HLA-DPB1 Reactive T Cell Receptors for Adoptive Immunotherapy in Allogeneic Stem Cell Transplantation.

HLA-DPB1 antigens are mismatched in about 80% of allogeneic hematopoietic stem cell transplantations from HLA 10/10 matched unrelated donors and were shown to be associated with a decreased risk of leukemia relapse. We recently developed a reliable in vitro method to generate HLA-DPB1 mismatch-reactive CD4 T-cell clones from allogeneic donors. Here, we isolated HLA-DPB1 specific T cell receptors (TCR DP) and used them either as wild-type or genetically optimized receptors to analyze in detail the reactivity of transduced CD4 and CD8 T cells toward primary AML blasts. While both CD4 and CD8 T cells showed strong AML reactivity in vitro, only CD4 T cells were able to effectively eliminate leukemia blasts in AML engrafted NOD/SCID/IL2Rγc-/- (NSG) mice. Further analysis showed that optimized TCR DP and under some conditions wild-type TCR DP also mediated reactivity to non-hematopoietic cells like fibroblasts or tumor cell lines after HLA-DP upregulation. In conclusion, T cells engineered with selected allo-HLA-DPB1 specific TCRs might be powerful off-the-shelf reagents in allogeneic T-cell therapy of leukemia. However, because of frequent (common) cross-reactivity to non-hematopoietic cells with optimized TCR DP T cells, safety mechanisms are mandatory.

Antibody-based therapies in patients with acute lymphoblastic leukemia.

The use of multiagent combination chemotherapy regimens results in cure rates of >90% for children and ∼40% for adults with acute lymphoblastic leukemia (ALL) but is associated with extensive toxicity and disappointingly low efficacy in relapsed patients. ALL blast cells express several surface antigens, including CD20, CD22, and CD19, which represent valuable targets for immunotherapy. Monoclonal antibodies, antibody-drug conjugates, and bispecific T-cell-engaging antibodies targeting these antigens offer novel mechanisms of action. Within the last several years, the anti-CD20 antibody rituximab has been added to chemotherapy for newly diagnosed patients <60 years with CD20+ pre-B ALL and significantly improved the 2-year event-free survival from 52% to 65%. In adults with relapsed or refractory CD22+ ALL, the antibody-drug conjugate inotuzumab ozogamicin resulted in a complete response rate of 81% and median overall survival of 7.7 months with reduced toxicity compared with standard chemotherapy. Similarly, the bispecific T-cell-engaging antibody blinatumomab yielded a complete response rate of 44% and a median overall survival of 7.7 months in an extensively treated ALL population. Moreover, ∼80% of ALL patients in complete remission with evidence of minimal residual disease (MRD) achieved a complete MRD response following treatment with blinatumomab. These results highlight the tremendous promise of antibody-based treatment approaches for ALL. Ongoing and future research is critical to further define the role of the various immunotherapies in the frontline treatment of ALL. Additional challenges include the optimal sequencing of the available antibodies in the relapsed setting as well as their integration with stem cell transplant and chimeric antigen receptor T-cell therapy.

Exonal switch down-regulates the expression of CD5 on blasts of acute T cell leukaemia.

To date, CD5 expression and its role in acute T cell lymphoblastic leukaemia (T-ALL) have not been studied closely. We observed a significant reduction in surface expression of CD5 (sCD5) on leukaemic T cells compared to autologous non-leukaemic T cells. In this study, we have shown the molecular mechanism regulating the expression and function of CD5 on leukaemic T cells. A total of 250 patients suffering from leukaemia and lymphoma were immunophenotyped. Final diagnosis was based on their clinical presentation, morphological data and flow cytometry-based immunophenotyping. Thirty-nine patients were found to be of ALL-T origin. Amplification of early region of E1A and E1B transcripts of CD5 was correlated with the levels of surface and intracellular expression of CD5 protein. Functional studies were performed to show the effect of CD5 blocking on interleukin IL-2 production and survival of leukaemic and non-leukaemic cells. Lack of expression of sCD5 on T-ALL blasts was correlated closely with predominant transcription of exon E1B and significant loss of exon E1A of the CD5 gene, which is associated with surface expression of CD5 on lymphocytes. High expression of E1B also correlates with increased expression of cytoplasmic CD5 (cCD5) among leukaemic T cells. Interestingly, we observed a significant increase in the production of IL-2 by non-leukaemic T cells upon CD5 blocking, leading possibly to their increased survival at 48 h. Our study provides understanding of the regulation of CD5 expression on leukaemic T cells, and may help in understanding the molecular mechanism of CD5 down-regulation.

T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is biologically distinct from its B lymphoblastic (B-ALL) counterpart and shows different kinetic patterns of disease response. Although very similar regimens are used to treat T-ALL and B-ALL, distinctions in response to different elements of therapy have been observed. Similar to B-ALL, the key prognostic determinant in T-ALL is minimal residual disease (MRD) response. Unlike B-ALL, other factors including age, white blood cell count at diagnosis, and genetics of the ALL blasts are not independently prognostic when MRD response is included. Recent insights into T-ALL biology, using modern genomic techniques, have identified a number of recurrent lesions that can be grouped into several targetable pathways, including Notch, Jak/Stat, PI3K/Akt/mTOR, and MAPK. With contemporary chemotherapy, outcomes for de novo T-ALL have steadily improved and now approach those observed in B-ALL, with approximately 85% 5-year event-free survival. Unfortunately, salvage has remained poor, with less than 25% event-free and overall survival rates for relapsed disease. Thus, current efforts are focused on preventing relapse by augmenting therapy for high-risk patients, sparing toxicity in favorable subsets and developing new approaches for the treatment of recurrent disease.

Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients.

Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called "damage-associated molecular patterns," DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response.

Significance of CD34/CD123 expression in detection of minimal residual disease in B-ACUTE lymphoblastic leukemia in children.

BACKGROUND: MRD is seen as the major cause of disease relapse. So, it gives important feedback about conventional treatment success and helps in selecting therapeutic alternatives. We aimed to compare the expression of CD34/CD123 on normal B-cell precursors in bone marrow ("hematogones") and on leukemic blasts in B-acute lymphoblastic leukemias (B-ALL) pediatric cases by flowcytometric analysis. Our study conducted on 20 children as a control and 30 B-ALL children cases at diagnosis and after 28days of induction therapy. We found that the less mature hematogones (dim CD45+) that express CD34 lack CD123 expression, whereas the more mature hematogones (moderate CD45+) lack CD34 but always express CD123. In contrast with this discordant pattern of CD34 and CD123 expression in hematogones, blasts in 24 of 30 cases (80%) of B-ALL showed concordant expression pattern of the 2 antigens: 63% (19 of 30) cases expressed both antigens, whereas 17% (5 of 30) expressed neither. Our study concluded that these distinct patterns of CD34/CD123 expression on hematogones (discordant) and B-ALL blasts (concordant) are useful in differentiating small populations of residual blasts from hematogones after induction therapy to detect MRD.

Occurrence of Donor Cell-derived Lymphoid Blast Crisis 24 Years Following Related Bone Marrow Transplantation for Chronic Myeloid Leukemia.

We herein report a unique case of donor cell leukemia (DCL), as donor cell-derived lymphoid blast crisis of chronic myeloid leukemia (CML) was observed 24 years after related bone marrow transplantation for CML in the chronic phase. Short tandem repeat testing of the leukemic blast sample revealed full donor chimerism, strongly indicative of DCL. The original donor is healthy with a normal complete blood cell count for the past 24 years. This rare case may provide a precious opportunity to consider not only the underlying mechanism of DCL, but also the pathogenesis of CML.

Characteristics of the TCR Vß repertoire in imatinib-resistant chronic myeloid leukemia patients with ABL mutations.

Diversity in the T cell receptor (TCR) repertoire provides a miniature defense ability for the T cell immune system that may be related to tumor initiation and progression. Understanding the T cell immune status of leukemia patients is critical for establishing specific immunotherapies. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vß T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vß repertoire in 4 cases with imatinib-resistant CML in blast crisis (BC-CML) with abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase domain mutations (KDMs). Examination of TCR V expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) and GeneScan analysis. Significantly skewed TCR Vß repertoires were observed in BC-CML patients with different KDMs, and 4 to 8 oligoclonally expanded TCR Vß subfamilies could be identified in each sample. Intriguingly, a relatively highly expanded Vß9 clone with the same length as complementarity- determining region 3 (CDR3) (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the only CML patient in myeloid blast crisis (MBC-CML). In conclusion, restricted TCR Vß repertoire expression and decreased clone complexity was a general phenomenon observed in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency of these patients. In addition, clonally expanded Vß9 T cell clones may indicate a specific immune response to leukemia-associated antigens in LBC-CML patients.

IL-17/IL-10 double-producing T cells: new link between infections, immunosuppression and acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is an incurable disease with fatal infections or relapse being the main causes of death in most cases. In particular, the severe infections occurring in these patients before or during any treatment suggest an intrinsic alteration of the immune system. In this respect, IL-17-producing T helper (Th17) besides playing a key role in regulating inflammatory response, tumor growth and autoimmune diseases, have been shown to protect against bacterial and fungal pathogens. However, the role of Th17 cells in AML has not yet been clarified. METHODS: T cell frequencies were assessed by flow cytometry in the peripheral blood of 30 newly diagnosed AML patients and 30 age-matched healthy volunteers. Cytokine production was determined before and after culture of T cells with either Candida Albicans or AML blasts. Statistical analyses were carried out using the paired and unpaired two-tailed Student's t tests and confirmed with the non parametric Wilcoxon signed-rank test. RESULTS: A strong increase of Th17 cells producing immunosuppressive IL-10 was observed in AML patients compared with healthy donors. In addition, stimulation of AML-derived T cells with a Candida albicans antigen induced significantly lower IFN-γ production than that observed in healthy donors; intriguingly, depletion of patient Th17 cells restored IFN-γ production after stimulation. To address the role of AML blasts in inducing Th17 alterations, CD4+ cells from healthy donors were co-cultured with CD33+ blasts: data obtained showed that AML blasts induce in healthy donors levels of IL-10-producing Th17 cells similar to those observed in patients. CONCLUSIONS: In AML patients altered Th17 cells actively cause an immunosuppressive state that may promote infections and probably tumor escape. Th17 cells could thus represent a new target to improve AML immunotherapy.

Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia.

The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off­target effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and their survival beyond remission. This minimal residual disease is the major cause of relapse and is responsible for a 5-year survival rate of only 24%. More specific and efficient approaches are therefore required to eradicate malignant cells while leaving healthy cells unaffected. In this study, we generated scFv antibodies that bind specifically to the surface of AML blast cells and AML bone marrow biopsy specimens. We isolated the antibodies by phage display, using subtractive whole-cell panning with AML M2­derived Kasumi­1 cells. By selecting for internalizing scFv antibody fragments, we focused on potentially novel agents for intracellular drug delivery and tumor modulation. Two independent methods showed that 4 binders were internalized by Kasumi-1 cells. Furthermore, we observed the AML­selective inhibition of cell proliferation and the induction of apoptosis by a recombinant immunotoxin comprising one scFv fused to a truncated form of Pseudomonas exotoxin A (ETA'). This method may therefore be useful for the selection of novel disease-specific internalizing antibody fragments, providing a novel immunotherapeutic strategy for the treatment of AML patients.

CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia.

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

Predicting early blast transformation in chronic-phase chronic myeloid leukemia: is immunophenotyping the missing link?

BACKGROUND: Flow cytometry (FC) is a commonly requested test in the workup of leukocytosis in community practices. The role of FC in chronic-phase chronic myeloid leukemia (CP-CML) is unknown. We hypothesized that finding aberrant cells with FC in CP-CML may predict early blast-phase (BP) transformation. METHODS: Results for FC performed at the time of diagnosis for adult and pediatric patients with CP-CML who were referred to our institution were reviewed, and they were correlated with outcomes. RESULTS: FC was performed at the time of diagnosis for 110 of 233 patients (47%) with CP-CML. Aberrant populations, representing a median of 2% (range, 0.3%-15%), were detected with FC in 30% of patients (33 of 110): 2 of these 33 patients expressed lymphoid markers, and 31 expressed aberrant myeloid markers. Patients received imatinib (85%), dasatinib (12%), or nilotinib (3%) as their first-line treatment. With a median follow-up of 43 months (range, 2-113 months), chronic myeloid leukemia transformed to BP in 5 of the 33 patients. The 2 patients with lymphoid markers and the 3 of 31 patients with aberrant myeloid markers experienced a transformation to lymphoid BP at a median of 11 months (range, 4-72 months) after the initiation of tyrosine kinase inhibitor therapy. Although both cases with detectable lymphoid markers rapidly progressed to lymphoid BP, the positive predictive value of BP transformation by the detection of myeloid aberrant cells with FC was only 10% (3 of 31). CONCLUSIONS: In contrast to aberrant myeloid markers, the detection of lymphoid markers by FC at the time of the diagnosis of CP-CML appears to be associated with early progression to lymphoid BP.

A novel self-lipid antigen targets human T cells against CD1c(+) leukemias.

T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c(+) acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c(+) human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy.

Down-regulation of signal transducer and activator of transcription 3 improves human acute myeloid leukemia-derived dendritic cell function.

Signal transducer and activator of transcription (STAT) 3 inhibits dendritic cell (DC) differentiation and is constitutively activated in blasts of approximately half of AML patients. We investigated the correlation between STAT3 activity, DC maturation and the ability to stimulate T-cells in primary acute myeloid leukemia (AML)-derived DCs. STAT3 knock-down by shRNAmir increased the ability of AML-DCs to stimulate T-cells. Treatment of AML-DC with arsenic trioxide, but not AG490, JSI-124 or NSC-74859, led to a more mature phenotype and enhanced T-cell stimulation, while having minimal effect on normal DC. We conclude that AML-DCs have improved immunogenicity after reducing STAT3.

KIR2DS1-dependent acquisition of CCR7 and migratory properties by human NK cells interacting with allogeneic HLA-C2+ DCs or T-cell blasts.

Natural killer (NK) cells may capture the CCR7 chemokine receptor from allogeneic CCR7(+) cells by trogocytosis and acquire migrating properties in response to lymph node chemokines. This event is negatively regulated by inhibitory killer Ig-like receptors (KIRs) and NKG2A. In this study, we analyzed the role of the HLA-C2-specific activating receptor KIR2DS1 in the process of CCR7 uptake by NK cells interacting with different allogeneic CCR7(+) cells. Co-incubation of KIR2DS1(+) fresh NK cells or NK-cell clones with HLA-C2(+) CCR7(+) lymphoblastoid cell lines resulted in increased CCR7 uptake. Remarkably, KIR2DS1 expression represented a major advantage for acquiring CCR7 from HLA-C2(+) allogeneic dendritic cells (DCs) and T-cell blasts. These findings have important implications in haploidentical hematopoietic stem cell transplantation in which donor-derived (alloreactive) KIR2DS1(+) NK cells, upon CCR7 acquisition, become capable of migrating toward lymph nodes, where they may kill patient DCs and T cells, preventing graft-versus-host and host-versus-graft reactions.

A novel nonradioactive CFDA assay to monitor the cellular immune response in myeloid leukemia.

BACKGROUND: Donor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, AML is a highly diverse disease and the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach. OBJECTIVE: To monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system to assess the antileukemia immunity by flow cytometry, correlated with [3H]-thymidine uptake. METHODS: The myeloid blasts derived from five patients with AML relapsed post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML progenitor cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail (50ng/mL of SCF; 25ng/mL of IL-3; 100ng/mL of GM-CSF; 100ng/mL of G-CSF; 2U/mL of EPO; 0.47g/L of transferrin; and 5×10(-5)mmol/L of 2-ME). The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake. The simultaneous use of TO-PRO-dye and calibrate beads allowed not only the cell viability to be known but also allowed quantification of the effector function. RESULTS: Here, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with our nonradioactive, CFDA-based assay provided comparable results with the classic [3H]-thymidine assay with an even lower ratio of effector to target cells. CONCLUSION: Taken together, the novel, nonradioactive, CFDA-based assay was a robust tool to monitor the antileukemic immune response after DLT in myeloid leukemias.

Characterization of plasminogen binding to NB4 promyelocytic cells using monoclonal antibodies against receptor-induced binding sites in cell-bound plasminogen.

The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17) that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20-26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

Cytometric evaluation of transferrin receptor 1 (CD71) in childhood acute lymphoblastic leukemia.

Transferrin receptor 1 (CD71) is a transmembrane glycoprotein responsible for cellular iron uptake. Higher expression of CD71 has been identified as a negative prognostic marker for numerous solid tumor types and for some lymphomas. The aim of this study was to evaluate CD71 expression on acute lymphoblastic leukemia (ALL) cells and to follow its possible clinical correlations. Sixty one patients, aged 1-17 years and diagnosed with ALL, were enrolled in the study. CD71 expression was analyzed on the bone marrow blastic cells by flow cytometry. CD71 expression on the leukemic blasts was diversified; in most patients, all blastic cells showed expression of CD71, but levels of expression varied. CD71 expression was statistically higher on T-lineage leukemias. Within the B lineage ALL, a significant difference in CD71 expression existed between precursor B ALL and mature B-ALL, which showed higher CD71 expression. CD71 expression positively correlated with Hgb concentration at diagnosis. Initial risk group assessment and therapy response were not correlated with CD71 expression, although disease free and overall survival times tended to be shorter in patients with B-lineage leukemias with initial high CD71 expression.