy of mAb-targeted therapy for EOC. In order to expand applications for T cell-based immunotherapy and to enhance ADCC, we developed novel effector T cells engineered to express Fc binding immune receptors (FcIR) containing human Fc receptor of low FcγRIIIA (CD16), intermediate FcγRIIA (CD32) or high affinity FcγRI (CD64) molecules fused to intracellular TCR and co-stimulatory signaling domains in order to enable cytotoxic T cells to mediate strong mAb-directed cytotoxicity against antigen-expressing tumor cells. Following FcIR gene transduction, all forms of FcγRs were efficiently expressed on the surface of primary human T cells which allowed these cells to be armed with mAb. Trastuzumab-armed FcIR T cells specifically recognized HER2+ cancer cells, as did unarmed FcIRs but only when the cancer cells were first pre-bound with Trastuzumab. Addition of a CD28 cytoplasmic domain juxtaposed to the TCR CD3z signaling moiety increased IFN-g secretion by FcIR-28z transduced T cells following encounter with antigen-bound mAb on the cancer cell surface. Notably, T cells expressing a high affinity FcIRI (CD64) demonstrated the greatest specific anti-tumor reactivity in comparison to cells expressing FcγRIIA (CD32) or FcγRIIIA (CD16) FcIRs. In addition, FcIRI (CD64) T cells exhibited stronger specific lytic activity than NK cells, even at low antibody concentrations. Further, co-administration of FcIRI (CD64) FcIR-28z T cells with immunotherapeutic mAb, Trastuzumab, exerted strong antitumor activity in vivo, completely eliminating HER2+ tumor. In summary, our results show that ADCC can be enhanced by human T cells engineered to express an FcIR and that this novel approach may overcome issues of resistance to mAb-targeted therapies including those utilizing Trastuzumab. Citation Format: Katarzyna Urbanska, Mathilde Poussin, Caitlin Stashwick, Jenessa B. Smith, Daniel J. Powell Jr. Overcoming resistance to antibody targeted therapy in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1433.

Amino-terminal fragments of laminin γ2 chain stimulate migration of metastatic breast cancer cells by interacting with CD44.

Laminin γ2 (Lmγ2) chain, a subunit of the basement membrane protein laminin-332, is regarded as a typical cancer invasion marker. The overexpression of Lmγ2 chain by invasive cancer cells correlates with poor prognosis of cancer patients, and its forced expression in human cancer cells promotes their invasive growth in a nude mouse model. However, its actual roles in cancer progression, as well as the mechanism of its proinvasive effect, remain unclear. CD44 is known to be an important cancer stem cell marker and support cancer progression and stem cell functions. Here we demonstrate that amino-terminal fragments of Lmγ2 interact with CD44 on the membrane of breast cancer cells. Lmγ2 highly bound to the metastatic cell line MDA-MB-231 but poorly to the benign cell line MCF-7. The membrane receptor for Lmγ2 on MDA-MB-231 cells was identified to be the standard form of CD44 (CD44s) by co-immunoprecipitation, affinity chromatography and direct protein interaction assay. Lmγ2 interacted with CD44s through EGF-like repeat 2/3 in the Lmγ2 amino-terminus. Amino-terminal fragments of Lmγ2 induced the phosphorylation of CD44 cytoplasmic domain and stimulated migration of the cancer cells in a CD44-dependent manner. This migration was blocked by inhibitors of TGF-β receptor I (TGF-βRI) kinase. These results suggest that two important tumor markers, Lmγ2 and CD44, cooperate for cancer progression and possibly for cancer stem cell functions. TGF-βRI may be involved in the Lmγ2/CD44 interaction.

CD44 and Hyaluronan Promote the Bone Morphogenetic Protein 7 Signaling Response in Murine Chondrocytes

Cell-matrix interactions promote cartilage homeostasis. We previously found that Smad1, the transcriptional modulator of the canonical bone morphogenetic protein 7 (BMP-7) pathway, interacted with the cytoplasmic domain of CD44, the principal hyaluronan receptor on chondrocytes. To elucidate the physiologic function of CD44-Smad1 interactions, as well as the role of hyaluronan, we studied the response of chondrocytes isolated from CD44(-/-) and BALB/c (wild-type [WT]) mice to stimulation with BMP-7. In primary murine chondrocytes, CD44 expression was decreased by small interfering RNA (siRNA) transfection or was enhanced by plasmid transfection. Pericellular hyaluronan was removed by hyaluronidase treatment, or its endogenous synthesis was inhibited. Changes in response to BMP-7 stimulation were evaluated by Western blotting of Smad1 phosphorylation and aggrecan messenger RNA (mRNA) expression. Chondrocytes from CD44(-/-) mice and WT mice transfected with CD44 siRNA were less responsive than untransfected chondrocytes from WT mice to BMP-7. CD44(-/-) mouse chondrocytes transfected with pCD44 showed increased sensitivity to BMP-7. Significant increases in aggrecan mRNA were observed in WT mouse chondrocytes in response to 10 ng/ml of BMP-7, whereas at least 100 ng/ml of BMP-7 was required for CD44(-/-) mouse chondrocytes. However, in chondrocytes from CD44(-/-) and WT mice, hyaluronidase treatment decreased cellular responses to BMP-7. Treatment of both bovine and murine chondrocytes with 4-methylumbelliferone to reduce the synthesis of endogenous hyaluronan confirmed that hyaluronan promoted BMP-7 signaling. Taken together, these investigations into the mechanisms underlying BMP-7 signaling in chondrocytes revealed that while hyaluronan-dependent pericellular matrix is critical for BMP-7 signaling, the expression of CD44 promotes the cellular response to lower concentrations of BMP-7.

4-1BBL costimulation enables CD4 T cells expressing a CD28/zeta chimeric antigen receptor to eliminate aggressive leukemia. (P4338)

Abstract Adoptive T-cell therapy using chimeric antigen receptor (CAR) engineered T cells is a promising cancer treatment. First generation CARs include an scFv targeting a surface antigen and the CD3ζ chain to direct tumor cell killing. CAR transduced T cells however need appropriate costimulation to proliferate and persist. Here we assessed in a B-cell leukemia xenograft model, the anti-tumor activities of human T cells transduced with an anti-CD19 CAR harboring the zeta chain alone (19z1+), fused to the CD28 or 4-1BB cytoplasmic domain (1928z+), (19BBz+), along with 4-1BBL (19z1-4-1BBL+), or with CD28 and 4-1BBL (1928z-4-1BBL+). We observed total tumor regression in NALM-6 bearing NSG mice treated with 1928z-4-1BBL+ T cells and no or partial regression in the other T-cell groups, as measured by bioluminescence imaging. 1928z-4-1BBL+ T cells showed the highest bone marrow accumulation and persisted the most. Experiments utilizing purified T cell subsets revealed that naïve and central memory 1928z-4-1BBL+ CD4+ T cells are sufficient to trigger leukemia eradication and are necessary to support CD8+ T-cell in vivo expansion. 1928z-4-1BBL+ CD4+ T cells displayed reduced apoptosis and increased cytotoxicity compared to 1928z+ CD4+ T cells. Thus, constitutive 4-1BBL expression and CAR-mediated CD28 signaling afforded the most efficient costimulatory combination to generate long-lived T cells capable of eradicating aggressive leukemia.

Mapping the interaction between the cytoplasmic domains of HIV‐1 viral protein U and human CD4 with NMR spectroscopy

Viral protein U (VpU) of HIV-1 plays an important role in downregulation of the main HIV-1 receptor CD4 from the surface of infected cells. Physical binding of VpU to newly synthesized CD4 in the endoplasmic reticulum is an early step in a pathway leading to proteasomal degradation of CD4. In this study, regions in the cytoplasmic domain of VpU involved in CD4 binding were identified by NMR spectroscopy. Amino acids in both helices found in the cytoplasmic region of VpU in membrane-mimicking detergent micelles experience chemical shift perturbations upon binding to CD4, whereas amino acids between the two helices and at the C-terminus of VpU show no or only small changes, respectively. The topology of the complex was further studied with paramagnetic relaxation enhancement. Paramagnetic spin labels were attached at three sequence positions of a CD4 peptide comprising the transmembrane and cytosolic domains of the receptor. VpU binds to a membrane-proximal region in the cytoplasmic domain of CD4.

C-terminal Hydrophobic Region in Human Bone Marrow Stromal Cell Antigen 2 (BST-2)/Tetherin Protein Functions as Second Transmembrane Motif

BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.

Solution NMR Spectroscopy and Protein Interaction Studies of Membrane Proteins in Nanodiscs

Insolubility of integral membrane proteins (IMPs) in buffer often prevents their investigation with biophysical methods developed for soluble proteins. First, solution NMR of large proteins or complexes is limited by slow rotational diffusion. IMPs in cellular membranes or liposomes are beyond the size limit of solution NMR. Therefore, detergent micelles or bicelles are often used for solubilization and NMR studies of IMPs. Unfortunately, detergents may destabilize the native structure or compromise the activity of proteins. Nanodiscs are a lipid-based, detergent free, relatively small and very promising new model membrane system. We inserted a recombinantly produced and 15N, 13C-labeled fragment of human CD4 into nanodiscs. This polypeptide comprises the membrane-spanning and the cytoplasmic domains of CD4. Our NMR data demonstrate the feasibility of solution NMR spectroscopy on IMPs in nanodiscs. Second, surface plasmon resonance (SPR) is predominantly utilized in interaction studies of soluble proteins. Incorporation of IMPs into nanodiscs provides a close to native environment to the membrane protein and results in a water-soluble proteolipid particle that might be amenable to standard SPR-based methodology. We reconstituted a decahistidine-tagged IMP into nanodiscs and studied binding between the nanodisc-inserted IMP and a PentaHis monoclonal antibody (mAb) immobilized on the surface of a CM5-sensorchip. For comparison, we also determined the affinity of the decahistidine-tagged soluble domain of the same IMP toward the immobilized PentaHis mAb. Binding affinities were almost identical in both cases. However, the association and dissociation rate constants were found to differ, which is in agreement with the distinct diffusion coefficients of the soluble analyte particles. Our data indicate that nanodisc-inserted IMPs can serve as analyte in interaction studies of membrane proteins.

CD4 dimerization requires two cysteines in the cytoplasmic domain of the molecule and occurs in microdomains distinct from lipid rafts

CD4 is engaged in APC-mediated T-cell activation and serves as the primary receptor for HIV. CD4 dimerization and location in specific microdomains has been previously suggested to control its pathophysiological activity. In this study, we investigated (i) whether the CD4 cytoplasmic domain contributes to its dimerization by evaluating the dimerization of mutants, bearing deletions or point mutations in their cytoplasmic tail, (ii) whether CD4 monomers and dimers segregate in distinct microdomains by subcellular fractionation, and (iii) how CD4 dimerization is affected by T-cell activation or HIV-1 viral proteins. Our results indicated that within the cytoplasmic tail of CD4, two cysteines played a crucial role in the dimers formation, since point mutations or truncation upstream of these residues prevented dimerization. The solubility of CD4 dimers and monomers in various detergents was different and CD4 dimers were poorly associated with lipid rafts, but strongly interacted with the tetraspanin CD81. Neither cytoskeleton-disrupting drugs nor cholesterol-sequestering agents had an effect on the CD4 dimerization indicating that dimers formation was independent of CD4 association with the cytoskeleton or lipid rafts. Finally, whereas T-cell activation poorly impact on CD4 dimerization, HIV-1 gp120 and Nef drastically reduced the ratio of CD4 dimers/monomers. Together, these findings demonstrate that two cysteines within the CD4 cytoplasmic tail are critical for dimerization, that CD4 dimers locate preferentially in microdomains distinct than classical lipid rafts, likely tetraspanin-enriched microdomains, and that CD4 dimers are implicated in the process of HIV infection.

CD44 interacts directly with Lck in a zinc-dependent manner

CD44 is a widely expressed cell adhesion molecule with functional similarities to the selectin and integrin adhesion molecules. CD44 has a lectin domain that binds hyaluronan, a component of the extracellular matrix. Interactions between CD44 and hyaluronan promote lymphocyte rolling under flow and cell-cell and cell-matrix adhesion. Attachment of lymphocytes to immobilized CD44 antibodies induces cell adhesion and spreading, which is dependent on Src family kinase activity. Both Lck and Fyn associate with CD44 in T cells. CD4 and CD8 associate with Lck via a zinc-dependent interaction that is inhibited by the divalent metal cation chelator, 1,10-phenanthroline. Here we show that both CD4 and CD44-mediated T cell spreading is abolished in the presence of 1,10-phenanthroline and their association with Lck is significantly reduced. In contrast, the co-immunoprecipitation of Fyn by CD44 was unaffected. The cytoplasmic domain of CD44 was required for divalent cation-dependent association of Lck, but not for its association with Fyn. Mutational analysis of CD44 revealed that cysteine residues were not essential for the interaction nor were the carboxy-terminal 41 amino acids. Progressive deletion of the remaining 31 amino acids of the CD44 cytoplasmic domain revealed the importance of this membrane proximal region for its association with Lck. Using purified recombinant proteins, we demonstrated a direct, zinc-inducible interaction between the cytoplasmic domain of CD44 and Lck but not Fyn. The zinc-inducible interaction required the first 13 amino acids of the cytoplasmic domain of CD44 and the non-catalytic regions of Lck. Taken together, we conclude that CD44 directly associates with Lck in a zinc-inducible manner and this is important for the transmission of CD44-mediated signaling events leading to T cell spreading.

Retrovirally expressed 4-1BBL dramatically increases proliferation and in vivo persistence of adoptively transferred CD19-targeted T cells, promoting complete tumor elimination. (131.16)

Abstract Human T cells transduced with a chimeric antigen receptor (CAR) composed of an antibody-derived scFv fragment and the CD3ζ chain can recognize and kill tumor cells on binding to the targeted membrane antigen. The co-transduction of a CAR along with CD80 and 4-1BBL induces auto-costimulation enabling high in vivo T cell expansion and tumor rejection in a prostate tumor model. Here we investigated this concept in T cells harboring either a first generation anti-CD19 CAR (19z1) or a second generation CAR (1928z), which additionally comprises the CD28 cytoplasmic domain. We compared the proliferation and anti-tumor activity of 19z1+, 19z1-4-1BBL+, 19z1-CD80+4-1BBL+, 1928z+ and 1928z-4-1BBL+ transduced T cells in a pro B cell leukemia model (NALM-6). Seven days after IV injection into NALM-6 bearing NOD/SCID/IL2Rγnull mice, 1928z-4-1BBL+ CD8 and CD4 T cells accumulated in the bone marrow and the spleen 100 times more than 19z1+ T cells and 10 times more than 1928z+ T cells, corroborating in vitro proliferative responses to repeated antigen exposure. An increased proportion of central memory CD8 T cells was observed in all groups treated with 4-1BBL-expressing cells. Within 15 days, in vivo bioluminescence imaging showed total tumor regression in the 1928z-4-1BBL+ T cell-treated group and partial regression in the other groups. Thus, constitutive 4-1BBL expression drives high proliferation of anti-tumor CD8 T cells, promoting central memory T cell persistence and tumor eradication.

Roles of the TRAF2/3 Binding Site in Differential B Cell Signaling by CD40 and Its Viral Oncogenic Mimic, LMP1

The EBV protein, latent membrane protein 1 (LMP1), is a functional mimic of the cellular receptor CD40, but signals to B lymphocytes in an amplified and sustained manner compared with CD40. LMP1 contributes to the development of B cell lymphoma in immunosuppressed patients, and may exacerbate flares of certain autoimmune diseases. The cytoplasmic domain of LMP1 binds the signaling adaptor TRAF2 with lower avidity than the cytoplasmic domain of CD40, and TRAF2 is needed for CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. We thus tested the hypothesis that relative affinity for TRAF2, and/or distinct sequence differences in the TRAF2/3 binding sites of CD40 vs LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3, and their distinct signaling characteristics. CD40 and LMP1 mutants in which the TRAF binding site sequences were swapped were examined, testing TRAF binding and degradation, and induction of B cell activation. Results revealed that TRAF binding affinity and TRAF binding site sequence dictate a distinct subset of CD40 vs LMP1 signaling properties. Examination of TRAF binding, degradation, cytokine production, IgM secretion, and the activation of c-Jun kinase and NF-kappaB revealed that some events are dictated by TRAF binding site sequences, others are partially regulated, and still others are independent of the TRAF binding site sequence.

A novel polymorphism of the human CD40 receptor with enhanced function

AbstractCD40 signaling is critical for innate and adaptive immunity against pathogens, and the cytoplasmic domain of CD40 is highly conserved both within and between species. A novel missense single nucleotide polymorphism (SNP) in the cytoplasmic domain of CD40 at position 227 (P227A) was identified, which resides on a conserved ancestral haplotype highly enriched in persons of Mexican and South American descent. Functional studies indicated that signaling via human (h) CD40-P227A stably expressed in several B-cell lines led to increased phosphorylation of c-Jun, increased secretion of the pro-inflammatory cytokines interleukin (IL)–6 and TNF-α, and increased Ig production, compared with wild-type hCD40. Cooperation between hCD40-P227A signaling and B-cell receptor (BCR)– or Toll-like receptor 9 (TLR9)–mediated signaling was also enhanced, resulting in elevated and synergistic production of IL-6 and Ig. We have thus identified a novel genetic variant of hCD40 with a gain-of-function immune phenotype.