Mixed Chimerism, Lymphocyte Recovery, and Evidence for Early Donor-Specific Unresponsiveness in Patients Receiving Combined Kidney and Bone Marrow Transplantation to Induce Tolerance

Background We have previously reported operational tolerance in patients receiving HLA-mismatched combined kidney and bone marrow transplantation (CKBMT). We now report on transient multilineage hematopoietic chimerism and lymphocyte recovery in five patients receiving a modified CKBMT protocol, and evidence for early donor-specific unresponsiveness in one of these patients. Methods Five patients with end-stage renal disease received CKBMT from HLA-mismatched, haploidentical living related donors following modified non-myeloablative conditioning. Polychromatic flow cytometry (FCM) was used to assess multilineage chimerism where evaluable and lymphocyte recovery post-transplant. Limiting dilution analysis was used to assess helper-T-lymphocyte reactivity to donor antigens. Results Transient multilineage mixed chimerism was observed in all patients but chimerism became undetectable by 2 weeks post-CKBMT. A marked decrease in T and B lymphocyte counts immediately following transplant was followed by gradual recovery. Initially recovering T cells were depleted of CD45RA+/CD45RO− “naïve-like” cells, which have shown strong recovery in two patients and CD4/CD8 ratios increased immediately following transplant but then declined markedly. NK cells were enriched in the peripheral blood of all patients following transplant. For Subject 2, a pre-transplant limiting dilution assay revealed T helper cells recognizing both donor and third-party PBMCs. However, the anti-donor response was completely undetectable by Day 24, while third-party reactivity persisted. Conclusion These results characterize the transient multilineage mixed hematopoietic chimerism and recovery of lymphocyte subsets in patients receiving a modified CKBMT protocol. The observations are relevant to the mechanisms of donor-specific tolerance in this patient group

ASP4058, a Novel Agonist for Sphingosine 1-Phosphate Receptors 1 and 5, Ameliorates Rodent Experimental Autoimmune Encephalomyelitis with a Favorable Safety Profile

<div><p>Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P₁–S1P₅). S1P₁ is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P₁. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2<i>S</i>)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1<i>H</i>-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P₁ and S1P₅. ASP4058 preferentially activates S1P₁ and S1P₅ compared with S1P_{2, 3, 4} in GTPγS binding assays <i>in vitro</i>. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.</p></div

Plasma concentrations of ASP4058 and blood concentration of fingolimod phosphate during and after intravenous infusion of rats.

<p>Plasma concentrations of ASP4058 and blood concentration of fingolimod phosphate during and after intravenous infusion of rats.</p

Chemical structure of ASP4058 (C19H12F6N4O2.HCl).

<p>Chemical structure of ASP4058 (C19H12F6N4O2.HCl).</p

Plasma or blood concentrations of ASP4058 and fingolimod-P after repeated dosing.

<p>ASP4058 (0.1 mg/kg) or fingolimod (0.1 mg/kg) was administered once-daily for 14 days in male Lewis rats. Plasma concentration of ASP4058 and blood concentration of fingolimod phosphate (fingolimod-P) in rats were measured just before the last administration, 0.25 (for fingolimod-P) or 0.5 (for ASP4058), 1, 3, 8, and 24 h after the last administration. Data represent the mean ± S.E. (<i>n</i> = 5).</p

Effects of ASP4058 and fingolimod-P on heart rate and mean blood pressure in conscious rats.

<p>Vehicle, ASP4058 (1 or 3 mg/kg) or fingolimod phosphate (fingolimod-P) (0.01, 0.03, or 0.1 mg/kg) were continuously administered for 10 min using an infusion pump (1 ml/kg/min) through a catheter inserted into the femoral vein, and the effects of ASP4058 and fingolimod-P on heart rate and mean blood pressure were determined (ASP4058, A and C, respectively; fingolimod-P, B and D, respectively). Heart rates and mean blood pressures were recorded for 20 min after the start of infusion. Heart rate was determined at 1-min intervals, and mean blood pressures were determined 0, 2, 4, 6, 8, 10, 15, and 20 min after the start of infusion. All values represent the mean ± S.E. for 5 rats per group, except for the 20-min time points of the groups treated with 1 mg/kg of ASP4058 (mean ± S.E. for 4 rats) or 0.1 mg/kg fingolimod phosphate (mean ± S.E. for 3 rats).</p

Effects of ASP4058 or fingolimod phosphate on bronchoconstriction in anesthetized rats.

<p>To assess the effect of compound on base-line airway pressure, ASP4058 (0.3 mg/kg/min) or fingolimod phosphate (fingolimod-P) (0.003, 0.03, 0.3 mg/kg/min) were administered by continuous intravenous infusion, and airway pressure was measured just before the initiation of compound infusion (baseline) and approximately 20 min after the initiation of compound infusion, which is sufficient time to reach a plateau. Results are shown as the percent change from baseline value and represent the mean ± S.E. The number of animals in each group is shown in parentheses. **<i>P</i><0.01 compared with the vehicle-treated group (Dunnett's multiple comparison test).</p

Agonistic effects of ASP4058 and fingolimod phosphate on human S1P receptor subtypes.

<p>Agonistic effects of ASP4058 and fingolimod phosphate on human S1P receptor subtypes.</p