Eomes+T-betlow CD8+ T Cells Are Functionally Impaired and Are Associated with Poor Clinical Outcome in Patients with Acute Myeloid Leukemia

Abstract Acute myeloid leukemia (AML) is a heterogenous group of diseases characterized by a clonal proliferation of myeloid progenitors. Its poor prognosis with conventional chemotherapy justifies seeking for adjuvant immunotherapeutic approaches to eliminate minimal residual disease. We evaluated an immunotherapeutic strategy that bypass the need for epitope identification and the limitation due to HLA restriction. Naturally processed peptides were extracted by acid elution from AML cells at diagnosis, and loaded on mature dendritic cells (mDCs) derived from autologous monocytes obtained when the patients were in complete remission (CR). We evaluated i) the feasibility to elute naturally processed peptides from AML cells at diagnosis, ii) the capacity of mDCs loaded with eluted peptides (mDC/EP) to stimulate specific T cell lines in vitro. We showed that stimulation by mDC/EP was able to generate anti-leukemic T cells lines from PBMC of 6 AML patients in CR. CD4+ and CD8+ T cells were isolated from T cell lines of 5 patients and analyzed for their proliferation, INF-γ production and cytotoxicity in response to autologous or allogeneic AML targets, or to normal autologous PBMC. We showed that both CD4+ and CD8+ leukemia-specific T cells were generated in vitro by mDC/EP stimulations since proliferation of CD4+ T cells, IFN-γ secretion by CD4+ and CD8+ T cells and cytotoxicity mediated by CD8+ T cells were induced in response to stimulation with autologous AML cells. Furthermore, we could not detect auto-immune recognition of autologous normal PBMC, consistent with the specificity of the T cell response induced by mDC/EP. These results provide the proof of concept for using mDC/EP to vaccinate patients with poor-risk AML, and will soon be evaluated in a phse I/II clinical trial.

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Clonal Involvement of the CD34+CD33− Progenitor Population by Leukemic Cells Harboring FLT3/ITD Correlates with High Risk of Relapse in Pediatric Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v106.11.227.227 ◽

2005 ◽

Vol 106 (11) ◽

pp. 227-227

Author(s):

Jessica A. Wright ◽

Todd A. Alonzo ◽

Robert B. Gerbing ◽

William G. Woods ◽

Beverly J. Lange ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

High Risk ◽

Clinical Outcome ◽

Myeloid Leukemia ◽

Poor Clinical Outcome ◽

Free Survival ◽

Allelic Ratio ◽

Risk Of Relapse ◽

Acute Myeloid

Abstract Internal tandem duplication of the FLT3 gene (FLT3/ITD) has been associated with high risk of relapse in acute myeloid leukemia (AML) yet nearly 25–30% of the patients with FLT3/ITD have long-term disease free survival with conventional chemotherapy. We hypothesized that FLT3/ITD AML patients with poor clinical outcome may have disease that involves less mature hematopoietic precursors than patients with favorable outcome. To test this hypothesis, we isolated less mature, CD34+CD33− and more mature, CD34+CD33+ precursor cells from 24 pediatric AML patients enrolled on Children’s Cancer Group clinical trials CCG-2891 and 2961 previously identified as having a FLT3/ITD. Granulocyte/monocyte colonies (CFU-GM) were grown in methylcellulose, harvested, and analyzed for the presence of FLT3/ITD after 14 days of growth. Twenty patients yielded sufficient cells and growth of CFU-GM colonies for analysis. FLT3/ITD was detected in CFU-GM colonies derived from CD34+CD33+ cells in all patient samples (median 80% of colonies tested per patient, range 6–100%). In contrast, FLT3/ITD was detected in CFU-GM colonies derived from CD34+/CD33− cells in only 11 of the 20 patient samples (median 46% of colonies tested per patient, range 6–100%). Of the 9 patient samples without FLT3/ITD involvement of CD34+CD33− colonies, 8 achieved a CR, 6 of whom are long-term survivors, and one patient died of non-leukemic causes. In contrast, of the 11 patients with CD34+CD33− cell involvement, 9 either failed to achieve CR or relapsed after achieving CR, and 2 died of non-leukemic causes. Actuarial progression-free survival at 4 years from diagnosis for the patients with and without FLT3/ITD in the CD34+CD33− population was 0% vs. 68% respectively (p=0.017). As allelic ratio of the FLT3/ITD has been used to define high-risk patients within the FLT3/ITD cohort, we determined the FLT3/ITD allelic ratio in our study population and correlated it with the presence of FLT3/ITD in the CD34+CD33− population. Ten of the 11 (91%) of the patient samples with FLT3/ITD involvement of the progenitor cells had high allelic ratio compared to 5 of 9 (56%) of the patients without early cell involvement. Together these data suggest that clonal dominance of FLT3/ITD containing leukemia cells at the CD34+CD33− stage of hematopoietic development is correlated with a high risk of relapse. Further studies are required to determine whether clonal dominance at this hematopoietic stage is a variable that, independent of high allelic ratio, accounts for the poor clinical outcome seen in a subset of FLT3/ITD positive AML patients.

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Expression of Tumor-Associated Antigens (TAAs) in Acute Myeloid Leukemia (AML) Correlated with Specific T Cell Responses and Survival.

Blood ◽

10.1182/blood.v108.11.414.414 ◽

2006 ◽

Vol 108 (11) ◽

pp. 414-414

Author(s):

Jochen Greiner ◽

Michael Schmitt ◽

Li Li ◽

Krzysztof Giannopoulos ◽

Katrin Bosch ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Clinical Outcome ◽

Immune Responses ◽

Dna Microarray ◽

Myeloid Leukemia ◽

T Cell Responses ◽

Cell Responses ◽

Acute Myeloid

Abstract Several Tumor-associated antigens (TAAs) are expressed in acute myeloid leukemia (AML) and elicit specific immune responses of CD8 positive T cells. These specific T cell responses against leukemic blasts expressing TAAs might play a critical role in the control of minimal residual disease (MRD) in AML. Therefore, we investigated whether TAAs inducing specific immune responses in AML patients were associated with the clinical outcome. A DNA-microarray analysis of 116 AML samples was performed to correlate expression of TAAs to the clinical outcome. In these AML patients specific T cell responses to TAAs were assessed by ELISPOT analysis, tetramer staining and chromium release assays. Quantitative RT-PCR based validation of our results demonstrated the power of DNA microarray technology. We found a significant correlation of high mRNA expression of the TAA G250/CA9 with a longer overall survival (P=0.022), a trend for better outcome in patients with high expression levels of PRAME (P=0.103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1 we found no correlation with clinical outcome of AML patients. Moreover, co-expression of RHAMM/HMMR, PRAME and G250/CA9 provided a favorable prognostic effect (P=0.005). We found specific T cell responses at high frequency for these three antigens in AML patients. Positive immune reactions were detected in 8/17 (47%) AML patients for RHAMM/HMMR-R3-derived, in 7/10 (70%) for PRAME-P3-derived, and in 6/10 (60%) for newly characterized G250/CA9-G2-derived peptides. We detected a significant increased immune response of AML patients in complete remission compared to AML patients with refractory disease (P<0.001). Furthermore, we could demonstrate specific lysis of T2 cells and AML blasts presenting these epitope peptides RHAMM/HMMR-R3, PRAME-P3 and G250/CA9-G2. In conclusion, the expression of the TAAs RHAMM/HMMR, PRAME and G250/CA9 can induce strong anti-leukemic immune responses of CD8 positive T cells possibly enabling the control of MRD in AML patients. Thus, the antigens RHAMM/HMMR, PRAME and G250/CA9 represent interesting target structures for polyvalent immunotherapeutic approaches in AML.

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Identification and characterization of epitopes of the receptor for hyaluronic acid–mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2–positive patients with acute myeloid leukemia

Blood ◽

10.1182/blood-2004-12-4787 ◽

2005 ◽

Vol 106 (3) ◽

pp. 938-945 ◽

Cited By ~ 78

Author(s):

Jochen Greiner ◽

Li Li ◽

Mark Ringhoffer ◽

Thomas F. E. Barth ◽

Krzysztof Giannopoulos ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Hyaluronic Acid ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

T Cell Epitopes ◽

Computer Algorithms ◽

Cd8 Cells ◽

Acute Myeloid

AbstractThe receptor for hyaluronic acid–mediated motility (RHAMM/CD168) has been described as a leukemia-associated antigen. To define T-cell epitopes of RHAMM/CD168 toward specific immunotherapies for acute myeloid leukemia (AML), 10 potential HLA-A2–binding RHAMM/CD168 peptides (R1 to R10) were synthesized based on computer algorithms and screened by enzyme-linked immunospot (ELISPOT) analysis using CD8+ T cells isolated from peripheral blood (PB) of patients with AML and healthy donors. We found that CD8+ cells from 7 of 13 (54%) patients with AML presensitized with peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) specifically recognized T2 cells pulsed with R3 (39%) or R5 (15%) peptide. In contrast, only 4 of 21 (19%) healthy volunteers had CD8+ cells reactive with R3- or R5-pulsed T2 cells after presensitization. The presence of R3 peptide–specific effector T cells in the peripheral blood of patients with AML could be confirmed by staining as HLA-A2/R3 peptide tetramer+ CCR7-CD45RA+ cells. In chromium-51 release assays, peptide-primed CD8+ T cells from patients with AML were able to lyse RHAMM/CD168 peptide–pulsed T2 cells, AML blasts, and dendritic cells generated thereof (AML DCs). Transfection of COS7 cells with RHAMM/CD168 cDNA revealed that peptides R3 and R5 are naturally processed epitopes of RHAMM/CD168 that are presented in an HLA-A2–restricted manner. In summary, RHAMM/CD168 is a promising target for immunotherapies in patients with AML, and we have therefore initiated a clinical vaccination trial with R3 peptide. Because RHAMM/CD168 is also expressed in various other hematologic malignancies and solid tumors, vaccines targeting this antigen may have even wider application.

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