Are weakly binding bridges present in resting intact muscle fibers?

Several experimental results (Schoenberg, M. 1988. Biophys. J. 54:135-148) have shown that the force response of relaxed skinned muscle fibers to fast stretches arises from the presence of cross-bridges rapidly cycling between attached and detached states. These bridges were identified with the M.ATP<-->AM.ATP and M.ADP.Pi<-->AM.ADP.Pi states seen in solution and are commonly referred to as weakly binding bridges. In this paper we have investigated the possibility that weakly binding bridges are also present in resting intact muscle fibers. The force response to fast stretches can be accounted for by assuming the presence in the fiber of a viscous and a viscoelastic passive component arranged in parallel. None of these components has the properties previously attributed to weakly binding bridges. This shows that in intact resting fibers there is no mechanical evidence of attached cross-bridges, suggesting that, under physiological conditions, either the M.ATP or M.ADP.Pi states have a negligibly small affinity for actin or the AM.ATP and AM.ADP.Pi cross-bridge states are unable to bear tension and contribute to fiber stiffness.     

CAR T cells transform to trucks: chimeric antigen receptor–redirected T cells engineered to deliver inducible IL-12 modulate the tumour stroma to combat cancer

Adoptive T cell therapy recently achieved impressive efficacy in early-phase clinical trials; this significantly raises the profile of immunotherapy in the fight against cancer. A broad variety of tumour cells can specifically be targeted by patients' T cells, which are redirected in an antibody-defined, major histocompatibility complex-unrestricted fashion by endowing them with a chimeric antigen receptor (CAR). Despite promising results for some haematologic malignancies, the stroma of large, established tumours, the broad plethora of infiltrating repressor cells, and cancer cell variants that had lost the target antigen limit their therapeutic efficacy in the long term. This article reviews a newly described strategy for overcoming some of these shortcomings by engineering CAR T cells with inducible or constitutive release of IL-12. Once redirected, these T cells are activated, and released IL-12 accumulates in the tumour lesion where it promotes tumour destruction by at least two mechanisms: (1) induction of an innate immune cell response towards those cancer cells which are invisible to redirected T cells and (2) triggering programmatic changes in immune-suppressive cells. Given the enormous complexity of both tumour progression and immune attack, the upcoming strategies using CAR-redirected T cells for local delivery of immune-modulating payloads exhibited remarkable efficacy in pre-clinical models, suggesting their evaluation in clinical trials.     

Manufacturing chimeric antigen receptor T cells from cryopreserved peripheral blood cells: time for a collect-and-freeze model?

Background aimsChimeric antigen receptor (CAR)-modified T-cell therapy has revolutionized outcomes for patients with relapsed/refractory B-cell malignancies. Despite the exciting results, several clinical and logistical challenges limit its wide applicability. First, the apheresis requirement restricts accessibility to institutions with the resources to collect and process peripheral blood mononuclear cells (PBMCs). Second, even when utilizing an apheresis product, failure to manufacture CAR T cells is a well-established problem in a significant subset. In heavily pre-treated patients, prior chemotherapy may impact T-cell quality and function, limiting the ability to manufacture a potent CAR T-cell product. Isolation and storage of T cells shortly after initial cancer diagnosis or earlier in life while an individual is still healthy are an alternative to using T cells from heavily pre-treated patients. The goal of this study was to determine if a CAR T-cell product could be manufactured from a small volume (50 mL) of healthy donor blood.MethodsCollaborators at Cell Vault collected 50 mL of whole peripheral venous blood from three healthy donors. PBMCs were isolated, cryopreserved and shipped to the Medical College of Wisconsin. PBMCs for each individual donor were thawed, and CAR T cells were manufactured using an 8-day process on the CliniMACS Prodigy device with a CD19 lentiviral vector.ResultsStarting doses of enriched T-cell numbers ranged from 4.0 × 10⁷ cells to 4.8 × 10⁷ cells, with a CD4/CD8 purity of 74–79% and an average CD4:CD8 ratio of 1.4. On the day of harvest, total CD3 cells in the culture expanded to 3.6–4.6 × 10⁹ cells, resulting in a 74- to 115-fold expansion, an average CD4:CD8 ratio of 2.9 and a CD3 frequency of greater than 99%. Resulting CD19 CAR expression varied from 19.2% to 48.1%, with corresponding final CD19+ CAR T-cell counts ranging from 7.82 × 10⁸ cells to 2.21 × 10⁹ cells. The final CAR T-cell products were phenotypically activated and non-exhausted and contained a differentiated population consisting of stem cell-like memory T cells.ConclusionsOverall, these data demonstrate the ability to successfully generate CAR T-cell products in just 8 days using cryopreserved healthy donor PBMCs isolated from only 50 mL of blood. Notably, numbers of CAR T cells were more than adequate for infusion of an 80-kg patient at dose levels used for products currently approved by the Food and Drug Administration. The authors offer proof of principle that cryopreservation of limited volumes of venous blood with an adequate starting T-cell count allows later successful manufacture of CAR T-cell therapy.     

“Self” to cytotoxic T cells has to be 1 000 or less high affinity nonapeptides per MHC antigen

In order to serve as the effective target of a relevant cytotoxic T-cell receptor, the same peptide fragment has to occupy at least 0.1% of the class I major histocompatibility complex (MHC) antigen sites on the plasma membrane. Because of this need, I contend that the thymic educator cell of self to cytotoxic T cells can suppress autoreactive T-cell clones only with regard to at the most, 1000 self nonapeptides per a given allelic form of class I MHC antigens; e. g., HLA-A2. Each allelic form of class I MHC antigen apparently developed the preferential binding affinity toward a specific set of nonapeptides. The requirement for preferential binding can either be permissive or stringent. In the case of human HLA-A2, those nonapeptides having either Leu or Met at the second position and mainly Val, but occasionally Leu at the ninth position are preferred. Since both Leu and Val are very common residues, the typical somatic cell type readily supplies nearly 3000 high affinity host nonapeptides preferred by HLA-A2. Of those, the tolerance can be induced, at the most, to only 1000 nonapeptides. In view of this, permissive class I MHC antigens such as HLA-A2 carefully avoid high affinity nonapeptides in viral proteins, for their status as to self or nonself is uncertain, and they choose second choice nonapeptides as T epitopes. In sharp contrast to human HLA-A2, mouse H-2D^b represents the stringent class I MHC antigens. In order to show the high binding affinity toward H-2D^b, nonapeptides are required to carry Asn at position 5 and Met or Ile at the equally critical position 9. Inasmuch as Asn and Met are rare residues and Ile, too, is not a common residue, the typical somatic cell type can supply only several hundred host nonapeptides having the high binding affinity toward H-2D^b. Under the circumstance, there is no problem in memorizing the selfness of all of them. Accordingly, T epitopes are almost invariably chosen from the high affinity nonapeptides that are present in their viral proteins.     

T cell-intrinsic factors contribute to the differential ability of CD8+ T cells to rapidly secrete IFN-γ in the absence of antigen

A subset of CD44(hi)CD8(+) T cells isolated from C57BL/6/J (B6) mice, but not BALB/c/By/J (BALB/c) mice, rapidly secrete IFN-γ within 16 h of infection with Listeria monocytogenes. This Ag-independent response requires the presence of both IL-12 and IL-18. Previous studies showed that dendritic cells from B6 mice produced more Th1-type cytokines such as IL-12 than did those from BALB/c mice in response to L. monocytogenes infection. In this report, we demonstrate that the microenvironment in L. monocytogenes-infected BALB/c mice is sufficient to induce responsive B6 CD8(+) T cells to rapidly secrete IFN-γ. Furthermore, BALB/c CD8(+) T cells did not rapidly secrete IFN-γ even when they were exposed to high concentrations of IL-12 plus IL-18 in vitro. In the presence of IL-12 and IL-18, B6 CD44(hi)CD8(+) T cells upregulated expression of the receptor subunits for these cytokines more rapidly than did BALB/c T cells. In comparing particular subsets of memory phenotype CD8(+) T cells, we found that virtual memory cells, rather than true Ag-experienced cells, had the greatest level of impairment in BALB/c mice. These data suggest that the degree of cytokine-driven bystander activation of CD8(+) T cells that occurs during infection depends on both APCs and T cell-intrinsic properties that can vary among mouse strains.     

Are T cells at the origin of B cell lymphomas?

Lymphoma pathogenesis is at least in some cases related to transformed B cells (BCs) arising from germinal centre reactions (GCRs). In this article possible deregulations of GCRs are investigated using in silico simulations. It is found that the final differentiation of BCs as regulated by helper T cells (TCs) is the best candidate mechanism for such a deregulation. This shifts the paradigm of BC lymphoma pathogenesis from BC transformations to an emphasized role of TC-BC interactions.     

Is sodium current present in human sinoatrial node cells?

Pacemaker activity of the sinoatrial node has been studied extensively in various animal species, but is virtually unexplored in man. As such, it is unknown whether the fast sodium current (I_Na) plays a role in the pacemaker activity of the human sinoatrial node. Recently, we had the unique opportunity to perform patch-clamp experiments on single pacemaker cells isolated from a human sinoatrial node. In 2 out of the 3 cells measured, we observed large inward currents with characteristics of I_Na. Although we were unable to analyze the current in detail, our findings provide strong evidence that I_Na is present in human sinoatrial node pacemaker cells, and that this I_Na is functionally available at potentials negative to -60 mV.     

Regulatory T cells: friend or foe in immunity to infection?

Homeostasis in the immune system depends on a balance between the responses that control infection and tumour growth and the reciprocal responses that prevent inflammation and autoimmune diseases. It is now recognized that regulatory T cells have a crucial role in suppressing immune responses to self-antigens and in preventing autoimmune diseases. Evidence is also emerging that regulatory T cells control immune responses to bacteria, viruses, parasites and fungi. This article explores the possibility that regulatory T cells can be both beneficial to the host, through limiting the immunopathology associated with anti-pathogen immune responses, and beneficial to the pathogen, through subversion of the protective immune responses of the host.     

Sensitization of T cells to apoptosis—A role for ROS?

T cell homeostasis is achieved by balancing the production and proliferation of T cells with their apoptotic cell death. Activation of naïve T cells by antigen in the context of MHC results in the massive expansion of antigen-specific T cells and the production of reactive oxygen species (ROS). Following expansion, the majority of the T cells die via apoptosis, while a small number of them survive and differentiate into memory T cells. This cell fate decision is crucial to our understanding of how autoimmunity is avoided and how immunity is maintained. It has become increasingly clear that ROS can affect this cell fate decision by sensitizing T cells to apoptosis. Interestingly, ROS have effects on both intrinsic and extrinsic apoptosis pathways through modulation of expression of the major molecules in these pathways, Bcl-2 and FasL. In this review, we will focus on the pro-apoptotic effects of ROS and mechanisms by which they regulate the death of T cells.     

Is the delayed-type hypersensitivity observed after a low dose of antigen mediated by helper T cells?

Unprimed murine spleen cells, when cultured at different densities but in the presence of the same concentration of antigen, are induced to mount different classes of response. Three modes of behavior are found. A low density does not support the induction of any response, a medium density supports a transient IgM and substantial delayed-type hypersensitivity (DTH) response, and a high density only supports a sustained IgM response. This in vitro system has been used to show that a low density of cells, when complemented with irradiated specific T cells, can mount a DTH response, and thus behave as a medium density of cells. These observations show that the induction of DTH requires helper T cells, and that a medium density, in contrast to a low density, allows sufficient collaboration to obtain a DTH response. The observation that a high density only mounts a sustained humoral response suggests that the formation of more helper T cell-dependent signals than the number generated at a medium density may be required to induce a sustained humoral response as well as the suppression of DTH. This hypothesis is supported by the findings that the response by a medium density of cells is dramatically affected by the addition of irradiated antigen-specific helper T cells; the DTH response is specifically suppressed and a sustained humoral response is observed. These results show that the induction of a humoral response is more T cell dependent than the induction of a cell-mediated response and provides an in vitro means for switching a cell-mediated response to a humoral one in an antigen-specific manner. Observations are also presented to show that the production of antibody by a high density of cells is not a prerequisite for the suppression of DTH reactivity.     

Can the dual-functional capability of CIK cells be used to improve antitumor effects?

Cytokine-induced killer (CIK) cells, which display both potent anti-tumor ability of T lymphocytes and non-major histocompatibility complex (MHC) restricted killing tumor cells capacity of natural killer (NK) cells are capable of recognizing and lysing a broad array of tumor targets. They have begun to be used in clinical care with good prospects for treatment success. CIK cells are a heterogeneous cell population that contain CD3⁺CD56⁺ cells, CD3⁻CD56⁺ natural killer (NK) cells and CD3⁺CD56⁻ T cells on which much attention has been focused. This review will summarize the connections and differences among CD3⁺CD56⁺CIK cells, CD3⁻CD56⁺ NK cells and CD3⁺CD56⁻ T cells in the following aspects: the main cell surface molecule, killing mechanism, and clinical applications so that treatment with CIK cells can be optimized and further to enhance the antitumor effect.     

Ovarian cancer stem cells: Can targeted therapy lead to improved progression-free survival?

Despite significant effort and research funds, epithelial ovarian cancer remains a very deadly disease. There are no effective screening methods that discover early stage disease; the majority of patients are diagnosed with advanced disease. Treatment modalities consist primarily of radical debulking surgery followed by taxane and platinum-based chemotherapy. Newer therapies including limited targeted agents and intraperitoneal delivery of chemotherapeutic drugs have improved disease-free intervals, but failed to yield longlasting cures in most patients. Chemotherapeutic resistance, particularly in the recurrent setting, plagues the disease. Targeting the pathways and mechanisms behind the development of chemoresistance in ovarian cancer could lead to significant improvement in patient outcomes. In many malignancies, including blood and other solid tumors, there is a subgroup of tumor cells, separate from the bulk population, called cancer stem cells(CSCs). These CSCs are thought to be the cause of metastasis, recurrence and resistance. However, todate, ovarian CSCs have been difficult to identify, isolate, and target. It is felt by many investigators that finding a putative ovarian CSC and a chemotherapeutic agent to target it could be the key to a cure for this deadly disease. This review will focus on recent advances in this arena and discuss some of the controversies surrounding the concept.