Real world evaluation of dual-zone ICD and CRT-D programming compared to single-zone programming: the ALTITUDE REDUCES study

Tachycardia Detection, ICD, CRT‐D Devices, Appropriate and Inappropriate Shock. Introduction: We evaluated the frequency of appropriate and inappropriate shocks and survival in patients using dual‐zone programming versus single‐zone programming. Methods and Results: For the ALTITUDE REDUCES study, patients were followed for 1.6 ± 1.1 years. The 12‐month incidence of any shock was lower for dual‐versus single‐zone programmed detection at rates ≤170 bpm and between 170–200 bpm (P < 0.001). Appropriate shock rates at 1 year were also lower with dual‐zone programming in these rate intervals (single zone 9.1%, 5.4%, P < 0.001, dual zone 6.7%, 4.7%, P < 0.02). There were no detectable differences between single‐ and dual‐zone shock incidence at detection rates ≥ 200 bpm (P = 0.14). Inappropriate shock incidence was less with dual‐ versus single‐zone detection at all detect rates <200 bpm, but not at rates ≥200 bpm (P < 0.001, P = 0.37). The lowest risk of appropriate and inappropriate shock was associated with dual‐zone programming and detection rates ≥200 bpm (2.1%). Dual‐zone detection was associated with more nonsustained and diverted therapy episodes but these patients did not have an increased risk of death compared to patients with single‐zone programming. Patients programmed to low detection rate, single‐zone detection and shock‐only therapy also had the highest preshock mortality risk (P = 0.05). Conclusions: Shock incidence is lowest with either single‐ or dual‐zone detection ≥200 bpm. For detection rates <200 bpm, dual‐zone programming is associated with a reduction in the incidence of total shocks, appropriate shocks, and inappropriate shocks. (J Cardiovasc Electrophysiol, Vol. 22, pp. 1023‐1029, September 2011)     

Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation

Mechanisms of dominant tolerance have evolved within the mammalian immune system to prevent inappropriate immune responses. CD4(+)CD25(+) regulatory T (T(reg)) cells have emerged as central constituents of this suppressive activity. By using multiphoton intravital microscopy in lymph nodes (LNs) of anesthetized mice, we have analyzed how cytotoxic T lymphocytes (CTLs) interact with antigen-presenting target cells in the presence or absence of activated T(reg) cells. Nonregulated CTLs killed their targets at a 6.6-fold faster rate than regulated CTLs. In spite of this compromised effector activity, regulated CTLs exhibited no defect in proliferation, induction of cytotoxic effector molecules and secretory granules, in situ motility, or ability to form antigen-dependent conjugates with target cells. Only granule exocytosis by CTLs was markedly impaired in the presence of T(reg) cells. This selective form of regulation did not require prolonged contact between CTLs and T(reg) cells but depended on CTL responsiveness to transforming growth factor-beta. CTLs quickly regained full killing capacity in LNs upon selective removal of T(reg) cells. Thus, T(reg) cells reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill.     

Enhancement of antigen-specific Treg vaccination in vivo

The conversion of naive T cells into Treg can be achieved in vivo by delivery of antigen under subimmunogenic conditions. Here we have examined several drugs for their ability to enhance the conversion process in vivo and have found that the rapamycin analog everolimus potently enhances Treg conversion by interfering with T-cell costimulation, reducing cell division and thereby activation of DNA methyltransferase 1 as well as by reducing T-cell activation through the ATP-gated P2×7 receptor controlling Ca2⁺ influx. The resulting Tregs exhibit increased stability of Foxp3 expression even when generated in TGFβ-containing media in vitro. Thus the mammalian target of rapamycin (mTOR) inhibitor everolimus in addition to inhibiting immune responses enhances Treg conversion by several distinct pathways. The converted Tregs can be further expanded by injection of IL-2/IL-2ab complexes. These complexes also increase the number of CD25⁺Foxp3⁻ cells that, however, do not represent cytokine secreting effector cells but anergic cells, some of which can secrete IL-10 and can themselves be considered regulatory T cells as well. The combined use of everolimus and IL-2/IL-2ab complexes in vivo makes it feasible to achieve highly effective antigen-driven conversion of naive T cells into Treg and their expansion in vivo and thereby the described protocols constitute important tools to achieve immunological tolerance by Treg vaccination.     

Nanoparticle-enabled innate immune stimulation activates endogenous tumor-infiltrating T cells with broad antigen specificities

Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive, although they often show signs of prior clonal expansion. Hypothesizing that this may be due to peripheral tolerance, we formulated nanoparticles containing innate immune stimulants that we found were sufficient to activate self-specific CD8⁺ T cells and injected them into two different mouse tumor models, B16F10 and MC38. These nanoparticles robustly activated and/or expanded antigen-specific CD8⁺ tumor-infiltrating T cells, along with a decrease in regulatory CD4⁺ T cells and an increase in Interleukin-17 producers, resulting in significant tumor growth retardation or elimination and the establishment of immune memory in surviving mice. Furthermore, nanoparticles with modification of stimulating human T cells enabled the robust activation of endogenous T cells in patient-derived tumor organoids. These results indicate that breaking peripheral tolerance without regard to the antigen specificity creates a promising pathway for cancer immunotherapy.

Cancer immunotherapy has held out the promise of harnessing a patient’s own immune system to attack cancer cells for many years. While checkpoint inhibitors and chimeric antigen receptor T cells have triggered long-term remissions in some patients, many more do not respond or have a recurrence, hence the need for additional approaches (1–5). Tumor-infiltrating T cells (TILs), which are found in approximately one-third of the most common tumor types (6, 7), are often either self-antigen or mutated antigen (neoantigen) specific (8, 9), although recent analyses have also documented T cells specific for infectious diseases (10, 11). Self-antigen–specific T cells are particular characteristics of tumor types with lower tumor mutational burden and poorer outcomes (12–14), suggesting that activating these T cells might be particularly beneficial for immunotherapy. Although for many years, it has been thought that almost all self-antigen–specific T cells were deleted in the thymus due to negative selection (15, 16), our own studies of healthy human blood donors (17, 18) and parallel work in mice (19) have shown that only a fraction (∼70 to 0%) of the self-specific T cells is actually eliminated. We also observed profound differences in the activation requirements in the self-antigen–specific vs. foreign antigen-specific T cells—while the latter could be stimulated with cognate antigen plus anti-CD28 and Interleukin (IL)-2, the former could not. Since anti-CD3 could stimulate both cells in vitro, the results suggested that the self-specific T cells were not anergic, but rather, lacked some other signals. Reasoning that these signals were likely related to those triggered by an active infection, as suggested by the classical work of Ohashi and colleagues (20) and others, we surveyed a range of agonists for pattern recognition receptors (PRRs). We found that just two, the lipoprotein (Pam3CysSerLys4 [Pam3CSK4]), an agonist for the Toll-like receptor 1/2 heterodimer (TLR1/2), and muramyl dipeptide (L18-MDP), an agonist for the cytosolic nucleotide-binding oligomerization domain receptor 2 (NOD2), were sufficient to stimulate self-antigen–specific CD8⁺ T cells in vitro as efficiently as foreign antigen-specific CD8⁺ T cells, where both were in the presence of IL-2 and anti-CD28. These results support the hypothesis that self-specific T cells are held in a nonreactive state unless activated by an infection, with its mobilization of the innate immune system.In our previous human colon carcinoma studies (21, 22), both CD4⁺ and CD8⁺ TILs showed extensive clonal expansion, indicating that they had been very active at one point but likely were no longer. Consistent with this aborted response phenomenon is earlier work showing that in metastatic melanoma patients, tumor-associated “self”-antigen–specific CD8⁺ T cells recognizing peptides from melanoma antigen recognized by T cells 1 (MART-1), gp100, and tyrosinase are present in the circulation in much higher frequency than baseline but in an inactive or only partially active state compared with virus-specific T cells in the same patients (12). Also suggesting an arrest in activity are the results of cell transfer experiments, where Yee et al. (23) transferred large numbers of MART-1–specific CD8⁺ T cells in melanoma patients and observed only short-term activity arrested after a few days.Thus, we hypothesized that tumor-infiltrating self-specific and perhaps even neoantigen-specific CD8⁺ T cells could be in a quiescent state similar to that of self-specific T cells in the periphery, and breaking this type of peripheral tolerance might activate these cells and trigger antitumor immunity. However, if given systemically, many of these agents will elicit severe inflammatory toxicities (24, 25). The systemic activation of self-specific CD8⁺ T cells in particular would also likely induce autoimmunity (26). To address these issues, we developed a biodegradable nanoparticle (NP) platform, covalently conjugated with an anti-CD28 antibody (Ab) and capable of delivering IL-2, Pam3CSK4, and L18-MDP in a sustained manner to achieve continuous local stimulation in the tumor microenvironment and minimize the side effects that would likely result from systemic exposure.Using this NP, we first demonstrated that self-specific CD8⁺ T cells isolated from either peripheral blood mononuclear cells (PBMCs) of healthy humans or the tumor-draining lymph nodes of B16F10 melanoma-inoculated mice could be expanded in vitro with NP-enabled stimulation in the presence of relevant self-peptides. Second, we showed that the repeated injection of these NPs into mice bearing either the B16F10 melanoma or MC38 colon carcinoma could generally activate both self-antigen and neoantigen-specific TILs, leading to significant retardation of tumor growth and prolongation of survival in these treated mice. Some mice totally rejected tumors and were completely resistant to tumor reinoculation, indicating that their immune system has acquired systemic “memory.” Evidence suggests that this antitumor activity is, at least in part, due to a reduction in the number and suppressive activity of regulatory T cells (Tregs) and the maturation of antigen-presenting cells (APCs). We further demonstrated that this approach could stimulate human TILs in patient-derived tumor organoids from three different cancer types, even those unresponsive to checkpoint blockades. Altogether, these results indicate that breaking peripheral tolerance with a combination of innate immune stimulants to activate both self-antigen– and neoantigen-specific TILs represents a broadly applicable and effective strategy for cancer immunotherapy.     

Notch1-dependent lymphomagenesis is assisted by but does not essentially require pre-TCR signaling

Overexpression of intracellular Notch plays an important role in the generation of human acute lymphoblastic T cell leukemia (T-ALL). In mouse models, it was shown that Notch-dependent T-ALL required pre-TCR signaling. Here we show that pre-TCR signaling is required to condition mice for Notch-dependent transformation but that it is not required to sustain malignant growth of T-ALL. In contrast to previous studies, we found that disease development does not require pre-TCR but that it can be accelerated in Rag2(-/-) mice by transient mimicking of pre-TCR signals.     

Allelic exclusion of the T cell receptor beta locus requires the SH2 domain-containing leukocyte protein (SLP)-76 adaptor protein.

Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.     

Deletion of autospecific T cells in T cell receptor (TCR) transgenic mice spares cells with normal TCR levels and low levels of CD8 molecules

Transgenic mice that carry on a large fraction of their T cells an alpha/beta T cell receptor that recognizes the male antigen in the context of H-2Db molecules were constructed. An mAb specific for the transgenic receptor was developed and used to analyze T cell subsets in male transgenic H-2b mice. The vast majority of immature CD4+8+ T cells that express the transgenic TCR were deleted in the male transgenic mouse. Nevertheless, the majority of T cells spared by this deletion process expressed a high level of the transgenic TCR. These T cells, however, had an abnormal CD4/CD8 phenotype in that they expressed either no CD8 molecules or only low levels.     

Peripheral T Cell Survival Requires Continual Ligation of the T Cell Receptor to Major Histocompatibility Complex–Encoded Molecules

In the thymus, T cells are selected according to their T cell receptor (TCR) specificity. After positive selection, mature cells are exported from primary lymphoid organs to seed the secondary lymphoid tissue. An important question is whether survival of mature T cells is an intrinsic property or requires continuous survival signals, i.e., engagement of the TCR by major histocompatibility complex (MHC) molecules in the periphery, perhaps in a similar way as occurring during thymic positive selection. To address this issue we used recombination-activating gene (Rag)-deficient H-2^b mice expressing a transgenic TCR restricted by I-E^d class II MHC molecules. After engraftment with Rag^−/− H-2^d fetal thymi, CD4⁺8⁻ peripheral T cells emerged. These cells were isolated and transferred into immunodeficient hosts of H-2^b or H-2^d haplotype, some of the latter being common cytokine receptor γ chain deficient to exclude rejection of H-2^b donor cells by host natural killer cells. Our results show that in the absence, but not in the presence, of selecting MHC molecules, peripheral mature T cells are short lived and disappear within 7 wk, indicating that continuous contact of the TCR with selecting MHC molecules is required for survival of T cells.Positive selection of T cells in the thymus requires the interaction of the TCR on immature thymocytes with self-MHC molecules expressed on thymic epithelial cells (1–3). This process is dependent on the continual interaction of TCRs to MHC molecules since the transfer of thymocytes exhibiting only the first signs of positive selection (i.e., TCR upregulation and CD69 expression) into hosts lacking appropriate MHC molecule expression results in the loss of the transferred cells (4, 5). The presence of certain intermediate stages of T cell development in different gene-deficient mice also indicated that T cells were selected by a multistep process (6–8). Nevertheless, T cells do acquire functional maturity within the thymus since they can be stimulated to divide (9–11), as was found for recent thymic emigrants (12).After the export to secondary lymphoid organs, some naive T cells do not divide but are long lived with an intermitotic lifespan of the order of 8 wk in euthymic mice, and much longer in athymic mice (13, 14). These experiments were performed in mice expressing the same MHC molecules in peripheral lymphoid tissue that induced positive selection in the thymus. Recently, some evidence was presented that MHC molecules in the peripheral lymphoid organs may have a role in sustaining long-term survival of mature T cells (15). We have addressed the same question in a different experimental system and, even though we agree that MHC molecules do support the survival of mature T cells, we have obtained results that differ from those obtained in the previous report (15). In our experiments, we have grafted recombination activating gene (Rag)¹-deficient mice of H-2^b haplotype that express a transgenic I-E^d-restricted TCR with thymi from fetal H-2^d Rag^−/− mice. This allowed the positive selection of CD4⁺8⁻ T cells expressing the transgenic TCR. These cells were transferred into immunodeficient hosts of either H-2^b or H-2^d haplotype, some of the latter being deficient in expression of the common cytokine receptor γ chain (IL-2Rγ^−/−) and therefore devoid of NK cells (16, 17). The results show that H-2^d-restricting MHC molecules are required for survival of the transferred T cells in secondary lymphoid tissue.