Rapid Lymphocyte Recovery Research Articles

Oral administration of Euglena gracilis paramylon ameliorates chemotherapy-induced leukocytopenia and gut dysbiosis in mice

Euglena gracilis (EUG) is a food supplement rich in beta-glucans, which are stored in the form of granules called paramylon. We determined whether EUG improved chemotherapy-induced leukocytopenia and dysbiosis. Mice were orally administered EUG prior to gemcitabine treatment. Analyses of the blood cell count, leukocyte population in the spleen, granulocyte/macrophage–colony-stimulating factor (GM–CSF) production by splenocytes, and fecal microbiome were conducted. The recovery of total leukocytes, neutrophils, and monocytes was accelerated after a single gemcitabine treatment. A more rapid lymphocyte recovery rate was observed after four gemcitabine treatments. No difference was observed in the percentage of T, B, or myeloid cells or in the expression of Dectin-1 in the spleens of the gemcitabine and EUG/gemcitabine groups. The EUG/gemcitabine group showed an enhanced GM–CSF production by lipopolysaccharides-stimulated splenocytes. Next-generation sequencing revealed that gemcitabine-induced dysbiosis was alleviated. This study demonstrated that EUG-derived beta-glucans could act as a biological response modifier as well as prebiotics for ameliorating chemotherapy-induced adverse effects.

Potential for Dendritic Cell Stimulated De Novo Anti-Leukaemia CD4 T Cell Response in the Bone Marrow Tumour Microenvironment during Induction Chemotherapy

In acute lymphoblastic leukaemia (ALL), T cell anergy induced by tumour antigen presentation without co-stimulation contributes to immunological escape and disease progression. Cross priming of T cells by dendritic cells (DCs) may overcome this escape mechanism, restoring T cell activation and promoting a successful anti-tumour immune response. Remission induction chemotherapy induces rapid cytoreduction of leukaemia and has the modifying potential to affect effector-target cell ratios and tumour cell recognition. DCs may take up necrotic and apoptotic tumour cells, process tumour antigens, and present these to T cells alongside the costimulatory molecules required for effective priming. Further, lymphocyte recovery during remission induction chemotherapy has been linked with improved prognosis, leading to postulation that selective lymphocyte expansion may occur as an immune response against tumour cells. In this study, we extensively characterised paediatric B cell ALL patient immune cells and plasma cytokines, at diagnosis and throughout remission induction chemotherapy in peripheral blood (PB) and the bone marrow (BM) tumour microenvironment.17 paediatric patients diagnosed with precursor B cell ALL were enrolled in this study. Matched PB and BM aspirate tissue samples were collected at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). Mononuclear cells and plasma samples were obtained by density gradient centrifugation. DCs, monocytes, B cells, natural killer (NK) cells, T cells, invariant natural killer T cells, cytokine induced killer cells and leukaemic blasts were extensively characterised by flow cytometry in matched PB and BM aspirate samples at diagnosis, day 8, day 15 and day 29 of induction therapy. Plasma cytokines from diagnosis and day 29 samples were analysed by Luminex multi-plex immunoassay.PB absolute lymphocyte counts (ALCs) at induction days 8 and 15 were lower than those recorded at diagnosis and lymphocyte recovery was observed at day 29. An equivalent pattern of lower mononuclear cell yields at days 8 and 15 with increased yields at day 29 was recorded in PB and BM samples processed for flow cytometry. All major immune cell populations were identified in PB and BM at all time points. BM DCs were significantly increased at day 29 compared to earlier time points and compared to matched day 29 PB DCs (Figure 1A). Naïve (CCR7+, CD45RA+) T cells dominated the peripheral and BM T cell pools at all time points with only low numbers of effector (CCR7-, CD45RA+), effector memory (CCR7-, CD45RA-) and central memory (CCR7+, CD45RA-) T cells recorded. However, activated (HLA-DR+) naïve CD4 T cells were significantly increased in the BM at day 29 compared to matched PB (Figure 1B) concordant to increased BM DCs. BM soluble cytokine analysis confirmed the presence of IL-1β, TNFα, IL-2, IL-4, IL-12p70 and IFNα in day 29 samples which may provide stimulus in the BM microenvironment for maturation of DCs and subsequent priming and activation of T cells.Immunological alterations in the BM tumour microenvironment may be reflective of an altered capacity to mount an effective anti-tumour immune response. Particularly, T cell reconstitution following induction chemotherapy may have important implications in childhood leukaemia, especially if recovering T cells are able to mediate anti-leukaemia effects. Rapid lymphocyte recovery has previously been associated with improved survival, and has been shown to be accounted for by increasing T cell numbers following induction chemotherapy in paediatric ALL. Here, while ALC recovery was recorded at day 29, extensive immune phenotyping of PB samples confirmed that recovery of T cell numbers is related to an increase in naïve T cells. T cell expansion associated with a specific immune response would be expected to occur in the effector and memory T cell compartments and the increased naïve T cell numbers observed here likely relate to homeostatic proliferation or increased thymic output. However, BM DCs were increased at day 29 alongside increased activation in naïve CD4 BM T cells, which may represent initiation of an anti-leukaemia T cell response. In vitro studies to investigate patient T cell response to autologous tumour cells are required as confirmation and are currently ongoing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Normal Lymphocytes at Diagnosis and during Induction Chemotherapy in Childhood Acute Lymphoblastic Leukaemia in Peripheral Blood and Bone Marrow Are Predominantly CD3+, CD4+ or CD8+, CCR7+, CD45RA+, HLA-DR-

Tumour immune surveillance requires both the recognition of malignant cells as immunogenic and the functional cytotoxic capacity of an effector cell. Key cytotoxic effector cells are T lymphocytes, capable of mediating tumour cell death through granule exocytosis and death receptor signalling. Whether chemotherapy can modify immune surveillance in haematological malignancies is not yet well understood. Remission induction chemotherapy induces rapid cytoreduction of leukaemia and has the modifying potential to affect effector-target ratios and tumour cell recognition. Professional antigen presenting cells may take up the necrotic and apoptotic tumour cells, process tumour antigens, and present these to T lymphocytes alongside the costimulatory molecules required for effective priming. Thus, the rapid leukaemia cytoreduction of induction therapy may allow the immune system to exert sufficient control as to sustain remission, or to achieve tumour elimination. In this study, we aimed to characterise circulating T lymphocytes and those from the bone marrow (BM) tumour microenvironment, of paediatric B cell acute lymphoblastic leukaemia (ALL) patients at diagnosis and throughout remission induction chemotherapy.17 paediatric patients diagnosed with precursor B cell ALL at Alder Hey Children's Hospital between August 2014 and February 2015 were enrolled in this study. Diagnosis, risk group assignment and treatment were performed according to the UKALL2011 multicentre trials. Philadelphia chromosome positive B-ALL or ALL in patients with Down syndrome were excluded. This study was approved by Norfolk Research Ethics Committee (REC 14/EE/0211) and informed consent was obtained from patients and/or their legal guardians in accordance with the Declaration of Helsinki. 87 paediatric BM and peripheral blood (PB) tissue samples were collected from enrolled patients at time points co-ordinating with treatment protocol (diagnosis, and induction days 8 and/or 15 and 29). CD4+ and CD8+ T cells, invariant natural killer T cells (iNKTs), cytokine induced killer T cells (CIKs) and γd T cells were stained with fluorochrome conjugated monoclonal antibody panels to allow subset analysis and activation state determination by six-colour flow cytometry.The relative frequencies of all major T cell populations; CD4+, CD8+, iNKTs, CIKs and γd T cells, were similar in PB and BM aspirate at diagnosis. CD4+ cells accounted for approximately 53% of circulating PB T cells and 47% of BM T cells. CD8+ cells accounted for approximately 38% of circulating PB T cells and 42% of BM T cells, the remaining 9% of circulating T cells and 11% of BM T cells were made up of combined iNKTs, CIKs and γd T cells. Naïve CD4+ and naïve CD8+ cells dominated both the circulating PB and BM T cell pools at diagnosis and throughout induction chemotherapy (Figure 1). Naïve CD4+ cells accounted for approximately 42% of circulating T cells and approximately 36% of BM T cells at diagnosis (Figure 1A). While naïve CD8+ cells accounted for approximately 21% of circulating T cells and 20% of BM T cells at diagnosis (Figure 1B). No significant changes in the relative frequencies of naïve CD4+ and CD8+ T cells were observed through induction days 8 and 15, although by induction day 29 the relative frequency of activated naïve CD4+ cells (HLA-DR+) was increased in the BM T cell pool compared to circulating naïve CD4+ cells in PB.T cell reconstitution following induction chemotherapy may have important implications in childhood leukaemia, especially if recovering T cells are able to mediate anti-leukaemia effects. Rapid lymphocyte recovery has been associated with improved survival and is accounted for by increasing numbers of T lymphocytes following induction therapy in paediatric ALL. Whether anti-leukaemia T cell cytotoxicity develops following induction therapy remains to be elucidated although there is the potential for a cytotoxic response to develop in both CD4+ and CD8+ T cell subsets. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunotoxin Enables Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) can cure non-malignant hemoglobinopathies, congenital immunodeficiencies and, perhaps, AIDS, diseases that affect millions of people worldwide; yet, it's use is limited outside of malignancies largely because allogeneic transplantation can be complicated by serious conditioning-related toxicities and graft-versus-host disease (GVHD). The advent of highly efficient gene editing technology promises to enable autologous HSCT while avoiding GVHD. However, conditioning-related toxicities will remain a significant barrier to patient accrual unless addressed.Current conditioning approaches rely on genotoxic methods such as total body irradiation (TBI) and/or chemotherapy. These non-targeted methods induce cytopenias, adversely affect niche cells and may damage other organ systems including germ cells. To fully realize the curative potential of HSCT, the development of mild-conditioning methods that preserve immunity while enabling efficient donor engraftment is essential. Efforts to develop mild non-genotoxic approaches (e.g. CD117 antagonist ACK2 antibody) have thus far been met with efficacy limited to immunocompromised animals with very poor engraftment in immunocompetent settings.While extensively explored in cancer therapy, protein-based internalizing immunotoxins (ITs) that induce cell death by inhibiting protein synthesis have not been explored as a conditioning strategy. By creating saporin-based ITs against various antigens present on HSCs, we identified an immunotoxin against CD45 receptor (CD45-IT) that potently depleted hematopoietic cells in vitro (40-70 picomolar IC50) and achieved efficient HSC depletion (>98% depletion in vivo)in fully immunocompetent mice when administered as a single dose. Following CD45-IT administration, transplantation of whole bone marrow donor cells enabled efficient donor engraftment (>90% chimerism). A wide engraftment window was tolerated as transplantation anywhere between 2-12 days post CD45-IT resulted in equivalent engraftment. Immune reconstitution was multi-lineage, durable (maintained for >15 months) and transferrable to secondary recipients, indicative of true stem cell engraftment. Using sickle cell disease mice, we demonstrated CD45-IT conditioning enabled >90% healthy donor cell engraftment resulting in complete disease correction (n=18 sickle mice).We next compared the toxicities induced by CD45-IT and conventional TBI (sub-lethal) in non-transplanted mice over a 120-day period. CD45-IT was non-lethal as mice survived long-term without requiring transplantation (n=12 mice). In contrast to TBI, CD45-IT had significantly less adverse effects on marrow hematopoietic progenitors, vascular integrity and overall marrow cellularity, which recovered faster than in TBI-treated mice. While peripheral B and T-cells were efficiently depleted by both CD45-IT and TBI immediately after treatment, unusually rapid lymphocyte recovery was observed following CD45-IT (12-18 days for T- and B-cell recovery) in comparison to TBI (48 days for recovery). Histology of thymi revealed CD45-IT does not induce thymic atrophy (unlike TBI) as the cortical region continued to support T-cell development as confirmed by T-cell receptor excision circle (TREC) analysis.Whereas TBI reduced peripheral neutrophil counts versus control, CD45-IT induced a rapid expansion of neutrophils (3-fold higher than naïve control). To test innate immunity, we performed a systemic challenge of candida albicans (a clinically relevant fungal strain). Candida challenge in TBI-conditioned mice proved lethal (100% death in 3 days, p value < 0.0001, n=10 mice). In contrast, CD45-IT treated mice were significantly immune to candida challenge with comparable survival to naïve controls (60% survival for CD45-IT, n=10 mice per group).To summarize, a protein-based immunotoxin targeting CD45 enables efficient HSCT in fully immunocompetent animals. The targeted immunotoxin avoids numerous toxicities associated with current genotoxic conditioning approaches and preserves innate immunity, thymic integrity and enables quicker recovery of adaptive immune cells while avoiding loss of vascular integrity, undesirable anemia, and prolonged cytopenias. Given these advantages, CD45-IT may be useful for conditioning of non-malignant transplant patients. DisclosuresHoggatt:Harvard University: Patents & Royalties.

Early NK Cell Proliferation After Umbilical Cord Blood Transplantation Is Associated With Superior Disease-Free Survival Due To Reduced Leukemia Relapse

Compared to traditional chemotherapy, allogeneic hematopoietic cell transplantation (allo-HCT) has the potential of triggering graft vs. leukemia (GVL) reactions that make this procedure uniquely curative. Despite this, relapse remains the most common cause of treatment failure. Early after allo-HCT the donor immune system reconstitutes within the host and natural killer (NK) cells are the earliest lymphocyte population to recover. Previous studies by us and other investigators have linked rapid lymphocyte recovery and/or high NK cell numbers early after transplant with less relapse. Mechanistically, the reasons for this are unknown. We hypothesized that NK cell proliferation would be associated with allo-HCT outcomes. In a large data set with short-term follow-up we compared stem cell source to NK cell proliferation at Day 28 after transplantation (using Ki67+ staining). In patients undergoing autologous (n=117), sibling (n=57), single umbilical cord blood (sUCB) (n=62) or double umbilical cord blood (dUCB) (n=50) transplantation there were significant differences in the median NK cell proliferation (2.1%, 3.3%, 3.8%, and 19.2%, respectively, p&lt;0.0001). These results suggest that NK cell proliferation is increased when patients are transplanted with stem cell sources that have an increased number of HLA mismatches (dUCB). We next examined factors extrinsic to the NK cells to determine whether differences in the four patient groups were due to these factors. Regulatory T cells (Tregs) are known to negatively regulate the proliferation and activation of a variety of cell types, including NK cells. We utilized the percentage and absolute number of Tregs (defined as CD4+CD25highFoxP3+CD127low) at Day 28 (available for a subset of the above patient samples) and correlated it with the percentage of proliferating NK cells at Day 28 after allogeneic transplant (n=212). The median percentage of Tregs within the lymphocyte fraction was 1.49%. In patients with higher than 1.49% Tregs, the mean NK cell proliferation was 10.8% +/-16.4. In contrast, patients with lower than 1.49% Tregs had a mean NK cell proliferation of 21.2% +/-24.3 (p=0.0004). A nearly identical trend was observed for the absolute numbers of Tregs, suggesting that Tregs may play an extrinsic role in NK cell proliferation after allo-HCT. To further address differences in lymphocyte proliferation and clinical outcomes, we used Ki67 staining to assess T cell (CD4+ and CD8+) and NK cell (CD3-CD56+) proliferation at Day 28 in a different subset of patients undergoing dUCB transplant with acute leukemia or MDS (80%) or other malignant disorders using either myeloablative (42%) or reduced intensity conditioning (58%) and a median of one year follow-up (n=53). There was no association of transplant outcomes with T cell (CD4 or CD8) proliferation. In the NK cell compartment there was a wide range in the percentage of proliferating NK cells (0-86%), with a median of 20%. Patients were segregated into high (&gt;20%) and low (&lt;20%) NK cell proliferating groups and assessed for cytokine levels and transplant outcomes. At Day 28 soluble IL15 levels were not different between high and low NK cell proliferating patients. There was no significant association between NK cell proliferation and the probability or time to neutrophil recovery (p=0.15) or treatment related mortality (p=0.88). Excluding the 14 patients who developed aGVHD prior to day of NK cell assessment (Day 28), the high NK proliferating group had a trend toward a higher cumulative incidence of aGVHD (46% vs. 25%, p=0.17). Using multivariable analysis to control for a variety of patient and disease-associated variables, patients with high NK cell proliferation had significantly better disease-free survival (HR=0.29, 95%CI=0.12-0.71, p=0.01) (Figure 1). Strikingly, patients with high NK cell proliferation were 4-times less likely to relapse (HR=0.24, 95%CI=0.08-0.77, p=0.02). Collectively, these studies show that early NK cell proliferation is associated with superior outcomes after dUCB transplantation, due to reduced relapse, and that this is partly modulated by Tregs. Prospective strategies to increase NK cell proliferation may be of therapeutic benefit to improve transplant outcomes.Figure 1Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant.Figure 1. Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant. Disclosures: Wagner: Novartis: Research Funding. Miller:Coronado Biosciences: Scientific Advisory Board Other.

Elevated Absolute Lymphocyte Counts Following Acute Graft Vs. Host Disease Treatment Is Associated with Therapeutic Responses and Lower Non-Relapse Mortality Following Umbilical Cord Blood Transplant,

Abstract 4085Acute graft vs. host diseases (aGVHD) is a life threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). In a large series of patients undergoing allo-HCT (n=863) we recently demonstrated that following the initiation of corticosteroids, a clinical response at day 28 was associated with an increased likelihood of overall treatment response and a reduction in treatment related mortality (TRM) (MacMillan, Blood, 2010). We have also shown that patients who have rapid lymphocyte recovery following umbilical cord blood transplantation (UCBT) have improved outcomes (Burke, BBMT, 2010). To date, no study has addressed the influence of absolute lymphocyte count (ALC) on aGVHD treatment responses. We hypothesized that high ALC might predict favorable responses to aGVHD therapy. To test this hypothesis we reviewed the ALC at the time of aGVHD diagnosis and weekly following the initiation of corticosteroid therapy (48 mg/m2 × 14 days followed by a 10% taper over 8 weeks). ALCs were collected on patients who had clinical laboratory data available in the electronic medical record and who had available data on clinical response to corticosteroid therapy. There were 211 patients transplanted at our center from 2001–2007 who fit the above criteria. Median age was 42 years (range 0.2–69 yrs). Thirty nine patients (19%) were <18 years of age and 135 (64%) were >35 years old. The majority of patients underwent UCBT (n=134, 64%) and most others received a sibling PBSC transplant (n=71, 34%). Myeloablative conditioning was used in 55% of patients. GVHD prophylaxis was mainly with CSA/MMF (n=142, 67%) or MTX/CSA (n=42, 22%). Median time to aGVHD onset was 37 days (11–92). Patterns of aGVHD included skin only (n=91, 43%), gut only (n=36, 17%) or multi-organ involvement (n=82, 39%). At the time of GVHD diagnosis, ALC was not predictive of response to therapy. Likewise, there was no association with the ALC at D7 (after the start of steroid treatment) and therapeutic response. The D14 post-treatment ALC showed an association with clinical response at D28 (a time point previously associated with long-term responses and NRM). Patients with an ALC of 0–0.14, 0.15–0.34 and >0.35 at D+14 after corticosteroid therapy had a 40%, 54% and 68% chance of a clinical response at D28 (p>0.01). This translated into a reduction in non-relapse mortality for patients with higher ALC (0–0.14 vs. >0.15; 33% [19–47%] vs 23% [17–29%], p=0.04). Subgroup analysis showed that these observations were mainly driven by the myeloablatve UCB group. Treating ALC as a continuous variable and using a repeated measures approach, we observed that for every unit increase in ALC between the day of diagnosis and D14, there was a 2.26 higher increased likelihood of having a complete clinical response at D28 (p>0.001). Similar results were seen for NRM (OR=0.89 [0.81–9.8], p=0.01). In multivariate analysis, repeated measures of ALC (from D0-14) remained significant for both D28 clinical response (p=0.001) and NRM (p=0.05). Patients with steroid refractory aGVHD (n=17) were treated with ATG. Because steroid refractory aGVHD has poor outcomes and because ATG can negatively impact ALC, we removed these patients from the analysis to determine the impact of ALC. In this subgroup analysis, ALC at D14 after aGVHD treatment remained associated with D28 clinical response in multivariate analysis (OR=3.01, 95%CI[1.24–7.35], p=0.02). Using repeated analysis, the change in ALC from aGVHD treatment day D0 to 14 was associated with D28 treatment response (OR=4.42 [1.88–10.37], p<0.001). Collectively, these results show that absolute ALC at D14 and increases in ALC from the day of treatment to day 14were associated with D28 clinical response and NRM. ALC maybe a simple and cost effective method to monitor response to aGVHD therapy. Disclosures:No relevant conflicts of interest to declare.

Accelerated lymphocyte reconstitution and long-term recovery after transplantation of lentiviral-transduced rhesus CD34 + cells mobilized by G-CSF and plerixafor

Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor produces significant mobilization of CD34(+) cells in rhesus macaques. We sought to evaluate whether these CD34(+) cells can stably reconstitute blood cells with lentiviral gene marking. We performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34(+) cells transduced with a lentiviral vector, and these data were compared with those of G-CSF and stem cell factor mobilization. G-CSF and plerixafor mobilization resulted in CD34(+) cell yields that were twofold higher than yields with G-CSF and stem cell factor. CD123 (interleukin-3 receptor) expression was greater in G-CSF and plerixafor-mobilized CD34(+) cells when compared to G-CSF alone. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages, similar to animals who received G-CSF and stem cell factor-mobilized grafts. Lymphocyte engraftment was accelerated in animals receiving the G-CSF and plerixafor-mobilized CD34(+) cells. One animal in the G-CSF and plerixafor group developed cold agglutinin-associated skin rash during the first 3 months of rapid lymphocyte recovery. One year after transplantation, all animals had 2% to 10% transgene expression in all blood cell lineages. G-CSF and plerixafor-mobilized CD34(+) cells accelerate lymphocyte engraftment and contain hematopoietic stem cell capable of reconstituting multilineage blood cells. These findings indicate important differences to consider in plerixafor-based hematopoietic stem cell mobilization protocols in rhesus macaques.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

AbstractAbstract 1449Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Early Lymphocyte Recovery and Outcomes after Umbilical Cord Blood Transplantation (UCBT) for Hematologic Malignancies

Rapid lymphocyte recovery after bone marrow or peripheral blood transplantation is associated with improved survival. However, the impact of early lymphocyte recovery has not been examined after umbilical cord blood transplant (UCBT). We evaluated lymphocyte recovery in 360 consecutive patients with hematologic malignancy that underwent UCBT between 2001 and 2007. Uniform myeloablative (MA), reduced intensity conditioning (RIC) and graft-versus-host disease prophylaxis regimens were used. In multivariate analysis, an absolute leukocyte count (ALC) >200 × 10(6)/L at day 30 (n = 73) after MA conditioning was associated with superior 2-year overall survival (OS) (73% versus 61%; P = .02) (relative risk [RR]: 2.29; 95% confidence interval [CI]: 1.15-4.56), progression-free survival (PFS) (68% versus 54%; P = .05) (RR: 1.96; 95% CI: 0.99-3.86) and less transplant-related mortality (8% versus 28%, P < .01) (RR: 4.38; 95% CI: 1.65-11.60) compared to ≤200 × 10(6)/L (n = 43). Similarly, an ALC >200 × 10(6)/L at day 42 (n = 105) after RIC was associated with superior 2-year OS (59% versus 41%, P < .01) (RR: 2.10; 95% CI: 1.3-3.41) and PFS (46% versus 36%, P = .05) (RR: 1.58; 95% CI: 1.01-2.49) compared to ≤200 × 10(6)/L (n = 55). There was no significant relationship between ALC and relapse. Rapid lymphocyte recovery early after UCBT predicts better survival in patients with hematologic malignancies.

Impact of Cytomegalovirus (CMV) Reactivation after Umbilical Cord Blood Transplantation

This study investigated the impact of pretransplant cytomegalovirus (CMV) serostatus and posttransplant CMV reactivation and disease on umbilical cord blood transplant (UCBT) outcomes. Between 1994 and 2007, 332 patients with hematologic malignancies underwent UCBT and 54% were CMV seropositive. Pretransplant recipient CMV serostatus had no impact on acute or chronic graft-versus-host disease (aGVHD, cGVHD), relapse, disease-free survival (DFS), or overall survival (OS). There was a trend toward greater day 100 treatment-related mortality (TRM) in CMV-seropositive recipients (P=.07). CMV reactivation occurred in 51% (92/180) of patients with no difference in myeloablative (MA) versus reduced-intensity conditioning (RIC) recipients (P=.33). Similarly, reactivation was not influenced by the number of UCB units transplanted, the degree of HLA disparity, the CD34(+) or CD3(+) cell dose, or donor killer cell immunoglobulin-like receptor (KIR) gene haplotype. Rapid lymphocyte recovery was associated with CMV reactivation (P=.02). CMV reactivation was not associated with aGVHD (P=.97) or cGVHD (P=.65), nor did it impact TRM (P=.88), relapse (P=.62), or survival (P=.78). CMV disease occurred in 13.8% of the CMV-seropositive patients, resulting in higher TRM (P=.01) and lower OS (P=.02). Thus, although recipient CMV serostatus and CMV reactivation have little demonstrable impact on UCB transplant outcomes, the development of CMV disease remains a risk, associated with inferior outcomes.

Rapid Lymphocyte Recovery And Intravenous Immunoglobulin Independence In Children With Severe Combined Immunodeficiency (SCID) Undergoing Allogeneic Stem Cell Transplantation With Reduced Intensity Conditioning (RIC)

Allogeneic SCT is curative for patients with SCID. We evaluated a RIC SCT approach in these patients for engraftment, toxicity and immune reconstitution. Between 2000 and 2008, RIC transplant was performed in 14 SCID patients (8 males and 6 females; median age 5 months (range 1.1–98.3)). Median time from diagnosis to transplant was 1.8 months (range 1.0–96.3). Donor sources included: HLA-matched related donor peripheral blood stem cells (PBSC) (n = 1), unrelated donor PBSC (7), related cord blood (CB) (1) and unrelated CB (6).

Rapid T Cell Recovery and Possible Auto-GVHD Following Adoptive Transfer of Ex-Vivo Costimulated Autologous T Cells at Day +2 Post-Transplant.

Retrospective studies suggest that rapid lymphocyte recovery following autologous stem cell transplants (SCT) may be associated with better outcomes. Previously we showed that adoptive transfer of in-vivo vaccine-primed and ex-vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at about day 14 post-transplant increased CD4 and CD8 T cell counts at day 42 post-transplant and induced pneumococcal conjugate vaccine-directed T and B-cell responses [Rapoport et al, Nature Medicine, 2005]. In 2 current studies, we are further investigating the impact of ex-vivo costimulated autologous T cells on vaccine responses after SCT. In the first study, we are investigating whether a similar strategy of pre- and post-transplant immunizations along with an early infusion of vaccine-primed ex-T can induce responses to a putative tumor vaccine composed of 4 HLA-A2-restricted peptides derived from survivin and hTERT in pts undergoing SCT for myeloma. In the second (randomized) trial, the impact of early ex-T on immune recovery and vaccine reponses is being tested in pediatric neuroblastoma pts. Compared to the previous study, two methodologic changes were made:i.The target number of T cells infused was raised 5-fold to 5 x 1010 (109/kg)ii.T cells were infused on day + 2 to take greater advantage of homeostatic expansion mechanisms.Patients were monitored for delayed hematopoietic recovery because of this switch to early ex-T and the fact that survivin and hTERT are also expressed in hematopoietic stem cells. At the time of submission, 16 adult and 30 pediatric patients have been enrolled on these trials of whom 11 and 21, respectively, are evaluable for post-transplant hematopoietic and T-cell recovery. On the myeloma trial, the mean # of T cells infused was 3.95 x 1010 with 96% viability and a CD4/CD8 ratio of 1.8:1. At day 14 post-transplant, the median CD4 count was 1951/mcl (range 651–7668) and the median CD8 count was 4117/mcl (range 1499–39,354). The median # days to achieve an absolute neutrophil count (ANC) > 500 was 12 (range 11–14) and the median # days to achieve a PLT count >20,000/mcl was 13 days (range 0–28). Similarly, in the pediatric cohort, median CD4 and CD8 counts at day 30 were 1500 and 2100/mcl, respectively, compared to 22 and 14 in a group of pts who did not receive d+2 ex-T, with no impact on engraftment. 1 adult and 3 pediatric pts also developed an “engraftment syndrome” characterized by GHVD-like features with or without fever. The adult pt with day 14 CD4 and CD8 counts of 2,724 and 11,571 cells/mcl had clinical and histologic features of (autologous) gut GVHD. 3 pediatric pts developed pruritic rashes clinically and pathologically indistiguishable from GVHD within 14 d of ex-T infusion, with fever seen in 1. In the adult and 1 pediatric pt, steroid treatment led to complete resolution of symptoms. These combined data sets demonstrate that robust CD4 and CD8 T cells counts can be achieved as early as day 14 post-SCT when adults or children receive ex-T at day +2 post-SCT without exogenous IL-2 or other cytokine support. It appears that a subset of patients develop a T cell “engraftment syndrome” similar to autologous GVHD. The mechanisms responsible for this rapid immune cell recovery are currently under investigation.

T-cell reconstitution and expansion after hematopoietic stem cell transplantation: ‘T’ it up!

Adoptive immunotherapy is the isolation and infusion of antigen-specific or nonspecific lymphocytes. Adoptive therapy with T cells may have a role in replacing, repairing, or enhancing immune function damaged by cytotoxic therapies, and rapid lymphocyte recovery may improve outcome after autologous and allogeneic stem cell transplantation (SCT). Recently, a plethora of information on the basic mechanisms of T-cell biology and regulation of cellular immune responses has emerged, permitting the development of new forms of adoptive cell therapy. Efficient ex vivo culture method for T-cell subsets affords the possibility of adoptive transfer of T cells engineered with enhanced capacity for central memory, effector cytotoxicity, Th1, Th2, veto cell, and T regulatory functions. Studies show that homeostatic T-cell proliferation is important for effective adoptive immunotherapy and pretreatment with chemotherapy may enhance the effects of infused T cells. Replicative senescence, in part due to telomere erosion, likely limits successful adoptive immunotherapy, though it may be possible to maintain T-cell pools by enforced expression of telomerase. Clinical trials now demonstrate that it is possible to enhance immune reconstitution after SCT with cytokines or infusions of ex vivo costimulated expanded T cells. These data all support the premise that adoptive therapy can accelerate reconstitution of cellular immunity with enhanced antitumor effects following SCT.

A Phase I/II Study of Xcellerated T Cells™ after Autologous Peripheral Blood Stem Cell Transplantation in Patients with Multiple Myeloma.

Background: Previous studies have demonstrated a correlation between survival and lymphocyte recovery following autologous transplantation in subjects with multiple myeloma and other malignancies (Porrata et al., Blood 2001). We initiated a trial in the transplant setting to evaluate the activity of T cells activated and expanded ex vivo with the Xcellerate™ Process, which uses anti-CD3 and anti-CD28 antibody-coated magnetic beads (Xcyte™ -Dynabeads®).Methods: Following induction therapy, patients underwent leukapheresis to collect peripheral blood mononuclear cells for the Xcellerate Process. Patients then underwent stem cell mobilization and collection, followed by high dose melphalan (200 mg/m2). Three days following peripheral blood stem cell infusion, subjects received 50–100 x 109 Xcellerated T Cells.Results: 36 subjects were treated. The median last f/u visit is 180 days post-transplant (range 90–450). A WaveBioreactor-based Xcellerate III Process, which was instituted in the last 18 subjects, resulted in 249 ± 90 fold (mean ± SD) T cell expansion. There were 93.6 ± 0.8 x 109 cells infused, which were 97.6 + 4.0% T cells. There were no Grade 3 or 4 acute infusional toxicities. Days of neutropenia and thrombocytopenia were 5 (3–43) and 4.5 (0–128) respectively [median (range)]. There were a median of 2 (range 0–14) units of packed red blood cell transfusions in 18/31 (58%) of subjects and a median of 0 (range 0–22) platelet transfusions in 15/31 (48%) of subjects. There were serious or Grade 3 infections in 5/29 (17%) of subjects, and mucositis in 5/29 (17%) of subjects (all ≤ Grade 2). Median days of hospitalization were 16 (range 10–70). Lymphocyte recovery was rapid, with counts reaching > 500/mm3 generally within 1–2 days following T cell infusion. Historically, lymphocyte recovery to > 500/mm3 usually does not occur for 3 or more weeks post-transplant. The rapid lymphocyte recovery included both CD4+ and CD8+ T cells. The mean (± SEM) CD4+ T cell count at 90 days post-transplant was 1,210 ± 80/mm3, significantly higher than that for historical controls receiving the same treatment regimen without Xcellerated T Cells (198 ± 72). The T cell receptor repertoire measured 25 days after the Xcellerated T Cell infusion demonstrated a normal pattern (n = 4/5). This is in contrast to the severe skewing of T cell receptor diversity observed in myeloma subjects following standard autologous stem cell transplantation (Mariani et al, BJH 2001). In 35 evaluable patients, preliminary results demonstrated 6% CRs, 46% VGPRs, 34% PRs, and 11% with PD, using the M-protein at diagnosis as reference. There have been no reported deaths to date.Conclusions: In multiple myeloma subjects, administration of Xcellerated T Cells following high-dose chemotherapy and autologous stem cell transplantation leads to rapid lymphocyte recovery and appears to restore a normal T cell receptor repertoire. The majority of subjects achieve clinical responses in the autologous transplant setting.

A phase I/II study of Xcellerated T Cells after autologous peripheral blood stem cell transplantation in patients with multiple myeloma

2540 Background: Previous studies have demonstrated an association between survival & lymphocyte recovery following autologous transplantation (Porrata et al., Blood 2001). We have initiated a trial in the transplant setting to evaluate the activity of T cells activated and expanded ex vivo with the Xcellerate Process, which uses anti-CD3 and anti-CD28 antibody coated magnetic beads. Methods: Following induction therapy, patients undergo leukapheresis to collect peripheral blood mononuclear cells for the Xcellerate Process. Patients then undergo stem cell mobilization and collection, followed by high dose melphalan (200 mg/m2). Three days following peripheral blood stem cell infusion, patients receive 50–100 x 109 Xcellerated T Cells. Results: 36 patients have been treated with median f/u of 143 days post transplant (range 10–353). Using a new bioreactor process instituted during the trial, T cells expanded 268 ± 101 fold (mean + SD), with 93.6 ± 0.8 x 109 cells infused, & final product 98.5 + 2.3% T cells (n = 19). There have been no Grade 3 or 4 acute infusional toxicities. Days of neutropenia and thrombocytopenia have been 5 (3–43) and 4 (0–88) respectively [median (range)] . Lymphocyte recovery has been rapid, with counts reaching > 500/mm3 generally within 1–2 days following T cell infusion. Historically, lymphocyte recovery to > 500/mm3 usually does not occur for 3 or more weeks post-transplant. The T cell repertoire measured 25 days following T cell infusion demonstrates a more normal pattern than prior to T cell infusion (n = 5). This is in contrast to the severe skewing of T cell receptor diversity normally seen in myeloma patients following autologous stem cell transplantation (Mariani et al, BJH 2001). In 24 patients with median f/u of 135 (45–270) days, preliminary results demonstrate 13% CRs, 42% VGPRs, 33% PRs, and 13% with PD, using the pre-induction M-protein as reference. Conclusions: Xcellerated T Cell therapy leads to rapid lymphocyte recovery and appears to restore a normal T cell receptor repertoire. Additional follow-up is needed to determine if this will translate to improved clinical outcomes. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Xcyte Therapies, Inc. Xcyte Therapies, Inc. Xcyte Therapies, Inc.

Response duration and recovery of CD4+ lymphocytes following deoxycoformycin in interferon-alpha-resistant hairy cell leukemia: 7-year follow-up.

The long-term outcome of patients with hairy cell leukemia resistant to interferon-alpha (IFN-alpha) following treatment with deoxycoformycin (DCF) was examined, and the kinetics of recovery of lymphocyte subsets and factors influencing the rate of recovery investigated. Between May 1986 and May 1989, 15 patients with histologically confirmed hairy cell leukemia resistant to IFN-alpha received DCF 4 mg/m2 every 2 weeks with 12 cycles planned. All 15 patients were evaluable for response and have been followed for a median of 88 months (range, 72 to 106 months) from the start of therapy. Fourteen patients responded to DCF, all attaining complete remission (CR) (response rate 93%; 95% confidence interval, 69% to 100%). Seven patients have developed recurrent disease after 45 to 74 months. Using the method of Kaplan and Meier, the median remission duration is 74 months and, at 8 years, 46% (95% confidence interval, 33% to 59%) of patients are projected to be in ongoing CR. The seven relapsing patients have responded to treatment with 2-chlorodeoxyadenosine (2-CdA) and all 15 patients remain alive. After DCF, nadir CD4+ and CD8+ lymphocyte counts were significantly lower than prior to therapy (P < 0.0001 and P = 0.05, respectively), but returned to baseline levels during follow-up. Median times to attainment of the lower limit of the normal range of CD4+ and CD8+ lymphocytes were 54 and 36 months, respectively. Those patients who had previously undergone splenectomy (n=7) had higher baseline CD4+ (P= 0.073) and CD8+ (P= 0.043) lymphocyte counts and more rapid recovery of both CD4+ (P= 0.027) and CD8+ lymphocyte counts (P = 0.016) than non-splenectomized patients. One elderly patient (age, 78 years) was diagnosed with subsequent malignancy. No late opportunistic infections were observed. Resistance to IFN-alpha does not impair subsequent responsiveness of patients with hairy cell leukemia to treatment with DCF. Responses are durable and without evidence of long-term sequelae. CD4+ and CD8+ lymphocyte subsets recover slowly without clinical manifestations of immunodeficiency. Splenectomized patients appear to have higher baseline lymphocyte counts and more rapid lymphocyte recovery following treatment with DCF.