Treg-deficient Mice Research Articles

Natural Regulatory T Cells Limit Angiotensin II–Induced Aneurysm Formation and Rupture in Mice

Abdominal aortic aneurysm is an inflammatory disease leading to destructive vascular remodeling and ultimately to lethal aortic rupture. Despite its frequent association with atherosclerosis, compelling studies have shown striking differences and potentially opposite roles of T-cell helper responses in aneurysm as compared with atherosclerosis, casting doubt on the relevance and suitability of T-cell-targeted therapies in this context. Here, we show that selective depletion of T regulatory (Treg) cells using a CD25-specific monoclonal antibody significantly enhances the susceptibility of C57Bl/6 mice to angiotensin II-induced abdominal aortic aneurysm and promotes aortic rupture (n=25-44 mice/group). Similar results are observed in angiotensin II-treated Cd80(-/-)/Cd86(-/-) or Cd28(-/-) mice with impaired Treg cell homeostasis (n=18-23 mice/group). Treg cell depletion is associated with increased immune cell activation and a blunted interleukin (IL)-10 anti-inflammatory response, suggesting an immunoinflammatory imbalance. Interestingly, Il-10(-/-) mice (n=20 mice/group) show increased susceptibility to angiotensin II-induced abdominal aortic aneurysm and aortic rupture and are insensitive to Treg cell depletion. Finally, reconstitution of Cd28(-/-) Treg-deficient mice with Treg cells (n=22 mice/group) restores a balance in the immunoinflammatory response, rescues the animals from increased susceptibility to aneurysm, and prevents aortic dissection. These results identify a critical role for Treg cells and IL-10 in the control of aneurysm formation and its progression to rupture and suggest that therapies targeting Treg responses may be most suited to treat aneurysmal disease.

FRI0270 Development of systemic lupus-like disease in regulatory T cells (TREG)-deficient scurfy mice.

BackgroundSystemic lupus erythematosus (SLE) is a severe systemic autoimmune disease with involvement of inner organs; autoreactive (CD4+) T cells, B cells and autoantibodies are crucial in its pathophysiology. CD4+FoxP3+ Treg,...

Scope and mechanisms of peripherally generated regulatory T cell mediated suppression in inflammatory contexts (P1031)

Abstract The peripheral Treg population, which prevents fatal autoimmunity in mice and man, consists of Tregs that develop in the thymus (tTregs) and Tregs derived from naïve T cells in the periphery (pTregs). Mice with a specific deficiency in pTregs do not develop the spontaneous multi-organ autoimmunity observed in Treg deficient mice. Instead, specific deficiency in pTregs results in spontaneous Th2 inflammation at mucosal surfaces. While loss of pTreg cells under homeostatic conditions favors Th2 inflammation, we hypothesized that pTregs also participate in control of other types of inflammation, such as induced Th17 inflammation. In a mouse model that is prone to development of spontaneous intestinal Th17 disease, we observed that the absence of pTregs significantly exacerbated Th17 inflammation in intestinal tissues without affecting Th2 inflammation. Next we investigated the potential mechanisms of pTreg mediated T-helper type-specific immune regulation. Unlike tTregs, which enter the periphery as a dedicated suppressor T cell subset, pTregs arise from a precursor population that likely has broad T-helper lineage potential. Here we explore the alternative differentiation of would-be pTregs unable to induce Foxp3 to assess the role of pTreg differentiation as a mechanism of “T-helper immune detraction”. Thus, we explore if Foxp3 induction in the periphery is essential to control a range of inappropriate inflammatory responses and the mechanisms by which this is achieved.

Regulation of pathogenic GL7-expressing helper T cells by Tregs and CD137 (P4111)

Abstract Dissecting the mechanisms that support autoantibody production is essential to our understanding of autoimmune diseases such as lupus. Recent work from our laboratory has shown that regulatory T cells (Tregs) play a key role in suppressing autoantibody production by helping to maintain B cell anergy. Our long-term goal is to understand how Tregs regulate B cell anergy. Our studies indicate that helper T cells play a key role as an intermediate between Treg and anergic B cells. Specifically, the absence of Tregs rapidly (within 4 days) drives the expansion of CD4+GL7+ helper T cells. This gross population can be further divided into 2 populations: germinal center follicular T cells (GC-TFH) and a novel non-TFH subset termed TAN cells. These GL7+ helper T cell subsets deliver potent signals to naïve and anergic B cells, inducing significant antibody production from both populations. Recent studies have begun to identify the signals that drive the expansion of GC-TFH and TAN cells in the absence of Tregs. We have shown that treatment of Treg-deficient mice with an agonistic anti-CD137 antibody reduces the expansion of CD4+GL7+ helper T cells (both GC-TFH and TAN cells). This treatment also reduced the expansion of class-switched B cells and abolished autoantibody production. Collectively, our studies indicate that CD137 signaling plays a key role in the expansion of pathogenic helper T cell subsets that support autoimmunity in the absence of Tregs.

CD4+CD25+Foxp3+ Regulatory T Cells in the Pathophysiology of Immune Thrombocytopenia

Regulatory T cells characterized by CD4, CD25, and transcription factor forkhead box P3, called Tregs, are a subpopulation of CD4(+) T cells specialized for immune suppression. Tregs contribute to maintenance of peripheral immune tolerance, and their defects are thought to play a role in the pathogenesis of various autoimmune diseases. Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and reduced platelet production, resulting in decreased platelet count. Recently, a series of studies in adults and children with ITP have found that the frequency of Tregs is reduced in circulation, bone marrow, and spleen, and Treg function is impaired. Treg dysregulation is improved after platelet count is recovered by treatment with dexamethasone, rituximab, or thrombopoietin receptor agonists. In addition, a critical role of Tregs in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in functional Tregs. Thrombocytopenia observed in Treg-deficient mice is mediated through production of IgG anti-platelet autoantibodies, which is analogous to human ITP. Further studies evaluating mechanisms of Treg dysregulation in ITP patients are necessary to elucidate the pathogenesis of ITP and develop novel therapeutic strategies that suppress anti-platelet autoimmune response.

Regulation of Interferon Gamma Signaling by Suppressors of Cytokine Signaling and Regulatory T Cells

Regulatory T cells (Tregs) play an indispensable role in the prevention of autoimmune disease, as interferon gamma (IFNγ) mediated, lethal auto-immunity occurs (in both mice and humans) in their absence. In addition, Tregs have been implicated in preventing the onset of autoimmune and auto-inflammatory conditions associated with aberrant IFNγ signaling such as type 1 diabetes, lupus, and lipopolysaccharide (LPS) mediated endotoxemia. Notably, suppressor of cytokine signaling-1 deficient (SOCS1−/−) mice also succumb to a lethal auto-inflammatory disease, dominated by excessive IFNγ signaling and bearing similar disease course kinetics to Treg deficient mice. Moreover SOCS1 deficiency has been implicated in lupus progression, and increased susceptibility to LPS mediated endotoxemia. Although it has been established that Tregs and SOCS1 play a critical role in the regulation of IFNγ signaling, and the prevention of lethal auto-inflammatory disease, the role of Treg/SOCS1 cross-talk in the regulation of IFNγ signaling has been essentially unexplored. This is especially pertinent as recent publications have implicated a role of SOCS1 in the stability of peripheral Tregs. This review will examine the emerging research findings implicating a critical role of the intersection of the SOCS1 and Treg regulatory pathways in the control of IFN gamma signaling and immune system function.

Regulatory T cells shape the effector and memory T cell response to virus infection in the tissues (170.15)

Abstract Regulatory T cells (Tregs) are well known for their role in dampening the immune responses to self-antigens and thereby helping to prevent autoimmune disease. However, they must also permit immune responses to occur against foreign infectious agents. Using a mouse model of West Nile Virus (WNV) infection, we examined Treg kinetics, trafficking, and activation at both acute and memory time points. We showed that Treg numbers increase in both the secondary lymphoid organs and CNS after infection, and become activated similar to effector T cells. Using the Foxp3DTR knock-in mice, we have performed experiments to study the course of WNV infection in the presence or absence of Tregs. We found that compared to ”normal” mice, Treg deficient mice had increased antigen-driven production of IFN-g from both CD4 and CD8 T cells in both the spleen and CNS. Additionally, Treg deficient mice had an increased frequency of poly-functional T cells. In mice lacking Tregs, there were greater numbers of short lived effector CD8+ cells at acute infection time points in both the spleen and brain. At a memory time point, there were significantly fewer antigen-specific T cells present in the CNS of mice deficient in Tregs at the time of infection compared to Treg sufficient mice. Studies are currently underway to examine the role of Tregs in CD4 memory using this Treg depletion model.

Critical role of CD4+CD25+ regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia

Autoimmune response suppression by regulatory T cells (Tregs) helps to maintain peripheral immune tolerance, and defects in this mechanism are thought to play a role in the pathogenesis of various autoimmune diseases. In patients with immune thrombocytopenia, naturally occurring CD4(+)CD25(+) Tregs are both functionally impaired and reduced in number. Thisstudy was undertaken to investigate Tregs' role in preventing immune thrombocytopenia in mice. Treg-deficient mice were prepared by inoculation of Treg-depleted CD4(+)CD25(-) Tcells isolated from BALB/c mice into syngeneic nude mice intravenously. Platelet count, proportion of reticulated platelets, platelet-associated IgG, platelet-associated anti-platelet antibodies, and IgG anti-platelet antibody production in splenocyte cultures were examined by flow cytometry. Of 69 Treg-deficient mice, 25 (36%) spontaneously developed thrombocytopenia that lasted at least 5 weeks. The platelet-associated IgG level and proportion of reticulated platelets were elevated in the thrombocytopenic mice. Platelet eluates and splenocyte culture supernatants prepared from thrombocytopenic mice, but not from nonthrombocytopenic mice, contained IgG antibodies capable of binding to intact platelets. Simultaneous transfer of Tregs completely prevented the onset of thrombocytopenia, but Treg transfer after the onset of thrombocytopenia had no apparent effect. Treatment with IgG anti-cytotoxic Tlymphocyte-associated antigen 4 antibody canceled this Treg-governed suppressive effect.In summary, these results indicate that Tregs play a critical role in preventing murine autoantibody-mediated thrombocytopenia by engaging cytotoxic T lymphocyte-associated antigen4.

T Helper Type 1-Bias in a Regulatory T Cell-Deficient Mouse Model for Immune Thrombocytopenia

Abstract 524 Background:Immune thrombocytopenia (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. Recently, we have found that approximately one third of Treg-deficient mice spontaneously develop thrombocytopenia with increased platelet-associated IgG and proportion of reticulated platelets. Platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contain IgG antibodies capable of binding to intact platelets, which are not detected in non-thrombocytopenic mice. The main target of anti-platelet autoantibodies is GPIb, and some mice also produce anti-GPIIIa antibodies. However, detailed mechanisms that elicit ITP during immune reconstitution through homeostatic proliferation in the absence of Tregs remain uncertain. Purpose:To evaluate T-helper (Th) cell balance that promotes anti-platelet autoantibody response in a Treg-deficient mouse model for ITP. Methods:Treg-deficient mice were prepared by inoculation of Treg-depleted CD4+ T cells obtained from BALB/c mice into syngeneic T cell-deficient nude mice. Platelet count was determined using flow cytometry 4 weeks after inoculation, and Treg-deficient mice with platelet count < 0.33 × 106/ul were regarded as ITP mice. Treg-deficient mice without thrombocytopenia were also used as a control. To evaluate cytokine profiles of Th cells, proportions of Th subsets in the freshly prepared splenic CD4+ T cells were evaluated by intracellular staining for IFN-γ, IL-4, and IL-17 followed by flow cytometry. Th1, Th2, and Th0 cells were defined as IFN-γ+IL-4−, IFN-γ−IL-4+, IFN-γ+IL-4+ cells, respectively, and Th17 and Th1/17 cells were defined as IFN-γ−IL-17+ and IFN-γ+IL-17+, respectively. In addition, CD4+ T cells were isolated from splenocytes using magnetic activated cell sorting, and were stimulated with phorbol 1,2-myristate 1,3-acetate and ionomycin for 4 days. The culture supernatants were subjected to a cytokine bead array to measure levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF). Finally, to determine IgG subclasses of anti-platelet autoantibodies, splenocyte culture supernatants were incubated with platelets derived from BALB/c mice, followed by incubation with fluorescence-conjugated antibodies to IgG1, IgG2a, IgG2b, or IgG3. Then, the antibodies bound to platelets was detected by flow cytometry. Results:Fourteen ITP mice and 8 control mice were used at 6–8 weeks after inoculation. The proportions of Th1, Th2, and Th0 cells did not differ significantly between ITP and control mice, while the Th1/Th2 ratio was significantly increased in ITP mice than in control mice (8.3 versus 3.2, p < 0.01). The proportions of Th17 and Th1/17 cells were comparable between ITP and control mice. There was no difference in the in vitro production levels of cytokines except IL-4, which was lower in ITP mice compared to control mice (140 versus 600 pg/ml, p = 0.02). Increase in the IFN-γ/IL-4 ratio was noted in the culture supernatants from ITP mice, compared to those from control mice (15.6 versus 9.2, p = 0.04). The Th1/Th2 ratio detected by flow cytometric measurement and the IFN-γ/IL-4 ratio in in vitro cultures were correlated with each other (r = 0.85, p < 0.01). IgG subclasses of anti-platelet autoantibodies were heterogeneous among individual ITP mice, but IgG2a was the predominant subclass in the majority of ITP mice. Interestingly, a high Th1/Th2 ratio was associated with production of IgG2b anti-platelet antibodies, while the mice with a low Th1/Th2 ratio produced IgG1 anti-platelet antibodies. Conclusions:These findings suggest that induction of IgG anti-platelet autoantibody response in Treg-deficient mice is associated with Th1 bias, which is analogous to the Th balance in patients with primary ITP. The Th1/Th2 balance may modulate the autoimmune responses during expansion of CD4+ T cells in the absence of Tregs. Disclosures:No relevant conflicts of interest to declare.

Emergence of polyclonal autoantibody responses to multiple platelet surface glycoproteins in regulatory T cell-deficient mice. (115.28)

Abstract Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to play an important role in preventing pathogenic autoimmune responses. Recently, we have found that approximately one third of mice deficient of CD4+CD25+Foxp3+ Tregs spontaneously develop thrombocytopenia with increased platelet-associated IgG, analogous to patients with immune thrombocytopenia. To identify platelet autoantigens in Treg-deficient mice, plasma samples from thrombocytopenic and non-thrombocytopenic Treg-deficient mice were subjected to immunoblots using platelets as an antigen in reduced and non-reduced conditions. IgG antibodies to platelet surface glycoproteins, such as GPIb, GPIIb, and GPIIIa, were detected specifically in thrombocytopenic mice in various combinations. IgG antibodies to native platelets were further assessed by flow cytometry using platelet eluates and splenocyte culture supernatants. IgG antibodies capable of binding to native platelets were detected in thrombocytopenic, but not in non-thrombocytopenic mice. Antibody reactivity was cancelled in the majority of samples when platelets derived from GPIb-deficient or GPIIIa-deficient mice were used instead of wild-type platelets as an antigen. In summary, Treg deficiency alone leads to emergence of polyclonal IgG autoantibody responses to native and denatured platelet surface glycoproteins, supporting a critical role of CD4+CD25+Foxp3+ Tregs in suppressing induction of autoimmunity.

A Critical Role of CD4+CD25+ Regulatory T Cells In Prevention of Murine Autoantibody-Mediated Thrombocytopenia

AbstractAbstract 381Immune thrombocytopenic purpura (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. Since CD4+CD25+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens, it has been believed that Treg dysfunction contributes to the development of a various forms of human autoimmune disorders. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. However, it remains unclear how Treg alteration is involved in the pathophysiology of ITP.Recently, we have found that a group of Treg-deficient mice develop autoantibody-mediated thrombocytopenia. For preparation of Treg-deficient mice, Treg-depleted T cells were prepared from BALB/c splenocytes by serial purification steps consisting of a positive selection of CD4+ T cells and a negative selection of CD25+ cells using magnetic bead-based cell sorting, and were transferred into syngeneic T cell-deficient nude mice via tail vein. Treg-depleted T cell fraction transferred contained >99% CD4+CD25− cells, and was confirmed to lack expression of Foxp3, a typical Treg marker. Three weeks after transfer, approximately one third of the recipient mice spontaneously developed thrombocytopenia, which sustained for > 20 weeks. Thrombocytopenic mice represented elevated platelet-associated IgG and increased proportion of reticulated platelets, but non-thrombocytopenic mice did not. In addition, platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contained IgG antibodies capable of binding to intact platelets, which were not detected in non-thrombocytopenic mice. The presence of anti-platelet antibodies and increased platelet turnover observed in thrombocytopenic Treg-deficient mice are analogous to ITP patients. Treg-deficient mice prepared by transfer of a less number of Treg-depleted T cells resulted in reduced prevalence of thrombocytopenia, suggesting that onset of thrombocytopenia depends on the number of conventional T cells transferred. Treg-deficient mice are known to frequently develop autoimmune gastritis, another autoimmune disease mediated by IgG anti-parietal cell antibodies, but anti-parietal cell antibodies were almost equally detected in plasma from thrombocytopenic and non-thrombocytopenic mice (70% versus 60%). Transplantation of Tregs together with Treg-depleted T cells completely prevented the onset of thrombocytopenia, but Treg transplantation was not effective as a treatment once thrombocytopenia occurred. To further investigate how Tregs exert the regulatory function, Treg-depleted T cells and Tregs were simultaneously transferred in the presence of antibodies that blocked engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA4). This treatment cancelled Treg function and resulted in development of thrombocytopenia in recipient nude mice, while mock treatment with control antibodies had no effect.In summary, these results together indicate that CD4+CD25+Foxp3+ Tregs play a critical role in preventing the development of murine autoantibody-mediated thrombocytopenia, in part, through CTLA4 engagement. Disclosures:No relevant conflicts of interest to declare.

Tregs control the development of symptomatic West Nile virus infection in humans and mice

West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4+ Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA+) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans.

Regulatory T Cells Suppress Innate Immunity in Kidney Ischemia-Reperfusion Injury

Both innate and adaptive mechanisms participate in the pathogenesis of kidney ischemia-reperfusion injury (IRI), but the role of regulatory immune mechanisms is unknown. We hypothesized that the anti-inflammatory effects of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) protect against renal IRI. Partial depletion of Tregs with an anti-CD25 mAb potentiated kidney damage induced by IRI. Reducing the number of Tregs resulted in more neutrophils, macrophages, and innate cytokine transcription in the kidney after IRI but did not affect CD4(+) T cells or B cells. We performed adoptive transfer of lymph node cells from wild-type mice or FoxP3-deficient Scurfy mice into T cell- and B cell-deficient RAG-1 knockout mice to generate mice with and without FoxP3(+) Tregs, respectively. FoxP3(+) Treg-deficient mice accumulated a greater number of inflammatory leukocytes after renal IRI than mice containing Tregs. To confirm that a lack of Tregs potentiated renal injury, we co-transferred isolated Tregs and Scurfy lymph node cells; Treg repletion significantly attenuated IRI-induced renal injury and leukocyte accumulation. Furthermore, although adoptive transfer of wild-type Tregs into RAG-1 knockout mice was sufficient to prevent kidney IRI, transfer of IL-10-deficient Tregs was not. Taken together, these results demonstrate that Tregs modulate injury after kidney IRI through IL-10-mediated suppression of the innate immune system.

Oestrogen modulates experimental autoimmune encephalomyelitis and interleukin‐17 production via programmed death 1

The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17beta-oestradiol (E(2)) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4(+) FoxP3(+) regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E(2) at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E(2) treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E(2) treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E(2) failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E(2)-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.

REGULATORY T-CELLS SUPPRESS THE DEVELOPMENT OF THE 2-HIT RESPONSE AFTER INJURY

Introduction: We recently reported that injury induces increased CD4 regulatory T cell (Treg) activity and that Tregs play a role in controlling the host inflammatory response to burn injury. In this study, we address the potential for Tregs to actively influence the development of enhanced innate immune system activation and the associated predisposition to the 2-hit response after burn injury. Methods: This was studied by testing the effects of in vivo Treg cell depletion on the responsiveness of sham versus burn mice to re-purified bacterial lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) specific agonist. Groups of male, C57BL/6J mice were depleted of Tregs by anti-CD25 antibody treatment or were given control antibody at 3 days before sham or burn injury. To examine mortality, mice were challenged with 5 mg/kg or 10 mg/kg of LPS by intraperitoneal injection at 7 days after sham or burn injury. The influence of Treg deficiency on TLR4 reactivity in vivo was determined by measuring plasma and organ cytokine levels after LPS challenge. Parallel studies tested the influence of Treg depletion on TLR4-induced cytokine production by spleen cells in vitro. Results: We found that burn mice that were Tregdeficient at the time of injury showed significantly higher mortality to LPS challenge at 7 days after injury (p<0.05, Log rank test). Burn-injured Treg-deficient mice had higher plasma and lung levels of TNFα, IL-6, and MCP-1after LPS challenge than control burn mice, sham mice, and Treg-deficient sham mice (P < 0.001, ANOVA). LPS-stimulated spleen cells prepared from Treg-deficient burn-injured mice also produced higher levels of TNFα and MCP-1 than control burn-injured mice, sham mice, and Treg-deficient sham mice. Conclusion: Taken together, these findings indicate that Tregs play an active role in suppressing the innate inflammatory response to injury. We also demonstrate that in their absence the injured-host becomes more predisposed to a heightened 2-hit response.