A new approach for eradication of residual lymphoma cells by host nonreactive anti-third-party central memory CD8 T cells.

Generation of T cells endowed with graft-versus-leukemia (GVL) and depleted of graft-versus-host (GVH) activity represents a highly desirable goal in bone marrow transplantation (BMT). Here, we demonstrate that donor anti-third-party CD8 T cells with central memory phenotype (Tcm) exhibit marked GVL reactivity through a unique T-cell receptor-independent mechanism. Thus, in a residual disease mouse model, Tcm therapy following autologous BMT led to significant survival prolongation, with 30% to 40% of the treated mice displaying long-term tumor-free survival. A more impressive finding was that infusion of donor Tcm in an allogeneic model rapidly eliminated residual lymphoma cells and led to long-term survival of 100% in the absence of GVH disease. Collectively, the strong GVL reactivity of anti-third-party Tcm, coupled with their demonstrated enhancement of bone marrow allografting, suggests that the use of Tcm therapy in conjunction with allogeneic T-cell-depleted BMT could be of particular benefit in patients with B-cell malignancies who cannot tolerate intensive myeloablative conditioning.

CTLs respond with activation and granule secretion when serving as targets for T-cell recognition.

Cytotoxic T lymphocytes (CTLs) suppress T cell responses directed against their antigens regardless of their own T cell receptor (TCR) specificity. This makes the use of CTLs promising for tolerance induction in autoimmunity and transplantation. It has been established that binding of the CTL CD8 molecule to the major histocompatibility complex (MHC) class I α3 domain of the recognizing T cell must be permitted for death of the latter cell to ensue. However, the signaling events triggered in the CTL by this molecular interaction in the absence of TCR recognition have never been clarified. Here we use single-cell imaging to study the events occurring in CTLs serving as targets for recognition by specific T cells. We demonstrate that CTLs actively respond to recognition by polarizing their cytotoxic granules to the contact area, releasing their lethal cargo, and vigorously proliferating. Using CTLs from perforin knockout (KO) mice and lymphocyte specific kinase (Lck) knockdown with specific small interfering RNA (siRNA), we show that the killing of the recognizing CD8 T cell is perforin dependent and is initiated by Lck signaling in the CTL. Collectively, these data suggest a novel mechanism in which the entire cascade generally triggered by TCR engagement is "hijacked" in CTLs serving as targets for T cell recognition without TCR ligation.

Induction of tolerance to bone marrow allografts by donor-derived host nonreactive ex vivo-induced central memory CD8 T cells.

Enabling engraftment of allogeneic T cell-depleted bone marrow (TDBM) under reduced-intensity conditioning represents a major challenge in bone marrow transplantation (BMT). Anti-third-party cytotoxic T lymphocytes (CTLs) were previously shown to be endowed with marked ability to delete host antidonor T cells in vitro, but were found to be less effective in vivo. This could result from diminished lymph node (LN) homing caused by the prolonged activation, which induces a CD44(+)CD62L(-) effector phenotype, and thereby prevents effective colocalization with, and neutralization of, alloreactive host T cells (HTCs). In the present study, LN homing, determined by imaging, was enhanced upon culture conditions that favor the acquisition of CD44(+)CD62L(+) central memory cell (Tcm) phenotype by anti-third-party CD8(+) cells. These Tcm-like cells displayed strong proliferation and prolonged persistence in BM transplant recipients. Importantly, adoptively transferred HTCs bearing a transgenic T-cell receptor (TCR) with antidonor specificity were efficiently deleted only by donor-type Tcms. All these attributes were found to be associated with improved efficacy in overcoming T cell-mediated rejection of TDBM, thereby enabling high survival rate and long-term donor chimerism, without causing graft-versus-host disease. In conclusion, anti-third-party Tcms, which home to recipient LNs and effectively delete antidonor T cells, could provide an effective and novel tool for overcoming rejection of BM allografts.

Epidemiology, longitudinal treatment patterns and outcomes of chronic lymphocytic leukemia in Israel.

This study aims to describe chronic lymphocytic leukemia (CLL) epidemiology, treatment patterns and outcomes in a 2.3-million-member healthcare provider database (Maccabi Healthcare Services, Israel). Newly-diagnosed CLL patients (1999-2017) were followed through 31/3/2018. A total of 1857 newly-diagnosed CLL patients were included. Annual incidence was 5.82 per 100,000 population. Median overall survival (OS) was 12.7 (95%CI: 11.8-13.5) years since diagnosis. Approximately 1/3 initiated treatment within 5 y. A statistical trend (p = 0.066) for improved OS over time was observed among younger patients (age <70 y) treated in 2009-2017 vs. 1999-2008). Among patients treated since 2009 (n = 411; median age = 68y), fludarabine-cyclophosphamide-rituximab (FCR), bendamustine-rituximab and obinutuzumab ± chlorambucil accounted for 19.5%, 12.2% and 11.4% of first line, respectively. Median (95%CI) time to next treatment and OS were 3.1(2.6-3.6) and 7.0(6.3-7.7) years, respectively. CLL incidence in Israel is comparable to developed countries. Real-world data suggest a trend of improved survival over the last decade among patients treated before age 70.

Distinct extracellular-matrix remodeling events precede symptoms of inflammation.

Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases.

Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.

Tumor macrophages are pivotal constructors of tumor collagenous matrix.

Tumor-associated macrophages (TAMs) promote tumor development, invasion, and dissemination by various mechanisms. In this study, using an orthotopic colorectal cancer (CRC) model, we found that monocyte-derived TAMs advance tumor development by the remodeling of its extracellular matrix (ECM) composition and structure. Unbiased transcriptomic and proteomic analyses of (a) TAM-abundant and -deficient tumor tissues and (b) sorted tumor-associated and -resident colonic macrophage subpopulations defined a distinct TAM-induced ECM molecular signature composed of an ensemble of matricellular proteins and remodeling enzymes they provide to the tumor microenvironment. Remarkably, many of these ECM proteins are specifically increased in human CRC versus healthy colon. Specifically, we demonstrate that although differentiating into TAMs, monocytes up-regulate matrix-remodeling programs associated with the synthesis and assembly of collagenous ECM, specifically collagen types I, VI, and XIV. This finding was further established by advanced imaging showing that TAMs instruct the deposition, cross-linking, and linearization of collagen fibers during tumor development, especially at areas of tumor invasiveness. Finally, we show that cancer-associated fibroblasts are significantly outnumbered by TAMs in this model and that their expression of collagen XIV and I is reduced by TAM deficiency. Here, we outline a novel TAM protumoral function associated with building of the collagenous ECM niche.