Leukemia Antigen Research Articles

Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8.

T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demon-strated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.

A label-free immunosensor for detecting common acute lymphoblastic leukemia antigen (CD10) based on gold nanoparticles by quartz crystal microbalance

Rapid and sensitive detection of the common acute lymphoblastic leukemia antigen (CD10) is crucial for diagnosing and prognosing hematopoietic tumors and several carcinomas. In the present study, a label-free immunosensor for detecting CD10 by Quartz Crystal Microbalance (QCM) was developed. The detection system has a good selectivity based on the biospecific interactions between the CD10 antigen and antibody, and the sensitivity of the immunosensor was further improved for Au NPs not only carried more antibodies but also acted as mass enhancers. Experimental results confirmed that this developed method could be conveniently used for the detection of CD10 without labeling, and CD10 could be measured quantitatively in the concentration range from 1.0×10−8 to 1.0×10−11M.

Regenerating blasts masquerading as relapse in a patient with ALL following G-CSF therapy.

TO THE EDITOR: Granulocyte colony-stimulating factor (G-CSF)-associated blastocytosis with a lack of an associated left shift poses a challenge for accurate assessment of the underlying disease [1]. In particular, blastocytosis after chemotherapy for acute leukemia may mimic a recurrence of underlying leukemia. Only a few cases of myeloblastosis in AML or ALL or of leukoerythroblastosis in AML after G-CSF therapy have been described anecdotally in the literature [2,3,4]. Herein, we report a case of myeloblastosis in ALL within 2 days of G-CSF administration masquerading as early relapse, causing a diagnostic dilemma. This was compounded by a striking lack of telltale evidence that G-CSF treatment was effective, such as increased granulation and a left shift. A 25-year-old man diagnosed with common acute lymphoblastic leukemia antigen [CALLA] positive, precursor B-cell ALL (Fig. 1A) completed his first phase of induction chemotherapy with methotrexate, cyclophosphamide, and cytarabine. His day 36 bone marrow showed 2.8% blasts with positive minimal residual disease (MRD) (0.046%). To accelerate recovery from persistent neutropenia, he was treated with G-CSF; an adequate response was noted, with a total leukocyte count (TLC) of 2.9×109/L. On day 70, he completed his second phase of induction chemotherapy and was started on G-GSF therapy for severe neutropenia (TLC: 0.76×109/L). On day 73, his peripheral blood smear showed marked leukopenia with a predominance of lymphocytes and 2% blasts without any left shift or increased granulations. This lack of prototypical G-CSF response along with previous MRD positivity and presence of 2% blasts led to a suspicion of relapse, and bone marrow examination was advised. Marrow aspirate showed hypocellular marrow with 14% blasts, and focal areas showed up to 25% blasts (Fig. 1B), along with a lack of a maturing myeloid component, which suggested relapse. Trephine biopsy, in contrast, showed hypocellularity with only a few scattered CD34 positive blasts. This necessitated flow cytometric evaluation, where the blasts showed negativity for B lymphoblastic markers and expressed myeloid markers, including CD13, CD33, and CD34, suggesting that they were regenerating myeloblasts (Fig. 1C-F). No further therapy was administered and the patient was kept on close hematological follow-up. The counts recovered after 4 days, with the typical responses of a left shift and increased granulation. Follow-up marrow examination after complete recovery of blood counts, 10 days after discontinuing G-CSF therapy, revealed cellular marrow elements with 4% blasts (regenerating) and negative MRD (<0.01%). Fig. 1 (A) Diagnostic blasts with a high nucleocytoplasmic ratio and scant agranular cytoplasm (Giemsa stain, ×1,000). (B) Regenerating blasts are larger with moderate granular cytoplasm (Giemsa stain, ×1,000). (C-F) Immunophenotyping showing ... Post-G-CSF blastocytosis is a problematic and confusing issue that is difficult to differentiate from disease recurrence and hemopoietic recovery. In our case, there were regenerating blasts (>20%) in the bone marrow, with an absence of telltale G-CSF-induced changes, such as presence of immature granulocytes, monocytosis, or prominent granulations on peripheral smear. To conclude, the regenerating blast percentage may increase after G-CSF therapy to >20%, without concomitant increases in other mature myeloid precursors. The lag time before complete response to G-CSF therapy may be as long as 10 days, as seen in our case, posing potential problems in management and a challenge in therapeutic decisions. A careful morphological assessment of the blasts at the time of diagnosis, aided by awareness of these G-CSF-associated aberrant patterns and flow cytometric characterization can help guide management decisions.

Peptide/MHC Tetramer-Based Sorting of CD8+ T Cells to a Leukemia Antigen Yields Clonotypes Drawn Nonspecifically from an Underlying Restricted Repertoire

Peptide/MHC Tetramer-Based Sorting of CD8+ T Cells to a Leukemia Antigen Yields Clonotypes Drawn Nonspecifically from an Underlying Restricted Repertoire

In Vitro Generation of Cytotoxic CD4 Lymphocyte Response against Autologous Acute Myeloid Leukemia

Relapsed and refractory disease shortens the survival of acute myeloid leukemia (AML) patients. Therefore, additional therapy apart from conventional chemotherapy and stem cell transplantation is urgently needed, especially in eradicating residual leukemic stem cells. The identification of leukemia associated antigens, and the observation that administration of allogeneic T cells may mediate graft versus leukemia effect paved the way to develop various immunotherapy strategies. Effective provision of both tumor antigen and co-stimulation are essential for strategies aimed at enrolling cytotoxic T- lymphocytes to eradicate leukemic cells. In this study, we attempted to up-regulate co- stimulators on leukemic cells (termed “dendritisation” to mimic dendritic cells in providing strong co-stimulation) and couple with inherent leukemic antigens to generate specific antileukemic T-lymphocytes against autologous blast cells. Our study shows evidence of distinct cytotoxicity of CD4 positive T lymphocytes against autologous blast cells. Dendritised AML blasts were able to function with antigen presenting capability, while still phenotypically existing as blast cells. This was also achievable for a wide range of subtypes of AML, making it a potential immunotherapy against AML.

Flow Cytometry-Based Maturity Score As a Novel Prognostic Parameter in AML

Background: Based on cytogenetic and molecular phenotypes, the European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). Single leukemic blast cell antigens were also shown to have prognostic value, but the influence of AML blast maturity within the different ELN risk groups is largely unknown.Methods: Our study includes 300 AML patients, who were diagnosed at our institution between 01/2003 and 04/2012 and in whom morphologic, immunophenotypic, cytogenetic and molecular data from primary diagnosis were available. A flow cytometric maturity score was developed in order to distinguish “mature” AML (AML-ma) from “immature” AML (AML-im) by means of a quantitative analysis of the expression of early progenitor cell antigens (CD34, CD117, and TdT). Clinical outcome analysis was performed both in the whole cohort and in the different ELN risk groups using Chi Square and Fisher’s Exact Test (CR/CRi) and Logrank Test (RFS, OS).Results: A mature AML blast phenotype (AML-ma) was associated with a significantly longer RFS (7.5 vs. 4.3 years) and OS (5.3 vs. 2.7 years) than an immature phenotype (AML-im) (p < 0.001). Low-risk phenotypes according to ELN (NPM1, PML-RARA) significantly accumulated in the AML-ma group, whereas high-risk aberrations (monosomal and complex aberrant karyotypes, -5 or del(5q), -7) had a higher frequency in AML-im.Interestingly, the differences in RFS and OS were maintained in a subgroup analysis within the different ELN risk groups. Statistical significance was obtained in the “intermediate risk” group with AML-ma being superior to AML-im (RFS: 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS: 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). In contrast, there was no correlation between AML blast maturity and CR/CRi rates.Conclusions: Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome, even within the different ELN risk groups. Particularly for “intermediate risk” patients according to ELN, the score may be useful in post remission therapy decision-making. It remains to be clarified whether these results reflect differences in genetic instability and/or a different frequency of leukemia stem cells in AML-ma and AML-im. [Display omitted] [Display omitted] RFS (A) and OS (B) of AML patients with mature (AML-ma) and immature (AML-im) blast phenotype within the ELN “intermediate risk” group. AML-ma show a significantly longer RFS and OS compared to AML-im. DisclosuresNo relevant conflicts of interest to declare.

Novel TCR-like Bi-Specific T Cell Engaging Antibody Targeting the PR1/HLA-A2 Myeloid Leukemia Antigen

PR1 is a HLA-A2 restricted immune-dominant peptide derived from proteinase 3 and neutrophil elastase - leukemia associated self-antigens aberrantly over-expressed in myeloid leukemia. The persistent PR1/HLA-A2 specific cytotoxic T cell responses are associated with prolonged remission from myeloid leukemia, suggesting that PR1/HLA-A2 would be a potent target for T cell based immunotherapy. Previously, we have successfully developed a high affinity T cell receptor (TCR)-like antibody specific for the PR1/HLA-A2 complex (h8F4), and demonstrated that h8F4 mediated specific lyses of myeloid leukemia cells invitro via antibody dependent cell mediated cytotoxicity (ADCC), and significantly reduced leukemic blasts invivo in murine xenograft model of treatment refractory acute myeloid leukemia. We hypothesize that the therapeutic efficacy of h8F4 would be enhanced by combining T cell mediated cytotoxicity through a novel antibody engineering technology, called, Bi-Specific T cell Engager (BiTE) specific for T cells and tumor associated antigens. The BiTE molecules redirect effector function of polyclonal T cells against the tumor leading to lyses of tumor cells, and early clinical data show promising therapeutic efficacy of this approach. In the current study, we constructed Bi-Specific T cell Engaging antibody: h8F4-BiTE, a single chain Fragment of variable domain (scFv) of h8F4 conjugated with scFv of anti-CD3 antibody – OKT3, and produced h8F4-BiTE proteins using eukaryotic expression system. The h8F4-BiTE proteins retained the similar binding specificity to PR1/HLA-A2 as compared to the parent antibody (h8F4), demonstrated by flowcytometry based binding assays with a series of alanine substituted PR1 peptide/HLA-A2 complexes on T2 cells. The 8F4-BiTE bound to native and endogenous PR1/HLA-A2 complexes presented by HLA-A2 positive or transduced myeloid cell lines such as K562, THP-1, and HL-60. In addition, h8F4-BiTE showed comparable binding affinity to CD3 moiety of T cells. Lastly, h8F4-BiTE proteins activated T cells in a PR1/HLA-A2 specific and dose dependent manner, shown by increased expression of activation markers such as CD69 when co-cultured with PR1-pulsed T2 target cells in various concentrations of h8F4-BiTE proteins. Importantly, the PR1/HLA-A2 specific activation of T cells by h8F4-BiTE proteins was equally efficient among all subsets of T cells regardless of their phenotypes, e.g., CD4 vs CD8, and naïve vs memory, suggesting that h8F4-BiTE would be a highly potent inducer of cytotoxic T cells against PR1/HLA-A2 positive myeloid leukemia. Moreover, we generated multiple h8F4-BiTE variants with specific mutations in frameworks and/or complement determining regions (CDRs), and compared binding affinity and function to wildtype h8F4-BiTE. The h8F4-BiTE variants indeed improved specificity to PR1/HLA-A2 complex by complete elimination of residual non-specific binding to HLA-A2, and increased the binding affinity to CD3 by 10-folds resulting in dramatic 50 fold increase in potency to activate T cells. In conclusion, we have developed a novel immunetherapeutics: h8F4-BiTE targeting PR1/HLA-A2 myeloid leukemia antigen. We demonstrated high binding affinity and specificity of h8F4-BiTE to both PR1/HLA-A2 on myeloid leukemia cells and CD3 on T cells and in vitro activation of T cells in the presence of PR1/HLA-A2 expressing target cells in a dose dependent manner. Finally, we improved antigen specificity and functional activity of h8F4-BiTE via selective site-directed mutagenesis. Further preclinical evaluation of therapeutic efficacy is thus warranted using animal in vivo animal treatment model with NOD/SCID/IL2Rγ −/− mice xenografted with human myeloid leukemia cells. This unique TCR-like therapeutic agent adds an additional mechanism of action to PR1/HLA-A2 specific antibody (h8F4) and promises to enhance T cell function against myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Timely Controlled T Cell Receptor Expression of Genetically Engineered Precursor T Cells Requires Early Transgene Induction for Leukemia Control after Hematopoietic Stem Cell Transplantation

BACKGROUND: The co-transplantation of hematopoietic stem cells (HS) with those that have been engineered to express tumor-reactive T cell receptors (TCRs) and differentiated into precursor T cells (preTs) may optimize tumor reduction. Since expression of potentially self-(tumor-) reactive TCRs will lead to negative selection upon thymic maturation, we investigated whether preTs forced to express a leukemia-reactive TCR under the control of a tetracycline-inducible promoter would allow timely controlled TCR expression thereby avoiding thymic negative selection. METHODS: Using lentiviral vectors, murine LSK cells were engineered to express a Tetracycline-inducible TCR directed against a surrogate leukemia antigen. TCR-transduced LSK cells were co-cultured on T cell development-supporting OP9-DL1 cells to produce preTs. Lethally-irradiated B6/NCrl recipients received syngeneic T cell-depleted bone marrow and 8 × 106 syngeneic or allogeneic (B10.A) TCR-engineered preTs. An otherwise lethal leukemia cell (C1498) challenge was given 28 days later. RESULTS: After in vivo maturation and gene induction up to 70% leukemia free survival was achieved in recipients of syngeneic TCR-transduced preTs (p<0.001) as shown in figure 1A. Importantly, transfer of allogeneic gene-manipulated preTs increased the survival of recipients (p<0.05) without inducing graft versus host disease (GVHD). Non-transduced preTs provided significantly lower leukemia protection being not significantly superior to the PBS controls. The progenies of engineered preTs gave rise to effector and central memory cells providing protection even after repeated leukemia challenge (Figure 1B and 1C). In vitro transduction and consecutive expansion of mature T cells required at least 40 × 106 cells/recipient to mediate similar anti-leukemia efficacy, risking the development of severe GVHD if of mismatched origin, and providing no long-term protection. Importantly, while transgene induction starting immediately after transplant forced CD8+ T cell development and was required to obtain a mature T cell subset of targeted specificity, late induction favored CD4 differentiation and failed to produce a leukemia-reactive population due to missing thymic positive selection. CONCLUSION: Co-transplanting TCR gene-engineered preTs is of high clinical relevance since small numbers of even mismatched HS can be transduced at a reasonable cost, expanded in vitro, stored if needed, and provide potent and long lasting leukemia protection. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The immune receptor Tim-3 mediates activation of PI3 kinase/mTOR and HIF-1 pathways in human myeloid leukaemia cells

The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

AbstractThe efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

Engineering therapeutic cancer vaccines that activate antitumor immunity.

Vaccination represents one the most effective methods of preventing disease. Because dendritic cells (DCs) are the most efficient antigen presenting cells, exploiting their plasticity is likely to yield improved therapeutic vaccines. Herein, we applied a novel DC-based vaccine (i.e., DC loaded with leukemia antigens that have been transfected with an IL-10 siRNA capable of coordinately activating DCs via TLR7/8) in a rat model of acute myeloid leukemia. Leukemic rats treated with this new vaccine had less leukemic cell mass in their bone marrows and less extramedullar dissemination of the leukemic disease examined postmortem compared with rats given the control vaccine. Collectively, the new strategy demonstrates the possible usefulness of dual siRNAs as an immunomodulatory drug with antileukemic properties.

Differential ion mobility spectrometry coupled to tandem mass spectrometry enables targeted leukemia antigen detection.

Differential ion mobility spectrometry (DIMS) can be used as a filter to remove undesired background ions from reaching the mass spectrometer. The ability to use DIMS as a filter for known analytes makes DIMS coupled to tandem mass spectrometry (DIMS–MS/MS) a promising technique for the detection of cancer antigens that can be predicted by computational algorithms. In experiments using DIMS–MS/MS that were performed without the use of high-performance liquid chromatography (HPLC), a predicted model antigen, GLR (FLSSANEHL), was detected at a concentration of 10 pM (20 amol) in a mixture containing 94 competing model peptide antigens, each at a concentration of 1 μM. Without DIMS filtering, the GLR peptide was undetectable in the mixture even at 100 nM. Again, without using HPLC, DIMS–MS/MS was used to detect 2 of 3 previously characterized antigens produced by the leukemia cell line U937.A2. Because of its sensitivity, a targeted DIMS–MS/MS methodology can likely be used to probe for predicted cancer antigens from cancer cell lines as well as human tumor samples.

CD8αα+ Innate-Type Lymphocytes in the Intestinal Epithelium Mediate Mucosal Immunity

Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.

Dendritic cells and acute myeloid leukemia

Dendritic cells can be derived from leukemia cells and normal precursor cells in the patients with acute myeloid leukemia (AML). Dendritic cells may capture leukemia antigen in bone marrow or lymph nodes, and present leukemia common antigen to stimulate proliferation of specific CD8(+) T cells, playing anti-leukemia effect. Dendritic cells for clinical and experimental use are transformed from leukemia cells and peripheral blood mononuclear cells and loaded in vitro with leukemia -specific or tumor common antigen, play a therapeutic role after reinfusion. This article reviews dendritic cells in the immunotherapy of AML.

Ultra Low-Dose IL-2 for GVHD Prophylaxis after Allogeneic Hematopoietic Stem Cell Transplantation Mediates Expansion of Regulatory T Cells without Diminishing Antiviral and Antileukemic Activity

GVHD after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Because Tregs express high levels of the interleukin (IL)-2 receptor, they may selectively expand in vivo in response to doses of IL-2 insufficient to stimulate T effector T-cell populations, thereby preventing GVHD. We prospectively evaluated the effects of ultra low-dose (ULD) IL-2 injections on Treg recovery in pediatric patients after alloSCT and compared this recovery with Treg reconstitution post alloSCT in patients without IL-2. Sixteen recipients of related (n = 12) or unrelated (n = 4) donor grafts received ULD IL-2 post hematopoietic stem cell transplantation (HSCT; 100,000-200,000 IU/m(2) ×3 per week), starting <day 30 and continuing for 6 to 12 weeks. No grade 3/4 toxicities were associated with ULD IL-2. CD4(+)CD25(+)FoxP3(+) Tregs increased from a mean of 4.8% (range, 0%-11.0%) pre IL-2 to 11.1% (range, 1.2%-31.1%) following therapy, with the greatest change occurring in the recipients of matched related donor (MRD) transplants. No IL-2 patients developed grade 2-4 acute GVHD (aGVHD), compared with 4 of 33 (12%) of the comparator group who did not receive IL-2. IL-2 recipients retained T cells reactive to viral and leukemia antigens, and in the MRD recipients, only 2 of 13 (15%) of the IL-2 patients developed viral infections versus 63% of the comparator group (P = 0.022). Hence, ULD IL-2 is well tolerated, expands a Treg population in vivo, and may be associated with a lower incidence of viral infections and GVHD.

Development of a rice-based peptide vaccine for Japanese cedar and cypress pollen allergies

Peptide immunotherapy using dominant T-cell epitopes is a safe treatment alternative to conventional subcutaneous injection of natural crude allergen extract, which is sometimes accompanied by anaphylactic shock. For Japanese cedar pollinosis (JCP), hybrid peptides composed of six to seven major T-cell epitopes (7Crp peptide) from the causative allergens Cry j 1 and Cry j 2 have been developed on the basis of different human leukemia antigen class II restrictions, because of the diversity of patients' genetic backgrounds. However, other dominant T-cell epitopes that are produced in some patients are not covered by these peptides. To develop a more universal peptide vaccine for JCP, we generated transgenic rice seeds containing seven new T-cell epitopes (Crp3) in addition to the T-cell epitopes used in the 7Crp peptide. Next, we co-expressed unique T-cell epitopes (6Chao) from the Japanese cypress pollen allergens Cha o 1 and Cha o 2 in transgenic rice seeds, with 7Crp and Crp3. These transgenic rice seeds, containing many highly homologous T-cell epitopes derived from cedar and cypress allergens, are expected to be applicable to a wide range of patients suffering from these pollen allergies.

The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors

Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms' tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity.

Adoptive T-cell therapy for Leukemia.

Allogeneic stem cell transplantation (alloSCT) is the most robust form of adoptive cellular therapy (ACT) and has been tremendously effective in the treatment of leukemia. It is one of the original forms of cancer immunotherapy and illustrates that lymphocytes can specifically recognize and eliminate aberrant, malignant cells. However, because of the high morbidity and mortality that is associated with alloSCT including graft-versus-host disease (GvHD), refining the anti-leukemia immunity of alloSCT to target distinct antigens that mediate the graft-versus-leukemia (GvL) effect could transform our approach to treating leukemia, and possibly other hematologic malignancies. Over the past few decades, many leukemia antigens have been discovered that can separate malignant cells from normal host cells and render them vulnerable targets. In concert, the field of T-cell engineering has matured to enable transfer of ectopic high-affinity antigen receptors into host or donor cells with greater efficiency and potency. Many preclinical studies have demonstrated that engineered and conventional T-cells can mediate lysis and eradication of leukemia via one or more leukemia antigen targets. This evidence now serves as a foundation for clinical trials that aim to cure leukemia using T-cells. The recent clinical success of anti-CD19 chimeric antigen receptor (CAR) cells for treating patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia displays the potential of this new therapeutic modality. In this review, we discuss some of the most promising leukemia antigens and the novel strategies that have been implemented for adoptive cellular immunotherapy of lymphoid and myeloid leukemias. It is important to summarize the data for ACT of leukemia for physicians in-training and in practice and for investigators who work in this and related fields as there are recent discoveries already being translated to the patient setting and numerous accruing clinical trials. We primarily focus on ACT that has been used in the clinical setting or that is currently undergoing preclinical testing with a foreseeable clinical endpoint.

Differential Ion Mobility Spectrometry Coupled to Tandem Mass Spectrometry Enables Targeted Leukemia Antigen Detection

Differential Ion Mobility Spectrometry Coupled to Tandem Mass Spectrometry Enables Targeted Leukemia Antigen Detection