scholarly journals Tocilizumab As a Novel Immunomodulatory Regimen for Hemophilia Gene Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1853-1853
Author(s):  
Klaudia Kuranda ◽  
Christine Weisshaar ◽  
Yifeng Chen ◽  
Corinne Smith ◽  
Heena Beck ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Factor Ix ◽  
Clotting Factor ◽  
Control Group ◽  
Aav Vector ◽  
Entire Study

Abstract Hemophilia is an X-linked bleeding disorder typically resulting in deficiency of factor VIII (FVIII) or factor IX (FIX) due to mutations in the F8 or F9 genes, respectively. Data from clinical trials have shown that the investigational delivery of functional F8 or F9 gene by recombinant adeno-associated virus (AAV) to hepatocytes can substantially eliminate the need for infusions of clotting factor in hemophilia patients. Despite major advances, the durability and redosing of these investigational gene therapies have been limited by the host immune response against the AAV capsid (Verdera et al. Mol Ther 2020). Currently, an oral corticosteroid, prednisone, is commonly used to prevent cytotoxic T cells from killing AAV-transduced hepatocytes and to sustain the production of transgenic clotting factor. However, in some instances, transgene expression was lost despite prednisone administration and prolonged use of prednisone can be associated with adverse effects. Previously, we demonstrated that the anti-capsid humoral response depends on interleukin 6 (IL-6) secretion from human monocyte-related dendritic cells (Kuranda et al. JCI 2018). IL-6 signaling in response to AAV was also observed in human non-parenchymal liver cells in vitro, animal gene transfer models and AAV-based gene therapy trial for hemophilia B (Konkle et al. Blood 2021). Here, we investigated the effects of the IL-6 signaling blockade as a possible targeted approach to modulate AAV vector immunogenicity in hemophilia gene therapy using a non-human primate (NHP) model. Spk100 AAV capsid was used to deliver the human F9 gene. To prevent IL-6 signaling, we used a monoclonal antibody, tocilizumab (TCZ), which blocks the IL-6 receptor. TCZ is currently approved for use in several forms of arthritis and cytokine release syndrome. Ten male cynomolgus monkeys received an intravenous injection of Spk100-hFIX vector (4x10 12 vg/kg) and 5 of those animals received a single dose of TCZ (8 mg/kg) the day prior to vector administration and were monitored for 13 weeks. As assessed by an array of clinical and anatomic pathology parameters, the investigational use of gene therapy combined with prophylactic TCZ administration was safe and well-tolerated. TCZ did not interfere with vector biodistribution, liver transduction or the transgenic FIX production. Spk100-hFIX alone modestly increased IL-6 secretion from NHP peripheral blood mononuclear cells (PBMC) in vitro but, following the vector infusion in animals, plasma IL-6 levels did not change significantly. Overall, cytokine secretion in response to Spk100 capsid and hFIX protein was lower in PBMCs isolated from TCZ-treated animals compared to cells obtained from control animals. As expected, animals administered Spk100-hFIX alone developed anti-capsid antibodies. The administration of TCZ was associated with a lower level of anti-Spk100 IgM, IgG and neutralizing antibodies (NAb). While the blockade of IL-6 signaling was effective for about for 14 days post-vector infusion, the lower level of anti-Spk100 antibodies persisted for the entire study duration. In the TCZ group, 4 out of 5 animals had NAb titers equal to or below 1:10, theoretically compatible with vector readministration. In contrast, 4 out of 5 animals in the control group developed high titer NAbs following vector infusion. Importantly, TCZ reduced the detection of the capsid-specific TNFα-positive T cells that were observed in animals after vector infusion. Finally, livers from sacrificed animals were used to prepare liver non-parenchymal cells for ex vivo phenotyping. Compared to the control animals, liver-resident immune cells from the TCZ-treated group had increased basal IL-10 secretion, while liver sinusoidal endothelial cells (albumin-CD45-CD31+CD146+MHCII+) had lower surface levels of MHC class II molecules, suggesting an anti-inflammatory milieu in the TCZ-treated livers. Our results show that short-term prophylactic blockade of IL-6 signaling was safely used in NHPs and has the potential to reduce immune responses commonly observed post AAV vector infusion. These results support the continued investigation of tocilizumab as a targeted immunomodulatory regimen in liver gene therapy for hemophilia. Disclosures Kuranda: Spark Therapeutics: Current Employment. Weisshaar: Spark Therapeutics: Current Employment. Chen: Spark Therapeutics: Ended employment in the past 24 months. Smith: Spark Therapeutics: Current Employment. Beck: Spark Therapeutics: Current Employment. Kahle: Spark Therapeutics: Current Employment. Mingozzi: Spark Therapeutics: Current Employment.

G-CSF Activated Granulocytes Inhibit Experimental Graft-versus-Host Disease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4989-4989
Author(s):  
Zilton F.M. Vasconcelos ◽  
Julia Farache ◽  
Bruna M. Santos Grad ◽  
Tereza S. Palmeira Grad ◽  
Luis Fernando Bouzas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Cell Activity ◽  
Control Group ◽  
Low Density ◽  
Graft Versus Host ◽  
The One

Abstract Acute Graft versus host diseas (aGVHD) is a major complication of stem cell transplantation. The disease is mediated by T cells and a higher incidence/severity would be expected when higher numbers of T cells are inoculated. However, the incidence of aGVHD in PBST, which carries about 10 times more T cells then BMT, is not higher than the one found in later. This finding indicates a modulatory role for G-CSF over T cell activity. We had previously shown that T cells from G-CSF treated PBSC donors do not produce g-IFN nor IL-4 and that this inhibition was mediated by low density, G-CSF activated, granulocytes. In order to test if in fact G-CSF activated granulocytes could inhibit disease, we first checked if G-CSF could generate low density granulocytes, in vivo and in vitro. Indeed, either in vivo(21mg /day - 5 days) or in vitro (150 ng -12hs) with G-CSF generates low density granulocytes which co-purify with the mononuclear cells in the ficoll® gradient. Moreover, as we had shown in humans, these low density cells, inhibit the production of g-IFN by anti-CD3 activated T cells on flow cytometry studies (17%-T cells alone versus 3% T cells with granulocytes 1:1). Radiation quimaeras were set with (B6 X BALB/c)F1 as hosts reconstituted with T cell depleted C57Bl6 bone marrow, in the presence or absence of nylon wool selected spleen cells (NWSC), as T cell source, from normal or G-CSF treated mice. As previously shown by others, NWSC from G-CSF treated mice diminishes the incidence of acute disease on day 20 post-transplant, from 75 to 25%. In order to investigate if this inhibition was dependent on the activated granulocytes present in the NWSC from G-CSF treated mice, granulocytes were depleted with anti-GR1 and complement. In this case, the incidence of disease is the same or even higher (75% experiment#1 and 100% in experiment #2) than the one observed on the control group (NWSC from control mice). These results strongly suggest that activated granulocytes could indeed inhibit aGVHD. We then generated activated granulocytes in vitro, by treating spleen derived high density granulocytes with 150ng of G-CSF for 12 hs. After the incubation period, a new ficoll® gradient was performed and the low density cells were obtained. T cell contamination on the second gradient was eliminated by anti-CD4 and CD8 complement lysis. These activated granulocytes were inoculated together with NWSC from control mice in the radiation quimaeras at a 1:1 ratio. In this case 100% disease inhibition was observed when compared to the positive control group, where 75% of the animals got sick. Our data indicate that activated granulocytes are the major mediators of the G-CSF immunossupressive effects and that these cells can be used as a novel immune modulator in clinical transplantation to prevent acute GVHD.

Specific Cytotoxicity and Clonal Expansion of TCR Vβ Subfamily T Cells Induced by PML-RARα Peptide.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3904-3904
Author(s):  
Yangqiu Li ◽  
Ji Tang ◽  
Lijian Yang ◽  
Shaohua Chen ◽  
Yubing Zhou
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Statistical Significance ◽  
Clonal Expansion ◽  
Cell Receptor ◽  
Control Group ◽  
Effector Cells ◽  
Specific Cytotoxicity

Abstract The analysis of T cell receptor (TCR) Vβ repertoire is one of the sensitive methods to identify the clonal expansion T cells which response to tumor associated antigens. Understanding the clonality and restricted usage of TCR Vβ repertoire of expanded T-cells induced by PML-RARα peptide may be useful in helping design the new immunotherapeutic strategy specifically for acute promyelocytic leukemia (APL).The aim of the present study was to investigate the specific cytotoxicity and clonality of TCR Vβ repertoire in cord blood T cells induced by PML-RARα peptide (LSSCITQGKAIETQSSSSEE) in vitro. Cord blood mononuclear cells were amplified by IL-2, anti-CD3 and anti-CD28 antibody with different concentration (16.7μg /ml, 33.3μg /ml or 50μg /ml respectively) of synthetic PML-RARα peptide. The induced T cells were collected at different time points after culture (3, 6, 9, 10, 12 or 15 days). The expression and clonality of TCR Vβ subfamilies within induced T cells were analyzed by using RT-PCR and genescan technique. The cytotoxicity of induced T cells was detected by LDH release assay. The results showed that the best condition for T cells induction and amplication was at a concentration with 16.7μg /ml of PML-RARα peptide and at a culture duration with 10 to 15 day. TCR Vβ repertoire analysis showed that restricted expression and cloanl expansion of TCR Vβ subfamily cord blood T cells could be identified after induction by PML-RARα peptide. Clonal expanded T cells were found in Vβ13, Vβ14 and Vβ16 subfamlies respectively. The induced T cells were showed to have the specific cytotoxicity for NB4 cell line (effector cells: tagerted cells=20:1), the cytotoxicity rates were 49.65±6.7% (p<0.05) at day 10th and 73.13±8.42% (p<0.01) at day 15th after culture, which show statistical significance in compare to the control group (without PML-RARα induction). In conclusions, the PML-RARα peptide could induce the clonal expansion T cells from cord blood in vitro, which may have specific cytotoxicity for PML-RARα+ cells.

The Effect of Adrenaline Infusions on Blood Coagulation in Normal and Haemophilia B Dogs

1966 ◽  
Vol 15 (03/04) ◽  
pp. 349-364 ◽  
Author(s):  
A.H Özge ◽  
H.C Rowsell ◽  
H.G Downie ◽  
J.F Mustard
Keyword(s):  
Body Weight ◽  
Factor Viii ◽  
Factor Ix ◽  
Clotting Factor ◽  
Blood Ph ◽  
Order Of Magnitude ◽  
Dose Dependent ◽  
Generation Test ◽  
Plasma Activity

SummaryThe addition of trace amounts of adrenaline to whole blood in plasma in vitro increased factor VIII, factor IX and whole plasma activity in the thromboplastin generation test. This was dose dependent.Adrenaline infusions less than 22 (μg/kg body weight in normal dogs accelerated clotting, increased factor IX, factor VIII and whole plasma activity in the thromboplastin generation test and caused a fall in blood pH. In a factor IX deficient dog, there was no increase in factor IX activity. After adrenaline infusions, however, the other changes occurred and were of the same order of magnitude as in the normal. Adrenaline in doses greater than 22 μg/kg body weight did not produce as great an effect on clotting in normal or factor IX deficient dogs. The platelet count in the peripheral blood was increased following the infusion of all doses of adrenaline. These observations suggest that the accelerating effect of adrenaline on clotting is not mediated through increase in activity of a specific clotting factor.

Levels interleukine-4 and interleukin-17 (IL-4, IL-17) of blood in patients with habitual recurrent pregnancy loss, which occurred in a loop in vitro fertilization

2016 ◽  
pp. 137-139
Author(s):  
K.P. Golovatyuk ◽  
Keyword(s):  
In Vitro Fertilization ◽  
Conditioned Medium ◽  
Mononuclear Cells ◽  
Recurrent Miscarriage ◽  
Interleukin 4 ◽  
Control Group ◽  
Interleukin 17 ◽  
Vitro Fertilization ◽  
Blood Mononuclear Cells

The objective: was to investigate the levels of cytokines IL-4 and IL-17 in serum and conditioned medium cultures of blood mononuclear cells (MNC) and evaluation association between their products and miscarriage, which occurred in IVF cycles. Patients and methods. We observed 240 patients with recurrent miscarriage, came in IVF cycles, and 100 apparently healthy fertile women in the control group. The concentrations of IL-4 and IL-17 in serum and conditioned medium of MNC cultures were determined. Results. The levels of IL-4 in the serum and conditioned medium in spontaneous and stimulated mitogen secretion was not significantly different from those in the control group, whereas IL-17 levels were higher than those in the control group serum, in conditioned media of stimulated and non-stimulated MNCs. Conclusion. Disregulation of activity of circulating blood mononuclear cells in women with recurrent miscarriage that followed IVF, is accompanied by increased secretion of IL-17 and almost constant production of IL-4 on the back of high stimulation index of production of these cytokines. Key words: in vitro fertilization, miscarriage, interleukin-4, interleukin-17, serum stimulated and non-stimulated mononuclear blood.

scholarly journals THU0046 A PIPELINE TO STUDY ANTIGEN-SPECIFIC CD4+ T CELLS IN RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 235.1-236
Author(s):  
R. Kumar ◽  
N. Yoosuf ◽  
C. Gerstner ◽  
S. Turcinov ◽  
K. Chemin ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Tenascin C ◽  
Tetramer Staining ◽  
Tcr Repertoire ◽  
Citrullinated Antigen

Background:Autoimmunity to citrullinated autoantigens forms a critical component of disease pathogenesis in rheumatoid arthritis (RA). Presence of anti-citrullinated protein antibodies (ACPAs) in patients has high diagnostic value. Recently, several citrullinated antigen specific CD4+T cells have been described. However, detailed studies of their T-cell receptor usage and in-vivo profile suffer from the disadvantage that these cells are present at very low frequencies. In this context, we here present a pipeline for TCR repertoire analysis of antigen-specific CD4+T cells from RA patients, including both citrulline and influenza (control) specificities using in-vitro peptide challenge induced-cell expansion.Objectives:To enable studies of the T cell repertoire of citrullinated antigen-specific CD4+T cells in rheumatoid arthritisMethods:Peripheral blood mononuclear cells (PBMCs) (n=7) and synovial fluid mononuclear cells (SFMCs) (n=5) from HLA-DR*0401-postive RA patients were cultured in the presence of citrullinated Tenascin C peptide cocktails or influenza peptides (positive control). Citrulline reactive cells were further supplemented with recombinant human IL-15 and IL-7 on day 2. All cultures were replenished with fresh medium on day 6 and rIL-2 was added every 2 days from then. Assessment of proportion of peptide-HLA-tetramer positive cells was performed using flow cytometry whereby individual antigen-specific CD4+T cells were sorted into 96-well plates containing cell lysis buffer, followed by PCR-based alpha/beta TCR sequencing. TCR sequencing data was demultiplexed and aligned for TCR gene usage using MiXCR. Some tetramer positive cells were sorted into complete medium containing human IL-2 and PHA for expansion of antigen-specific cells. Cells were supplemented with irradiated allogenic PBMCs (30 times number of antigen specific cells). Clones of antigen specific CD4+T cells were further subjected to tetramer staining to confirm expansion of cells.Results:As evidenced by increase in frequency of tetramer positive CD4+T cells, in vitro peptide stimulation resulted in expansion of both influenza specific (Fig. 1a) and citrullinated antigen specific (Fig. 1b) CD4+T cells. Polyclonal in-vitro expansion of tenascin C tetramer positive sorted cells followed by tetramer staining further confirmed antigen specificity and enrichment for antigen specific CD4+T cells after polyclonal stimulation (Fig.1c). TCR repertoire analysis in PB and SF dataset from the first patient showed clonal expansion of influenza specific cells in both sites. Synovial fluid had more diversity of expanding clones as compared to paired PB, with few expanded clones being shared among SF and PB. We observed a more diverse TCR repertoire in citrulline specific CD4+T cells. We also observed sharing of TCR alpha chains among different citrulline specific CD4+T cell clones.Fig. 1In-vitroexpansion of antigen specific CD4+T cells:Conclusion:This method provides a highly suitable approach for investigating TCR specificities of antigen specific CD4+T cells under conditions of low cell yields. Building on this dataset will allow us to assess specific features of TCR usage of autoreactive T cells in RA.PBMCs were cultured in presence of (a) influenza (HA, MP54) and (b) citrullinated tenascin peptides. The proportion of antigen specific CD4+T cells was assessed using HLA-class II tetramer staining. We observed an increase in frequency of (a) Infleunza specific cells (red dots in upper left and lower right quadrants) and (b) citrullinated tenascin C specific cells (red dots in lower right quadrant), at day 13 post culture as compared to day 3. (c) Sorting of citrullinated tenascin specific CD4+T cells, followed by PHA expansion resulted in visible increase in proportion of citrullinated tenascin specific CD4+T cells.Disclosure of Interests:Ravi kumar: None declared, Niyaz Yoosuf: None declared, Christina Gerstner: None declared, Sara Turcinov: None declared, Karine Chemin: None declared, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

scholarly journals The effect of serum on monocyte tissue factor generation

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 707-714 ◽  
Author(s):  
RL Edwards ◽  
D Perla
Keyword(s):  
T Cells ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
High Serum ◽  
Peripheral Blood Mononuclear ◽  
Serum Factors ◽  
Normal Peripheral Blood ◽  
Stimulatory Activity ◽  
Normal Hemostasis

Abstract Human monocytes generate the procoagulant tissue factor (MTF) following exposure to a variety of immune stimuli in vitro. The generation of MTF is modified by T cells, lymphokines, and immunoregulatory lipoproteins, and recent studies have shown that MTF can be activated in an immune- specific manner following exposure to antigen. We have examined the role of serum factors in the regulation of MTF generation. Low concentrations (less than 1%) of heat-inactivated normal human serum greatly enhanced MTF generation in cultures of normal peripheral blood mononuclear cells. The stimulatory effect was observed in cultures of both unstimulated cells and cells exposed to bacterial lipopolysaccharide. Stimulation was not observed at high serum concentrations (greater than 10%) and could not be explained by endotoxin contamination or activation of the assay system. Stimulatory activity was present in plasma and BaSO4-adsorbed plasma as well as autologous and allogeneic serum, was not abolished by removal of serum lipoproteins, and did not require the presence of T cells for its expression. Sera from 28 different normal volunteers were screened for stimulatory activity and demonstrated a wide variation in potency. These results suggest that a potent factor is present in sera that enhances the expression of MTF activity in vitro. This factor is distinct from previously described lipoprotein regulators and may play a role in the initiation of coagulation in both normal hemostasis and pathologic states.

scholarly journals Viral Protein VP4 Is a Target of Human Antibodies Enhancing Coxsackievirus B4- and B3-Induced Synthesis of Alpha Interferon

2005 ◽  
Vol 79 (22) ◽  
pp. 13882-13891 ◽  
Author(s):  
Wassim Chehadeh ◽  
Pierre-Emmanuel Lobert ◽  
Pierre Sauter ◽  
Anne Goffard ◽  
Bernadette Lucas ◽  
...  
Keyword(s):  
Healthy Subjects ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Insulin Dependent Diabetes Mellitus ◽  
Alpha Interferon ◽  
Dependent Diabetes Mellitus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peripheral Blood Mononuclear ◽  
Coxsackievirus B4

ABSTRACT Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-α) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-α were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56°C from CVB4E2 (VP4CVB4) and CVB3 (VP4CVB3) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis. There was no cross-reaction between VP4CVB4 and VP4CVB3 in the inhibiting effect. IFN-α levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4CVB4). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-α levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis by PBMC.

Effects of glutamine on the immune system: influence of muscular exercise and HIV infection

1995 ◽  
Vol 79 (1) ◽  
pp. 146-150 ◽  
Author(s):  
T. Rohde ◽  
H. Ullum ◽  
J. P. Rasmussen ◽  
J. H. Kristensen ◽  
E. Newsholme ◽  
...  
Keyword(s):  
Proliferative Response ◽  
Mononuclear Cells ◽  
Natural Killer Cell Activity ◽  
Killer Cell ◽  
Cell Activity ◽  
Control Group ◽  
Dependent Manner ◽  
Killer Cell Activity ◽  
Hiv Seropositive

Glutamine increased the proliferative response and the lymphokine-activated killer cell activity of blood mononuclear cells isolated from normal healthy subjects (n = 6) in a dose-dependent manner, with optimum at 0.3–1.0 mM. The relative fraction of CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells was not changed by glutamine at a concentration of 0.6 mM, except in the phytohemagglutinin-stimulated proliferation experiment where the fraction of CD4+, and therefore CD3+ cells, increased. The natural killer cell activity was not influenced by glutamine. Human immunodeficiency virus (HIV)-seropositive subjects (n = 8) who performed concentric bicycle exercise for 1 h at 75% of maximal O2 consumption had an overall lower phytohemagglutinin-stimulated proliferative response, compared with the HIV-seronegative control group (n = 7). The proliferation during exercise was lower in both the HIV-seropositive and the HIV-seronegative group. Addition of glutamine in vitro did not normalize the lower proliferation in the HIV-seropositive group or the attenuated proliferation seen during exercise in both groups.

scholarly journals The Anthrax Vaccine Adsorbed Vaccine Generates Protective Antigen (PA)-Specific CD4+ T Cells with a Phenotype Distinct from That of Naïve PA T Cells

2008 ◽  
Vol 76 (10) ◽  
pp. 4538-4545 ◽  
Author(s):  
William W. Kwok ◽  
Junbao Yang ◽  
Eddie James ◽  
John Bui ◽  
Laurie Huston ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Protective Antigen ◽  
Ex Vivo ◽  
Cytokine Profile ◽  
T Cell Epitopes ◽  
Peripheral Blood Mononuclear ◽  
Anthrax Vaccine ◽  
Cellular Immune

ABSTRACT Cellular immune responses against protective antigen (PA) of Bacillus anthracis in subjects that received the anthrax vaccine adsorbed (AVA) vaccine were examined. Multiple CD4+ T-cell epitopes within PA were identified by using tetramer-guided epitope mapping. PA-reactive CD4+ T cells with a CD45RA− phenotype were also detected by direct ex vivo staining of peripheral blood mononuclear cells (PBMC) with PA-specific tetramers. Surprisingly, PA-specific T cells were also detected in PBMC of nonvaccinees after a single cycle of in vitro PA stimulation. However, PA-reactive CD4+ T cells in nonvaccinees occurred at lower frequencies than those in vaccinees. The majority of PA-reactive T cells from nonvaccinees were CD45RA+ and exhibited a Th0/Th1 cytokine profile. In contrast, phenotyping and cytokine profile analyses of PA-reactive CD4+ T cells from vaccinees indicated that vaccination leads to commitment of PA-reactive T cells to a Th2 lineage, including generation of PA-specific, pre-Th2 central memory T cells. These results demonstrate that the current AVA vaccine is effective in skewing the development of PA CD4+ T cells to the Th2 lineage. The data also demonstrated the feasibility of using class II tetramers to analyze CD4+ cell responses and lineage development after vaccination.

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