CD3+/CD19+ Depleted Matched and Mismatched Unrelated Donor Hematopoietic Stem Cell Transplant with Targeted T Cell Addback Is Associated with Excellent Outcomes in Pediatric Patients with Nonmalignant Hematologic Disorders

Unrelated donor hematopoietic stem cell transplantation (HSCT) is increasingly being used to cure nonmalignant hematologic diseases (NMHD) in patients who lack HLA matched related donors. Both graft rejection and graft-versus-host disease (GVHD) remain major barriers to safe and effective transplant for these patients requiring unrelated donors. Partial T cell depletion combined with peripheral stem cell transplantation (pTCD-PSCT) has the potential advantages of providing a high stem cell dose to facilitate rapid engraftment, maintaining cells that may facilitate engraftment, and decreasing GVHD risk compared with T cell–replete HSCT. Here, we report a single-institution, retrospective experience of unrelated donor pTCD-PSCT for pediatric patients with NMHD. From 2014 to 2017, 12 pediatric patients with transfusion-dependent NMHD underwent matched unrelated donor (MUD) or mismatched unrelated donor (MMUD) pTCD HSCT in our center using disease-specific conditioning. Donor PSCs underwent CD3⁺ T cell and CD19⁺ B cell depletion using CliniMACS, followed by a targeted addback of 1?×?10⁵ CD3⁺ T cells/kg to the graft before infusion. All 12 patients demonstrated rapid trilinear engraftment. At a median follow-up of 740days (range, 279 to 1466), all patients were alive with over 92% total peripheral blood donor chimerism and without transfusion dependence or recurrence of their underlying hematologic disease. Immune reconstitution was rapid and comparable with T cell–replete HSCT. No patients developed severe acute GVHD (grades III to IV) or chronic extensive GVHD, and all patients had discontinued systemic immune suppression. Viral reactivations were common, but no patient developed symptoms of life-threatening infectious disease. Our data indicate that MUD and MMUD pTCD-PSCTs are safe and effective approaches that enable rapid engraftment and immune reconstitution, prevent severe GVHD, and expand availability of HSCT to any patients with NMHD who have closely MUDs.     

CD34+-Selected Stem Cell Boost without Further Conditioning for Poor Graft Function after Allogeneic Stem Cell Transplantation in Patients with Hematological Malignancies

We retrospectively analyzed outcomes of a CD34⁺-selected stem cell boost (SCB) without prior conditioning in 32 patients (male/22; median age of 54 years; range, 20 to 69) with poor graft function, defined as neutrophils ≤1.5 x 10⁹/L, and/or platelets ≤30 x 10⁹/L, and/or hemoglobin ≤8.5 g/dL). The median interval between stem cell transplantation and SCB was 5 months (range, 2 to 228). The median number of CD34⁺ and CD3⁺ cells were 3.4 x 10⁶/kg (.96 to 8.30) and 9 x 10³/kg body weight (range, 2 to 70), respectively. Hematological improvement was observed in 81% of patients and noted after a median of 30 days (range, 14 to 120) after SCB. The recipients of related grafts responded faster than recipients of unrelated grafts (20 versus 30 days, P = .04). The cumulative incidence of acute (grade II to IV) and chronic graft-versus-host disease (GVHD) after SCB was 17% and 26%, respectively. Patients with acute GVHD received a higher median CD3⁺ cell dose. The 2-year probability of overall survival was 45%. We suggest that SCB represents an effective approach to improve poor graft function post transplantation, but optimal timing of SCB administration, anti-infective, and GVHD prophylaxis needs further evaluation.     

CD19+CD5+ B Cells and B1-Like Cells Following Allogeneic Hematopoietic Stem Cell Transplantation

We and others have previously reported the expansion of CD5⁺CD19⁺ B cells after allogeneic hematopoietic stem cell transplantation. Recently, the equivalent of B1 cells in mice has been described in humans as CD20⁺CD27⁺CD43⁺CD70^- B cells. In this article, we report that although 39% of CD5⁺CD19⁺ cells were CD43⁺ in controls, >75% of CD5⁺CD19⁺ cells were CD43⁺ in patients independent of the presence or absence of chronic graft-versus-host disease (GVHD) (P = .0001). CD5⁺CD19⁺ B cell, CD5⁺CD43⁺CD19⁺ B cell, and CD27⁺CD43⁺ B cell counts were significantly lower in the patients with previous chronic GVHD, and this effect of GVHD was similar in both CD5⁺ and CD5^- within the CD27⁺CD43⁺ B cell subset. Our results strongly suggest that the previously reported expansion of the CD5⁺CD19⁺ population might be related to an expansion of the CD27⁺CD43⁺ B cell subset and that CD27⁺CD43⁺ B cell reconstitution is impaired in patients with chronic GVHD.     

Partially CD3+-Depleted Unrelated and Haploidentical Donor Peripheral Stem Cell Transplantation Has Favorable Graft-versus-Host Disease and Survival Rates in Pediatric Hematologic Malignancy

Most children who may benefit from stem cell transplantation lack a matched related donor. Alternative donor transplantations with an unrelated donor (URD) or a partially matched related donor (PMRD) carry an increased risk of graft-versus-host-disease (GVHD) and mortality compared with matched related donor transplantations. We hypothesized that a strategy of partial CD3⁺/CD19⁺ depletion for URD or PMRD peripheral stem cell transplantation (PSCT) would attenuate the risks of GVHD and mortality. We enrolled 84 pediatric patients with hematologic malignancies at the Children's Hospital of Philadelphia and the Children's Hospital of Wisconsin between April 2005 and February 2015. Two patients (2.4%) experienced primary graft failure. Relapse occurred in 23 patients (27.4%; cumulative incidence 26.3%), and 17 patients (20.2%) experienced nonrelapse mortality (NRM). Grade III-IV acute GVHD was observed in 18 patients (21.4%), and chronic GVHD was observed and graded as limited in 24 patients (35.3%) and extensive in 8 (11.7%). Three-year overall survival (OS) was 61.8% (95% confidence interval [CI], 50.2% to 71.4%) and event-free survival (EFS) was 52.0% (95% CI, 40.3% to 62.4%). Age ≥15 years was associated with decreased OS (P= .05) and EFS (P= .05). Relapse was more common in children in second complete remission (P = .03). Partially CD3⁺-depleted alternative donor PSCT NRM, OS, and EFS compare favorably with previously published studies of T cell-replete PSCT. Historically, T cell-replete PSCT has been associated with a higher incidence of extensive chronic GVHD compared with limited chronic GVHD, which may explain the comparatively low relapse and NRM rates in our study cohort despite similar overall rates of chronic GVHD. Partial T cell depletion may expand donor options for children with malignant transplantation indications lacking a matched related donor by mitigating, but not eliminating, chronic GVHD.     

Phase II Study of CD4+-Guided Pentostatin Lymphodepletion and Pharmacokinetically Targeted Busulfan as Conditioning for Hematopoietic Cell Allografting

One limitation of reduced-intensity preparative regimens is potential for graft failure. We have developed a regimen that targets CD4⁺ lymphodepletion to ensure early and durable engraftment. The primary endpoint was achievement of ≥50% CD3⁺ donor chimerism by day +28. Forty-two patients (median age, 53 years; range, 29 to 73 years) received pentostatin 4 mg/m² i.v. on days −28, −21, and −14 when the CD4⁺ cell count was >100 cells/μL and on days −4 and −3 regardless of CD4⁺ level. Rituximab 375 mg/m² was administered to patients with CD20⁺ malignancies on days −21, −14, −7, +1, and +8. Busulfan 200 mg/m² i.v. was administered on days −4 and −2 at a dose to target a cumulative AUC dose of 16,000 (±10%) μmol·min/L. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus plus methotrexate in 86% of patients. Donors were matched-related (47%), matched unrelated (43%), or mismatched unrelated (10%). Chronic lymphocytic leukemia (45%) and follicular non-Hodgkin lymphoma (14%) were the most common diagnoses. Disease status at initiation of the preparative regimen was complete remission in 22%, partial response in 55%, and stable/progression in 24%. The median percent CD4⁺ cell count decrease from baseline (day −28) was 52% to day −21, 66% to day −14, 62% to day −7, and 91% to day 0. At day +28, all 42 patients (100%) had ≥50% CD3⁺ donor chimerism. No patient experienced graft failure. Overall response rate was 82% (complete remisson, 67%). The day +100 cumulative incidence of grade II-IV acute GVHD was 59% (grade III-IV acute GVHD, 19%), and the 2 year cumulative incidence of chronic GVHD was 69% (moderate/severe, 58%). Nonrelapse mortality was 2% at day +100 and 17% at 2 years. Two-year PFS was 55%, and OS was 68%. This regimen ensures durable engraftment, is effective against persistent disease, and results in relatively low mortality from causes other than relapse.     

Presalvage International Staging System Stage and Other Important Outcome Associations in CD34+-Selected Allogeneic Hematopoietic Stem Cell Transplantation for Multiple Myeloma

Despite ongoing therapeutic advances, multiple myeloma (MM) remains largely incurable, and outcomes in patients who develop resistance to immunomodulatory drugs or proteasome inhibitors remain grim. Allogeneic hematopoietic cell transplantation (alloHCT) is an alternative option that may offer potential for cure. Although rates of transplantation-related morbidity and mortality have decreased in recent years, weighing this approach's potential benefits against nontransplantation therapies demands a thoroughly informed pre-alloHCT assessment. Here we assess the impact of pre-alloHCT variables on important clinical outcomes in a large cohort of relapsed refractory MM (RRMM) CD34⁺-selected alloHCT recipients. We included all patients with MM who underwent CD34⁺-selected alloHCT at our center between June 2010 and December 2015. Patients were conditioned with busulfan (0.8 mg/kg × 10), melphalan (70 mg/m² × 2), and fludarabine (25 mg/m² × 5), followed by infusion of a CD34⁺-selected peripheral blood stem cell graft, without post-alloHCT graft-versus-host disease (GVHD) prophylaxis. The 73-patient cohort had a median age of 55 years (range, 37 to 66 years). Overall survival (OS) and progression-free survival (PFS) rates were 70% and 53%, respectively, at 1 year (95% confidence interval [CI], 58% to 79% and 41% to 64%) and 50% and 30%, respectively, at 3 years (95% CI, 38% to 62% and 19% to 41%). The cumulative incidence of relapse was 25% at 1 year (95% CI, 15% to 35%) and 47% at 3 years (95% CI, 35% to 58%). Nonrelapse mortality at 1 year was 22% (95% CI, 13% to 32%). The cumulative incidence of grade II-IV acute GVHD (aGVHD) was 7% at 100 days (95% CI, 3% to 14%), and that of any chronic GVHD (cGVHD) was 8% at 1 year (95% CI, 3% to 16%). International Staging System (ISS) stage II-III assessed before salvage therapy was associated with poorer 3-year OS (30% versus 54%; P = .037) and 3-year PFS (9% versus 33%; P = .013), and increased 3-year relapse incidence (72% versus 39%; P = .004). Older age and GVHD before 6 months (aGVHD grade II-IV or cGVHD of any grade) were also associated with poorer OS, and a greater number of pre-alloHCT lines of therapy was also associated with increased relapse incidence. Our findings reinforce that CD34⁺-selected alloHCT can achieve prolonged disease control and long-term survival in high- risk, heavily treated refractory MM populations. We also identified numerous pre-alloHCT variables associated with OS, PFS, and relapse. Amongst these, presalvage ISS stage II-III was consistently associated with poorer survival and relapse outcomes. Given the lack of established alternate therapies for patients with RRMM, we advocate the identification of adverse pre-alloHCT variables to inform alloHCT decision making rather than to exclude patient cohorts from this potentially curative treatment option.     

CD34+ Cell Selection versus Reduced-Intensity Conditioning and Unmodified Grafts for Allogeneic Hematopoietic Cell Transplantation in Patients Age >50 Years with Acute Myelogenous Leukemia and Myelodysplastic Syndrome

Reduced-intensity conditioning (RIC) and T cell depletion (TCD) through CD34⁺ cell selection without the use of post-transplantation immunosuppression are 2 strategies used to reduce nonrelapse mortality (NRM) in older patients after allogeneic hematopoietic cell transplantation (allo-HCT). To compare the efficacy of the RIC and TCD approaches, we evaluated the outcomes of patients age >50 years with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) who underwent allo-HCT from an HLA-matched donor with one of these strategies. Baseline characteristics were comparable in the patients receiving TCD (n?=?204) and those receiving RIC (n?=?151), except for a higher proportion of unrelated donors (68% versus 40%; P?<?.001) and a higher comorbidity burden (Hematopoietic Cell Transplantation Comorbidity Index [HCT-CI] ≥3: 51% versus 38%; P?<?.001) in the TCD cohort. Analysis of outcomes at 3 years showed a higher chronic graft-versus-host disease (GVHD)/relapse-free survival (CRFS) (51% versus 7%; P?<?.001), lower incidences of grade II-IV acute GVHD (18% versus 46% at day +180) and chronic GVHD (6% versus 55% at 3 years; P?<?.001), and a lower incidence of relapse (19% versus 33% at 3 years; P?=?.001) in the TCD group compared with the RIC group. Relapse-free survival (RFS), overall survival (OS), and NRM were similar in the 2 groups. Combining transplantation approach (RIC versus TCD) and comorbidity burden (HCT-CI 0-2 versus ≥3), patients with an HCT-CI score of 0-2 seemed to benefit from the TCD approach. In conclusion, in this retrospective study, the use of a CD34⁺ cell-selected graft and a myeloablative conditioning regimen was associated with higher CRFS and similar RFS and OS compared with unmodified allo-RIC in patients age >50 years with AML and MDS.     

Combined CD4+ Donor Lymphocyte Infusion and Low-Dose Recombinant IL-2 Expand FOXP3+ Regulatory T Cells following Allogeneic Hematopoietic Stem Cell Transplantation

CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4⁺ lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4⁺DLI alone (P = .001). FOXP3 expression correlated with absolute CD4⁺CD25⁺ cell counts. Moreover, expanded CD4⁺CD25⁺ T cells displayed normal suppressive function and treatment with CD4⁺DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4⁺ cellular therapy may provide a mechanism to expand Treg in vivo.     

Optimized Timing of Post-Transplantation Cyclophosphamide in MHC-Haploidentical Murine Hematopoietic Cell Transplantation

Post-transplantation cyclophosphamide (PTCy) reduces the risks of severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Yet, the standard clinical dose and timing of PTCy were partly extrapolated from MHC-matched skin allografting models and were partly empirical. Here we investigated the impact of differential dosing and timing of PTCy on its efficacy in preventing GVHD in a murine MHC-haploidentical HCT model. Administration of PTCy on days +3/+4 was superior to administration on days +1/+2, +5/+6, or +7/+8, whereas low-dose (10 mg/kg/day) PTCy on days +1/+2 actually led to accelerated death. Although the optimal timing of PTCy dosing was day +2 or +3 in the skin allografting models, in our MHC-haploidentical HCT model, PTCy on days +2/+3 was inferior to PTCy on days +3/+4 at lower doses. PTCy administered on days +3/+4, +4/+5, or +3/+5 were similarly efficacious. Single-day versus 2-day dosing schedules demonstrated that PTCy is maximally effective when given on day +4. Flow cytometric analysis showed that optimal PTCy dosing schedules both decreased alloreactive CD4⁺CD25⁻Foxp3⁻ T cell proliferation at day +7 and allowed preferential CD4⁺CD25⁺Foxp3⁺ T cell reconstitution at day +21, suggesting that this combination may be a potential predictive biomarker of successful GVHD prevention by PTCy. These results show that the dose, timing, and cumulative exposure of PTCy all are critical for its efficacy in preventing GVHD. We are currently investigating the clinical relevance of these findings in a protocol seeking to optimize PTCy dose and timing and test these T cell endpoints as candidate biomarkers of successful GVHD prevention by PTCy.     

IL-7 Is the Limiting Homeostatic Factor that Constrains Homeostatic Proliferation of CD8+ T Cells after Allogeneic Stem Cell Transplantation and Graft-versus-Host Disease

Immune reconstitution after allogeneic hematopoietic stem cell transplantation relies primarily on homeostatic proliferation (HP) of mature T lymphocytes, but this process is typically impaired during graft-versus-host disease (GVHD). We previously showed that low IL-7 levels combined with lack of dendritic cell (DC) regeneration constrain CD4⁺ T cell HP during GVHD. However, it is not clear whether these alterations to the peripheral CD4⁺ T cell niche also contribute to impair CD8⁺ T cell regeneration during GVHD. We found that IL-7 therapy was sufficient for restoring CD8⁺ T cell HP in GVHD hosts while forcing DC regeneration with Flt3-L had only a modest effect on CD8⁺ T cell HP in IL-7 treated mice. Using bone marrow chimeras, we showed that HP of naïve CD8⁺ T cells is primarily regulated by MHC class I on radio-resistant stromal cells, yet optimal recovery of CD8⁺ T cell counts still requires expression of MHC class I on both radio-resistant and radio-sensitive hematopoietic cells. Thus, IL-7 level is the primary limiting factor that constrains naïve CD8⁺ T cell HP during GVHD, and accessibility of MHC class I on stromal cells explains how IL-7 therapy, as a single agent, can induce robust CD8 ⁺ T cell HP in the absence of DCs.     

Growth and Differentiation Advantages of CD4+OX40+ T Cells from Allogeneic Hematopoietic Stem Cell Transplantation Recipients

OX40 (CD134), an activation-induced costimulatory molecule, is mainly expressed on CD4⁺ T cells. Several reports, including previous reports from our laboratory, suggest that OX40-mediated signaling plays an important role in the development of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (Allo HSCT). Here, we show that peripheral blood CD4⁺OX40⁺ T cells are a unique cell subset as they possess the homing receptors of lymph nodes, and some of them have an exceptional capacity to produce high levels of interleukin-2 (IL-2) upon the stimulation through T cell receptors. Stimulation with IL-7 acts selectively on CD4⁺OX40⁺ T cells not only to induce antigen-independent growth but also to increase the frequency of cells with IL-2-producing potential. Simultaneous, but not sequential, ligation of the T cell receptor and OX40 induces CD4⁺OX40⁺ T cells to produce far more IL-2, which causes them to proliferate abundantly and differentiate readily into Th1- or Th2-biased effector memory T cells, especially in Allo HSCT recipients. Although not all the CD4⁺OX40⁺ T cells had IL-2-producing capacity, Allo HSCT recipients with chronic GVHD (cGVHD) had a significantly higher frequency of IL-2-producing OX40⁺ cells in their peripheral blood CD4⁺ T cell subset than Allo HSCT recipients without cGVHD. Collectively, CD4⁺OX40⁺ T cells with IL-2-producing potential are expected to be privileged for growth and differentiation in lymph nodes upon antigen presentation, suggesting that they might be involved in the process of inducing or maintaining cGVHD.     

CD34 Selected Cells for the Treatment of Poor Graft Function after Allogeneic Stem Cell Transplantation

Poor graft function (PGF) is characterized by pancytopenia and a hypoplastic marrow, with complete donor chimerism, usually without severe graft-versus-host disease (GVHD). We report 41 patients with PGF, treated with granulocyte colony-stimulating factor–mobilized CD34 selected cells, at a median interval from transplant of 140 days, without conditioning and without GVHD prophylaxis. Donors were HLA matched siblings (n = 12), unrelated donors (n = 18), or mismatched family members (n = 11). The median number of infused CD34⁺ cells was 3.4 × 10⁶/kg. The rate of trilineage recovery was 75%: 83% for HLA matched siblings and 72% for unrelated and mismatched family members (P = .3). The cumulative incidence of acute grade II GVHD was 15%, and no patient developed de novo chronic GVHD. The actuarial 3-year survival is 63%: 76% and 25% for patients with or without trilineage recovery. These data confirm the role of CD34⁺ selected cells from the same donor in the treatment of PGF and warrant the request for a second donation also when the donor is unrelated.     

Depletion of Naïve Lymphocytes with Fas Ligand Ex Vivo Prevents Graft-versus-Host Disease without Impairing T Cell Support of Engraftment or Graft-versus-Tumor Activity

Graft-versus-host disease (GVHD) can be prevented by Fas-mediated selective depletion of host-sensitized donor lymphocytes ex vivo. We tested the hypothesis that Fas-mediated depletion of lymphocytes in the absence of host-specific antigenic stimulation can alleviate GVHD. Brief exposure (24 hours) of unstimulated donor lymphocytes to Fas ligand (FasL) ex vivo results in balanced apoptosis of CD8⁺ and CD4⁺ subsets with preferential depletion of CD62L and CD69, increased T regulatory fractions, and sustained responses to stimulation. This procedure ameliorates weight loss and improves the clinical and histologic score of skin and gastrointestinal GVHD with and without concurrent transplantation of hematopoietic progenitors and irrespective of conditioning-induced tissue injury. Although FasL-resistant donor T cells are less potent effectors of GVHD, they facilitate hematopoietic progenitor engraftment when infused with or after the graft and retain the potential to elaborate graft-versus-tumor reactions. These findings in a preclinical model together with the known trophic effects of FasL on primitive hematopoietic progenitors suggest that brief ex vivo incubation of hematopoietic grafts with FasL may improve the outcome and safety of clinical T cell–replete allogeneic and haploidentical transplants.     

Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen‐driven T cell activation

Background It has been suggested that allergic diseases are caused by defective suppression of allergen‐specific Th2 cells by CD4⁺CD25⁺ regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25‐expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127^lo, allowing discrimination between these distinct T cell subpopulations. Objective The aim of this study was to study whether the putative regulatory subset defined as CD4⁺CD25⁺CD127^lo was involved in grass pollen‐reactive T cell responses. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non‐atopic controls out of season. Grass pollen‐induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4⁺CD25⁺, CD4⁺CD25⁺CD127^hi or CD4⁺CD25⁺CD127^lo T cells. Results Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non‐atopic controls. Depletion of all CD25⁺ T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4⁺CD25⁺CD127^lo cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non‐atopic group. Conclusion Our study showed that T cells from grass pollen‐allergic patients and non‐atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.     

Impact de la composition du greffon sur le devenir des patients après une allogreffe de cellules souches hématopoïétiques : corrélation entre proportion des lymphocytes T CD4 du greffon exprimant le CCR7 et la survenue d’une GVH aiguë

In a previous prospective study on 62 patients who underwent an HLA-matched allogeneic stem cell transplantation, we have observed that proportion of donor-derived CCR7⁺/CD4⁺ T cells in the graft provided a predictive indicator of acute GVHD without interfering on chronic GVHD and relapse rate. Here we present our results on a confirmatory cohort of 137 consecutive patients. Indeed patients who received more than 76% of CCR7⁺/CD4⁺ T cells in the graft developed more often acute GVHD be it of low or high grade than those who did not. Determination of the CCR7⁺/CCR7^neg ratio of CD4⁺ T cells in the graft provides a predictive indicator of acute GVHD and could help to define strategies of partial selective T cell depleted transplantation.     

Impact of a Low CD34+ Cell Dose on Allogeneic Peripheral Blood Stem Cell Transplantation

Although the CD34⁺ cell dose in allogeneic peripheral blood stem cell transplantation (PBSCT) is considered to be associated with transplantation outcomes, a lower acceptable threshold has not been defined. We retrospectively analyzed 2919 adult patients with hematologic malignancies who underwent related PBSCT in Japan between 2001 and 2014. According to the number of CD34⁺ cells in the graft, we categorized 2494 patients in the standard group (2 to 5 × 10⁶ cells/kg), 377 patient in the low group (1 to 2?×?10⁶ cells/kg), and 48 patients in the very low group (<1?×?10⁶ cells/kg). Compared with the standard group, the low and very low groups showed delayed neutrophil recovery (93.8%, 89.5%, and 78.3%, respectively at day +28; P?<?.001) and platelet recovery (69.3%, 53.0%, and 45.5%, respectively at day +28; P?<?.001). The 2-year overall survival (OS) in the 3 groups was 45.5%, 45.3%, and 29.8%, respectively, with inferior survival in the very low group. However, a higher percentage of high-risk patients may account for the inferior survival in the very low group, and no significant difference in OS was found in a multivariate analysis. There were no differences in relapse, nonrelapse mortality, or the development of graft-versus-host disease among the 3 groups. In conclusion, allogeneic PBSCT with low CD34⁺ cell doses of 1 to 2?×?10⁶ cells/kg gives acceptable results, whereas further investigations are needed to evaluate the effects of lower doses of <1?×?10⁶ cells/kg owing to the smaller number and the higher percentage of patients with adverse prognostic factors in this cohort.     

Donor Lymphocyte Infusion for Relapsed Hematological Malignancies after Allogeneic Hematopoietic Cell Transplantation: Prognostic Relevance of the Initial CD3+ T Cell Dose

The impact of donor lymphocyte infusion (DLI) initial cell dose on its outcome is known in patients with chronic myeloid leukemia but limited in patients with other hematological malignancies. In this retrospective study, we evaluated the effect of initial DLI CD3⁺ cell dose on graft-versus-host disease (GVHD) and overall survival after DLI given for relapse of any hematological malignancies after allogeneic hematopoietic cell transplantation (HCT) with high- or reduced-intensity conditioning. The cohort included 225 patients. Initial DLI CD3⁺ cell dose per kilogram of recipient body weight was ≤1 × 10⁷ (n = 84; group A), >1.0 to <10 × 10⁷ (n = 58; group B), and ≥10 × 10⁷ (n = 66; group C). The initial cell dose was unknown for the remaining 17 patients. Cumulative incidence rates of GVHD at 12 months after DLI were 21%, 45%, and 55% for groups A, B, and C, respectively. Multivariate analysis showed that initial DLI CD3⁺ cell ≥10 × 10⁷ dose per kilogram is associated with an increased risk of GVHD after DLI (P = .03). Moreover, an initial DLI CD3⁺ cell dose of 10 × 10⁷ or higher did not decrease the risk of relapse and did not improve overall survival. Thus, these results support the use of less than 10 × 10⁷ CD3⁺ cell per kilogram as the initial cell dose of DLI for treatment of persistent or recurrent hematological malignancy after HCT.     

Transient Treg‐cell depletion in adult mice results in persistent self‐reactive CD4+ T‐cell responses

Depletion of Foxp3⁺CD4⁺ regulatory T cells (Treg) in adults results in chronic inflammation and autoimmune disease. However, the impact of transient Treg‐cell depletion on self‐reactive responses is poorly defined. Here, we studied the effect of transient depletion of Treg cells on CD4⁺ T‐cell responses to endogenous self‐antigens. Short‐term ablation of Treg cells in mice resulted in rapid activation of CD4⁺ T cells, increased percentage of IFN‐γ⁺ and Th17 cells in lymphoid organs, and development of autoimmune gastritis. To track self‐reactive responses, we analyzed the activation of naïve gastric‐specific CD4⁺ T cells. There was a dramatic increase in proliferation and acquisition of effector function of gastric‐specific T cells in the stomach draining LNs of Treg‐cell‐depleted mice, compared with untreated mice, either during Treg‐cell depletion or after Treg‐cell reconstitution. Moreover, the hyperproliferation of gastric‐specific T cells in the Treg‐cell‐ablated mice was predominantly antigen‐dependent. Transient depletion of Treg cells resulted in a shift in the ratio of peripheral:thymic Treg cells in the reemerged Treg‐cell population, indicating an altered composition of Treg cells. These findings indicate that transient Treg‐cell depletion results in ongoing antigen‐driven self‐reactive T‐cell responses and emphasize the continual requirement for an intact Treg‐cell population.     

Allogeneic Th1 Cells Home to Host Bone Marrow and Spleen and Mediate IFNγ-Dependent Aplasia

Bone marrow graft failure and poor graft function are frequent complications after hematopoietic stem cell transplantation and result in significant morbidity and mortality. Both conditions are associated with graft-versus-host disease (GVHD), although the mechanism remains undefined. Here we show, in 2 distinct murine models of GVHD (complete MHC- and class II-disparate) that mimic human peripheral blood stem cell transplantation, that Th1 CD4⁺ cells induce bone marrow failure in allogeneic recipients. Bone marrow failure after transplantation of allogeneic naïve CD4⁺ T cells was associated with increased CD4⁺ Th1 cell development within bone marrow and lymphoid tissues. Using IFNγ-reporter mice, we found that Th1 cells generated during GVHD induced bone marrow failure after transfers into secondary recipients. Homing studies demonstrated that transferred Th1 cells express CXCR4, which was associated with accumulation within bone marrow and spleen. Allogeneic Th1 cells were activated by radiation-resistant host bone marrow cells and induced bone marrow failure through an IFNγ-dependent mechanism. Thus, allogeneic Th1 CD4⁺ cells generated during GVHD traffic to hematopoietic sites and induce bone marrow failure via IFNγ-mediated toxicity. These results have important implications for prevention and treatment of bone marrow graft failure after hematopoietic stem cell transplantation.     

Immune Cytopenias after Ex Vivo CD34+-Selected Allogeneic Hematopoietic Cell Transplantation

Immune-mediated cytopenias (ICs), such as immune thrombocytopenia and immune hemolytic anemia, are among the adverse events after allogeneic hematopoietic cell transplantation (allo-HCT). Previous reports suggest that in vivo T cell depletion may increase the incidence of IC after allo-HCT. We evaluated whether a strategy that reduces functional donor T cells via ex vivo CD34⁺-selection associates with the development of IC in a cohort of 408 patients who underwent allo-HCT for hematologic malignancy. The cumulative incidence of IC at 6, 12, and 36 months after the 30-day landmark post-HCT was 3.4%, 4.9%, and 5.8%, respectively. Among 23 patients who developed IC, 7 died of relapse-related mortality and 4 of nonrelapse mortality. A median 2 types of treatment (range, 1 to 5) was required to resolve IC, and there was considerable heterogeneity in the therapies used. In univariable analyses, a hematologic malignancy Disease Risk Index (DRI) score of 3 was significantly associated with an increased risk of IC compared with a DRI of 1 or 2 (hazard ratio [HR], 4.12; P = .003), and IC (HR, 2.4; P = .03) was associated with increased risk of relapse. In a multivariable analysis that included DRI, IC remained significantly associated with increased risk of relapse (HR, 2.4; P = .03). Our findings show that IC events occur with relatively similar frequency in patients after ex vivo CD34⁺-selected allo-HCT compared with unmodified allo-HCT, suggesting that reduced donor T cell immunity is not causative of IC. Moreover, we noted a possible link between its development and/or treatment and increased risk of relapse.