M2 macrophage-derived exosomal long non-coding RNA AGAP2-AS1 enhances radiotherapy immunity in lung cancer by reducing microRNA-296 and elevating NOTCH2.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play vital roles in human diseases. We aimed to identify the effect of the lncRNA AGAP2 antisense RNA 1 (AGAP2-AS1)/miR-296/notch homolog protein 2 (NOTCH2) axis on the progression and radioresistance of lung cancer. Expression of AGAP2-AS1, miR-296, and NOTCH2 in lung cancer cells and tissues from radiosensitive and radioresistant patients was determined, and the predictive role of AGAP2-AS1 in the prognosis of patients was identified. THP-1 cells were induced and exosomes were extracted, and the lung cancer cells were respectively treated with silenced AGAP2-AS1, exosomes, and exosomes upregulating AGAP2-AS1 or downregulating miR-296. The cells were radiated under different doses, and the biological processes of cells were assessed. Moreover, the natural killing cell-mediated cytotoxicity on lung cancer cells was determined. The relationships between AGAP2-AS1 and miR-296, and between miR-296 and NOTCH2 were verified. AGAP2-AS1 and NOTCH2 increased while miR-296 decreased in radioresistant patients and lung cancer cells. The malignant behaviors of radioresistant cells were promoted compared with the parent cells. Inhibited AGAP2-AS1, macrophage-derived exosomes, and exosomes overexpressing AGAP2-AS1 or inhibiting miR-296 facilitated the malignant phenotypes of radioresistant lung cancer cells. Furthermore, AGAP2-AS1 negatively regulated miR-296, and NOTCH2 was targeted by miR-296. M2 macrophage-derived exosomal AGAP2-AS1 enhances radiotherapy immunity in lung cancer by reducing miR-296 and elevating NOTCH2. This study may be helpful for the investigation of radiotherapy of lung cancer.

Stem cell transplantation impairs dendritic cell trafficking and herpesvirus immunity.

Long-term survivors after hematopoietic stem cell transplantation are at high risk of infection, which accounts for one-third of all deaths related to stem cell transplantation. Little is known about the cause of inferior host defense after immune cell reconstitution. Here, we exploited a murine syngeneic BM transplantation (BMT) model of late infection with murine gammaherpesvirus 68 (MHV-68) to determine the role of conventional DC (cDC) trafficking in adaptive immunity in BMT mice. After infection, the expression of chemokine Ccl21 in the lung is reduced and the migration of cDCs into lung draining lymph nodes (dLNs) is impaired in BMT mice, limiting the opportunity for cDCs to prime Th cells in the dLNs. While cDC subsets are redundant in priming Th1 cells, Notch2 functions in cDC2s are required for priming increased Th17 responses in BMT mice, and cDC1s can lessen this activity. Importantly, Th17 cells can be primed both in the lungs and dLNs, allowing for increased Th17 responses without optimum cDC trafficking in BMT mice. Taken together, impaired cDC trafficking in BMT mice reduces protective Th1 responses and allows increased pathogenic Th17 responses. Thus, we have revealed a previously unknown mechanism for BMT procedures to cause long-term inferior immune responses to herpes viral infection.

Protease-Activated Receptor 1 Deletion Causes Enhanced Osteoclastogenesis in Response to Inflammatory Signals through a Notch2-Dependent Mechanism.

We found that protease-activated receptor 1 (PAR1) was transiently induced in cultured osteoclast precursor cells. Therefore, we examined the bone phenotype and response to resorptive stimuli of PAR1-deficient (knockout [KO]) mice. Bones and bone marrow-derived cells from PAR1 KO and wild-type (WT) mice were assessed using microcomputed tomography, histomorphometry, in vitro cultures, and RT-PCR. Osteoclastic responses to TNF-α (TNF) challenge in calvaria were analyzed with and without a specific neutralizing Ab to the Notch2-negative regulatory region (N2-NRR Ab). In vivo under homeostatic conditions, there were minimal differences in bone mass or bone cells between PAR1 KO and WT mice. However, PAR1 KO myeloid cells demonstrated enhanced osteoclastogenesis in response to receptor activator of NF-κB ligand (RANKL) or the combination of RANKL and TNF. Strikingly, in vivo osteoclastogenic responses of PAR1 KO mice to TNF were markedly enhanced. We found that N2-NRR Ab reduced TNF-induced osteoclastogenesis in PAR1 KO mice to WT levels without affecting WT responses. Similarly, in vitro N2-NRR Ab reduced RANKL-induced osteoclastogenesis in PAR1 KO cells to WT levels without altering WT responses. We conclude that PAR1 functions to limit Notch2 signaling in responses to RANKL and TNF and moderates osteoclastogenic response to these cytokines. This effect appears, at least in part, to be cell autonomous because enhanced osteoclastogenesis was seen in highly purified PAR1 KO osteoclast precursor cells. It is likely that this pathway is involved in regulating the response of bone to diseases associated with inflammatory signals.

Notch signaling pathway suppresses CD8+ T cells activity in patients with lung adenocarcinoma.

Evolution and progression of cancer always leads to CD8+ T cells dysfunction/exhaustion. Controversy remains as to the role of Notch signaling pathway in CD8+ T cells regulation in tumorigenesis. Thus, the aim of this study was to investigate the immunomodulatory activity of Notch signaling pathway to peripheral and lung-resident CD8+ T cells in patients with lung adenocarcinoma. Forty-eight lung adenocarcinoma patients and twenty healthy individuals were enrolled in the current study, and CD8+ T cells were purified from both peripheral bloods and bronchoalveolar lavage fluids. Notch receptor mRNA expression was semi-quantified by real-time PCR. Cytolytic and noncytolytic activity of CD8+ T cells evaluated in direct and indirect contact co-culture with A549 cells in response to Notch signaling inhibition by measuring of lactate dehydrogenase release and cytokines production. Expression of Fas ligand (FasL), perforin, and granzyme B were also assessed by flow cytometry. Notch2 mRNA expression was elevated in both peripheral and lung-resident CD8+ T cells in lung adenocarcinoma patients, however, did not correlated with tumor stages or epidermal growth factor receptor mutation. Peripheral CD8+ T cells from healthy individuals exhibited stronger cytotoxicity in direct contact co-culture system, which was not influenced by Notch signaling inhibition. Moreover, suppression of Notch signaling augmented cytotoxicity of peripheral and lung-resident CD8+ T cells from lung adenocarcinoma patients in direct contact co-culture system, and promoted interferon-γ production in both systems. This process was accompanied by increased expression of FasL and perforin within CD8+ T cells. The current data revealed a potential immunosuppressive property of Notch signaling pathway to CD8+ T cells probably via inhibition of FasL and perforin in lung adenocarcinoma patients.

Notch signaling pathway dampens tumor-infiltrating CD8+ T cells activity in patients with colorectal carcinoma.

CD8+ T cells play critical role in controlling the metastasis and prognosis of cancer. Controversy remains as to the contribution of Notch signaling pathway in modulation of CD8+ T cells activity and development of tumorigenesis. Thus, the aim of the current study was to investigate the immunoregulatory role of Notch signaling pathway to peripheral and tumor-infiltrating CD8+ T cells in patients with colorectal carcinoma. A total of 46 patients with colorectal carcinoma and 20 health individuals were enrolled, and CD8+ T cells were purified from both peripheral bloods and carcinoma specimens. Cytolytic and noncytolytic functions of CD8+ T cells in response to Notch signaling inhibition were evaluated by measurements of lactate dehydrogenase release and proinflammatory cytokines production in both direct and indirect contact co-culture system to target HT29 cells. Cellular proliferation and inhibitory receptors expression in CD8+ T cells were also assessed by CCK-8 method and flow cytometry. There was no remarkable difference in percentage of CD8+ T cells between healthy individuals and patients with colorectal carcinoma. Notch1/2 and Hes1/5 mRNAs were elevated expressed in tumor-infiltrating CD8+ T cells in patients with colorectal carcinoma, however, did not correlated with tumor differentiation or stages. CD8+ T cells from healthy individuals presented stronger cytotoxicity, which was not affected by Notch signaling inhibitor. Inhibition of Notch signaling pathway not only promoted cytotoxicity of tumor-infiltrating CD8+ T cells, but also enhanced proinflammatory cytokines (including IFN-γ, TNF-α, IL-1ß, IL-6, and IL-8) production by CD8+ T cells from patients with colorectal carcinoma. This process was accompanied by decreased expression of PD-1 in CD8+ T cells without influencing cellular proliferation. Our results indicated a potential immunosuppressive property of Notch signaling pathway, which dampened both cytolytic and noncytolytic functions of CD8+ T cells probably via induction of PD-1 in patients with colorectal carcinoma.

CD19 regulates ADAM28-mediated Notch2 cleavage to control the differentiation of marginal zone precursors to MZ B cells.

As the first line of defence, marginal zone (MZ) B cells play principal roles in clearing blood-borne pathogens during infection and are over-primed in autoimmune diseases. However, the basic mechanisms underlying MZ B-cell development are still unclear. We found here that CD19 deficiency blocked the differentiation of marginal zone precursors (MZP) to MZ B cells, whereas CD19 expression in CD19-deficient MZP rescues MZ B-cell generation. Furthermore, CD19 regulates Notch2 cleavage by up-regulating ADAM28 expression in MZP. Finally, we found that CD19 suppressed Foxo1 expression to promote ADAM28 expression in MZP. These results suggest that CD19 controls the differentiation of MZP to MZ B cells by regulating ADAM28-mediated Notch2 cleavage. Thus, we demonstrated the basic mechanisms underlying the differentiation of MZP to MZ B cells.

Bruton's Tyrosine Kinase Synergizes with Notch2 To Govern Marginal Zone B Cells in Nonobese Diabetic Mice.

Expansion of autoimmune-prone marginal zone (MZ) B cells has been implicated in type 1 diabetes. To test disease contributions of MZ B cells in NOD mice, Notch2 haploinsufficiency (Notch2(+/-)) was introduced but failed to eliminate the MZ, as it does in C57BL/6 mice. Notch2(+/-)/NOD have MZ B cell numbers similar to those of wild-type C57BL/6, yet still develop diabetes. To test whether BCR signaling supports Notch2(+/-)/NOD MZ B cells, Bruton's tyrosine kinase (Btk) deficiency was introduced. Surprisingly, MZ B cells failed to develop in Btk-deficient Notch2(+/-)/NOD mice. Expression of Notch2 and its transcriptional target, Hes5, was increased in NOD MZ B cells compared with C57BL/6 MZ B cells. Btk deficiency reduced Notch2(+/-) signaling exclusively in NOD B cells, suggesting that BCR signaling enhances Notch2 signaling in this autoimmune model. The role of BCR signaling was further investigated using an anti-insulin transgenic (Tg) BCR (125Tg). Anti-insulin B cells in 125Tg/Notch2(+/-)/NOD mice populate an enlarged MZ, suggesting that low-level BCR signaling overcomes reliance on Notch2. Tracking clonotypes of anti-insulin B cells in H chain-only VH125Tg/NOD mice showed that BTK-dependent selection into the MZ depends on strength of antigenic binding, whereas Notch2-mediated selection does not. Importantly, anti-insulin B cell numbers were reduced by Btk deficiency, but not Notch2 haploinsufficiency. These studies show that 1) Notch2 haploinsufficiency limits NOD MZ B cell expansion without preventing type 1 diabetes, 2) BTK supports the Notch2 pathway in NOD MZ B cells, and 3) autoreactive NOD B cell survival relies on BTK more than Notch2, regardless of MZ location, which may have important implications for disease-intervention strategies.

Rescue of notch-1 signaling in antigen-specific CD8+ T cells overcomes tumor-induced T-cell suppression and enhances immunotherapy in cancer.

An impaired antitumor immunity is found in patients with cancer and represents a major obstacle in the successful development of different forms of immunotherapy. Signaling through Notch receptors regulates the differentiation and function of many cell types, including immune cells. However, the effect of Notch in CD8(+) T-cell responses in tumors remains unclear. Thus, we aimed to determine the role of Notch signaling in CD8(+) T cells in the induction of tumor-induced suppression. Our results using conditional knockout mice show that Notch-1 and Notch-2 were critical for the proliferation and IFNγ production of activated CD8(+) T cells and were significantly decreased in tumor-infiltrating T cells. Conditional transgenic expression of Notch-1 intracellular domain (N1IC) in antigen-specific CD8(+) T cells did not affect activation or proliferation of CD8(+) T cells, but induced a central memory phenotype and increased cytotoxicity effects and granzyme B levels. Consequently, a higher antitumor response and resistance to tumor-induced tolerance were found after adoptive transfer of N1IC-transgenic CD8(+) T cells into tumor-bearing mice. Additional results showed that myeloid-derived suppressor cells (MDSC) blocked the expression of Notch-1 and Notch-2 in T cells through nitric oxide-dependent mechanisms. Interestingly, N1IC overexpression rendered CD8(+) T cells resistant to the tolerogenic effect induced by MDSC in vivo. Together, the results suggest the key role of Notch in the suppression of CD8(+) T-cell responses in tumors and the therapeutic potential of N1IC in antigen-specific CD8(+) T cells to reverse T-cell suppression and increase the efficacy of T cell-based immunotherapies in cancer.

Involvement of Notch in activation and effector functions of γδ T cells.

Notch signaling plays a pivotal role in cell fate decision and lineage commitment of lymphocytes. Although the role of Notch in CD4(+) and CD8(+) αß T cells is well documented, there are no reports on how Notch signaling regulates effector functions of γδ T cells. γδ T cells are a minor fraction in the peripheral blood but are known to play a major role in defense against pathogens and tumors. In this study, we show that Notch receptors (mRNA and protein) are expressed in peripheral γδ T cells. Inhibition of Notch signaling by γ-secretase inhibitor inhibited the proliferation and IFN-γ secretion of γδ T cells in response to stimulation with phosphoantigens and anti-CD3 mAb. In the presence of γ-secretase inhibitor, the antitumor cytolytic ability of γδ T cells was inhibited with a decreased CD107a expression. Knockdown of Notch1 and Notch2 genes in γδ T cells using small interfering RNA inhibited their antitumor cytotoxic potential. Our study describes for the first time, to our knowledge, the role of Notch as an additional signal contributing to Ag-specific effector functions of γδ T cells.

Notch controls generation and function of human effector CD8+ T cells.

The generation of effector CD8(+) T cells with lytic capacity is crucial for tumor control. Dendritic cells (DCs) provide important signals to promote naive CD8(+) T cell priming and activation of effector T cells. Here, we report that the Notch pathway has an important role in both these processes in human CD8(+) T cells. Activated monocyte-derived DCs express Notch ligands Jagged1 and Delta-like4, whereas naive CD8(+) T cells express Notch2. The role for Notch signaling in CD8(+) T cell priming was determined using an ex-vivo model system in which tumor antigen-specific primary CD8(+) T cell responses were measured. Inhibition of Notch using γ-secretase inhibitors or soluble Delta-like4-Fc during activation reduced expansion of antigen-specific CD8(+) T cells, which was mirrored by decreased frequencies of interferon (IFN)γ-, tumor necrosis factor-α-, and granzymeB-producing CD8(+) T cells. Moreover, T cells primed when Notch signaling was prevented are functionally low-avidity T cells. In addition, Notch partially regulates established effector T cell function. Activation-induced Notch signaling is needed for IFNγ release but not for cytolytic activity. These data indicate that Notch signaling controls human CD8(+) T cell priming and also influences effector T cell functions. This may provide important information for designing new immunotherapies for treatment of cancer.

Notch2 and immune function.

Notch2 is expressed in many cell types of most lineages in the hematolymphoid compartment and has specific roles in differentiation and function of various immune cells. Notch2 is required for development of splenic marginal zone B cells and regulates differentiation of dendritic cells (DCs) in the spleen. Notch2 appears to play some specific roles in the intestinal immunity, given that the fate of mast cells and a subset of DCs is regulated by Notch2 in the intestine. Notch2 also has important roles in helper T cell divergence from naïve CD4 T cells and activation of cytotoxic T cells. Moreover, recent genetic evidence suggests that both gain-and loss-of-function abnormalities of Notch2 cause transformation of immune cells. Inactivating mutations are found in Notch2 signaling pathways in chronic myelomonocytic leukemia, while activating mutations are found in mature B cell lymphomas, which reflects the role of Notch2 in the developmental process of these cells.

Therapeutic antibody targeting of individual Notch receptors.

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.

Notch2 signaling is required for potent antitumor immunity in vivo.

CD8(+) T cells play a central role in cancer immunosurveillance, and the efficient induction of CTLs against tumor Ags is required for successful immunotherapy for cancer patients. Notch signaling directly regulates the transcription of effector molecules in CTLs. However, it remains unclear whether Notch signaling in CD8(+) T cells is required for antitumor CTL responses and whether modulation of Notch signaling can augment antitumor CTL responses. In this study, we demonstrate that signaling by Notch2 but not Notch1 in CD8(+) T cells is required for antitumor CTL responses. Notch2(flox/flox) mice crossed with E8I-cre transgenic (N2F/F-E8I) mice, in which the Notch2 gene is absent only in CD8(+) T cells, die earlier than control mice after inoculation with OVA-expressing EG7 thymoma cells. In contrast, Notch1(flox/flox) mice crossed with E8I-cre transgenic mice inoculated with EG7 cells die comparable to control mice, indicating that Notch2 is crucial for exerting antitumor CTL responses. Injection of anti-Notch2 agonistic Ab or delta-like 1-overexpressing dendritic cells augmented the antitumor response in C57BL/6 mice inoculated with EG7 cells. These findings indicate that Notch2 signaling in CD8(+) T cells is required for generating potent antitumor CTLs, thus providing a crucial target for augmenting tumor immune responses.

Notch activation enhances IFNgamma secretion by human peripheral blood and decidual NK cells.

NK cells specialize in killing tumor cells and virally infected cells and also possess non-cytotoxic functions, which include secretion of a variety of cytokines and growth factors. Their activity is mediated by a vast repertoire of inhibitory and activating NK receptors. Recently, it was demonstrated that ligation of the Notch receptor plays a significant role not only in T cell development but also in human T cell and mouse NK cell activation. However, the involvement of Notch triggering in human NK cell activity has not yet been determined. Here we show that Notch1 and Notch2, but not Notch3 and Notch 4, are expressed in human peripheral blood NK cells and in decidual NK cells. We demonstrate that in peripheral blood NK cells only the Notch ligand Delta4 could interact with Notch, whereas in decidual NK cells both Delta1 and Delta4 can interact with Notch. Finally, we show that Notch activation in these cells leads to increased secretion of IFNgamma. We therefore present here a new function of the Notch receptors as enhancers of peripheral blood NK cell and decidual NK cell functions.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

The follicular versus marginal zone B lymphocyte cell fate decision.

Bone marrow-derived B cells make an important cell fate choice to develop into either follicular B cells or marginal zone B cells in the spleen, which depends on signalling through the B cell receptor, Notch2, the receptor for B cell-activating factor and the canonical nuclear factor-kappaB pathway, as well as signals involved in the migration and anatomical retention of marginal zone B cells. Recent information discussed in this Review reconciles some of the controversies regarding the role of the B cell receptor in this cell fate decision and a clearer picture has also emerged regarding the anatomical location of ligands for Notch2 in the spleen. This cell fate decision could provide mechanistic insights that are relevant to other commitment events in lymphocytes.

Notch signaling confers antigen-presenting cell functions on mast cells.

BACKGROUND: Notch signaling is involved in cell fate determination along with the development of the immune system. However, very little is known about the role for Notch signaling in mast cells. OBJECTIVE: We investigated the role of Notch signaling in mast cell functions. METHODS: After mouse bone marrow-derived mast cells (BMMCs) or peritoneal mast cells (PMCs) were cocultured with mouse Notch ligand-expressing chinese hamster ovary cells for 5 days, we examined the mast cell surface expressions of MHC-II molecules and OX40 ligand (OX40L), Fc epsilon RI-mediated cytokine production, and the effects of the mast cells on proliferation and differentiation of naive CD4(+) T cells in vitro. RESULTS: We showed that BMMCs and PMCs constitutively expressed Notch1 and Notch2 proteins on the cell surface. We also found that Delta-like 1 (Dll1)/Notch signaling induced the expression of MHC-II and upregulated the expression level of OX40L on the surface of the mast cells. Dll1/Notch signaling augmented Fc epsilon RI-mediated IL-4, IL-6, IL-13, and TNF production by BMMCs. Dll1-stimulated MHC-II(+)OX40L(high) BMMCs promoted proliferation of naive CD4(+) T cells and their differentiation into T(H)2 cells producing IL-4, IL-5, IL-10, and IL-13. CONCLUSION: Dll1/Notch signaling confers the functions as an antigen-presenting cell on mast cells, which preferentially induce the differentiation of T(H)2.

Notch2 integrates signaling by the transcription factors RBP-J and CREB1 to promote T cell cytotoxicity.

The acquisition of cytotoxic effector function by CD8(+) T cells is crucial for the control of intracellular infection and tumor invasion. However, it remains unclear which signaling pathways are required for the differentiation of CD8(+) cytotoxic T lymphocytes. We show here that Notch2-deficient T cells had impaired differentiation into cytotoxic T lymphocytes. In addition, dendritic cells with lower expression of the Notch ligand Delta-like 1 induced the differentiation of cytotoxic T lymphocytes less efficiently. We found that the intracellular domain of Notch2 interacted with a phosphorylated form of the transcription factor CREB1, and together these proteins bound the transcriptional coactivator p300 to form a complex on the promoter of the gene encoding granzyme B. Our results suggest that the highly regulated, dynamic control of T cell cytotoxicity depends on the integration of Notch2 and CREB1 signals.