Recovery Of Lymphocyte Subsets Research Articles

Dynamic Effects of Thoracic Irradiation on Immune Status of Organs in and out of Radiation Field in Mice

Thoracic irradiation involves large blood vessels and hematopoietic tissues which was easily inducing lymphopenia. Lymphocytes are important components of immune response and lymphopenia is associated with poorer survival. However, the dynamic recovery of lymphocyte subsets after thoracic irradiation were rarely reported in the literature. This study focused on dynamic changes of lymphocytes subsets of mice after receiving thoracic irradiation in and out of radiation field. C57BL/6 male mice aged 6-8 weeks received 2Gy*5fx whole thoracic irradiation and were sacrificed on the 1 day, 7 days, 15 days, 25 days after irradiation. Peripheral blood was harvested for mature lymphocyte subsets (B cells, T cells, CD4+T cells, CD8+T cells, NK cells) detection by performing flow cytometry, while thymus, femur bone marrow and sternal bone marrow for hematopoietic stem cells (Long-term Hematopoietic Stem Cell [LT-HSC], Short-term Hematopoietic Stem Cell [ST-HSC]) and progenitor cells (Multipotent Progenitor [MPP], Common Lymphoid Progenitor [CLP]). Absolute counts method was used in flow cytometry and data analysis were performed by scientific 2-D graphing and statistics software. We found that the lymphocyte subsets were significantly reduced in peripheral blood of irradiated mice (all p<0.05). The mature lymphocyte subsets returned to normal 2-3 weeks after irradiation. After radiation, thymus weight was significantly decreased (p<0.0001). With the recovery of thymus weight, the number of hematopoietic stem cells returned to the baseline. For the femur bone marrow (out of the radiation field), hematopoietic stem cells and progenitor cells showed a proliferation tendency. MPP significantly increased in the 1day after irradiation (p = 0.0347) and LT-HSC increased in the 8 days (p = 0.0011). However, although ST-HSC and CLP were slightly increased after irradiation, there were no statistically significance. The sternal bone marrow (in the radiation field) reached the lowest value 1day after irradiation (all p<0.0001). Although hematopoietic stem cells and progenitor cells recovered gradually, but still far away from the level of the control group (all p<0.05). The recovery of lymphocytes in the peripheral blood might mainly come from the supplement of bone marrow out of the irradiation field, while bone marrow in the field suffered serious destruction which did slightly help for lymphocytes recovery.

Distinct Patterns in the Reconstitutions of Treg and T-Helper Subsets after Autologous Hematopoietic Stem Cell Transplantation in Juvenile-Onset Systemic Sclerosis Patients

Systemic sclerosis (SSc) is a refractory autoimmune disease characterized by diffuse fibrosis and vasculopathy affecting the skin, joints, and internal organs. Most immunosuppressive drugs have failed to show long-term benefit in randomized trials. In recent years, autologous hematopoietic stem cell transplantation (ASCT) has been performed to treat multiple sclerosis and SSc, however, the reconstitutions of immune components post-ASCT in SSc have not been fully described. Subjects and Methods: We tested a total of 13 samples from 4 consenting SSc patients (3 females, 1 male) receiving IRB-approved immunoablation (TBI 600cGy) with lung shielding and Cytoxan (n=3) or Thiotepa (n=1) +/- Alemtuzumab followed by autologous CD34+ selected, cryopreserved peripheral blood hematopoietic stem cells transplantation. Their median age was 18.5-years-old (range 15.2- to 21.4-years-old). Peripheral blood samples were collected at baseline pre-ASCT, and 3, 6, 12, 24-months post-ASCT. By flow cytometry, we examined lymphocyte and monocyte subset recovery with additional focus on the FOXP3+Treg and T-helper subset reconstitution. Results and Discussion: All 4 subjects experienced significant improvement in several clinical and quality of life parameters, especially in their skin disease evident within weeks after ASCT. T-cell lymphopenia was profound at 3-months; however, the recovery was evident by 6-months reaching pre-ASCT levels by 2-years post-ASCT (Fig 1A). Natural killer (NK) lymphocytes and monocytes attained the normal values by 3-months and after (Fig 1A). The FOXP3+Tregs that appeared post-ASCT displayed distinct features. There was an increased percentage of FOXP3+Tregs in total CD4+T cells during the early stage (3- to 6-months) post-ASCT (Fig 1B), leading to an increase in the ratio of Treg/Tcon (Fig 1B). The higher proportions of proliferating Tregs as measured by Ki67+FOXP3+CD4+/CD25+/CD127- T cells (Fig 1C) likely led to their increased numbers. This trend was sustained until the last evaluation at 2-years post-ASCT (Fig 1C). In contrast, CD4+Tcons showed a relatively lower proliferation rate, leading to a higher ratio of Ki67+Treg/Ki67+Tcon compared to pre-ASCT values (Fig 1C). Notably, there was an increased proportion of Tregs expressing the skin homing marker cutaneous lymphocyte-associated antigen (CLA) at 3-months lasting until the last test at 2-years post-ASCT (Fig 1D), whereas CLA+/CD4+Tcons were less plentiful. The reconstitutions of T-helper (Th) subsets were also examined. Due to lymphopenia at the early stage post-ASCT, Th subsets were identified according to their chemokine receptor expression (Mahnke et alCytometry 2013;83(5):439-440). Gradually decreased numbers of Th2, Th9, and Th17 were observed in the peripheral pool (Fig 1E) contributing to a decline of their proportions in total CD4+T (Fig 1F). Nevertheless, absolute number of Th1 and Th22 cells returned to levels seen pre-ASCT without any recurrence or flare of SSc symptoms. The CD45RO+ memory phenotype was dominant during the early stages post-ASCT in all subsets (data not shown). Summary: Successful reconstitution of lymphocytes and monocytes post-ASCT was observed in available SSc patients. Post-ASCT circulating FOXP3+Tregs displayed distinct features where the increased number led to an elevated Treg/Tcon ratio in the peripheral pool. Amongst Tregs, the skin homing CLA+ subset was further enriched correlating with improved skin scores in all patients. Improved peripheral proliferation and increased expression of CLA+ Tregs may provide protection from skin disease recurrence despite parallel Tcon reconstitution. The decrease of the Th2/Th9/Th17 subsets in the peripheral pool post-ASCT possibly contributes to the clinically less autoimmune phenotype. These results need to be validated in future patients. Acknowledgements: We thank all participating patients, nurses, physicians, and scientists in this study for their contributions. The study was supported by the "Idea Development Award" from the Department of Defense - Scleroderma Research Program (Award number W81XWH-21-1-0782 and W81XWH-21-1-0783). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune Recovery Post BCMA CAR-T: Implications for Infection Prophylaxis and Vaccinations

Immune Recovery Post BCMA CAR-T: Implications for Infection Prophylaxis and Vaccinations

Donor T Cells Genetically Modified with a Novel Suicide Gene (inducible Caspase 9, iC9) Expand and Persist over Time after Post-Allograft Infusion in Patients Given αβ T-Cell and B-Cell Depleted HLA-Haploidentical Allogeneic Stem Cell Transplantation (αβ haplo-HSCT) Contributing to Accelerate Immune Recovery

Background: αβ haplo-HSCT is an effective option for children with non-malignant disorders (Bertaina et al., Blood 2014) or acute leukemia (Locatelli et al. Blood 2017) and in need of an urgent allograft. In patients given this type of HSCT, recovery of adaptive immunity is, however, suboptimal. With the aim of accelerating reconstitution of adaptive immunity, we evaluated an innovative approach based on post-transplant infusion of titrated numbers of donor αβ T cells, transduced with the suicide gene iC9 (BPX-501 cells). Since the transduced gene contains sequences for the CD19 marker, BPX-501 cells are CD3+/CD19+ and, thus, can be easily tracked in peripheral blood. We report data on immune recovery of children transplanted at Bambino Gesù Children's Hospital, Rome, and enrolled in a multicenter prospective phase I-II trial on BPX-501 cells (sponsored by Bellicum Pharmaceuticals®, ClinicalTrials.gov identifier: NCT02065869).Patients and study design: Included in this analysis were 108 children. Median age at transplant was 5.6 years (range, 0.3-18) and the male/female ratio was 61/47. Patients were subdivided according to the original disorder in 5 groups: group A (53 children with acute leukemia), group B (22 with primary immune-deficiency, PIDs), group C (14 with erythroid disorders), group D (7 with Fanconi anemia) and group E (12 with other diseases). No patient was given any post-transplant graft-versus-host disease (GvHD) prophylaxis. Nine children were enrolled in the phase I portion of the trial consisting of 3 cohorts, receiving escalating doses of BPX-501 cells of 2.5x105, 5x105, and 1x106 cells/kg, respectively. The remaining 99 patients included in the phase II portion received 1x106 BPX-501 cells/kg. Per protocol, BPX-501 cells were to be infused within the first month after αβ haplo-HSCT. We evaluated the absolute number of circulating CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, and NK cells at 1, 2, 3, 6, 9, 12 and 24 months after αβ haplo-HSCT. We also analyzed the number of BPX-501 cells, αβ T cells and γδ T cells at the same time points. The impact of patient gender, age (0-2, 2-6, 6-12 and >12 years), original disease, use of total body irradiation (TBI) during conditioning regimen, occurrence of cytomegalovirus (CMV) reactivation, of acute GvHD and of leukemia relapse (only in group A patients), on recovery of different lymphocyte subsets was investigated, as well. Data were expressed as mean value+SEM (standard error of mean). Six patients given Rimiducid, the agent activating iC9, were censored at time of drug infusion.Results: After infusion, BPX-501 cells expanded progressively reaching a peak at 9 months after the allograft, when their mean value was 144+36/μL. In the 16 patients with 2-year follow-up, the mean number of BPX-501 cells was still 62+23/μL. Details on expansion/persistence of BPX-501 cells, as well as on recovery of CD3+, CD4+ and CD8+ T cells, are shown in Figure 1A and 1B, respectively. A mean number of CD3+ cells greater than 1.000/μL was reached at 6 months after transplantation. Figure 1C depicts the progressive recovery of B cells. NK and γδ T cells promptly recovered after the allograft; the number of αβ T cells overcomes that of γδ T cells at 2 months after transplant. CMV reactivation promoted a statistically better expansion of BPX-501 cells (see also Figure 1D). This effect was paralleled by an improved recovery of both CD3+ and αβ T cells in patients who experienced CMV reactivation. A statistically higher number of both CD3+ and αβ T cells was observed at 6 and 12 months after αβ haplo-HSCT in patients who did not receive TBI. Neither acute GvHD nor leukemia recurrence occurrence statistically affected the number of BPX-501 cells. Children with an age comprised between 0-2 years or with PIDs had a better recovery of both CD3+ and αβ T cells at 6, 9 and 12 months after the allograft.Conclusions: Our results indicate that BPX-501 cells infused after αβ haplo-HSCT expand in vivo and persist over time, contributing to fasten adaptive immunity recovery. Their peak of expansion is reached at 9 months after the allograft, but they are consistently detected also at 2 years after infusion. CMV reactivation is the main driver of BPX-501 cell expansion, this finding suggesting that BPX-501 cells cooperate to the viral clearance. Overall, the whole pattern of immune recovery of these 108 children is improved as compared with that of patients given αβ haplo-HSCT without BPX-501 cell infusion. [Display omitted] DisclosuresO'Neill:Bellicum Pharmaceuticals: Other: contractor. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders . Foster:Bellicum Pharmaceuticals: Employment, Other: stockholders .

Relationship between Changes of Lymphocyte Subsets and Early Hematologic Response in Immunosuppressive Therapy of Severe Aplastic Anemia Patients

To explore the relationship between the change of lymphocyte subsets before and after immunosuppressive therapy (IST) with disease severity of severe aplastic anemia (SAA) and hematologic response to IST. The clinical data of 94 patients with SAA/VSAA treated by r-ATG and CsA in our hospital from December 2009 to October 2011 was analyzed retrospectively. Among them, 26 patients who had sequential data of lymphocyte subsets and cytokines before and after treatment were enrolled. The relationship between lymphocyte subsets, cytokine level before IST and disease severity, as well as the relationship between changes if lymphocyte subsets, changes of cytokine and the HR after IST for 6 months was analyzed. There were no statistical differences in the ratio and absolute count of lymphocyte, the ratio and absolute count of each lymphocyte subsets, including CD3+T cells, CD3+CD4+T cells, CD3+CD8+T cells, CD3-CD16+1CD56+NK cells, and CD19+B cells, and the level of cytokines, such as IL-1, IL-2, IL-4, IL-6 and TNF-α before IST between SAA and VSAA groups. Also, there were no statistical difference in the levels of above-motional parameter at 3 and 6 months after IST. The ratio and absolute count of Lym, absolute count of CD3+T cells, absolute count of B cells and IL-2 level in response group after IST for 3 and 6 months was significant lower than those before IST. However, only ratio of Lym showed significant decrease after IST for 3 and 6 months in non-response group. After IST for 3 months, the absolute count of CD3+T and CD4+T cells in response group was significant higher than those in non-response group. The hematopoietic recovery and early hematologic remission may be affected by the intensity of immune suppression reflected from the changes of lymphocyte subsets and the immune reconstruction reflected from the recovery of lymphocyte subsets. The immune reconstruction is most significant within 3 months after IST.

Dynamic changes of lymphocyte subsets during multimodal treatment of patients with inoperable stage III NSCLC.

e21011 Background: Lymphocytopenia is associated with deterioration of patient survival in solid cancers treated with concurrent chemo/radiotherapy (cCRT). A prospective analysis of peripheral blood mononuclear cells during cCRT in inoperable stage III NSCLC patients was performed to determine dynamic changes of individual lymphocyte subsets. Methods: Twenty-one patients were prospectively enrolled in this study. Eighteen patients received platinum-based cCRT, seven of them received, additionally, concurrent and/or sequential immune checkpoint-inhibition (ICI). Thoracic irradiation (TRT) was delivered with median total dose of 62Gy (31 daily fractions of 2Gy) in all patients. Peripheral blood was collected 5-10 days before treatment (A1), three weeks after start of cCRT (A3), on the last day of TRT (RTend) and during follow-up. Samples were analyzed using polychromatic flow cytometry. Results are reported for time-points A1, A3 and RTend. Results: Sixteen patients met final analysis criteria, 50% of them received concurrent and/or sequential ICI. All patients developed severe lymphocytopenia; in 81% of them lymphocyte nadir was documented at A3. Lymphocyte subsets, B cells, T cells (CD4, CD8), regulatory T cells, and NK cells, decreased with medians between 99.9% and 59%. Lymphocyte nadir was independent of the absolute numbers of immune cells that a patient had before start of cCRT or whether additional ICI was applied. From A3 to RTend, all lymphocyte subsets started to recover in patients treated with cCRT alone, while they remained low in patients who received additional ICI. The ratios of CD4/CD8 and CD8/Treg cells did not change during treatment (A1 to RTend) and was not different between the patients treated with or without ICI. However, the fraction of PD-1 cells among CD8 T-cells decreased in patients treated with ICI and remained low until RTend (range 0.55%-13.8%). In contrast, in 50% of patients treated with cCRT alone, PD-1 T-cell among CD8 T-cells increased and remained high (range 6.8%-46.3%) until RTend. Conclusions: Delayed recovery of lymphocyte subsets in peripheral blood was observed in patients treated with cCRT combined with concurrent or sequential ICI. A decrease of PD-1 T-cells among CD8 T-cells was described exclusively in patients treated with additional ICI.

PF751 POST-TRANSPLANT CYCLOPHOSPHAMIDE AND SIROLIMUS IN HAPLOIDENTICAL TRANSPLANT WITH PBSC GRAFTS: RESULTS IN 151 PATIENTS

Background: The use of haploidentical hematopoietic stem cell transplantation (HSCT) with post- transplant cyclophosphamide (PT-Cy) and calcineurin inhibitors (CNI) as graft-versus-host disease (GVHD) prophylaxis is rapidly increasing. Aims: Herein we describe long-term outcomes of haploidentical HSCT from PBSC grafts, using intensified myeloablative conditioning, and GVHD prophylaxis with PT-Cy and sirolimus. Methods: From 2012 to 2017, 151 adult patients received haploidentical HSCT for high-risk hematological malignancies, mainly acute myeloid leukemia (n = 92). Disease risk index (DRI; Armand et al) was low-intermediate in 52, high in 64 and very high in 35 patients. At HSCT, 58% of patients were not in CR; 25% of patients received a previous allogeneic HSCT. Conditioning was myeloablative in 123 and reduced-intensity in 28 patients. It was based on treosulfan-fludarabine, and 116 patients receive a regimen intensified by a 2nd alkylating agent (melphalan or thiotepa). The majority of patients (n = 148) received unmanipulated PBSCs. All pts received PT-Cy (50 mg/kg/day) on days 3 and 4. Sirolimus was given from day 5, and withdrawn 3 months after HSCT. Mycophenolate mofetil (MMF) was initiated on day 5, and withdrawn on day 30. All patients were treated according to current institutional programs upon written informed consent for transplant procedures and for the use of medical records for research. Results: Median follow up was 26 months (3–68). Median PBSC graft CD34+ and CD3+ cell doses were respectively 6x10^6/Kg (range, 5–8) and 2x10^8/Kg (range, 0.8–4.8). The majority of patients reached the neutrophil (86%) and platelet (77%) engraftment within 30 and 90 days after HSCT, respectively. Post-HSCT recovery of lymphocyte subsets was broad and fast, reaching more than 100/ml CD3+ T cells within a median of 33 days (20–799). Clinically relevant CMV and HHV-6 infections occurred in 63 and 60 patients, respectively. Invasive fungal diseases were reported in 14 cases. The cumulative incidence of grades II-IV and III-IV acute GVHD at 100 days was 35% and 20%, respectively. The cumulative incidence of chronic GVHD was 42% at 3 years; we observed severe chronic GVHD only in 16% of pts. The cumulative incidences of relapse and non-relapse mortality (NRM) were respectively 35% and 27% at 3 years. Three-year overall survival (OS) was 44% and progression free survival (PFS) 38%. The composite end point of GVHD-free/relapse-free survival (GRFS) was 24% at 3 years. Multivariate analysis confirmed the effect of DRI on transplant outcomes, with worse PFS in high and very high compared to low-intermediate DRI population. Summary/Conclusion: Extended follow-up in 151 high-risk patients confirms that Sirolimus and PT-Cy association as GvHD prophylaxis is feasible in haploidentical HSCT based on unmanipulated PBSC graft; DRI is still a major issue for transplant long-term outcome.

Identification of Lymphocyte Subsets Associated with Outcomes in Patients with Hematological Malignancy Following Allogeneic Stem Cell Transplantation: A Single Institute Study

Background: Immune reconstitution after allogeneic stem cell transplantation (SCT) is a complicated process influenced by factors such as preconditioning regimens, graft-versus-host disease (GVHD) prophylaxis, and grafts. We studied the association between the kinetics of lymphocyte subsets and transplant outcome to clarify the differences in immune reconstitution after hematopoietic cell transplantation according to stem cell sources and its clinical significance.Patients and Methods: Clinical data were collected from patients' medical charts at Kanagawa Cancer Center, Yokohama, Japan. Patients with hematological malignancies aged ≥18 years old who underwent SCT between April 2009 and December 2017 were initially selected. Those who died or experienced disease relapse before day 100 post SCT were excluded. We measured absolute lymphocyte count (ALC) and lymphocyte subsets by flow cytometry with antibodies against CD2, CD3, CD4, CD8, CD11b, CD11c, CD16, CD25, CD29, CD56, CD57, CD45RA, and CD45RO on days 100, 180, 365, and 730 post SCT.Results: The final cohort included 314 patients (acute leukemia, n = 249; myelodysplastic syndrome, n = 44; chronic myelogenous leukemia, n = 9; malignant lymphoma, n = 6; and others, n = 6). The median age was 51 (range: 18- 69) years, with 184 males and 130 females. The disease risk at transplantation was standard in 209 and high in 105 patients. Myeloablative preconditioning was administered to 114 and reduced-intensity preconditioning to 200 patients. Bone marrow transplantation (BMT), peripheral blood SCT (PBSCT), and cord blood transplantation (CBT) were performed in 121, 57, and 136 patients, respectively. A calcineurin inhibitor with short-term methotrexate was mainly used for GVHD prevention. The median follow-up of surviving patients was 869 (range: 103-3074) days. The 2-year overall survival (OS), cumulative incidence of relapse (CIR), and non-relapse mortality (NRM) in BMT, PBSCT, and CBT were 62%, 68%, and 76% (P = 0.023); 33%, 38%, and 27% (P = 0.068); and 17%, 16%, and 13% (P = 0.82); respectively. The 2-year cumulative incidence of chronic GVHD was 43% in BMT, 45% in PBSCT, and 28% in CBT (P = 0.027).There were significant differences between lymphocyte subset recovery and stem cell sources (Table). ALC; CD20+ B cell; CD4+, CD4+CD29+, CD4+CD45RO+, CD4+CD45RO− , and CD4+CD45RA+ T cell subsets; and CD3−CD56+ and CD16+CD57− natural killer (NK) cell subsets were significantly elevated in CBT compared with BMT and PBSCT at day 100 post SCT. Conversely, CD8+CD11b+ and CD8+CD11b− T cell subsets and CD3+CD56+ NKT cells were significantly lower in CBT than in BMT and PBSCT on day 100. Univariate analysis revealed that lymphocyte subsets exhibiting higher levels of CD20+ B cell; CD16+CD57− and CD3−CD56+ NK cells; and CD4+CD25+, CD4+CD29+, CD4+CD45RA+, CD8+CD11b+, and CD8+CD11b− T cell subsets at day 100 were associated with a better 2-year OS. There were strong correlations with a lower CIR and higher CD16+CD57+ and CD16+CD57− NK cell levels . A higher incidence of chronic GVHD was associated with lower levels of CD16+CD57− NK cells and CD4+CD25+ T cells and with higher levels of CD8+CD11b+ T cells and CD8+CD11b− and CD3+CD56+ NKT cells. Further, a lower NRM correlated with higher levels of CD20+ B cells and CD8+CD11b− T cells.The lymphocyte subsets were used for multivariate analysis. Favorable factors for better OS were higher levels of CD16+CD57− NK cells [hazard ratio (HR), 0.62; 95% confidence index (CI), 0.38-0.81; P = 0.024] and CD20+ B cells (HR, 0.56; 95% CI, 0.31-0.0.98; P = 0.048). Prognostic factors for lower CIR were higher levels of CD16+CD57+ NK cells (HR, 0.51; 95% CI, 0.27-0.95; P = 0.034) and CD16+CD57− NK cells (HR, 0.52; 95% CI, 0.28-0.99; P = 0.048). A lower incidence of NRM was associated with higher levels of CD8+CD11b− T cells (HR, 0.18; 95% CI, 0.08-0.39; P < 0.001) and CD20+ B cells (HR, 0.24; 95% CI, 0.08-0.70; P = 0.0088). High levels of CD8+CD11b+ T cells were an independent predictor for a higher incidence of chronic GVHD (HR, 2.38; 95% CI, 1.22-4.95; P = 0.012).Conclusions: The distinct differences in immune reconstitution according to stem cell sources and lymphocyte subset analysis at day 100 post SCT are useful for predicting transplant outcome. Furthermore, the results suggest that characteristic immune recovery in CBT positively affects transplant outcome. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impaired thymopoiesis predicts for a high risk of severe infections after reduced intensity conditioning without anti-thymocyte globulin in double umbilical cord blood transplantation

Umbilical cord blood stem cell transplantation (UCBT) is associated with retarded hematopoietic recovery and immune reconstitution and a high infection-related morbidity and mortality, especially after conditioning including anti-thymocyte globulin (ATG). However, data on immune recovery, incidence of infections, and outcome in double UCBT (dUCBT) recipients receiving an ATG-free reduced intensity conditioning (RIC) are lacking. In this study, recovery of lymphocyte subsets, thymopoiesis, and its association with severe infections and clinical outcome was assessed in a group of 55 recipients of a dUCBT ATG-free RIC regimen. T cell recovery was severely protracted in the majority of patients. However, T cell receptor excision circle TREC+ T cells were detectable in 62% of patients at 3 months post-transplantation. A total of 128 common toxicity criteria grade 3-4 infections were observed in the first year post-transplantation. Non-relapse mortality at 12 months post-transplant was 16%, of which 78% infectious mortality. One-year overall survival was 73%. Patients who failed to recover thymopoiesis at 3 months post-transplantation were at a 3.3-fold higher risk of subsequent severe grade 3-4 infections.

213 - Lymphocyte Subset Recovery Pattern and Incidence of Infectious Disease after Allogeneic Hematopoietic Stem Cell Transplant

213 - Lymphocyte Subset Recovery Pattern and Incidence of Infectious Disease after Allogeneic Hematopoietic Stem Cell Transplant

Immune System Profiling Following Autologous Stem Cell Transplant for High-Risk Neuroblastoma Predicts Disease-Free Survival

Objective: Neuroblastoma (NB) is the most common extra cranial solid tumor in childhood. It accounts for approximately 7% of pediatric cancer diagnoses each year in the United States yet is responsible for 15% of pediatric cancer deaths annually. Occurring overwhelmingly in patients with high-risk (HR) disease.Measurable immune surveillance correlates with improved overall disease-free survival in patients with a myriad of cancer subtypes, including NB. Current therapeutic strategies in HR-NB incorporate manipulation of the host immune response, including autologous stem cell transplantation (autoSCT), cis-retinoic acid, and chimeric antibody targeting the tumor-associated antigen disialoganglioside (GD2). Understanding how such therapies affect the immune system and its relation to clinical response is of critical importance. We speculate that patients with higher proportions of regulatory T cells (Tregs) and slower recovery of other lymphocyte subsets will have higher rates of progressive disease and/or relapse.Methods: We reviewed the records of all University of Chicago patients between 2009 and 2015 who received autoSCT as part of upfront therapy for HR neuroblastoma. Data included complete blood count and peripheral blood lymphocyte flow cytometric (FACS) subset monitoring. Our analysis aimed to determine the association between lymphocyte subset kinetics following autoSCT with pre-transplant treatment, transplant conditioning, and disease-free/overall survival. Patients who had a follow-up of greater than one-year post-transplantation were included in the analysis.Results: Thirty-four patients (59% male, median age 5.6 years) received autoSCT between 2009 and 2015 for HR neuroblastoma. Seventy-six percent (n = 26) received either carboplatin, etoposide, melphalan (CEM) or busulfan/melphalan (BuMel) for conditioning. Among those patients, twenty-seven percent received MIBG prior to their autoSCT. The other patients received BSO/melphalan or tandem transplantations. Sixty-eight percent of these patients went on to receive chimeric antibody after their autoSCT irrespective of their conditioning regimen. Antibody therapy with IL-2 or GM-CSF was initiated at a median of 101 days (mean 98.9) post-autoSCT. All patients had normal neutrophil counts at 100 days post transplant, while the total absolute lymphocyte count (ALC), CD8+ T cells, NK cells and CD19+ B cells, were below normal levels in the majority of patients, (100%, 89%, 79% and 94% of patients respectively). Patients who received MIBG prior to autoSCT had a higher proportion of CD3+CD4+CD25+ T cells, the subset containing Tregs, among their total CD3+ population at 100 days post-transplant, (79.8% versus 32.8% p-value = 0.02). Following single autoSCT, patients with higher ALC and CD4+ cells had increased disease free survival in evaluations after six months post transplant. (Figure 1) CD8+ cells, NK cells and CD19+ cell counts were not significant in this analysis.Conclusions: Patients with HR NB have differential immune cell recovery following autoSCT that is dependent on the type of therapy received prior to autoSCT. Specifically, patients who received MIBG had markedly higher proportion of CD3+CD4+CD25+ T cell recovery compared to patients not receiving pre-autoSCT MIBG. This is the subset containing Tregs, which may indicate suppression of an anti-NB immune response long-term. Immune reconstitution is globally poor in all patients at the time of chimeric antibody immunotherapy that is initiated around day 100-post autoSCT. There are features of immune reconstitution that appear to correlate with improved outcomes, including elevated ALC, CD8+ and CD4+ cell counts when evaluated at least six months post transplantation. Additional characterization of T cell subsets is currently being performed to quantify the number of Treg cells in the CD3+CD4+CD25+ T cell subset population. Prospective evaluation of both immune cell recovery and functional evaluation of cells is required to confirm these findings, which may have a profound impact on how immune modulatory therapy is scheduled for those patients with high-risk neuroblastoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Donor Lymphocyte Infusions Correct Deficiency of Naive T Cells and Improve T-Cell Competence after Allogeneic Haematopoietic Stem Cell Transplantation with Lymphocyte Depletion

Lymphocyte-depletion effectively reduces risk of graft versus host disease (GvHD) after allogeneic haematopoietic stem cell transplantation (allo-HSCT), but risk of infections and malignant disease relapse remains high. We have previously reported that pre-emptive donor lymphocyte infusions (pDLI) given to patients after allo-HSCT for myeloid malignancies to reverse falling donor T-cell chimerism improve overall and relapse-free survival. GvHD rates after pDLI were not high and grade rarely severe. To investigate the basis for better outcome after pDLI, we have assessed recovery of lymphocyte subsets, T-cell receptor (TCR) diversity and T-cell functional competence after allo-HSCT with fludarabine and busulphan in cohorts of 59 patients (median age 59) given alemtuzumab for lymphocyte-depletion and 34 patients (median age 58) given anti-thymocyte globulin (ATG).Lymphocytes were significantly less depleted with ATG compared to alemtuzumab (Day 30: Median 3.9 x 108/liter versus 2.3x108/liter, P=0.03) but numbers for both ATG and alemtuzumab remained significantly below the normal range (median 2.34x109/liter for 11 aged-matched healthy volunteers) for at least one year (Day 360 P<0.005: Median 8.35 x 108/liter after ATG; median 1.04 x 109/liter after alemtuzumab). Lymphocyte subset composition was similar after ATG or alemtuzumab, and abnormal. Notable, the T-cell population comprised only memory and effector T cells early after HSCT. These cells expressed significantly higher levels of Ki67 than T cells from healthy volunteers (Day 30 P<0.005: Median CD4 T cells 41.3% Ki67+ after ATG, 66% after alemtuzumab compared to 2.51% for healthy volunteers; median CD8 T cells 18.5% Ki67+ after ATG, 50.8% after alemtuzumab compared to 2.58% for healthy volunteers). This marker is indicative of homeostatic proliferation likely driven by increased levels of IL7 and IL15 detected in the serum of patients early after HSCT compared to healthy volunteers (Day 30 P=0.066 and P<0.005 respectively). Higher frequency of T cells expressing the proliferation marker in patients treated with alemtuzumab was associated with high frequencies of T cells expressing the PD1 marker, indicative of exhaustion (Day 30 P<0.005: Median CD4 T cells 84.0% PD1+ after alemtuzumab compared to 6.35% for healthy volunteers; median CD8 T cells 49.1% PD1+ after alemtuzumab compared to 12.3% for healthy volunteers). Expression of PD1 by T cells was near normal in patients treated with ATG.Naïve T cells were typically absent for at least six months after HSCT following lymphocyte depletion with ATG or alemtuzumab, and any subsequent recovery was poor. In contrast, the naïve T-cell population increased rapidly in patients after pDLI (n=18). Six of these patients received pDLI early after HSCT (at 3-5 months) and naïve T-cell recovery was significantly enhanced at six months compared to patients that did not receive pDLI (Day 180 P<0.005: Median 19.25% naïve CD4 T cells compared to 1.36%; median 23.5% naïve CD8 T cells compared to 3.48%).Naïve T cells are the main source of repertoire diversity and responsible for responses to antigens not previously encountered. Analysis of the TCR β chain repertoire of five patients by deep sequencing revealed that pDLI boosts repertoire diversity. For example, unique TCR β chain sequences increased 31-fold in 150 days after pDLI compared to a 2-fold increase during a similar period for another patient that did not receive DLI. Furthermore, instances of emergence of public clonotypes specific for CMV or EBV that were not detected before DLI were seen in virus-positive patients whose donors were virus-negative. Emergence and rapid expansion of donor-derived clonotypes to frequencies up to 6.75% suggests that naïve T cells present in the DLI had been primed upon encounter with virus in the patient. In vitro stimulation with overlapping 15-mer peptide libraries for CMV antigens and EBV antigens followed by assessment of activation marker expression and interferon-γ, MIP-1β, and TNF-α production showed that virus-specific T-cell responses increased in magnitude and poly-functionality after DLI.These findings show that DLI replenishes naïve T cells and restores ability to respond to viral antigens previously unseen. By inference, this may extend to leukaemia antigens and underlie the reduced rate of malignant disease relapse seen in patients given pDLI. DisclosuresNo relevant conflicts of interest to declare.

Low-Dose Antithymocyte Globulin, Post-Transplant Cyclophosphamide and Sirolimus As Graft-Versus-Host Disease Prophylaxis in Unrelated Donor Transplants

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cure for several patients with high-risk hematological malignancies. Recent advances in supportive therapies have significantly reduced the treatment-related mortality over the last decades. Nevertheless graft-versus-host disease (GvHD) still represents a major complication, and research is needed to achieve further progress in this area of transplantation. Moreover, although initial studies suggested relative equivalence, recent and large prospective studies showed higher rates of acute and chronic GvHD, and worse survival in allo-HSCT from unrelated donors (URD) compared with HLA-identical sibling donors. Following our encouraging results in haplodentical setting with the more recently post-transplantation cyclophosphamide (PTCy) and sirolimus as GvHD prophylaxis (Cieri et al, 2015), in this study we investigated whether the incorporation of low dose anti-thymocyte globulin (ATG) to PTCy and sirolimus GvHD prophylactic approach, may alleviate some of the increased alloreactivity associated with URD transplants.We retrospectively evaluated a cohort of 21 haematological patients receiving URD allo-HSCT in the San Raffaele Haematology and BMT Unit, from 2013 to 2015.All subjects had high-risk hematologic malignancies, with the most common diagnosis being myeloid diseases (52%). A total of 11 patients had active disease at HSCT, while 9 were in hematologic remission. Nine patients scored high according to disease risk index defined by Armand et al, 10 scored intermediate, while only 2 had a low disease score.Myeloablative conditioning consisted of treosulfan (14 g/m2/day) on days -6 to -4, fludarabine (30 mg/m2/day) on days -6 to -2, and melphalan (70 mg/m2/day) on days -2 and -1, followed by T-replete G-CSF-mobilized PBSCs (n=19) or BM grafts (n=2). Whether 12 patients received stem cells from HLA- matched URD (fully 10/10 HLA matched), the remaining 9 had one-locus HLA-mismatched from their donors. During conditioning patients received ATG-Fresenius 5 mg/kg per day IV on day -4 to -2. In vivo B-cell depletion was obtained by rituximab (i.v. 375mg/m2 given as a single dose on day -1). Postgrafting immunosuppression consisted of PTCy (50 mg/kg/day) on days 3 and 4. Sirolimus was given orally from day 5, and monitored to maintain a target therapeutic plasma level of 5-14 ng/ml. According to chimeric status and evidence of GVHD, the dosage of sirolimus was tapered during the second month post-transplantation, ending in complete withdrawal within the sixth month after HSCT. Mycophenolate mofetil (MMF) was initiated orally at 10 mg/kg t.i.d. on day 5 after HSCT, and withdrawn on day 30.Median follow up was 247 days (range 29-486) from HSCT. All patients were engrafted in a median time of 17 days (range, 14-51 days), with full donor chimerism achieved by day 30. Poor graft function was observed in 11 patients. Post-HSCT recovery of lymphocyte subsets was lengthy as compared to other unmanipulated HSCT, with a median time to CD3>100/ml of 41 days (range, 22-389 days) by flow-cytometry, while 5 patients never reached immune reconstitution.All except one developed febrile neutropenia during aplasia. We observed a high rate of serious infections: severe pneumonia in 7 cases (5 required NIV, 4 were admitted to ICU), CMV reactivation in 13 (3 CMV disease), HHV6 reactivation in 10, BK virus haemorrhagic cystitis in 2 patients. Although the overall mortality associated with these complications was limited, as evidenced by a favourable NRM, it caused significant morbidity in patients and often required aggressive therapies, potentially contributing to poor graft function.The incidences of acute and chronic GVHD were low. A grade II-IV acute GvHD was reported in 10% patients, while five patients developed chronic GvHD (mild in 4 cases, only one moderate).Non-relapse mortality (NRM) at 100 days and 1 year was 4.8% and 20.7%, respectively. The estimated 1-year overall survival (OS) and relapse incidence were 51% and 22.5% respectively.Although limited by a small number of patients with relatively short follow-up, our study suggests that URD allo-HSCT with ATG, PTCy and sirolimus-based GvHD prophylaxis, can be very effective in preventing GvHD, but at the expense of delayed immune reconstitution and high rate of infectious complications, while warranting acceptable NRM and relapse incidence. DisclosuresBonini:MolMed S.p.A: Consultancy.

Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells

Haploidentical hematopoietic stem cell transplantation (HSCT) performed using bone marrow (BM) grafts and post-transplantation cyclophosphamide (PTCy) has gained much interest for the excellent toxicity profile after both reduced-intensity and myeloablative conditioning. We investigated, in a cohort of 40 high-risk hematological patients, the feasibility of peripheral blood stem cells grafts after a treosulfan-melphalan myeloablative conditioning, followed by a PTCy and sirolimus-based graft-versus-host disease (GVHD) prophylaxis (Sir-PTCy). Donor engraftment occurred in all patients, with full donor chimerism achieved by day 30. Post-HSCT recovery of lymphocyte subsets was broad and fast, with a median time to CD4 > 200/μL of 41 days. Cumulative incidences of grade II to IV and III-IV acute GVHD were 15% and 7.5%, respectively, and were associated with a significant early increase in circulating regulatory T cells at day 15 after HSCT, with values < 5% being predictive of subsequent GVHD occurrence. The 1-year cumulative incidence of chronic GVHD was 20%. Nonrelapse mortality (NRM) at 100 days and 1 year were 12% and 17%, respectively. With a median follow-up for living patients of 15 months, the estimated 1-year overall and disease-free survival (DFS) was 56% and 48%, respectively. Outcomes were more favorable in patients who underwent transplantation in complete remission (1-year DFS 71%) versus patients who underwent transplantation with active disease (DFS, 34%; P = .01). Overall, myeloablative haploidentical HSCT with peripheral blood stem cells (PBSC) and Sir-PTCy is a feasible treatment option: the low rates of GVHD and NRM as well as the favorable immune reconstitution profile pave the way for a prospective comparative trial comparing BM and PBSC in this specific transplantation setting.

Sirolimus and Post Transplant Cyclophosphamide (PT-Cy) Allow the Use of Haploidentical PBSC Grafts Inducing a Favorable Immune Reconstitution with Low Rates of GvHD: Results in 39 Patients

Introduction:Haploidentical hematopoietic stem cell transplantation (HSCT) performed using T–replete BM grafts and post-transplantation cyclophosphamide (PT-Cy) has gained much interest in the transplantation community for the excellent toxicity profile, achieving outcomes equivalent to those of HSCT performed using matched related or unrelated donors (Bashey, JCO 2013). Nonetheless, relapse remains a major challenge with the use of this non-myeloablative regimen (Luznik, BBMT 2008). Thus, we investigated whether the use of PBSC graft, an intensified myeloablative conditioning and sirolimus instead of tacrolimus might help increasing the therapeutic index of this procedure.Patients and Methods:Here we report the results on 39 patients treated between Nov 2012 and June 2014. The median age was 56 years (range, 21–78). Diagnoses included AML (21), ALL (5), CML-BC (1), MDS (1), NHL (5), and HD (6). The majority (62%) of patients were not in disease remission at time of HSCT. Eight patients (20%) received a prior allo-HSCT. For the remaining patients who underwent haplo-PTCy as a first allogeneic transplant, 19 patients (61%) scored high/very high according to the CIBMTR disease score, 12 (39%) scored intermediate, while no patient had a low disease score. The median Sorror Comorbidity Index was 3 (range: 0-9). Conditioning consisted of treosulfan (14 g/m2/day) on days –6 to –4, fludarabine (30 mg/m2/day) on days –6 to –2, and melphalan (70 mg/m2/day) on days –2 and –1, followed by T-replete G-CSF-mobilized PBSC. Postgrafting immunosuppression consisted of PT-Cy (50 mg/kg/day) on days 3 and 4, followed by mycophenolate mofetil for 30 days, and sirolimus for at least 3 months. Overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method. Cumulative incidence (CI) of non-relapse mortality (NRM) was calculated using relapse as a competing risk, for GvHD the competing risk was death.Results:PBSC grafts contained an infused median CD34/kg of 6.03 e6 (4.15–8.02 e7), CD3/kg 21.12 e7 (10.5–48.26 e7). All patients but 1 engrafted (97.5%), with a median time to neutrophil and platelet recovery of 18 days (range 13–45) and 19 days (9–65), respectively. Notably, the only patient with primary graft failure had a HLA-DP mismatch in HvG direction. Post-HSCT recovery of lymphocyte subsets was broad and fast: median time to CD3>100/ml was 28 days (range: 8-97), to CD4>200/ml 35 days (21-137) and to CD19>0/ml 34 days (24-137). Circulating T cells comprised naïve and memory subsets, with a recovery of CD31+ recent thymic emigrants (RTEs) starting from day 30. All patients had a significantly higher proportion of circulating RTEs at day 30, 90 and 180 compared to their pre-HSCT levels, suggesting an improvement in their thymic function after HSCT. We observed a significant early increase in circulating Tregs at day 15 post HSCT.With a median follow-up for living patients of 327 days, the 1-year cumulative CI of NRM and relapse were 15% and 32%, respectively. Two of the 9 relapses were HLA-loss variants (Vago, NEJM 2009) occurring at a median time of 277 days post-HSCT. Classical relapses had instead an earlier occurrence (median time to relapse: 92 days, range 37-134). CI of acute GvHD grade II-IV and III-IV at 6 months were 18% and 8%, while CI of chronic GvHD at 12 months was 19%.Estimated 1-year probability of OS and DFS were 58% and 51%. Outcomes were particularly favorable in patients transplanted in CR (1-year OS 93%, DFS 95%) as compared to those of patients transplanted with active disease (1-year OS 37%, DFS 29%; p=0.005 and p=0.001, respectively). Importantly, stratification according to CIBMTR score held similar results: in patients with intermediate score, 1-year OS was 91% vs. 33% in patients with high/very high score (p=0.015); similarly, 1-year DFS was 92% vs. 34% (p=0.009).Conclusions: PT-Cy coupled to sirolimus allow the use of haploidentical PBSC grafts with low rates of GvHD, and intensified myeloablative regimen is a valid option for patients with aggressive/advanced hematologic malignancies. The acceptable rates of GvHD and NRM as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory or cellular therapies to improve DFS in patients at high risk for relapse who would benefit from further treatment intensification early after transplant. DisclosuresBonini:MolMed S.p.A.: Consultancy.

Comparison of different rabbit ATG preparation effects on early lymphocyte subset recovery after allogeneic HSCT and its association with EBV-mediated PTLD

Rabbit antithymocyte globulin (ATG) is commonly used before allogeneic hematopoietic stem cell transplantation (allo-HSCT) to prevent graft-versus-host disease. Studies comparing the effect of different ATG preparations and dosages on immune reconstitution and risk for Epstein-Barr virus (EBV)-mediated post-transplant lymphoproliferative disorder (PTLD) are rare. In this retrospective study, we determined T and B cell subsets by flow cytometry after allo-HSCT in children, who received ATG-Genzyme (ATG-G, n = 15), ATG-Fresenius (ATG-F, n = 25) or no-ATG treatment (n = 19). Additionally, PCR-quantified EBV-genome copy counts were correlated with incidence of PTLD. We could confirm a dose-dependent impairment of CD8(+) and CD4(+) T cell regeneration by ATG-G, including naïve and memory CD4(+) T cells. No differences were seen between the currently applied dosages of 5-10 mg/kg ATG-G and 20-60 mg/kg ATG-F. Significantly delayed T cell subset reconstitution was determined only at high dosages of 20-60 mg/kg ATG-G compared to ATG-F. B cell reconstitution was comparably impaired in ATG-G- and ATG-F-treated patients. Although the incidence of EBV reactivation was similar in both ATG groups, EBV copy counts of >10(4) copies/10(5) peripheral blood mononuclear cells and the occurrence of PTLD were only found in ATG-G-treated patients. We conclude that high, but importantly not currently applied low dosages of ATG-G, impair thymic T cell regeneration and memory T cell immunity to a greater extent than ATG-F in pediatric patients. In addition, our results suggest an increased risk for EBV-PTLD when treated with ATG-G. Prospective studies are warranted to compare different ATG preparations with regard to the immune reconstitution and EBV-PTLD.

Lymphocyte Subset Recovery and Outcome after Autologous Hematopoietic Stem Cell Transplantation for Plasma Cell Myeloma

Rapid immune reconstitution—particularly of natural killer cells (NK cells)—after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with protection from relapse. Whether such an association also exists after autologous stem cell transplantation is less clear. We retrospectively assessed lymphocyte subsets after autologous HSCT in 114 patients and correlated lymphocyte recovery with outcome. CD8 T cell and NK cell counts recovered rapidly to pretransplantation levels, whereas B cell and CD4 T cell recovery were delayed. Compared with patients with low NK cells (<100/uL), high NK cell count at 1 month after HSCT was associated with significantly prolonged progression-free survival: for NK cells 100 to 200/uL hazard ratio [HR], .33 (95% confidence interval [CI]; .16 to .80; P = .004); for NK cells > 200/μL HR, .27 (95% CI, .13 to .58; P = .001). No significant protective effects were associated with rapid recovery of any other lymphocyte subset. None influenced overall survival (OS) or time to next treatment. Early NK cell recovery is associated with better progression-free survival after autologous HSCT. The failure to detect an effect on OS might be due to the salvage strategies available to these patients.

Post-Transplant Cyclophosphamide Haplo-HSCT Revised: Peripheral Blood Stem Cell Graft and Sirolimus To Enhance Immune Reconstitution and Graft Versus Leukemia Effect In Patients With Active Leukemia

IntroductionAchieving a cure for relapsed leukemia remains a challenge. Hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative provides a potentially curative treatment for such patients in urgent need. Based on previous studies on the feasibility and low toxicity profile of haploidentical HSCT with high-dose post-transplant Cyclophosphamide (PT-Cy), we planned to intensify this protocol for patients with active disease by the use of: 1) myeloablative conditioning, 2) peripheral blood stem cells (PBSCs) graft instead of bone marrow (BM), and 3) sirolimus instead of tacrolimus, for its direct anti-leukemic effect. Patients and MethodsHere we report the results on 18 patients treated between November 2012 and July 2013. The median age was 59 years (range, 30–78). Diagnoses included AML (10 refractory, 3 CR1, and 2 CR>1), ALL (2 refractory), and 1 CML in lymphoid blast crisis. The majority (78%) of patients were not in remission at the time of HSCT. Consistently, 13 (72%) patients scored high/very high accordingly to the CIBMTR disease score (Todisco et al., Leukemia 2013). Fourteen (78%) patients had a Sorror comorbidity score ≥ 3. Conditioning consisted of treosulfan (14 g/m2/day) on days –6 to –4, fludarabine (30 mg/m2/day) on days –6 to –2, and melphalan (70 mg/m2/day) on days –2 and –1, followed by T-repleted peripheral blood stem cells (PBSCs). Postgrafting immunosuppression consisted of PT-Cy (50 mg/kg/day) on days 3 and 4, followed by mycophenolate mofetil for 30 days, and sirolimus for 3 months. ResultsPBSC grafts contained an infused median TNC/kg of 10.5 e8 (range 5.5–20.7 e8), CD3/kg 18.4 e7 (range 10.5–40.9 e7) and a CD34/kg of 7.01 e6 (range 4.3–8.02 e6). All patients but 1 engrafted (94%), with a median time to neutrophil and platelet recovery of 18 days (range 14–26) and 19 days (range 9–51) respectively. Notably, the patient with primary graft failure had a HLA-DP mismatch in HvG direction. No patient experienced secondary graft failure. Post-HSCT recovery of lymphocyte subsets was broad and fast: the median CD3 count at day 30 after HSCT was 642/µl and exceeded 1000/µl by day 60, with a median value of CD4 T cell counts of 329/µl at day 30; NK cells reached a median value of 192/µl at day 30 and remained stable thereafter, while B cells steadily increased from a median value of 2/µl at day 30 to 48/µl at day 180. Circulating T cells comprised both naïve and memory subsets, with a recovery of CD31+ recent thymic emigrants (RTEs) starting from day 30. All patients had a significantly higher proportion of circulating RTEs at day 30, 90 and 180 compared to their pre-HSCT levels, suggesting an improvement in their thymic function after HSCT. Peri-engraftment syndrome (PES) occurred in 4 patients with a median time to onset of 15 days and was promptly resolved with short-course steroids. Interestingly, the percentage of circulating Ki-67+, dividing, effector memory CD8 T cells at day 8 post HSCT was able to accurately predict the occurrence of PES. Grade I/II skin acute GvHD occurred in 6 patients, while 4 patients experienced grade III/IV acute GvHD (22%). Acute GvHD were accompanied by a rise in circulating Ki-67+ CD8 T cells, while response to first line therapy resulted in a drop in Ki-67 expression. Chronic GVHD occurred in 2 of the 7 patients at risk, but only one patient required systemic therapy. Of the 16 patients at high risk, 7 had CMV reactivations. No patient had EBV-related PTLD. With a median follow-up of 100 days (range 15–260), 14 (78%) out of the 18 patients are alive. Only two patients died of non-relapse mortality (NRM, 11%). All patients in CR at time of transplant are alive and disease-free, while 8 (57%) out of 14 patients with active disease at transplant are alive and leukemia-free. In 4 patients tumor-specific T cells against PRAME or WT1 were detectable both in the peripheral blood and BM up to 180 days after HSCT, suggesting that PT-Cy did not hamper the GvL effect. ConclusionsMyeloablative haploidentical HSCT with PBSCs, PT-Cy and sirolimus is feasible and might be a valid option for patients with refractory/active leukemia. If confirmed in a larger cohort of patients and with a longer follow-up, these results suggest that the acceptable rates of GvHD and NRM as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory or cellular therapies to improve leukemia-free survival. Disclosures:Bonini:MolMed SpA: Consultancy.

Reconstitution of Lymphocyte Subsets and Outcomes After Matched and Mismatched Hematopoietic Stem-Cell Transplantation

Abstract 4485 Allogeneic stem-cell transplantation (ASCT) is curative for many malignant and nonmalignant hematological disorders. Limitations of expanding this form of treatment are related primarily to non-relapse mortality (NRM) associated with reconstituting the recipient's immune system. We aimed to study reconstitution of lymphocyte subsets after matched and mismatched transplantation. Lymphocyte subsets (absolute numbers of CD3, CD4, CD8, CD19, CD56, CD25, CD45RA, CD45RO) were evaluated by flow cytometry at 1,3,6 and 12 months (mo) post-ASCT. Lymphocyte recovery was determined using means at each time point and group differences were assessed using analysis of variance. Kaplan-Meier survival curves were used to estimate time-to-event outcome measures and the log-rank test was used to evaluate differences between groups. 100 patients (pts) were included in the study. 25 pts received a matched sibling donor transplant (MSD), 20 pts a 10/10 MUD, 18 pts a 9/10 MUD, 9 pts a T cell depleted haploidentical (TCD haplo), and 28 a T cell replete haploidentical donor (TCR haplo). 53 pts received bone marrow and 47 had peripheral blood stem cells. Most patients were treated for acute leukemia (AML 41, ALL 23), 16 MDS, 6 CML, 4 CLL, 5 lymphoma. Median age was 43 years (range: 20–71). The median follow-up was 13.6 mo. 60 pts were alive and disease-free at last follow-up and 28 pts died 75% due to relapse. Non-relapse mortality (NRM) was 6% for the entire cohort. Overall, alive pts (vs who died) had higher mean CD3 (615 vs 349, p=0.03 on day 90), CD8 (427 vs 187, p=0.03 on day 90), CD4 (391 vs 54, p=0.01, on day 365) cells, and lower mean CD56 cells (178 vs 300, p=0.01, on day 30) post ASCT. Pts who progressed (vs did not), had lower 1 year mean CD4 cells (123 vs 394, p=0.02), lower mean CD3 cells (359 vs 1147, p=0.06), with no differences in CD8, NK, and CD45RA. NRM was associated with higher mean NK numbers at 6 months (499 vs 188, p=0.01) and with lower mean CD3 at day 90 (184 vs 557, p=0.07). T-cell recovery occurred most rapidly in MSD transplants ([Figure1][1]C), and compared with 10/10 MUD had higher mean CD3 cells in the first mo (1085 vs 510, p=0.01), CD4 in the first 6 mo (310 vs 147, p<0.01 day 180) and CD4CD45RA in the first 6 mo (105 vs 31, p=0.02 on day 180). Interestingly, we have found that higher CD4CD25 cell numbers recovered early and most rapidly in the MSD transplants, which may partly explain a lower incidence of GVHD in this group. Overall, TCR haplos had a similar pattern of T-cell recovery and outcomes as 10/10 MUDs ([Figure1][1]C-E). There was a significant delay in CD3 recovery between MSD and TCR haplos in the first 3 mo (mean 779 vs 483, p=0.04 on day 90), CD4 (mean 270 vs 170, p=0.03 day 90) and in the first 6 months for CD45RA (mean 105 vs 28, p=0.01 on day 180), while these differences were significant only at day 30 for CD8 cells (mean 703 vs 88, p<0.01). No significant differences in T cell subsets were found between 10/10 MUD and TCR HaploSCT for CD3, CD4, CD8, CD45RA and CD4CD25 for any time points. Of all types of transplant, TCD haplos had most impaired T-cell reconstitution and worst outcomes ([Figure 1][1]A and [1][1]B). As previously reported it was characterized by rapid early NK cell recovery and delayed CD3, CD4, CD8 reconstitution. Interestingly, pts surviving 6–9 months post-transplant recovered CD3, C4, CD8 cells; however, naive T-cell recovery remained impaired for more than 1 year post-transplant, suggesting that T cell recovery comes predominantly from the memory T cell compartment. In conclusion, these results suggest that recovery of lymphocyte subsets may vary widely with the type of transplant, may correlate with outcomes, and it is important to be further explored in this setting. ![Figure][2] Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:yes

Immune reconstitution after allogeneic hematopoietic stem cell transplantation in children: a single institution study of 59 patients

PurposeLymphocyte subset recovery is an important factor that determines the success of hematopoietic stem cell transplantation (HSCT). Temporal differences in the recovery of lymphocyte subsets and the factors influencing this recovery are important variables that affect a patient's post-transplant immune reconstitution, and therefore require investigation.MethodsThe time taken to achieve lymphocyte subset recovery and the factors influencing this recovery were investigated in 59 children who had undergone HSCT at the Department of Pediatrics, The Catholic University of Korea Seoul St. Mary's Hospital, and who had an uneventful follow-up period of at least 1 year. Analyses were carried out at 3 and 12 months post-transplant. An additional study was performed 1 month post-transplant to evaluate natural killer (NK) cell recovery. The impact of pre- and post-transplant variables, including diagnosis of Epstein-Barr virus (EBV) DNAemia posttransplant, on lymphocyte recovery was evaluated.ResultsThe lymphocyte subsets recovered in the following order: NK cells, cytotoxic T cells, B cells, and helper T cells. At 1 month post-transplant, acute graft-versus-host disease was found to contribute significantly to the delay of CD16+/56+ cell recovery. Younger patients showed delayed recovery of both CD3+/CD8+ and CD19+ cells. EBV DNAemia had a deleterious impact on the recovery of both CD3+ and CD3+/CD4+ lymphocytes at 1 year post-transplant.ConclusionIn our pediatric allogeneic HSCT cohort, helper T cells were the last subset to recover. Younger age and EBV DNAemia had a negative impact on the post-transplant recovery of T cells and B cells.