Laura Snell

Endogenous 4-1BB Ligand Plays a Critical Role in Protection from Influenza-Induced Disease

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

T‐cell intrinsic effects of GITR and 4‐1BB during viral infection and cancer immunotherapy

Summary:  GITR [glucocorticoid inducible tumor necrosis factor receptor (TNFR)‐related protein] and 4‐1BB are costimulatory TNFR family members that are expressed on regulatory and effector T cells as well as on other cells of the immune system. Here we discuss the role of GITR and 4‐1BB on T cells during viral infections and in cancer immunotherapy. Systemic treatment with agonistic anti‐4‐1BB antibody leads to a number of immune system abnormalities, and clinical trials of anti‐4‐1BB have been terminated. However, other modes of 4‐1BB ligation may be less toxic. To date, similar toxicities have not been reported for anti‐GITR treatment of mice, although anti‐GITR antibodies can exacerbate mouse autoimmune models. Intrinsic effects of GITR and 4‐1BB on effector T cells appear to predominate over their effects on other cell types in some models. Despite their similarities in enhancing T‐cell survival, 4‐1BB and GITR are clearly not redundant, and both pathways are required for maximal CD8+ T‐cell responses and mouse survival following severe respiratory influenza infection. GITR uses TNFR‐associated factor (TRAF) 2 and TRAF5, whereas 4‐1BB recruits TRAF1 and TRAF2 to mediate survival signaling in T cells. The differential use of signaling adapters combined with their differential expression may explain the non‐redundant roles of GITR and 4‐1BB in the immune system.

A critical role for TNF receptor-associated factor 1 and Bim down-regulation in CD8 memory T cell survival.

The mechanisms that allow the maintenance of immunological memory remain incompletely defined. Here we report that tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 1, a protein recruited in response to several costimulatory TNFR family members, is required for maximal CD8 T cell responses to influenza virus in mice. Decreased recovery of CD8 T cells in vivo occurred under conditions where cell division was unimpaired. In vitro, TRAF1-deficient, antigen-activated T cells accumulated higher levels of the proapoptotic BH3-only family member Bim, particularly the most toxic isoform, BimS. In the presence of excess IL-15, memory phenotype T cells with similar surface phenotype and comparable levels of Bcl-2 family members could be generated from WT or TRAF1-deficient T cell receptor transgenic OT-I T cells. However, when the memory CD8 T cells were allowed to compete for survival signals in the absence of antigen in vivo, the TRAF1-deficient T cells showed decreased recovery compared with TRAF1-sufficient T cells. This defect in T cell recovery in vivo was alleviated by introduction of siRNA to down-modulate Bim in TRAF1-deficient memory T cells. These studies identify the TRAF1 signaling axis and Bim down-regulation as critical for CD8 memory T cell survival in vivo.

CD8 T Cell-Intrinsic GITR Is Required for T Cell Clonal Expansion and Mouse Survival following Severe Influenza Infection

The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-κB induction, resulting in increased Bcl-xL. In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphoctye survival, they clearly have nonredundant functions in protection from severe infection.

Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

Background: The T cell costimulatory molecule 4-1BB signals through TRAF1 and TRAF2. Results: TRAF1−/− T cells show hyperproliferation due to enhanced alternative NF-κB activation but have impaired classical NF-κB activation. Conclusion: TRAF1 enhances 4-1BB induced classical NF-κB activation but restricts alternative NF-κB activation in the absence of costimulation. Significance: This explains the contrasting roles attributed to TRAF1. T cells lacking TRAF1 hyperproliferate in response to T cell receptor signaling but have impaired signaling downstream of specific TNFR family members such as 4-1BB. Here we resolve this paradox by showing that while TRAF1 is required for maximal activation of the classical NF-κB pathway downstream of 4-1BB in primary T cells, TRAF1 also restricts the constitutive activation of NIK in anti-CD3-activated T cells. Activation of the alternative NF-κB pathway is restricted in unstimulated cells by a cIAP1/2:TRAF2:TRAF3:NIK complex. Using knockdown of NIK by siRNA we show that in activated CD8 T cells TRAF1 is also involved in this process and that constitutive activation of the alternative NF-κB pathway is responsible for costimulation independent hyperproliferation and excess cytokine production in TRAF1-deficient CD8 T cells compared with WT CD8 T cells. The T cell costimulatory molecule 4-1BB critically regulates the survival of activated and memory CD8 T cells. We demonstrate that stimulation through 4-1BB induces cIAP1-dependent TRAF3 degradation and activation of the alternative NF-κB pathway. We also show that while both TRAF1 and cIAP1 have non-redundant roles in suppressing the alternative NF-κB pathway in T cells activated in the absence of costimulation, activation of the classical NF-κB pathway downstream of 4-1BB requires TRAF1, whereas cIAP1 plays a redundant role with cIAP2. Collectively these results demonstrate that TRAF1 plays a critical role in regulating T cell activation both through restricting the costimulation independent activation of NIK in activated T cells and by promoting the 4-1BB-induced classical NF-κB pathway.

Measuring and managing patient expectations for breast reconstruction: impact on quality of life and patient satisfaction

The goal of postmastectomy breast reconstruction is to restore a woman’s body image and to satisfy her personal expectations regarding the results of surgery. Studies in other surgical areas have shown that unrecognized or unfulfilled expectations may predict dissatisfaction more strongly than even the technical success of the surgery. Patient expectations play an especially critical role in elective procedures, such as cancer reconstruction, where the patient’s primary motivation is improved health-related quality of life. In breast reconstruction, assessment of patient expectations is therefore vital to optimal patient care. This report summarizes the existing literature on patient expectations regarding breast reconstruction, and provides a viewpoint on how this field can evolve. Specifically, we consider how systematic measurement and management of patient expectations may improve patient education, shared medical decision-making and patient perception of outcomes.

Immediate tissue expander/implast breast reconstruction after salvage mastectomy for cancer recurrence following lumpectomy/irradiation.

Background: The objective of this study was to analyze early complications and long-term outcomes in patients undergoing salvage mastectomy and immediate tissue expander/implant reconstruction for cancer recurrence following breast conservation therapy (lumpectomy/irradiation). Methods: A review of all tissue expander/implant reconstructions performed by a single surgeon over an 11-year period from 1997 to 2008 was performed. Two patient cohorts were identified: (1) patients who underwent salvage mastectomy for a cancer recurrence following prior breast conservation therapy, and (2) patients who underwent primary mastectomy without a history of prior irradiation. The incidence of early complications and long-term outcomes were determined for each cohort. Results: Immediate, tissue expander/implant reconstruction was initiated in 1699 patients. One hundred twenty-one patients had a history of breast conservation therapy (lumpectomy/irradiation), and 1578 did not have a history of prior irradiation. The incidence of early complications was significantly higher in the irradiated cohort compared with that in the nonirradiated cohort (29.7 percent versus 15.5 percent; p ⩽ 0.001). The most common complication in both groups was mastectomy flap necrosis (18.0 percent in the irradiated group and 7.7 percent in the nonirradiated group; p < 0.001). Six hundred ninety-seven patients had long-term follow-up data available. Most previously irradiated patients had good or very good results, whereas most nonirradiated patients had excellent results (p = 0.04; Mann-Whitney U test). Conclusions: Carefully selected patients who have had prior breast conservation therapy who require salvage mastectomy can successfully complete postmastectomy tissue expander/implant reconstruction. The rate of early complications in this patient group is higher than in the nonirradiated cohort but remains acceptable. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

GITR-Dependent Regulation of 4-1BB Expression: Implications for T Cell Memory and Anti–4-1BB–Induced Pathology

The TNFR family member 4-1BB plays a key role in the survival of activated and memory CD8 T cells. However, the mechanisms that regulate 4-1BB re-expression on memory CD8 T cells after Ag clearance are unknown. In unimmunized mice, ∼10% of CD8 CD44hi memory T cells in the bone marrow (BM) and liver express 4-1BB, with minimal 4-1BB expression in spleen and lymph node. IL-2, IL-15, and IL-7 are collectively dispensable for 4-1BB expression on the memory CD8 T cells. Rather, T cell–intrinsic glucocorticoid-induced TNFR-related protein (GITR) contributes to 4-1BB expression on CD8 T cells upon their entry into the BM or liver. Consistent with its role in regulation of 4-1BB, GITR is required on memory CD8 T cells for their persistence in vivo. These findings reveal site-specific effects of the BM and liver microenvironment on CD8 memory T cells. Previous work has demonstrated that 4-1BB agonists given to unimmunized mice induce splenomegaly, hepatitis, and other immune system anomalies. Moreover, severe liver pathology has been observed in a subset of anti–4-1BB–treated melanoma patients. Remarkably, the absence of GITR in mice almost completely abrogates cellular expansions, splenomegaly, and liver inflammation associated with anti–4-1BB agonist treatment of unimmunized mice. In contrast, lack of CD8 T cells selectively improves liver pathology, but not splenomegaly in the mice. Thus, the regulation of 4-1BB expression by GITR on CD8 T cells, as well as on other cells, contributes to the pathological effects of anti–4-1BB in unimmunized mice.

IL-15–Dependent Upregulation of GITR on CD8 Memory Phenotype T Cells in the Bone Marrow Relative to Spleen and Lymph Node Suggests the Bone Marrow as a Site of Superior Bioavailability of IL-15

CD8 memory T cells are enriched in the bone marrow, a site where these cells are thought to receive homeostatic signals. However, the primary site where CD8 memory T cells receive their cytokine-induced homeostatic signals has recently come under debate. In this study, we demonstrate that the bone marrow contains a fraction of CD8 memory phenotype T cells with elevated expression of glucocorticoid-induced TNFR-related protein (GITR). In contrast, splenic and lymph node memory phenotype T cells have GITR levels similar to those on naive T cells. The bone marrow GITRhi memory T cells have a phenotype indicative of cytokine activation, with higher CD122 and lower CD127 than do the GITRbasal memory T cells. Remarkably, these bone marrow-specific GITRhi cells are almost completely ablated in the absence of IL-15, whereas TNFR2 and 4-1BB expression on the CD8 memory T cells are IL-15 independent. Furthermore, adoptively transferred splenic CD8 memory phenotype T cells show IL-15–dependent GITR upregulation upon entry into the bone marrow. This result implies that the selective appearance of GITRhi memory phenotype T cells in the bone marrow reflects the local microenvironment rather than a different subset of memory T cells. GITR−/− mice have a lower frequency of CD8 memory phenotype cells in the bone marrow, yet the GITR−/− cells hyperproliferate compared with those in wild-type mice. Taken together, these data suggest that GITR plays a role in the survival of CD8 memory phenotype T cells and that GITR upregulation represents a precise marker of cells that have responded to IL-15.

Contribution of 4–1BBL on radioresistant cells in providing survival signals through 4–1BB expressed on CD8+ memory T cells in the bone marrow

The persistence of memory lymphocytes is a critical feature of adaptive immunity. The TNF family ligand 4–1BBL supports the antigen‐independent survival of CD8+ memory T cells. Here, we show that mice lacking 4–1BB only on αβ T cells show a similar defect in CD8+ T‐cell recall responses, as previously shown in 4–1BBL‐deficient mice. We show that 4–1BB is selectively expressed on BM CD8+ but not CD4+ memory T cells of unimmunized mice. Its ligand, 4–1BBL, is found on VCAM‐1+ stromal cells, CD11c+ cells, and a Gr1lo myeloid population in unimmunized mice. Adoptive transfer of in vitro generated memory T cells into mice lacking 4–1BBL only on radioresistant cells recapitulates the defect in CD8+ T‐cell survival seen in the complete knockout mice, with smaller effects of 4–1BBL on hematopoietic cells. In BM, adoptively transferred DsRed CD8+ memory T cells are most often found in proximity to VCAM‐1+ cells or Gr1+ cells, followed by B220+ cells and to a much lesser extent near CD11c+ cells. Thus, a VCAM‐1+CD45− stromal cell is a plausible candidate for the radioresistant cell that provides 4–1BBL to CD8+ memory T cells in the BM.