scholarly journals Early CD4+ T-Cell Effector Alloreactivity Towards Multiple Mismatched HLA Class II Alleles Is Associated with Graft Predominance after Double Umbilical Cord Blood Transplantation (dUCBT)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 387-387
Author(s):  
Jan J. Cornelissen ◽  
Rebecca Wijers ◽  
Cornelis A.M. van Bergen ◽  
Judith Somers ◽  
Eric Braakman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Graft Failure ◽  
Class Ii ◽  
Median Number ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Kinetics Of

Abstract While dUCBT may be associated with less graft failure as compared to single UCBT, hematopoietic recovery following dUCBT generally originates from only a single graft, designated as the 'winning' graft. Graft predominance was suggested to be T-cell mediated, but is still incompletely understood. We recently showed that CD4+ T-cells rapidly expand after dUCBT and early CD4+ T-cell chimerism predicts for graft predominance (Somers et al BBMT 2012; Haematologica 2014). Given the frequent HLA class II allele mismatches between the 2 UCB units in dUCBT, we hypothesized that HLA class II-specific CD4+ T-cells from the 'winning' CBU may be responsible for rejection of the 'loser' CBU. In order to test that hypothesis, we evaluated whether 'wining' CD4+ T-cells specifically recognize individual HLA class II allele mismatches, expressed by the rejected graft. Patient T-cells were propagated in-vitro by 1. HLA unbiased polyclonal expansion (by K562 clone 2D11+CD3mAb and sequential addition of cytokines IL-7, Il-15 and IL-12), and 2. specific activation by HLA class II allele transduced HELA stimulator cells. In-vitro reactivity of propagated T cells was assessed in a co-culture with class II allele transduced HELA cells, and measured by analysis of T cell activation or effector markers by flow cytometry (FCM). 11 patients with poor-risk hematological malignancies were included, receiving 22 UCB units matched at HLA A, B, and DRB1 for 5/6 (n=7) or 4/6 (n=15) with the recipient. The median number (range) of class II allele mismatches between the 2 UCB units per transplant was 2 (1-6). In total, 33 different class II allele mismatches were tested, including 16 at HLA DRB1, 7 at DQB, and 10 at DP. Conditioning with TBI (4 Gy) combined with fludarabine and cyclophosphamide was applied and mycophenolate and ciclosporin were given for GVH-prophylaxis. All patients engrafted, at a median number of 26 days (range: 20-50) after transplantation (without G-CSF), and all developed complete single unit chimerism. Peripheral blood CD3+ T-cell numbers of samples taken at 1-6 months post UCBT were low (median: 0.207; range 0.030-0.699 x 10-9/L) with 74% (range, 8-96%) consisting of CD4+ T-cells. In all 11 patients, alloreactive CD4+ T-cells towards one or more mismatched class II alleles were detectable. In total, CD4+ alloreactivity towards 29 out of 33 (88%) mismatches was detected, including 15/16 for DRB1 (94%), 7/7 for DQ (100%), and 7/10 (70%) for DP alleles. All mismatched alleles but one (DPB1*04:01) elicited a CD4+ T-cell response. Stronger CD4+ T-cell reactivity was observed towards DRB1 and DQ. Analysis of activity towards matched, control alleles showed positivity in 2/11 (18%) combinations. Reactivity towards irrelevant third party alleles showed positivity in 3/17 (18%). The class II alloresponse was significantly higher for mismatched versus matched alleles (median fold increase over control: 7.4 (range 4.4-21.5) versus 1.4 (1.1-2.2), respectively). The highest alloreactive responses were observed in samples (n=14) taken at 1 month post UCBT (Figure). Alloreactive CD4+ T-cells upregulated CD137 and CD134, PD1 and the effector markers CD107 and Interferon-gamma. Collectively, these results demonstrate that specific effector alloreactivity by 'winner' CD4+ T-cells directed to multiple class II mismatched alleles was present in all patients, already at 1 month post UCBT. These results suggest that immediate cytotoxicity exerted by CD4+ T-cells from the 'winning' cord represent a novel mechanism of rapid rejection of the 'losing' unit after dUCBT following a non-ATG conditioning regimen. Furthermore, these observations suggest that the lower incidence of graft failure observed after dUCBT may, in part, result from an immunological, graft-potentiating effect evoked by mismatched class II alleles expressed by the rejected graft. Therefore, allele matching at HLA class II between the 2 units can be ignored in dUCBT. Class II mismatches between de UCB units might, alternatively, even be aimed for, but this should be confirmed in a prospective study. The potential graft versus leukemia effect of these alloreactive CD4+ T-cells is subject of ongoing investigation. Figure 1. Kinetics of anti-HLA class II allele CD4+ T-cell alloreactivity. Figure 1. Kinetics of anti-HLA class II allele CD4+ T-cell alloreactivity. Disclosures No relevant conflicts of interest to declare.

Human Alloreactive CD4+ T Cells as Potent Effector Cells and Sole Mediators of Anti-Tumor Responses in a NOD/SCID Mouse Model for Human Acute Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1245-1245 ◽  
Author(s):  
Sanja Stevanovic ◽  
Marieke Griffioen ◽  
Marianke LJ Van Schie ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Leukemic Cells ◽  
T Cell Clones ◽  
Hla Dr ◽  
Cell Clones

Abstract Donor lymphocyte infusion (DLI) following allogeneic stem cell transplantation (alloSCT) can be a curative treatment for patients with hematological malignancies. The therapeutic benefit of DLI is attributed to a graft versus leukemia (GvL) reactivity mediated by donor T cells recognizing allo-antigens on malignant cells of the patient. Donor T cells, however, often recognize allo-antigens which are broadly expressed in non-malignant tissues of the patient, thereby causing severe graft versus host disease (GvHD). In contrast to HLA class I molecules which are ubiquitously expressed on all nucleated cells, HLA class II molecules are predominantly expressed on cells of the hematopoietic system, and therefore CD4+ T cells may selectively mediate GvL reactivity without GvHD. Several clinical studies have indeed demonstrated that CD8-depleted DLI after alloSCT can lead to clinical remissions with reduced incidence of GvHD. Since in most of these studies DLI was contaminated with CD8+ T cells, it remained unclear whether CD4+ T cells alone are capable of mediating GvL reactivity. To assess the capacity of purified CD4+ T cells to solely exert GvL reactivity we compared the anti-tumor effects of CD4+ DLI and CD3+ DLI in a NOD/SCID mouse model of human acute leukemia. Iv injection of primary human leukemic cells from three different patients reproducibly resulted in engraftment of leukemia in mice, as monitored by peripheral blood analysis. Three weeks after inoculation of leukemic cells, established tumors were treated by infusion of human donor T cells. In mice treated with CD4+ DLI (5*106 CD4+ T cells), the emergence of activated (HLA-DR+) T cells coincided with rapid disappearance of leukemic cells, showing similar kinetics as for CD3+ DLI (consisting of 5*106 CD4+ T cells and 3*106 CD8+ T cells). To analyze the specific reactivity of T cells responsible for the anti-leukemic effect, we clonally isolated human CD45+ T cells during the anti-tumor response following CD4+ DLI in which the donor was matched for HLA class I and mismatched for the HLA-DR (DRB1*1301), -DQ (DQB1*0603) and –DP (DPB1*0301/0401) alleles of the patient. A total number of 134 CD4+ T cell clones were isolated expressing various different TCR Vbeta chains. Most of the isolated CD4+ T cell clones (84%) were shown to be alloreactive, as determined by differential recognition of patient and donor EBV-transformed B cells (EBV-LCL) in IFN-g ELISA. A substantial number of these CD4+ T cell clones also exerted cytolytic activity (17%), as demonstrated by specific reactivity with patient EBV-LCL but not donor EBV-LCL in a 10 hr 51Cr-release cytotoxicity assay. Further characterization of the specificity of 20 CD4+ T cell clones using blocking studies with HLA class II specific monoclonal antibodies illustrated HLA class II restricted recognition directed against HLA-DR (n=3), HLA-DQ (n=16) and HLA-DP (n=1) molecules of the patient. Of the 127 alloreactive CD4+ T cell clones, only 36 clones directly recognized primary leukemic cells of the patient. Flowcytometric analysis demonstrated that HLA class II, and in particular HLA-DQ, molecules were expressed at relatively low levels on patient leukemic cells as compared to patient EBV-LCL. Upregulation of HLA class II and costimulatory molecules on patient leukemic cells upon differentiation in vitro into leukemic antigen presenting cells (APC) resulted in recognition of patient leukemic cells by all alloreactive CD4+ T cell clones. Therefore, we hypothesize that the alloreactive CD4+ T cells have been induced in vivo by patient leukemic cells, which, upon interaction with T cells or other environmental factors, acquired an APC phenotype. In conclusion, our data show that alloreactive CD4+ T cells can be potent effector cells and sole mediators of strong antitumor responses in a NOD/SCID mouse model for human acute leukemia.

Identification of Four New HLA Class II Restricted Minor Histocompatibility Antigens Contributing to Graft Versus Leukemia Reactivity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3247-3247
Author(s):  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A.M. van Bergen ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
T Cell Clones ◽  
Cell Clones

Abstract Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

Wilms Tumor Protein-1-Derived 9-Mer Peptide Induces CD4 T-Cell Responses in an HLA-DR Restricted Manner

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4351-4351
Author(s):  
Shigeo Fuji ◽  
Julia Fischer ◽  
Markus Kapp ◽  
Thomas G Bumm ◽  
Hermann Einsele ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Cell Responses ◽  
Hla Dr ◽  
Ifn Γ

Abstract Abstract 4351 Wilms‘ tumor protein-1 (WT1) is one of the most investigated tumor-associated antigens (TAA) in hematological malignancies. CD8 T-cell responses against several WT1-derived peptides have been characterized and are known to contribute to disease control after allogeneic hematopoietic stem cell transplantation (HSCT). Also the identification of human leukocyte antigen (HLA) class II-restricted CD4 T-cell epitopes from WT1 is a challenging task of T-cell-based cancer immunotherapy to improve the effectiveness of WT1 peptide vaccination. We found a highly immunogenic WT1 peptide composed of only 9 amino acids having the ability to induce IFN-γ secretion in CD4 T-cells in an HLA DR-restricted manner. This finding is of great interest as it was generally accepted that HLA class II binding peptides are composed of at least 12 amino acids being recognized by CD4 T-cells, whereas HLA class I binding peptides are composed of 8–11 amino acids being recognized by CD8 T-cells (Wang et al Mol. Immunol. 2002). However, both HLA class I and class II molecules bind to primary and secondary peptide anchor motifs covering the central 9–10 amino acids. Thus, considering this common structural basis for peptide binding there is a possibility that the WT1 9-mer peptide binds to HLA class II molecules, and induces CD4 T-cell responses. IFN-γ induction in response to several WT1 9-mer peptides was screened in 24 HLA-A*02:01 positive patients with acute myeloid leukemia or myelodysplastic syndrome after allogeneic HSCT. Responses to one WT1 9-mer peptide were exclusively detected in CD3+CD4+ T-cells of 2 patients after allogeneic HSCT, but not in CD3+CD4+ T-cells of their corresponding HSC donors. CD4+ T-cell responses to this WT1 9-mer peptide exhibited high levels of functional avidity, as IFN-γ induction was detected after stimulation with 100 ng peptide per mL. Peptide-induced IFN-γ production was confirmed with IFN-γ ELISPOT assays and the HLA restriction of the T-cell response was determined by HLA blocking antibodies. The reaction was significantly blocked by anti-pan HLA class II antibody (85 % reduction), but neither by pan-HLA class I nor by anti-HLA A2 antibody. To identify the subtype of HLA class II molecule, blocking assays with antibodies against HLA-DP, HLA-DR and HLA-DQ were performed. IFN-γ induction was completely abrogated by anti-HLA-DR antibody (99 % reduction) (fig 1, p value of unpaired student‘s t-test <0.0001 for the medium control vs anti-pan HLA class II antibody or anti-HLA-DR antibody, respectively). To test whether IFN-γ was exclusively induced in CD4 T cells, CD4 or CD8 T-cells were depleted from PBMC. Whereas CD8 T-cell depletion did not affect IFN-γ induction, CD4 T-cell depletion completely abrogated the WT1 9-mer peptide induced response (fig 2). CD4 T-cells responding to the WT1 9-mer peptide were indicated to be functional cytotoxic T-cells with an effector CD4 T-cell phenotype. Longitudinal analyses demonstrated the persistence and functionality of WT1 9-mer specific CD4 T-cells in PBMC of patients even at day 1368 after allogeneic HSCT. These data indicate for the first time that a TAA-derived 9-mer peptide can induce HLA class II-restricted CD4 T-cell responses. Vaccination with the characterized WT1 9-mer peptide can enhance the induction and maintenance of not only CD4 but also indirect CD8 T-cell responses. Considering that CD4 T-cells play an important role in tumor rejection, the possibility that other TAA-derived 9-mer peptides having the potential to induce CD4 T-cell responses should be explored in other settings of tumor immunology as well to improve vaccination strategies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HLA Class II Defects in Burkitt Lymphoma: Bryostatin-1-Induced 17 kDa Protein Restores CD4+ T-Cell Recognition

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Azim Hossain ◽  
Jason M. God ◽  
Faisal F. Y. Radwan ◽  
Shereen Amria ◽  
Dan Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Burkitt Lymphoma ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Immune Recognition ◽  
Antigenic Peptides ◽  
Bryostatin 1

While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL) have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s) have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.

Class-II Associated Invariant Chain Peptide (CLIP) Expression on AML Blasts Adversely Affects Alloreactive CD4+ T Cell Recognition.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4888-4888
Author(s):  
Marvin M. van Luijn ◽  
Martine E.D. Chamuleau ◽  
Theresia M. Westers ◽  
James A. Thompson ◽  
Suzanne Ostrand-Rosenberg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Immune Surveillance ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Invariant Chain ◽  
Wild Type ◽  
Hla Dr

Abstract Although acute myeloid leukemia (AML) can be cured with intensive treatment including myeloablative chemotherapy and haematopoietic stem cell transplantation, relapses occur in the majority of cases. A common feature of tumor cells is their ability to escape immune surveillance through adapted intrinsic mechanisms. Thus, it is a great challenge to develop optimal strategies that direct a specific cellular immune response against residual AML blasts in vivo. As CD4+ T cells are needed to initiate a strong anti-leukemic CD8+ T cell response, the mechanism through which HLA class-II restricted (leukemia-specific) antigens are presented on AML blasts could be an essential factor in immune surveillance. Previously, we showed that the self peptide Class-II Associated Invariant Chain Peptide (CLIP) important in HLA class-II antigen presentation appeared to be disadvantageous, as its expression on AML blasts predicted a shortened disease-free survival (Chamuleau et al. Canc. Res.2004; 64(16):5546–50). We hypothesized that CLIP interferes with the presentation of specific tumor antigens on HLA class-II molecules, thereby preventing recognition of AML blasts by CD4+ T cells. To investigate whether CLIP expression indeed has a functional effect on leukemia-specific T cell activation in patients, an AML cell line model with CLIP+ and CLIP− leukemic blasts was set up. The Kasumi-1 and THP-1 AML cell lines were selected as both stained positive for extracellular HLA-DR (89%; MFI=31.3 and 91%; MFI=37.5 respectively) and CLIP expression (88%; MFI=37.2 and 91%; MFI=34.0 respectively) by flow cytometric analysis. These DR+CLIP+ cell lines were specifically silenced for Invariant Chain (Ii) expression using RNA interference to down-modulate CLIP presentation on the cell surface. Indeed, Ii siRNA-treated cells not only showed a significant decrease of intracellular Ii expression (MFI decrease of 87.7% for Kasumi-1 and 82.7% for THP-1), but also a marked downregulation of relative CLIP amount per HLA-DR molecule (fold decline in CLIP/DR ratio of 1.4 for Kasumi-1 and 2.0 for THP-1). Wild type (DR+CLIP+) and modulated (DR+CLIP−) cells of Kasumi-1 or THP-1 origin acted as stimulators for alloreactive CD4+ T cells in mixed leukocyte reactions using different stimulator to responder (S/R) ratios. Modulated DR+CLIP− Kasumi-1 and THP-1 cells induced a strong increase in alloreactive CD4+ T cell proliferation as compared to DR+CLIP+ wild type controls, both in an HLA-DR-specific and a S/R-dependent manner. At the highest S/R ratio, mean proliferation increases of 2.58-fold for Kasumi-1 (n=3) and 1.71-fold for THP-1 (n=2) were observed. These data support our hypothesis that the expression of CLIP on AML blasts plays an important role in immune surveillance, which might have impact on cellular immunotherapy with dendritic cell-based vaccines in AML.

Low CLIP Expression On Leukemic Blasts From AML Patients Is Essential for the Generation of Effective CD4+ T Cell Responses.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 996-996
Author(s):  
Marvin M. van Luijn ◽  
Martine E.D. Chamuleau ◽  
James A. Thompson ◽  
Suzanne Ostrand-Rosenberg ◽  
Theresia M. Westers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Flow Cytometric ◽  
Hla Dr ◽  
Class Ii Antigen

Abstract Abstract 996 Poster Board I-18 In patients with AML, the outgrowth of minimal residual disease (MRD) is considered as the major cause of relapse, whereby it is hypothesized that residual leukemic blasts are able to escape from immune surveillance. Since CD4+ T cells are critical for inducing effective anti-leukemic immunity, certain leukemic blasts might exhibit aberrant HLA class II antigen presentation that interferes with antigen-specific CD4+ T cell recognition. Increased binding of the class II-associated invariant chain self peptide (CLIP) to the HLA class II antigen-binding groove may thereby prevent the presentation of antigenic peptides. This study investigates both the clinical and functional role of CLIP expression on myeloid leukemic blasts. Blood and bone marrow samples from a cohort of 207 de novo AML patients were analyzed by flow cytometry for plasma membrane expression of CLIP and HLA-DR (DR). Significantly shortened disease-free and overall survival rates were found for patients with leukemic blasts characterized by a high amount of DR occupied by CLIP (relative CLIP amount). To explore the functional role of CLIP, we transduced blasts of the human Kasumi-1 and THP-1 myeloid leukemic cell lines with retroviral siRNAs specific for the Invariant Chain, a chaperone molecule that is critically involved in DR processing. Significant reductions in relative CLIP amount were found on blasts of both cell lines. Subsequently, CD4+ T cells derived from different healthy donors (n=3) were stimulated with either irradiated DR+CLIP- (Ii siRNA-treated) or DR+CLIP+ (wild type) THP-1 and Kasumi-1 blasts during mixed leukocyte reactions. In contrast to DR+CLIP+ blasts, DR+CLIP- blasts of both cell lines induced strong increases in allogeneic CD4+ T cell proliferation in a stimulator-to-responder dependent manner. To examine the effect of CLIP on CD4+ T cell induction in primary samples, we performed flow cytometric sorting experiments to select for CLIP- and CLIP+ leukemic blasts from different DR+ AML patients (n=5). CD4+ T cells collected from these same patients after achieving complete remission were isolated and stimulated with sorted CLIP- or CLIP+ leukemic blasts during four weeks of culture. In 2 of the 5 patients, marked proliferation of autologous remission CD4+ T cells stimulated with CLIP- leukemic blasts was observed in contrast to stimulation with CLIP+ leukemic blasts. In addition, in 4 of the 5 patients, flow cytometric analysis of CD4+ T cells showed that CLIP- leukemic blasts were able to induce both high CD25 and HLA-DR and low CD45RA and CD27 expression as compared to CLIP+ leukemic blasts, indicating increased activation of effector memory CD4+ T cells. Moreover, CD4+ T cells stimulated with CLIP- leukemic blasts also revealed strongly increased IFN-g/IL-4 ratios in contrast to CD4+ T cells stimulated with CLIP+ leukemic blasts, as determined by flow cytometry after PMA/ionomycin stimulation. This might imply skewing towards a more Th1 phenotype. In conclusion, these findings not only emphasize that the relative CLIP amount on leukemic blasts predicts clinical outcome, but also reveal that it is a critical factor for CD4+ T cell activation in AML. Hence, CLIP may serve as a target for immunomodulatory strategies to optimize HLA class II antigen presentation on AML whole-cell or DC vaccines and induce leukemia-specific CD4+ T cell immunity in patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4073-4082 ◽  
Author(s):  
Katharina N. Steinitz ◽  
Pauline M. van Helden ◽  
Brigitte Binder ◽  
David C. Wraith ◽  
Sabine Unterthurner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Structural Features ◽  
Cd4 T Cell ◽  
T Cell Epitopes ◽  
Mhc Class Ii Molecules

Abstract Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-cell epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Modification of Invariant Chain and CLIP Expression Increases the Immunogenicity of Leukemic Blasts to Potentiate Leukemia-Specific T Cell Reactivity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5430-5430
Author(s):  
Marvin M. van Luijn ◽  
Martine E.D. Chamuleau ◽  
James A. Thompson ◽  
Suzanne Ostrand-Rosenberg ◽  
Theresia M. Westers ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
T Cell Proliferation ◽  
Invariant Chain ◽  
Hla Dr

Abstract In patients suffering from AML, disease progression could be explained by the ability of leukemic blasts to escape immune surveillance. Since CD4+ T cells are indispensable for generating effective anti-leukemic immune responses, escaping leukemic blasts might exhibit aberrant HLA class II antigen presentation that interferes with antigen-specific CD4+ T cell activation. The Invariant Chain (Ii) is essentially involved in HLA class II processing, since it blocks endogenous antigen loading of HLA class II in the endoplasmic reticulum and mediates its transport to the lysosomal exogenous antigen-loading compartments. We previously showed that increased expression of the class II-associated invariant chain peptide (CLIP), a small remnant of Ii, on AML blasts predicts poor clinical outcome [Chamuleau et al., Cancer Research2004; 64]. This study was undertaken to modulate Ii and CLIP expression of leukemic blasts and examine the impact on leukemia-specific CD4+ T cell recognition. The THP-1 and Kasumi-1 AML cell lines were selected for Ii and CLIP modulation based upon their flow cytometrically determined DR+CLIP+Ii+ immunophenotype. Retroviral transduction of both THP-1 and Kasumi-1 with specific Ii siRNAs led to a clear decline in Ii expression, as MFI values dropped from 4.5 to 1.4 and 13.5 to 0.9, respectively, 6 weeks after transduction. Interestingly, the effect of Ii down-modulation on CLIP and HLA-DR expression levels differed between THP-1 and Kasumi-1 blasts. In THP-1, Ii down-modulation resulted in reduced CLIP expression (MFI values decreased from 35.9 to 14.0), while HLA-DR expression levels remained relatively constant. This yielded a marked reduction in the relative amount of CLIP presented by DR (decline from 1.12 to 0.52). In Kasumi-1, both CLIP and DR levels were markedly decreased by Ii down-modulation (MFI values declined from respectively 35.5 to 2.7 and 24.6 to 3.7). Although total DR expression was already reduced, the relative amount of CLIP presented by DR was even further reduced (decline from 1.49 to 0.78). These results might indicate that Ii and CLIP down-modulation enables HLA class II presentation of leukemia-associated antigens on these blast cell lines. Subsequently, DR+CLIP+Ii+ and DR+CLIP−Ii− blasts were compared in their capacity to induce allogeneic CD4+ T cell proliferation in mixed leukocyte reactions (MLRs). CD4+ T cells were obtained from different healthy donors and cultured in triplicate with irradiated blasts at various stimulator-to-responder (S/R) ratios. MLRs consisting of DR+CLIP−Ii− THP-1 blasts showed marked increases in CD4+ T cell proliferation in a S/R dependent manner compared to MLRs performed with DR+CLIP+Ii+ THP-1 blasts. These increases in CD4+ T cell proliferation (maximal 4.5-fold) correlated strongly with the decreased relative CLIP/DR amounts on THP-1 transductants. Similar increases in CD4+ T cell proliferation were observed when DR+CLIP−Ii− Kasumi-1 blasts were used as stimulator cells, also clearly correlating with the accompanying relative CLIP/DR amounts. The DR-specific L243 antibody totally abrogated CD4+ T cell proliferation, confirming HLA-DR restriction of the proliferative responses. These data demonstrate an essential role for Ii and CLIP expression of AML blasts in modifying T cell responsiveness and introduce Ii down-modulation as a potential immunotherapeutic strategy to activate leukemia-specific CD4+ T cells.

scholarly journals Contact-dependent delivery of IL-2 by dendritic cells to CD4 T cells in the contraction phase promotes their long-term survival

2019 ◽  
Vol 11 (2) ◽  
pp. 108-123
Author(s):  
Dan Tong ◽  
Li Zhang ◽  
Fei Ning ◽  
Ying Xu ◽  
Xiaoyu Hu ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Cd4 T Cell ◽  
Immune Memory ◽  
Term Survival

Abstract Common γ chain cytokines are important for immune memory formation. Among them, the role of IL-2 remains to be fully explored. It has been suggested that this cytokine is critically needed in the late phase of primary CD4 T cell activation. Lack of IL-2 at this stage sets for a diminished recall response in subsequent challenges. However, as IL-2 peak production is over at this point, the source and the exact mechanism that promotes its production remain elusive. We report here that resting, previously antigen-stimulated CD4 T cells maintain a minimalist response to dendritic cells after their peak activation in vitro. This subtle activation event may be induced by DCs without overt presence of antigen and appears to be stronger if IL-2 comes from the same dendritic cells. This encounter reactivates a miniature IL-2 production and leads a gene expression profile change in these previously activated CD4 T cells. The CD4 T cells so experienced show enhanced reactivation intensity upon secondary challenges later on. Although mostly relying on in vitro evidence, our work may implicate a subtle programing for CD4 T cell survival after primary activation in vivo.

scholarly journals African Green Monkey TRIM5α Restriction in Simian Immunodeficiency Virus-Specific Rhesus Macaque Effector CD4 T Cells Enhances Their Survival and Antiviral Function

2015 ◽  
Vol 89 (8) ◽  
pp. 4449-4456 ◽  
Author(s):  
Sumiti Jain ◽  
Matthew T. Trivett ◽  
Victor I. Ayala ◽  
Claes Ohlen ◽  
David E. Ott
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rhesus Macaque ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
T Cell Clones ◽  
Cell Clones ◽  
Immunodeficiency Virus ◽  
Antiviral Function

ABSTRACTThe expression of xenogeneic TRIM5α proteins can restrict infection in various retrovirus/host cell pairings. Previously, we have shown that African green monkey TRIM5α (AgmTRIM5α) potently restricts both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus mac239 (SIVmac239) replication in a transformed human T-cell line (L. V. Coren, et al., Retrovirology 12:11, 2015,http://dx.doi.org/10.1186/s12977-015-0137-9). To assess AgmTRIM5α restriction in primary cells, we transduced AgmTRIM5α into primary rhesus macaque CD4 T cells and infected them with SIVmac239. Experiments with T-cell clones revealed that AgmTRIM5α could reproducibly restrict SIVmac239replication, and that this restriction synergizes with an intrinsic resistance to infection present in some CD4 T-cell clones. AgmTRIM5α transduction of virus-specific CD4 T-cell clones increased and prolonged their ability to suppress SIV spread in CD4 target cells. This increased antiviral function was strongly linked to decreased viral replication in the AgmTRIM5α-expressing effectors, consistent with restriction preventing the virus-induced cytopathogenicity that disables effector function. Taken together, our data show that AgmTRIM5α restriction, although not absolute, reduces SIV replication in primary rhesus CD4 T cells which, in turn, increases their antiviral function. These results support priorin vivodata indicating that the contribution of virus-specific CD4 T-cell effectors to viral control is limited due to infection.IMPORTANCEThe potential of effector CD4 T cells to immunologically modulate SIV/HIV infection likely is limited by their susceptibility to infection and subsequent inactivation or elimination. Here, we show that AgmTRIM5α expression inhibits SIV spread in primary effector CD4 T cellsin vitro. Importantly, protection of effector CD4 T cells by AgmTRIM5α markedly enhanced their antiviral function by delaying SIV infection, thereby extending their viability despite the presence of virus. Ourin vitrodata support priorin vivoHIV-1 studies suggesting that the antiviral CD4 effector response is impaired due to infection and subsequent cytopathogenicity. The ability of AgmTRIM5α expression to restrict SIV infection in primary rhesus effector CD4 T cells now opens an opportunity to use the SIV/rhesus macaque model to further elucidate the potential and scope of anti-AIDS virus effector CD4 T-cell function.

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