CELLULAR DIFFERENTIATION OF THE IMMUNE SYSTEM OF MICE : III. SEPARATE ANTIGEN-SENSITIVE UNITS FOR DIFFERENT TYPES OF ANTI-SHEEP IMMUNOCYTES FORMED BY MARROW-THYMUS CELL MIXTURES

Marrow cell suspensions of unprimed donor mice have been transplanted into X-irradiated syngeneic hosts. 5–46 days later, bone cavities and spleens contained regenerated cells of the immune system which required interaction with thymocytes (from intact donors) and antigen (SRBC) to form antigen-sensitive units (ASU) and to generate mature immunocytes. These cells were capable of differentiating either into direct or indirect hemolytic plaque-forming cells (PFC). The precursors of PFC regenerated earlier than the other cell type necessary for immunocompetence, the antigen-reactive cell (ARC). The latter was not found until 10 or more days after transplantation. Availability of ARC was inferred from PFC responses elicited by grafted mice challenged with SRBC at varying intervals. In a second series of experiments, graded numbers of marrow cells (ranging from 107 to 5 x 107) were transplanted with 5 x 107 or 108 thymocytes into irradiated mice, and SRBC were given 18 hr later. After 9–12 days the recipient spleens contained all or some of the following immunocytes: direct and indirect PFC, and hemagglutinating cluster-forming cells. The frequency of each immune response varied independently of the others, but in relation to the number of marrow cells grafted. This was interpreted to indicate that ASU formed in irradiated mice by interaction of marrow and thymus cells were similar to those of intact mice. In particular, they were specialized for the molecular class (IgM or IgG) and function (lysis or agglutination) of the antibody to be secreted by their descendent immunocytes. Hence, class-differentiation appeared to be conferred upon ASU by their marrow-derived components

DISTINCT EVENTS IN THE IMMUNE RESPONSE ELICITED BY TRANSFERRED MARROW AND THYMUS CELLS : I. ANTIGEN REQUIREMENTS AND PROLIFERATION OF THYMIC ANTIGEN-REACTIVE CELLS

Marrow cells and thymocytes of unprimed donor mice were transplanted separately into X-irradiated syngeneic hosts, with or without sheep erythrocytes (SRBC). Antigen-dependent changes in number or function of potentially immunocompetent cells were assessed by retransplantation of thymus-derived cells with fresh bone marrow cells and SRBC; of marrow-derived cells with fresh thymocytes and SRBC; and of thymus-derived with marrow-derived cells and SRBC. Plaque-forming cells (PFC) of the direct (IgM) and indirect (IgG) classes were enumerated in spleens of secondary host mice at the time of peak responses. By using this two-step design, it was shown (a) that thymus, but not bone marrow, contained antigen-reactive cells (ARC) capable of initiating the immune response to SRBC (first step), and (b) that the same antigen complex that activated thymic ARC was required for the subsequent interaction between thymus-derived and marrow cells and/or for PFC production (second step). Thymic ARC separated from marrow cells but exposed to SRBC proliferated and generated specific inducer cells. These were the cells that interacted with marrow precursors of PFC to form the elementary units for plaque responses to SRBC, i.e. the class- and specificity-restricted antigen-sensitive units. It was estimated that each ARC generated 80–800 inducer cells in 4 days by way of a minimum of 6–10 cell divisions. On the basis of the available evidence, a simple model was outlined for cellular events in the immune response to SRBC

CELLULAR DIFFERENTIATION OF THE IMMUNE SYSTEM OF MICE : VI. STRAIN DIFFERENCES IN CLASS DIFFERENTIATION AND OTHER PROPERTIES OF MARROW CELLS

Marrow cells and 5 x 107 thymocytes of unprimed (C57BL/6 x DBA/2)F1, (C57BL/10 x WB)F1 and (C3H x C57BL)F1 donor mice were mixed in vitro and transplanted into X-irradiated syngeneic hosts. Upon injection of sheep erythrocytes, splenic plaque-forming cells (PFC) secreting IgM (direct PFC or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By grading the numbers of marrow cells, inocula were found that contained few immunocompetent cells reaching the recipient spleens, interacting with thymocytes or other accessory cells (or both), and generating PFC. The frequency of responses in BDF1 mice conformed to Poisson statistics, indicating that immunocompetent marrow cells participated in a single-hit interaction limiting PFC responses. The marrow cells assayed were not restricted for the antibody class (IgM versus IgG) to be secreted by mature PFC. Unrestricted marrow cells could have been either the precursors of PFC or accessory cells. Different results were obtained in BWF1 and C3BF1 mice. The frequency of responses in relation to the number of marrow cells grafted did not follow Poisson statistics, and the limiting cells were restricted for antibody class. Presumably, immunocompetent cells of these strains were more heterogeneous than those of BDF1 mice and participated in a multiplicity of cell-to-cell interactions. The strain differences reflected inherent properties of marrow cells and not influences of the environment in which PFC were produced. The results confirmed for bone marrow the heterogeneity of immunocompetent cells reported by others for spleen, and suggested that genetic factors such as "immune response" genes regulate cellular differentiation also for functions other than those related to antibody specificity

CELLULAR DIFFERENTIATION OF THE IMMUNE SYSTEM OF MICE : IV. LACK OF CLASS DIFFERENTIATION IN THYMIC ANTIGEN-REACTIVE CELLS

Thymocytes and marrow cells of unprimed donor mice were mixed in vitro and transplanted into X-irradiated syngeneic mice. 18 hr later, sheep erythrocytes were injected to induce immune responses. Splenic plaque-forming cells (PFC) secreting IgM (direct PFC) or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By transplanting graded and limiting numbers of thymocytes with 4 x 107 marrow cells, inocula were found which contained one or a few thymic antigen-reactive cells (ARC) reaching the recipient spleens, interacting with marrow cells, and inducing PFC formation. The frequency values of ARC inferred from direct and indirect plaque assays were very similar, 1 in ∼107 thymocytes. Furthermore, statistical analysis indicated that the formation of direct PFC was not independent of the formation of indirect PFC. This was interpreted to mean that ARC were not specialized themselves and did not determine the molecular class of antibody to be secreted after interaction with marrow cells. Spleens of thymus-marrow grafted mice containing one or two ARC and non-limiting numbers of marrow precursors of PFC (P-PFC), had direct and indirect PFC clustered in several focal areas. Assuming that each focal area represented the progeny of one P-PFC that had interacted with ARC, these results confirmed the statistical evidence for lack of class differentiation in thymic ARC, and also indicated that each ARC or its progeny cells interacted with more than one P-PFC of either class

CELLULAR DIFFERENTIATION OF THE IMMUNE SYSTEM OF MICE : V. CLASS DIFFERENTIATION IN MARROW PRECURSORS OF PLAQUE-FORMING CELLS

Marrow cells and thymocytes of unprimed donor mice were mixed in vitro and transplanted into X-irradiated syngeneic hosts. 18 hr later sheep erythrocytes were injected to induce immune responses. Splenic plaque-forming cells (PFC) secreting IgM (direct PFC) or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By transplanting graded and limiting numbers of marrow cells with 5 x 107 thymocytes, inocula were found that contained few precursors of PFC (P-PFC) reaching the recipient spleens, interacting with thymocytes, and generating PFC. However, the frequency of responses in relation to the number of grafted marrow cells did not follow Poisson statistics, presumably because the interaction of marrow cells with thymocytes was more complex than a single or a one-to-one cell event. The frequency of direct PFC responses was greater than that of indirect PFC responses in 13 of 15 groups of mice tested. This was interpreted as evidence for the existence of two classes of P-PFC, each of which was restricted to generate either direct or indirect PFC. The precursors of direct PFC were ∼ 15 times more frequent than those of indirect PFC. Since thymic antigen-reactive cells were not differentiated for antibody class, it follows that antigen-sensitive units reactive to sheep erythrocytes owe their class restriction to specialized marrow cells. Specialization of P-PFC may have arisen within marrow cell lines by differentiation, or may have been conferred upon P-PFC by interaction with other cells, including those of the irradiated host

A Phase III Trial of Tirasemtiv as a Potential Treatment for Amyotrophic Lateral Sclerosis

Objective: To assess the efficacy of tirasemtiv, a fast skeletal muscle troponin activator, vs. placebo in patients with amyotrophic lateral sclerosis. Methods: VITALITY-ALS (NCT02496767) was a multinational, double-blind, randomized, placebo-controlled clinical trial. Participants tolerating 2 weeks of open-label tirasemtiv (125 mg twice daily) were randomized 3:2:2:2 to placebo or one of three target tirasemtiv dose levels, using an escalating dosage protocol lasting 28 days. The primary outcome measure was changed in slow vital capacity (SVC) at 24 weeks. Secondary endpoints included a change in muscle strength and time to respiratory milestones of disease progression. Results: Of 744 participants, 565 tolerated open-label tirasemtiv and received randomized treatment. By 24 weeks, 23 (12.2%) placebo-treated participants discontinued study treatment vs. 129 (34.2%) randomized to tirasemtiv. SVC declined by 14.4% (95% CI: ˆ’16.8, ˆ’11.9) in the placebo group and 13.4% (95% CI: ˆ’15.3, ˆ’11.6) in the tirasemtiv group (p = 0.56). Secondary endpoints did not show significant differences. However, participants who tolerated tirasemtiv at their randomized dose showed a numeric trend toward a dose-related slowing of decline in SVC (p = 0.11). Dizziness, fatigue, nausea, weight loss, and insomnia occurred more frequently on tirasemtiv. Serious adverse events were similar across groups. Conclusions: Tirasemtiv did not alter the decline of SVC or significantly impact secondary outcome measures. Poor tolerability of tirasemtiv may have contributed to this result. However, participants tolerating their intended dose exhibited a trend toward treatment benefit on SVC, suggesting the underlying mechanism of action may still hold promise, as is being tested with a different fast skeletal muscle troponin activator (NCT03160898)