ALBERT

Description: Background: In allogeneic BMT patients, the presence of allo-reactive donor CD4+ T cells in the graft were reported to be the primary cause of GvHD. Moreover, donor T-cells are required to promote the stem cell engraftment and to decrease the disease relapse. A number of studies also reported that a subset of CD4+CD25+ T cells usually generated de novo from the thymus that expressed FoxP3 regulate the T cells allo-reactivity in vivo. Thus, to establish a therapeutically useful adoptive T-cells immunotherapy, we depleted the CD4+ T cells from the graft and transplanted along with T cell depleted (TCD) BM cells in clinically relevant parent to F1 experimental allogeneic BMT model. Our hypothesis is that CD4-depleted graft will not cause GvHD, preserve the thymic function, homeostatically produce donor BM-derived CD4+ T cells along with FoxP3+CD4+CD25+ regulatory T cells with beneficial anti-opportunistic infection and anti-tumor effects. Methods: We used a parent (C57BL/6) to (C57BL/6 X BALB/c)CB6F1 allogeneic BMT model with a combination of TCD BM and splenocytes as the hematopoietic graft. CD4+ or CD8+ cells were selectively depleted from the splenocytes of C57BL/6 donor mice using MACS column. 1×106 CD4-depleted splenocytes or a mixture of 2×106 CD8-depleted and 1×106 CD4-depleted splenocytes and/or grafts containing 10×106 unfractionated splenocytes along with 5×106 TCD BM cells harvested from the congeneic C57BL/6 donor mice, were adoptively transferred to lethally irradiated (11Gy) CB6F1 mice. GvHD was monitored twice weekly by weight loss and other clinical signs. After 50 days post transplant recipients mice were bled or sacrificed and lymphocytes isolated from blood and different organs were analyzed by multicolor FACS. Results: Within 50 days of transplant the recipients of CD4-depleted splenocytes had 100% survival without GvHD whereas recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes suffered from severe GvHD (%weight loss below 20%) with 50% survival. Surprisingly, very significantly expansion of total CD4+ T cells (37% ± 7% of lymphocytes, CD4:CD8 ratio 6:1) occurred in the blood of recipients of CD4-depleted splenocytes. In contrast the recipients of mixture of CD4- and CD8-depleted splenocytes DLI or whole splenocytes had only few CD4+ T cells (~2% ± 2% of lymphocytes, CD4:CD8 ratio 1:2). Over 90% of the CD4+ T cells in the blood of recipients of CD4-depleted splenocytes were from the donor BM and included significantly higher number of CD25+CD4+ T cells compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes. Similarly, significantly increased numbers of FoxP3+CD25+CD4+ regularity T cells were also found in the spleen and thymus of recipients of CD4-depleted splenocytes compared with the recipients of mixture of CD4- and CD8-depleted splenocytes or unfractionated splenocytes (p

Description: Background: Therapeutic options for steroid-refractory chronic graft-versus-host-disease (cGVHD) are limited. Extracorporeal photopheresis (ECP) is a photoimmune therapeutic modality to treat cGVHD that is tolerated relatively well, but its mechanism has not been fully defined. One model for the mechanism of ECP in cGVHD is dendritic cell (DC) depletion and T-cell modification (Alcindor, T, et al., BLOOD2001, 98:1622). We tested this hypothesis by determining the numbers of circulating DCs and T-cells prior to ECP and during therapy in patients with cGVHD, and correlating cell numbers with response. Methods: This study was IRB approved. We studied 25 adult pts (median age 43 yrs, range 23–71) with histories of hematological malignancies including NHL (n=7), AML (n=5), CML (n=5), ALL (n=3), MDS (n=3), Hodgkin’s lymphoma (n=1), and CLL (n=1), who developed cGVHD after allogeneic, HLA-matched HPCT. Ten pts had progressive, 9 pts had de novo, and 6 pts had interrupted cGVHD. Initial treatment of cGVHD included corticosteroids in all pts. At the time of ECP initiation, pts were either dependent upon corticosteroids for control of cGvHD (21 pts), or steroid-intolerant (4 pts). No pts had received ECP prior to this study. ECP was administered 2 consecutive days every week for the first 2 months, two times a week every other week for 2 months, and then two times a week once a month. In addition to ECP, pts received steroids (21), MMF (n=13), FK506 (n=15), cyclosporine (n=3), MTX (n=3), rapamycin (n=1), rituximab (n=1) or pentostatin (n=1). Sites of cGVHD included skin (n=25), oropharynx (n=7), liver (n=5), gut (n=4), lung (n=1), and eye (n=1). A good response was defined as having 〉 50% reduction in the corticosteroid dose within 4 months of starting ECP, with improved or stable lesions on skin and other sites. For steroid-intolerant pts, clinical parameters such as improvement in skin condition were used to identify responders. Peripheral blood mononuclear cells were analyzed before ECP began and every 2 months during ECP therapy. The numbers of plasmacytoid DCs (pDC, Lin− CD123+ CD11c− HLA-DR+), myeloid DCs (mDC, Lin− CD123− CD11c+ HLA-DR+), and CD4+ and CD8+ T-cells in blood were determined by flow cytometry. Results: Median follow up of the 25 pts was 47.1 months (range, 8.6–90.9) from the time of transplant. The median number of ECP treatments was 26 (range 2–68). Fourteen pts (56%) had good response, and 11 were non-responders. The median time between HPCT and onset of cGVHD was similar for responders (8.6 months, range 3.3–34.7) and non-responders (6.1, range 3.4–43.8, p=0.52). The median time between HPCT and ECP was also similar for the two groups (32.3 months, range 13.1–60.0, vs. 21.9 months, range 4.1–47.5, respectively, p=0.12). Responders had an estimated 2-yr survival of 88% after starting ECP, vs 18% for non-responders (p=0.004). Two responders died at 11.2 and 31.2 months after starting ECP, compared with 7 non-responders (median 4.4 months, range 2.8–22.1). Non-responders had a relative risk of death of 11.6 compared with responders (p=0.022). Average prednisone doses for responders and non-responders were comparable, averaging 24.3 and 41.8 mg/day, respectively (p=0.11). Responders had higher baseline numbers of pDCs (average 5.8 vs. 0.6 cells/mcL, p=0.025) and mDCs (average 15 vs. 3.8 cells/mcL, p= 0.01) compared with non-responders. Baseline CD4+ T-cell numbers were higher in responders compared with non-responders (average 623 vs. 178 cells/mcL, p=0.005), as were CD8+ T-cell numbers (712 vs. 251 cells/mcL, p=0.047). Contrary to the original hypothesis, there were no consistent changes in the numbers of circulating DCs and T-cells among responders over a 12-month period. Receiver-operator characteristics (ROC) analysis showed that baseline numbers of blood mDCs of 〉3.7 cells/mcL prior to ECP had 79% sensitivity and 82% specificity to predict response of cGvHD patients to ECP. Conclusion: Our results demonstrate that higher numbers of circulating DCs and T-cells predict response to ECP in pts with cGVHD. Response to ECP was significantly associated with improved survival in univariate and multivariate analyses (p

Description: We are investigating methods to reduce the graft-versus-host disease (GVHD) potential of donor T-cells while retaining graft-versus-leukemia (GVL) activity in allogeneic HSCT. Previous investigations by our group and others in have shown that naive CD4 T-cells induce severe acute GVHD, while memory CD4 T-cells do not induce GVHD but retain GVL activity in murine transplant models. These findings have led to studies for the development of methods to increase the number of memory T-cells available for transplant. The calcium ionophore, ionomycin, is a T-cell activating agent and mitogen. By increasing intracellular Ca2+ levels, ionomycin is induces T-cell activation through signaling mechanisms including phospholipase C activation, hydrolysis of phosphoinositides, and activation of protein kinase C. Differences in memory and naive T-cell responses to ionomycin have been attributed to resistance of memory T-cells to increases in Ca2+. Memory T-cells lack intracellular Ca2+ stores, and are also resistant to influx of Ca2+. Brief low dose ionomycin exposure (20min, 2μM) of T-cells, leading to increased density of naive T-cells, has previously been exploited as a method for separating memory and naive T-cells by Percoll gradient separation. Since ionomycin exposure induces T-cell activation through native Ca2+ dependent signaling mechanisms, we hypothesized that ionomycin-treated T-cells would shift to an activated/memory T-cell phenotype. Murine splenic T-cells were treated with 1.3μM ionomycin for 4hr. Memory and naive T-cell subsets and activation markers were analyzed by flow cytometry. 75% and 85% of untreated CD4 and CD8 T-cells, respectively, had the CD62L+ naive phenotype. These numbers were dramatically reduced to 7% and 17% after ionomycin exposure, representing a shift to the memory T-cell phenotype. Viability of T-cells was not significantly affected. The majority of remaining CD62L+ naive T-cells expressed activation markers CD25 and CD69. The fraction of CD4+CD25+Foxp3+ regulatory T-cells was also determined by intracellular staining of the transcription factor and co-expression of surface markers. CD4+CD25+Foxp3+ regulatory T-cells represented 4% of untreated CD4 T-cells and 3% of ionomycin-treated CD4 T-cells. While ionomycin has been used for many years in studies of T-cell activation, to our knowledge this is the first demonstration of a rapidly-induced shift of naive T-cells to a memory phenotype. A pilot experiment was conducted testing the GVHD activity of ionomycin-treated splenocytes (SP) in B6→ (B6 × Balb/C)CB6F1 recipients. 5 × 106 T-cell depleted bone marrow cells (TCD-BM) were transplanted along with 10 × 106 treated or untreated SP. Mice that received untreated SP all died from acute GvHD by 34 days after transplant, while all recipients of ionomycin-treated SP survived until the experiement was terminated at day 49 (average weight loss was 25%, data not shown). Continuing experients will refine the dose to further reduce GVHD symptoms and also test GVL activity of the treated cells. Treatment of donor T-cells with ionomycin may represent a clinically applicaple method to engineer donor lymphocyte infusions that are safer for HSCT patients. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49.

Description: Bortezomib, a proteasome inhibitor with efficacy in multiple myeloma, is associated with thrombocytopenia, the cause and kinetics of which are different from those of standard cytotoxic agents. We assessed the frequency, kinetics, and mechanism of thrombocytopenia following treatment with bortezomib 1.3 mg/m2 in 228 patients with relapsed and/or refractory myeloma in 2 phase 2 trials. The mean platelet count decreased by approximately 60% during treatment but recovered rapidly between treatments in a cyclic fashion. Among responders, the pretreatment platelet count increased significantly during subsequent cycles of therapy. The mean percent reduction in platelets was independent of baseline platelet count, M-protein concentration, and marrow plasmacytosis. Plasma thrombopoietin levels inversely correlated with platelet count. Murine studies demonstrated a reduction in peripheral platelet count following a single bortezomib dose without negative effects on megakaryocytic cellularity, ploidy, or morphology. These data suggest that bortezomib-induced thrombocytopenia is due to a reversible effect on megakaryocytic function rather than a direct cytotoxic effect on megakaryocytes or their progenitors. The exact mechanism underlying bortezomib-induced thrombocytopenia remains unknown but it is unlikely to be related to marrow injury or decreased thrombopoietin production.

Description: Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect. Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml. Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset. Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. Figure Figure