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This review is intended to reflect upon the current status and perspectives of cell-based immunotherapy at a time when the promise of extensive pre-clinical research has been translated into encouraging clinical responses. However, some of these have also been complicated by significant adverse reactions. As the field moves towards definitive late stage trials, with a growing interest from pharmaceutical companies, we realize that novel cell therapy strategies pose questions that are familiar to traditional drug development, along with new considerations due to the potential of T cells to persist long term and to expand after adoptive transfer. These questions address the safety of the product, the efficacy, the mode of action, and the anticipation of risks. From different perspectives, we intend to address exciting opportunities and safety concerns in current concepts of cellular immunotherapy., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The purpose of this analysis was to provide 6-year follow-up of the CLL3X trial, which studied reduced-intensity allogeneic hematopoietic stem cell transplantation (HSCT) in patients with poor-risk chronic lymphocytic leukemia (CLL), and to investigate the effect of TP53, SF3B1, and NOTCH1 mutations on HSCT outcome. For 90 allografted patients, 6-year overall survival (OS) was 58% and 6-year event-free survival (EFS) was 38%. TP53, SF3B1, and NOTCH1 mutations were found in 30%, 26%, and 14% of the trial population, respectively. By univariate and multivariate analyses, the mutational status of the TP53, SF3B1, and NOTCH1 genes had no significant effect on OS and EFS. Studies of minimal residual disease confirmed durability of CLL eradication in mutated patients. We conclude that HSCT can provide long-term disease control in patients with poor-risk CLL independent of the presence of TP53, SF3B1, and NOTCH1 mutations. The trial has been registered at the US National Cancer Institute as #EU-20554, NCT00281983.

The purpose of this prospective multicenter phase 2 trial was to investigate the long-term outcome of reduced-intensity conditioning allogeneic stem cell transplantation (alloSCT) in patients with poor-risk chronic lymphocytic leukemia. Conditioning was fludarabine/ cyclophosphamide-based. Longitudinal quantitative monitoring of minimal residual disease (MRD) was performed centrally by MRD-flow or real-time quantitative polymerase chain reaction. One hundred eligible patients were enrolled, and 90 patients proceeded to alloSCT. With a median follow-up of 46 months (7-102 months), 4-year nonrelapse mortality, event-free survival (EFS) and overall survival (OS) were 23%, 42%, and 65%, respectively. Of 52 patients with MRD monitoring available, 27 (52%) were alive and MRD negative at 12 months after transplant. Four-year EFS of this subset was 89% with all event-free patients except for 2 being MRD negative at the most recent assessment. EFS was similar for all genetic subsets, including 17p deletion (17p-). In multivariate analyses, uncontrolled disease at alloSCT and in vivo T-cell depletion with alemtuzumab, but not 17p-, previous purine analogue refractoriness, or donor source (human leukocyte antigen-identical siblings or unrelated donors) had an adverse impact on EFS and OS. In conclusion, alloSCT for poor-risk chronic lymphocytic leukemia can result in long-term MRD-negative survival in up to one-half of the patients independent of the underlying genomic risk profile. This trial is registered at http://clinicaltrials.gov as NCT00281983.

Forty patients were enrolled in this phase 2 study combining radioimmunotherapy (RIT) using yttrium-90-ibritumomab-tiuxetan (15 MBq [0.4 mCi]/kg) with reduced-intensity conditioning (RIC) using fludarabine (90 mg/m(2)) and 2 Gy total body irradiation followed by allogeneic hematopoietic cell transplantation (HCT) from related (n = 13) or unrelated (n = 27) donors for the treatment of advanced non-Hodgkin lymphoma. Diagnoses were follicular lymphoma (n = 17), chronic lymphocytic leukemia (n = 13), mantle cell lymphoma (n = 8), marginal zone lymphoma (n = 1), and lymphoplasmacytic lymphoma (n = 1). Median age was 55 years (range, 34-68 years). All patients were high risk with refractory disease or relapse after preceding autologous HCT. No additional toxicities attributable to RIT were observed. Engraftment was rapid and sustained. Incidences of acute graft-versus-host disease 2-4 and chronic graft-versus-host disease were 43% and 53%, respectively. Kaplan-Meier-estimated nonrelapse mortality was 45% at 2 years. Twenty-two of 40 patients (55%) are alive, resulting in a Kaplan-Meier-estimated 2-year survival of 51% for all, 67% for follicular lymphoma, 49% for chronic lymphocytic leukemia, and 37% for mantle cell lymphoma patients. The combined use of RIT with RIC is feasible with acceptable toxicity, even in elderly and heavily pretreated patients. This study is registered at www.clinicaltrials.gov as #NCT00302757.

From 2002 to 2007, 103 patients with primary myelofibrosis or postessential thrombocythemia and polycythemia vera myelofibrosis and a median age of 55 years (range, 32-68 years) were included in a prospective multicenter phase 2 trial to determine efficacy of a busulfan (10 mg/kg)/fludarabine (180 mg/m(2))-based reduced-intensity conditioning regimen followed by allogeneic stem cell transplantation from related (n = 33) or unrelated donors (n = 70). All but 2 patients (2%) showed leukocyte and platelet engraftment after a median of 18 and 22 days, respectively. Acute graft-versus-host disease grade 2 to 4 occurred in 27% and chronic graft-versus-host disease in 43% of the patients. Cumulative incidence of nonrelapse mortality at 1 year was 16% (95% confidence interval, 9%-23%) and significantly lower for patients with a completely matched donor (12% vs 38%; P = .003). The cumulative incidence of relapse at 3 years was 22% (95% confidence interval, 13%-31%) and was influenced by Lille risk profile (low, 14%; intermediate, 22%; and high, 34%; P = .02). The estimated 5-year event-free and overall survival was 51% and 67%, respectively. In a multivariate analysis, age older than 55 years (hazard ratio = 2.70; P = .02) and human leukocyte antigen-mismatched donor (hazard ratio = 3.04; P = .006) remained significant factors for survival. The study was registered at www.clinicaltrials.gov as #NCT 00599547.

This study investigated the immunogenicity of Wilms tumor gene product 1 (WT1)-peptide vaccination in WT1-expressing acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients without curative treatment option. Vaccination consisted of granulocyte-macrophage colony-stimulating factor subcutaneously days 1 to 4, and WT1.126-134 peptide and 1 mg keyhole limpet hemocyanin on day 3. The initial 9 patients received 4 vaccinations biweekly, then monthly, and the subsequent 10 patients received continual biweekly vaccination. Seventeen AML patients and 2 refractory anemia with excess blasts patients received a median of 11 vaccinations. Treatment was well tolerated. Objective responses in AML patients were 10 stable diseases (SDs) including 4 SDs with more than 50% blast reduction and 2 with hematologic improvement. An additional 4 patients had clinical benefit after initial progression, including 1 complete remission and 3 SDs. WT1 mRNA levels decreased at least 3-fold from baseline in 35% of patients. In 8 of 18 patients, WT1-tetramer(+) T cells increased in blood and in 8 of 17 patients in bone marrow, with a median frequency in bone marrow of 0.18% at baseline and 0.41% in week 18. This WT1 vaccination study provides immunologic, molecular, and preliminary evidence of potential clinical efficacy in AML patients, warranting further investigations.

Although ATG is frequently used in hematopoietic stem cell transplantation and solid organ transplantation, little is known on its effects on NK cells, which mediate important functions in post-transplantation immunology. We incubated peripheral blood lymphocytes of healthy donors with Thymoglobulin, Lymphoglobulin or ATG-Fresenius. Cell death and apoptosis of NK cells and T cells were determined by flow cytometry using propidium iodide and Annexin V. As expected, there were no significant differences between the different ATGs regarding their T cell toxicity. Surprisingly, we found profound differences between the different ATGs regarding their impact on NK cells: In clinically relevant concentrations Lymphoglobulin had less toxic effects on NK cells as compared to Thymoglobulin or ATG-Fresenius: the median percentages of apoptotic or necrotic NK cells in response to 1 mug/ml Lymphoglobulin, ATG-Fresenius and Thymoglobulin were 2%, 35% and 38%, respectively (p<0.001). This is the first report of differential effects of different ATGs on NK cells. Lymphoglobulin appears to be superior to Thymoglobulin or ATG Fresenius regarding the preservation of NK cell mediated immunity. Randomized trials addressing the impact of different ATGs on lymphocyte subpopulations in the clinical setting are urgently warranted.

Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C-mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.

There is considerable interest in immunotherapeutic approaches for lymphoma. The expression of proteinase inhibitor 9 (PI-9), a molecule that inactivates granzyme B, is considered an immune escape mechanism in lymphoma. Further, lymphomas frequently overexpress the antiapoptotic molecule bcl-2, which is able to inhibit perforin-dependent cytotoxic pathways. In this study, the impact of PI-9 and bcl-2 expression on the sensitivity of lymphomas to T- and natural killer (NK) cell-mediated cytotoxicity was analyzed. We found PI-9 expression in 10 of 18 lymphoma cell lines and in 9 of 14 primary lymphomas. Overexpression of bcl-2 was found in 8 of 18 cell lines and in 12 of 14 primary lymphomas. All lymphoma cells were sensitive to cytolysis by specific T cells and cytokine-activated NK cells, and no difference in sensitivity was observed with respect to PI-9 or bcl-2 expression. Cytolysis was mediated predominantly through perforin-dependent pathways despite expression of PI-9 and bcl-2. Interestingly, the majority of lymphoma cells were resistant to cytolysis by resting allogeneic NK cells. This was due to the failure of lymphomas to induce degranulation of resting NK cells. These results show that resistance to perforin-dependent pathways is not a relevant immune escape mechanism in lymphoma and therefore is unlikely to impair clinical outcome of immunotherapeutic approaches.

CD4+CD25+ regulatory T cells (Tregs) control immune responses to self- and foreign antigens and play a pivotal role in autoimmune diseases, infectious and noninfectious inflammation, and graft rejection. Since recent experimental studies have indicated that Tregs were able to ameliorate graft-versus-host disease (GvHD), we analyzed the number of infiltrating Tregs in the intestinal mucosa as one site of GvH reactivity using immunoenzymatic labeling to enumerate FOXP3+ T cells in 95 intestinal biopsies from 49 allografted patients in comparison with healthy controls and patients with infectious inflammation. While patients with cytomegalovirus (CMV)-colitis or diverticulitis showed a concomitant increase of CD8+ effectors and Tregs, acute and chronic GvHD were characterized by the complete lack of a counter-regulation indicated by a FOXP3+/CD8+ T-cell ratio identical to healthy controls. In contrast, specimens without histologic signs of GvHD demonstrated increased numbers of FOXP3+ per CD8+ T cells, indicating that the potential for Treg expansion is principally maintained in allografted patients. Our findings provide evidence that GvHD is associated with an insufficient up-regulation of Tregs in intestinal GvHD lesions. The determination of FOXP3+/CD8+ ratio can be a helpful tool to discriminate GvHD from infectious inflammation after allogeneic stem cell transplantation.

Adoptive immunotherapy using natural killer (NK) cells is currently under investigation, especially in situations where anti-neoplastic effect is needed but infusion of T cells is considered hazardous, such as in recipients of haematopoietic stem-cell transplantation (HSCT) from haploidentical donors. NK-cell therapy is mainly but not exclusively investigated in the setting of allogeneic stem-cell transplantation. NK cells may induce potent anti-leukaemic and possibly anti-rejection activity, and may even mitigate graft-versus-host disease (GvHD). It remains to be determined whether such effects are clinically important and whether or not they are mediated mainly or exclusively by KIR-HLA class I interactions. Recent advances in graft engineering has provided methods for isolating large numbers of purified NK cells. Several groups have shown that clinical-grade NK cells at doses up to 10(7)/kg may be collected and purified for the purpose of infusion to patients. Early results in a limited number of patients show that these cell doses may be administered without adverse events and possibly without inducing GvHD. Further study is required to determine whether such infusions will be useful in preventing graft rejection, exerting graft-versus-leukaemia effects, and/or hastening immune recovery.