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Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34+ hematopoietic stem cells from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34+ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

ASH Abstract. Intro Acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) are poor prognosis hematological malignancies characterized by abnormal hematopoiesis and dysfunctions of the hematopoietic stem cell system. Chemotherapy remains the standard of care but is associated with side effects and often high rates of relapse. Today, less than a third of patients diagnosed with AML are cured. Bispecific T-cell engagers (BiTEs) are promising immunotherapeutic agents intended for cancer treatment. BiTEs are small molecules constructed of two single chain variable fragments (scFv) connected in tandem by a flexible linker that acts by retargeting T-cells against tumor cells. One scFv binds to CD3, while the second scFv binds to a tumor-associated antigen. This structure and specificity allow a BiTE construct to physically link a T-cell to a tumor cell, stimulating effector cell activation ultimately leading to cytokine production and tumor killing. Material BiTEs against CD34/CD3 and relevant controls were constructed by recombinant DNA technology and purified from the supernatants of transfected CHO cells following standard procedures. The scFv domain binding to CD34 is positioned N-terminally, and the scFv binding to CD3e C-terminally followed by a hexa-histidine sequence. Results By co-culturing T-cells and target AML cells for 48 h in the presence of increasing concentrations of BiTE or controls, we observed that CD34-BiTE efficiently triggered T-cell-mediated depletion of the CD34 hi and CD34 low cell lines, while negative controls killed none of the target cell lines. Next, we examined the T-cell activation and proliferation. We observed that both CD4+ and CD8+ T-cells presented high levels of CD25/CD69 expressions when the CD34+ cell lines were co-cultured with T-cells in the presence of the CD34/CD3 BiTE. No unspecific activation was found when CD34- cell line was used as target cell. Since CD34 is constitutively expressed by HSCs, the CD34-specific BiTE may deplete not only CD34 +AML blasts but also healthy HSCs. To test this, T-cells and HSCs were purified from PBSC grafts and co-cultured in the presence of either CD34/CD3 BiTE or controls. After co-culture, a significant depletion of CD34 + HSCs was observed for the CD34/CD3 BiTE. To address the potential of the anti-CD34 BiTE in vivo, we next established a human CD34 + cell line in NSG mice per intravenous injection and randomized into three different groups and started treatment the day after. Two groups of mice received two consecutive cycles of one intraperitoneal injection of freshly isolated human T-cells followed by daily intravenous injections of either BiTE or control. The mice were euthanatized at day 21 by which the AML burden was measured, and T-cells quantified. No side effects of the treatment, including after BiTE administration, was observed. There were statistically significant reductions of leukemia burden in both bone marrow and spleen in mice receiving T-cells and BiTE compared to T-cells only and control. Conclusions We show that the CD34/CD3 BiTE is able to promote T-cell activation and killing of CD34-expressing target cells with high efficacy in vitro and in vivo, supporting the translation of this drug into clinical trials. In this scenario, the treatment with CD34-targeting BiTE prior to HSCT would trigger the patient's T-cells to deplete CD34 + leukemic blasts and HSCs. As consequence, this adjuvant treatment would decrease the use of cytotoxic and cytostatic conditioning drugs before HSCT, reducing life-threatening complications such as GvHD and infections. Disclosures Arruda: Anocca: Current Employment, Research Funding. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mattsson: MattssonAB medical: Current Employment, Current holder of individual stocks in a privately-held company. Onfelt: Desumo: Current Employment, Current holder of individual stocks in a privately-held company. Uhlin: XNK therapeutics: Current Employment, Current holder of stock options in a privately-held company.

Summary Background Bacille Calmette–Guerin (BCG) is the world's most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content peptide microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses. Methods The sera of BCG-vaccinated healthy adults were tested on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained at 56, 112, and 252 days after vaccination. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods. Results IgG recognition of host peptides exhibited a peak at day 56 post BCG vaccination in all study subjects tested, which diminished over time. Primarily, IgG responses exhibited increased reactivity to ion transporters (sodium, calcium channels), cytokine receptors (interleukin 2 receptor β (IL2Rβ), fibroblast growth factor receptor 1 (FGFR1)), other cell surface receptors (inositol, somatostatin, angiopoeitin), ribonucleoprotein, and enzymes (tyrosine kinases, phospholipase) on day 56. There was decreased IgG reactivity to transforming growth factor-beta type 1 receptor (TGFβR1) and, in agreement with the peptide microarray findings, immunohistochemical analysis of TB-infected lymph node samples revealed an overexpression of TGFβR in granulomatous lesions. Moreover, the vesicular monoamine transporter (VMAT2) showed increased reactivity on days 112 and 252, but not on day 56 post-vaccination. IgG to interleukin 4 receptor (IL4R) showed increased reactivity at 112 days post-vaccination, while IgG to IL2Rβ and FGFR1 showed decreased reactivity on days 112 and 252 as compared to day 56 post BCG vaccination. Conclusions BCG vaccination modifies the host's immune landscape after 56 days, but this imprint changes over time. This may influence the establishment of immunological memory in BCG-vaccinated individuals.

Anti-tuberculosis drug treatment is known to affect the number, phenotype, and effector functionality of antigen-specific T-cells. In order to objectively gauge Mycobacterium tuberculosis (MTB)-specific CD8+ T-cells at the single-cell level, we developed soluble major histocompatibility complex (MHC) class I multimers/peptide multimers, which allow analysis of antigen-specific T-cells without ex vivo manipulation or functional tests. We constructed 38 MHC class I multimers covering some of the most frequent MHC class I alleles (HLA-A*02:01, A*24:02, A*30:01, A*30:02, A*68:01, B*58:01, and C*07:01) pertinent to a South African or Zambian population, and presenting the following MTB-derived peptides: the early expressed secreted antigens TB10.4 (Rv0288), Ag85B (Rv1886c), and ESAT-6 (Rv3875), as well as intracellular enzymes, i.e., glycosyltransferase 1 (Rv2957), glycosyltransferase 2 (Rv2958c), and cyclopropane fatty acid synthase (Rv0447c). Anti-TB treatment appeared to impact on the frequency of multimer-positive CD8+ T-cells, with a general decrease after 6 months of therapy. Also, a reduction in the total central memory CD8+ T-cell frequencies, as well as the antigen-specific compartment in CD45RA - CCR7+ T-cells was observed. We discuss our findings on the basis of differential dynamics of MTB-specific T-cell frequencies, impact of MTB antigen load on T-cell phenotype, and antigen-specific T-cell responses in tuberculosis. (c) 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

Aerosols containing Mycobacterium tuberculosis (MTB) generated from the cough of patients with active pulmonary tuberculosis are the source of MTB infection. About 70% of individuals exposed to infected aerosols do not get infected, depending on the intensity and duration of MTB exposure. Only 40% of the rest of the individuals (about 10% of those originally exposed) develop primary tuberculosis, whereas the remaining 60% contain the infection with generation of a robust immune response leading to latent tuberculosis, which is regarded as a spectrum rather than a single entity. The mechanisms involved in this natural protection are not yet well understood. There is an increasing need to integrate all disparate observations into a coherent systems biology approach for a comprehensive understanding: we need to decipher the nature of success and failure in MTB infection in humans. New advances in cellular immunology will aid in achieving that goal. We review here the nature of MTB peptide generation, antigen presentation, and detection of major histocompatibility complex class I and II-presented T-cell epitopes. Cross-sectional thinking from lessons learned in the context of the major efforts to develop vaccines will help to dissect biologically relevant mechanisms that need to be translated into the clinical context of MTB infection with the aim to (1) better understand clinically relevant T-cell responses in individuals protected from tuberculosis disease and develop markers of immune protection and vaccine take, (2) characterize the nature of the immune response in individuals who are not able to contain MTB infection, and ultimately (3) characterize markers to gauge response to therapy.

High-tuberculosis (TB)-burden countries are located in sub-Saharan Africa. We examined the frequency of human leukocyte antigen (HLA) alleles, followed by recombinant expression of the most frequent HLA-A alleles, i.e., HLA-A*3001 and HLA-A*3002, to study differences in mycobacterial peptide presentation and CD8 + T-cell recognition. We screened a peptide library (9-mer peptides with an 8-amino-acid overlap) for binding, affinity, and off-rate of the Mycobacterium tuberculosis -associated antigen TB10.4 and identified only three TB10.4 peptides with considerable binding to HLA-A*3001. In contrast, 22 peptides bound to HLA-A*3002. This reflects a marked difference in the binding preference between the two alleles, with A*3002 tolerating a more promiscuous peptide-binding pattern and A*3001 accommodating only a very selective peptide repertoire. Subsequent analysis of the affinity and off-rate of the binding peptides revealed a strong affinity (8 nM to 7 μM) and moderate off-rate (20 min to 3 h) for both alleles. Construction of HLA-A*3001 and HLA-A*3002 tetramers containing selected binding peptides from TB10.4, including a peptide which was shared among both alleles, QIMYNYPAM (TB10.4 3-11 ), allowed us to enumerate epitope-specific T cells in HLA-A*3001- and HLA-A*3002-typed patients with active TB. HLA-A*3001 and HLA-A*3002 major histocompatibility complex-peptide complexes were recognized in individuals with active TB, irrespective of their homozygous HLA-A*3001 or HLA-A*3002 genetic background. The antigen-specific T cells exhibited the CD45RA + CCR7 + precursor phenotype and the interleukin-7 receptor (CD127), which were different from the phenotype and receptor exhibited by the parental CD8 + T-cell population.

The molecular definition of major histocompatibility complex (MHC) class I-presented CD8(+) T-cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T-cell-based diagnostics of tuberculosis (TB) and the measurement of TB vaccine-take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)-A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I-binding peptides from overlapping 9-mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I-binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N- and C-termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off-rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8(+) T-cell interaction with their nominal MHC class I-peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8(+) T cells from patients with active pulmonary TB. HLA-B alleles served as the dominant MHC class I restricting molecules for anti-Mtb TB10.4-specific CD8(+) T-cell responses measured in CD8(+) T cells from patients with pulmonary TB.

SummaryThe nature and longevity of the T-cell response directed against Mycobacterium tuberculosis (MTB) are important for effective pathogen containment. We analyzed ex vivo the nature of MTB antigen-specific T-cell responses directed against the MTB secreted antigens Rv0288, Rv1886c, Rv3875, the antigens Rv2958c, Rv2957, and Rv0447c (intracellular, non-secreted enzymes) in blood from Korean patients with active tuberculosis (TB). MTB-specific T-cell function was defined by intracellular cytokine production (interleukin (IL)-2, interferon gamma, tumour necrosis factor alpha, and IL-17) and by multimer-guided (HLA-A*02:01 and HLA-A*24:02) analysis of epitope-specific CD8+ T-cells, along with phenotypic markers (CD45RA and CCR7), CD107a, a marker for degranulation, and CD127 co-staining for T-cell differentiation and homing. Cytokine production analysis underestimated the frequencies of MTB antigen-specific T-cells defined by major histocompatibility complex (MHC) class I–peptide multimer analysis. We showed that MTB antigen-specific CD8+ T-cells exhibit a distinct marker profile associated with the nature of the MTB antigens, i.e., Rv0288, Rv1886c, and Rv3875-reactive T-cells clustered in the precursor T-cell compartment, whereas Rv2958c, Rv2957, and Rv0447c-reactive T-cells were associated with the terminally differentiated T-cell phenotype, in the patient cohort. Rv0288, Rv1886c, and Rv3875-specific CD8+ T-cells were significantly enriched for CD107a+ T-cells in HLA-A*02:01 (p

Human TCRαβ(+) CD4(-)CD8(-) double-negative (DN) T cells represent a minor subset in peripheral blood, yet are important in infectious diseases and autoimmune responses. We examined the frequency of DN T cells in 17 patients after allogeneic hematopoietic stem cell transplantation (aHSCT) at 1, 2, 3, 6, and 12 months post-aHSCT and show that these cells increase early after aHSCT and decrease with time after aHSCT. DN T cells reside in the terminally differentiated effector (CD45RA(+)CCR7(-)) T-cell population and are polyclonal, determined by T-cell receptor Vβ CDR3 analysis. Gene expression analysis of ex vivo sorted DN T cells showed a distinct set of gene expression, including interleukin-8, as compared with CD4(+) or CD8(+) T cells. DN T cells contributed to MHC class I-restricted EBV-directed immune responses, defined by antigen-specific cytokine production and by detection of HLA-A*02:01-restricted EBV BMLF-1 (GLCTLVAML), LMP-2A (CLGGLLTMV), and HLA-A*24:02-restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA3 (RYSIFFDY)-specific T cells. We created retroviral-transfected Jurkat cell lines with a Melan-A/MART-1-specific TCR(+) and the CD8α chain to study TCR(+) DN T cells in response to their nominal MHC class I/peptide ligand. We show that DN T cells exhibit increased TCRζ chain phosphorylation as compared with the TCR(+)CD8(+) transgenic T-cell line. DN T cells contribute to antigen-specific T-cell responses and represent an effector T-cell population that may be explored in immunotherapeutic approaches against viral infections or transformed cells.

The first cases of totally drug-resistant (TDR) tuberculosis (TB) were reported in Italy 10 years ago; more recently, cases have also been reported in Iran, India and South Africa. Although there is no consensus on terminology, it is most commonly described as 'resistance to all first- and second-line drugs used to treat TB'. Mycobacterium tuberculosis (M.tb) acquires drug resistance mutations in a sequential fashion under suboptimal drug pressure due to monotherapy, inadequate dosing, treatment interruptions and drug interactions. The treatment of TDR-TB includes antibiotics with disputed or minimal effectiveness against M.tb, and the fatality rate is high. Comorbidities such as diabetes and infection with human immunodeficiency virus further impact on TB treatment options and survival rates. Several new drug candidates with novel modes of action are under late-stage clinical evaluation (e.g., delamanid, bedaquiline, SQ109 and sutezolid). 'Repurposed' antibiotics have also recently been included in the treatment of extensively drug resistant TB. However, because of mutations in M.tb, drugs will not provide a cure for TB in the long term. Adjunct TB therapies, including therapeutic vaccines, vitamin supplementation and/or repurposing of drugs targeting biologically and clinically relevant molecular pathways, may achieve better clinical outcomes in combination with standard chemotherapy. Here, we review broader perspectives of drug resistance in TB and potential adjunct treatment options.

Meeting abstracts Expansion of antigen-specific T cells, from peripheral blood specific for tumor-associated antigens (TAAs) is a prerequisite for the advanced cellular therapy. Such antigen-specific T cells should express a Th1-functional phenotype and are able to enter tumor-tissue. We identified

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.

Background Previous exposures to flu and subsequent immune responses may impact on 2009/2010 pandemic flu vaccine responses and clinical symptoms upon infection with the 2009 pandemic H1N1 influenza strain. Qualitative and quantitative differences in humoral and cellular immune responses associated with the flu vaccination in 2009/2010 (pandemic H1N1 vaccine) and natural infection have not yet been described in detail. We designed a longitudinal study to examine influenza- (flu-) specific immune responses and the association between pre-existing flu responses, symptoms of influenza-like illness (ILI), impact of pandemic flu infection, and pandemic flu vaccination in a cohort of 2,040 individuals in Sweden in 2009–2010. Methods Cellular flu-specific immune responses were assessed by whole-blood antigen stimulation assay, and humoral responses by a single radial hemolysis test. Results Previous seasonal flu vaccination was associated with significantly lower flu-specific IFN-γ responses (using a whole-blood assay) at study entry. Pandemic flu vaccination induced long-lived T-cell responses (measured by IFN-γ production) to influenza A strains, influenza B strains, and the matrix (M1) antigen. In contrast, individuals with pandemic flu infection (PCR positive) exhibited increased flu-specific T-cell responses shortly after onset of ILI symptoms but the immune response decreased after the flu season (spring 2010). We identified non-pandemic-flu vaccinated participants without ILI symptoms who showed an IFN-γ production profile similar to pandemic-flu infected participants, suggesting exposure without experiencing clinical symptoms. Conclusions Strong and long-lived flu-M1 specific immune responses, defined by IFN-γ production, in individuals after vaccination suggest that M1-responses may contribute to protective cellular immune responses. Silent flu infections appeared to be frequent in 2009/2010. The pandemic flu vaccine induced qualitatively and quantitatively different humoral and cellular immune responses as compared to infection with the 2009 H1N1 pandemic H1N1 influenza strain.