Costimulated tumor-infiltrating lymphocytes are a feasible and safe alternative donor cell therapy for relapse after allogeneic stem cell transplantation.

Donor lymphocyte infusion (DLI), a standard relapse treatment after allogeneic stem cell transplantation (AlloSCT), has limited efficacy and often triggers GVHD. We hypothesized that after AlloSCT tumor-infiltrating donor lymphocytes could be costimulated ex vivo to preferentially activate/expand antitumor effectors. We tested the feasibility and safety of costimulated, tumor-derived donor lymphocyte (TDL) infusion in a phase 1 trial. Tumor was resected from 8 patients with B-cell malignancy progression post-AlloSCT; tumor cell suspensions were costimulated with anti-CD3/anti-CD28 Ab-coated magnetic beads and cultured to generate TDL products for each patient. Costimulation yielded increased proportions of T-bet(+)FoxP3(-) type 1 effector donor T cells. A median of 2.04 × 10(7) TDL/kg was infused; TDLs were well tolerated, notably without GVHD. Two transient positron emission tomography (PET) responses and 2 mixed responses were observed in these refractory tumors. TDL are a feasible, tolerable, and novel donor cell therapy alternative for relapse after AlloSCT.

Assessment of the hematopoietic cell transplantation comorbidity index in non-Hodgkin lymphoma patients receiving reduced-intensity allogeneic hematopoietic stem cell transplantation.

The hematopoietic cell transplantation comorbidity index (HCT-CI), a weighted index of 17 pretransplantation comorbidities, has been validated in nonmyeloablative and myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) studies, but it has not been specifically tested in patients with non-Hodgkin lymphoma (NHL) receiving reduced-intensity conditioning (RIC). We performed a retrospective analysis to assess the impact of the HCT-CI on outcomes of NHL patients treated with HSCT relative to treatment-related mortality (TRM), disease-related mortality (DRM), with a specific emphasis on overall survival (OS). Individual pretransplantation and disease-related factors also were analyzed with HCT-CI relative to their impact on OS. All patients were uniformly treated with an identical pretransplantation induction regimen and an identical RIC regimen (cyclophosphamide [Cy]/fludarabine [Flu]), and received T cell-replete allografts from HLA-matched siblings. The analysis included 63 NHL patients with a median HCT-CI score of 2 (range, 0 to 11). The HCT-CI (0 to 2 comorbidities vs 3+ comorbidities) demonstrated a potential association with TRM, but not with DRM, at 100 days (4.5% vs 26.3%) and at 1 year (13.6% vs 36.8%) posttransplantation. The factor most strongly associated with OS was response to pretransplantation chemotherapy (P= .0001), based on a composite measure. In a Cox model, pretransplantation chemotherapy response remained the most important factor (P< .0001) relative to OS, and there was a trend (P= .056) toward HCT-CI adding predictive value for OS. Although HCT-CI may be useful for predicting TRM, our data further underscore the importance of response to chemotherapy before transplantation as a predictor of overall transplantation outcome in NHL patients being considered for RIC allogeneic HSCT.

Age-dependent incidence, time course, and consequences of thymic renewal in adults.

Homeostatic regulation of T cells involves an ongoing balance of new T cell generation, peripheral expansion, and turnover. The recovery of T cells when this balance is disrupted provides insight into the mechanisms that govern homeostasis. In a long-term, single cohort study, we assessed the role of thymic function after autologous transplant in adults, correlating serial computed tomography imaging of thymic size with concurrent measurements of peripheral CD4(+) T cell populations. We established the age-dependent incidence, time course, and duration of thymic enlargement in adults and demonstrated that these changes were correlated with peripheral recovery of naive CD45RA(+)CD62L(+) and signal-joint TCR rearrangement excision circle-bearing CD4(+) populations with broad TCR diversity. Furthermore, we demonstrated that renewed thymopoiesis was critical for the restoration of peripheral CD4(+) T cell populations. This recovery encompassed the recovery of normal CD4(+) T cell numbers, a low ratio of effector to central memory cells, and a broad repertoire of TCR Vbeta diversity among these memory cells. These data define the timeline and consequences of renewal of adult thymopoietic activity at levels able to quantitatively restore peripheral T cell populations. They further suggest that structural thymic regrowth serves as a basis for the regeneration of peripheral T cell populations.

Dose-adjusted EPOCH-rituximab combined with fludarabine provides an effective bridge to reduced-intensity allogeneic hematopoietic stem-cell transplantation in patients with lymphoid malignancies.

PURPOSE: There is currently no standard chemotherapy regimen for patients with lymphoid malignancies being considered for reduced-intensity conditioning allogeneic hematopoietic stem-cell transplantation (RIC-alloHSCT). The ideal regimen would provide disease control and result in lymphocyte depletion to facilitate engraftment. To this end, we developed a novel regimen by adding fludarabine to dose-adjusted continuous-infusion etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin plus with or without rituximab (DA-EPOCH-F/R). PATIENTS AND METHODS: One hundred forty-seven patients with lymphoid malignancy (median age, 50 years) who had heavily pretreated (median prior regimens, three) and chemo-refractory (47%) disease were treated with DA-EPOCH-F/R before RIC-alloHSCT. Patients received one to three consecutive cycles until achieving lymphocyte depletion (CD4(+) count < 200/µL) or progressive disease. RESULTS: Overall response rate was 41%; 39% of patients had stable disease. Toxicity included grade 4 neutropenia in 65% and thrombocytopenia in 25% of patients. DA-EPOCH-F/R resulted in lymphocyte depletion (P < .001), which was inversely associated with serum interleukin (IL) 7 and IL-15 levels. Of 147 patients, 143 patients proceeded to RIC-alloHSCT. Patients with lower CD3(+) (P < .001), CD4(+) (P < .001), and CD8(+) (P < .001) T-cell counts after DA-EPOCH-F/R were more likely to achieve full donor lymphoid chimerism by day +14 after transplant. Relative to nonresponders to DA-EPOCH-F/R, patients with complete and partial response had increased event-free survival (77.4 v 4.8 months; P < .001) and overall survival (98.5 v 16.2 months; P < .001). CONCLUSION: DA-EPOCH-F/R safely provides tumor cytoreduction and lymphocyte depletion, thereby offering a bridge to RIC-alloHSCT in patients with aggressive lymphoid malignancies.

Correlation of pretransplant and early post-transplant response assessment with outcomes after reduced-intensity allogeneic hematopoietic stem cell transplantation for non-Hodgkin's lymphoma.

BACKGROUND: Chemotherapy sensitivity, defined simply as at least a partial response to chemotherapy, is an important outcome predictor for non-Hodgkin lymphoma (NHL) patients undergoing reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HCT). The authors hypothesized that further differentiation of chemotherapy sensitivity by specific response, complete remission (CR) versus partial remission (PR) versus stable disease (SD) versus progression of disease (PD), correlates with post-transplant outcomes. METHODS: The impact of pretransplant and early (28 days) post-transplant disease response on transplant outcomes was analyzed in 63 NHL patients treated with reduced-intensity allo-HCT. RESULTS: The 3-year event-free survival (EFS) and overall survival (OS) (median potential follow-up after reduced-intensity allo-HCT = 58 months) for all patients was 37% and 47%, respectively. The 3-year EFS based on pretransplant response was: CR = 50%; PR = 66%; SD = 18%; no patient with PD pretransplant reached 3-year follow-up. The 3-year OS based on pretransplant response was: CR = 63%; PR = 69%; SD = 45%. The 3-year EFS based on post-transplant response was: CR = 57%; PR = 32%; SD = 33%; no patient with PD post-transplant reached 3-year follow-up. The 3-year OS based on post-transplant response was: CR = 65%; PR = 43%; SD = 50%. In multivariate analyses, pretransplant response was the best predictor of EFS (P < .0001). Pretransplant response (P < .0001) and age (P = .0035) were jointly associated with OS. CONCLUSIONS: These data suggest that NHL patients with pretransplant SD, generally considered inappropriate candidates, may benefit from reduced-intensity allo-HCT, and patients with pretransplant PD should only receive this therapy in clinical trials.

Association of serum interleukin-7 levels with the development of acute graft-versus-host disease.

PURPOSE: Morbidity from acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic stem-cell transplantation (HSCT) to treat malignancy. Interleukin-7 (IL-7), the principal homeostatic cytokine for T cells, is required for acute GVHD in murine models. In contrast to inflammatory cytokines (eg, IL-2, tumor necrosis factor alpha), IL-7 has not been studied extensively in the clinical transplant setting relative to its relationship with acute GVHD. PATIENTS AND METHODS: We evaluated the association of serum IL-7 levels with acute GVHD in 31 patients who were uniformly treated in a prospective clinical trial with reduced-intensity allogeneic HSCT from human leukocyte antigen-identical siblings. GVHD prophylaxis consisted of cyclosporine and methotrexate. Serum IL-7 levels and lymphocyte populations were determined at enrollment, the day of transplantation before the allograft infusion, and at specified intervals through 12 months post-transplantation. RESULTS: As expected, IL-7 levels were inversely correlated with T-cell populations (P < .00001). Acute GVHD was significantly associated with higher IL-7 levels at day +7 (P = .01) and day +14 (P = .00003) post-transplantation as well as with the allograft CD34(+) cell dose (P = .01). IL-7 levels at day +14 also correlated with the severity of acute GVHD (P < .0001). In logistic regression models, these factors were highly sensitive (up to 86%) and specific (100%) for classifying whether patients developed acute GVHD. CONCLUSION: These data support preclinical observations that IL-7 plays a critical role in inducing acute GVHD and provide a rational basis for novel approaches to prevent and treat acute GVHD through modulation of the IL-7 pathway.

Host T cells affect donor T cell engraftment and graft-versus-host disease after reduced-intensity hematopoietic stem cell transplantation.

Mixed chimerism in the T cell compartment (MCT) after reduced-intensity stem cell transplantation (RIST) may influence immune repopulation with alloreactive donor T cells. We examined effects of host T cell numbers on donor T cell engraftment and recovery and on acute graft-versus-host disease (aGVHD) in a relatively homogeneous patient population with respect to residual host T cells through quantified immune depletion prior to RIST and to donor T cells by setting the allograft T cell dose of 1x10(5) CD3+ cells/kg. In this setting, 2 patterns of early donor T cell engraftment could be distinguished by day +42: (1) early and complete donor chimerism in the T cell compartment (FDCT) and (2) persistent MCT. FDCT was associated with lower residual host CD8+ T cell counts prior to transplant and aGVHD. With persistent MCT, subsequent development of aGVHD could be predicted by the direction of change in T cell donor chimerism after donor lymphocyte infusion, and no aGVHD occurred until FDCT was established. MCT did not affect recovery of donor T cell counts. These observations suggest that the relative number and alloreactivity of donor and host T cells are more important than the absolute allograft T cell dose in determining donor engraftment and aGVHD after RIST.

Phase I clinical trial of costimulated, IL-4 polarized donor CD4+ T cells as augmentation of allogeneic hematopoietic cell transplantation.

The primary objective of this clinical trial was to evaluate the safety, feasibility, and biologic effects of administering costimulated, interleukin (IL)-4 polarized donor CD4(+) T cells in the setting of HLA-matched sibling, T cell-replete allogeneic hematopoietic cell transplantation (HCT). Forty-seven subjects with hematologic malignancy received granulocyte colony-stimulating factor-mobilized allogeneic hematopoietic cell transplants and cyclosporine graft-versus-host disease (GVHD) prophylaxis after reduced intensity conditioning. Initial subjects received no additional cells (n = 19); subsequent subjects received additional donor CD4(+) T cells generated ex vivo by CD3/CD28 costimulation in medium containing IL-4 and IL-2 (administered day 1 after HCT at 5, 25, or 125 x 10(6) cells/kg). Studies after HCT included measurement of monocyte IL-1alpha and tumor necrosis factor alpha, detection of T cells with antitumor specificity, and characterization of T cell cytokine phenotype. The culture method generated donor CD4(+) T cells that secreted increased T helper 2 (Th2) cytokines and decreased T helper 1 (Th1) cytokines. Such Th2-like cells were administered without infusional or dose-limiting toxicity. The Th2 cohort had accelerated lymphocyte reconstitution; both cohorts had rapid hematopoietic recovery and alloengraftment. Acute GVHD and overall survival were similar in the Th2 and non-Th2 cohorts. Th2 cell recipients tended to have increased monocyte IL-1alpha and had increased tumor necrosis factor alpha secretion. CD8(+) T cells with antitumor specificity were observed in Th2 and non-Th2 cohorts. Post-transplantation T cells from Th2 cell recipients secreted IL-4 and IL-10 (Th2 cytokines) and IL-2 and interferon gamma (Th1 cytokines). Allograft augmentation with costimulated, IL-4-polarized donor CD4(+) T cells resulted in activated Th1, Th2, and inflammatory cytokine pathways without an apparent increase in GVHD.

Efficacy of reduced-intensity allogeneic stem cell transplantation in chemotherapy-refractory non-hodgkin lymphoma.

Chemotherapy sensitivity has been identified as an important prognostic factor in reduced-intensity allogeneic stem cell transplantation (RIST) for non-Hodgkin lymphoma (NHL). However, the effect of uniform salvage chemotherapy before RIST has not been studied prospectively. We examined whether the response to prospectively administered uniform salvage therapy (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and fludarabine) influenced the subsequent outcome of RIST in 28 patients with relapsed or refractory NHL. After RIST, overall survival (OS) at 36 months is 49%, whereas event-free survival (EFS) is 32%. In Cox model analyses, the response to chemotherapy was the best predictor of OS (P = .0006) and EFS (P = .0006) after RIST. Differentiating stable disease from progressive disease after salvage chemotherapy strengthened the association with survival. Among chemotherapy-sensitive patients, the median OS and EFS have not been reached. In patients with stable disease, OS and EFS at 24 months are 50% and 25%, respectively. In contrast, only 1 patient with progressive disease during salvage therapy survived longer than 12 months. These prospective data confirm the favorable prognosis for chemotherapy-sensitive NHL after RIST and suggest that chemotherapy resistance is not an absolute contraindication to RIST for NHL patients with stable disease during salvage therapy.

Establishment of early donor engraftment after reduced-intensity allogeneic hematopoietic stem cell transplantation to potentiate the graft-versus-lymphoma effect against refractory lymphomas.

Reduced-intensity allogeneic hematopoietic stem cell transplantation (alloHSCT), which typically results in mixed chimerism initially after transplantation, has had limited efficacy in chemotherapy-refractory lymphomas. We hypothesized that the rapid establishment of complete donor chimerism would potentiate a graft-versus-lymphoma effect. Fifteen patients with chemotherapy-refractory lymphoma initially received induction with a conventional chemotherapy regimen (etoposide, prednisone, vincristine, cyclophosphamide, adriamycin, fludarabine [EPOCH-F]) to deplete host T cells and provide disease control prior to alloHSCT. Patients then received conditioning with fludarabine and cyclophosphamide followed by alloHSCT from HLA-matched siblings. Graft-versus-host disease prophylaxis consisted of cyclosporine alone. EPOCH-F resulted in 73% of patients having partial responses or stable disease. EPOCH-F depleted host CD4(+) T cells from a median of 235 cells/microL to 56 cells/microL. Fourteen patients underwent alloHSCT, and all had >95% donor engraftment by day 14 after transplantation. The incidence of Grade II to III acute graft-versus-host disease was 71%. There were two therapy-related deaths. There were 8 partial responses and 3 complete responses (CRs) at day 28. Five additional CRs were observed at day 100 without withdrawal of cyclosporine or donor lymphocyte infusion. The rate of CRs for all 15 patients was 60%. The 1-year progression-free survival rate from time of study entry is 67% with only 1 relapse among 9 CRs. At a median potential follow-up of 28 months, the overall survival rate is 53%. These data demonstrate that a potent and durable graft-versus-lymphoma effect can occur against chemotherapy-refractory lymphomas and suggest that this effect may be associated with rapid, complete donor chimerism after reduced-intensity alloHSCT.

Clinical "cytokine storm" as revealed by monocyte intracellular flow cytometry: correlation of tumor necrosis factor alpha with severe gut graft-versus-host disease.

BACKGROUND & AIMS: Gut graft-versus-host disease (GVHD) contributes significantly to lethality after allogeneic hematopoietic stem-cell transplantation (HSCT). In murine models, macrophage secretion of interleukin 1alpha (IL-1alpha) and tumor necrosis factor alpha (TNF-alpha) contributes to gut GVHD pathogenesis. To help characterize whether human gut GVHD has similar biological characteristics, monocyte IL-1alpha and TNF-alpha production were evaluated after HSCT. METHODS: Patients with refractory hematologic malignancy (n = 17) underwent reduced-intensity conditioning, HLA-matched sibling HSCT, and cyclosporine A GVHD prophylaxis. After HSCT, monocyte IL-1alpha and TNF-alpha levels were measured using intracellular flow cytometry (IC-FCM), and results were correlated with clinical GVHD. RESULTS: Incidences of acute GVHD were none (n = 3), grades I-II (n = 9), or grades III-IV (n = 5; each case with stage 2-3 gut GVHD). Posttransplantation monocyte IL-1alpha production (percentage of CD14(+)IL-1(+) cells) increased significantly from 8.7% +/- 3.7% (week 2) to 40.3% +/- 7.3% (week 4; P = 0.0065) and was not associated with GVHD severity (P = 1.00). Conversely, increases in monocyte TNF-alpha were quantitatively reduced and temporally delayed, from 0.6% +/- 0.2% (week 2) to 3.6% +/- 1.4% (week 6; P = 0.076). Most importantly, elevation of monocyte TNF-alpha level correlated with increased gut GVHD severity (P = 0.0041); increases in monocyte TNF-alpha levels typically preceded the onset of gut GVHD symptoms. CONCLUSIONS: Human gut GVHD after reduced-intensity allogeneic HSCT is associated with monocyte cytokine secretion initially involving IL-1alpha, followed by TNF-alpha. Serial measurement of monocyte cytokines, in particular, TNF-alpha, by IC-FCM may represent a noninvasive method for GVHD monitoring, potentially allowing the identification of patients appropriate for early-intervention strategies.