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.

Complete Remission of Immunocytoma without Graft Versus Host Disease Caused by Allo-HLA-DP Specific T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3665-3665
Author(s):  
Caroline E. Rutten ◽  
Simone A.P. van Luxemburg-Heijs ◽  
Inge Jedema ◽  
Mirjam Heemskerk ◽  
Roelof Willemze ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Hematological Malignancies ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Leukemic Cells ◽  
T Cell Clones ◽  
Cell Clones

Abstract Mismatching for HLA-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a significant decreased risk of disease relapse, indicating that HLA-DP might be a target for a graft versus leukemia (GVL) effect in HLA-class II expressing hematological malignancies. To determine whether a specific GVL effect could be caused by allo-HLA-DP specific T cells, we analyzed the immune response in a patient with a refractory immunocytoma responding to donor lymphocyte infusion (DLI) after HLA-DP mismatched URD-SCT. Patient and donor were fully matched for HLA-A, -B, -C, -DR and -DQ, but differed for both HLA-DP alleles (donor HLA-DPB1*0402/0501; patient HLA-DPB*020102/0301). The patient received a T cell depleted URD-SCT after a non-myeloablative conditioning regimen, resulting in mixed chimerism (75% donor) without GVHD. Because of a hematological relapse, a single DLI was given 6 months after SCT, resulting in a profound anti-leukemic effect with only grade I GVHD, treated with topical corticosteroids. 6 weeks after DLI, malignant cells in peripheral blood (PB) had dropped from 72% to 47%. 7 weeks later, only 3% malignant cells were present, and after 4 months, complete remission and conversion to full donor chimerism in the absence of GVHD was observed. To determine whether allo-HLA-DP specific T cells were involved in the immune response, leukemia-reactive donor T cell clones were isolated from PB or bone marrow at different time points during the response to DLI. Patient derived T cells were overnight stimulated with irradiated leukemic cells harvested before transplantation, and clonal IFNγ producing T cells were sorted and expanded. 21 CD4+ T cell clones, 19 CD8+ T cell clones and 6 NK cell clones were tested for recognition of patient or donor derived cells as measured by IFNγ production and cytotoxic activity. The CD8+ or NK clones did not recognize patient leukemic cells. However, all 21 CD4+ clones produced INFγ in response to patient leukemic cells but not to donor cells. To determine whether these CD4+ T cell clones were capable of killing the leukemic cells, a CFSE based cytotoxicity assay was performed. 8 clones showed 30–90% lysis of the leukemic cell population. To further analyze the specificity of these CD4+ clones, blocking and panel studies were performed. Blocking with the HLA-DP specific mAb B7.21 abrogated IFNγ production by all clones, confirming HLA-DP restricted recognition. A panel study using 12 unrelated EBV-LCL expressing different HLA-DP alleles identified 18 clones specific for HLA-DPB1*0301, and 3 clones specific for HLA-DPB1*0201. To analyze the polyclonality of the immune response, the distribution of TCR Vβ chains was characterized by RT-PCR and sequence reactions. 7 different Vβs were found within the HLA-DPB1*0301 specific clones and 3 different Vβs within the HLA-DPB1*0201 specific clones. T cells using the same Vβ could be isolated at different time points during the clinical response, demonstrating the significance of this anti-HLA-DP response. In conclusion, we observed in a patient with an HLA-class II positive B cell malignancy a profound GVL effect without GVHD, caused by a polyclonal immune response comprising both T helper and cytotoxic CD4+ HLA-DP specific T cell clones directed against both HLA-DP alleles. These data indicate that in HLA-class II expressing hematological malignancies HLA-DP mismatched SCT may be preferable over a fully matched SCT making use of HLA-DP as a specific target for immunotherapy.

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.

HLA Class II Disparity Is Necessary and Sufficient for Induction of Effective Anti-Tumor Immunity by Donor Lymphocyte Infusion in a NOD/Scid Mouse Model for Human Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 648-648 ◽  
Author(s):  
Sanja Stevanovic ◽  
Marianke L.J. van Schie ◽  
Marieke Griffioen ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cell ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Leukemic Cells ◽  
T Cell Clones ◽  
Cell Clones

Abstract Abstract 648 Donor lymphocyte infusion (DLI) can be a curative treatment for patients with relapsed hematological malignancies after HLA matched allogeneic stem cell transplantation (alloSCT). However, curative responses in patients with acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphoid blastic phase (CML-BP) are infrequent after HLA matched DLI. This may be partly explained by the poor immunogenicity of these malignancies, since we previously demonstrated efficient induction of Graft-versus-Leukemia (GvL) immune responses in vitro and in vivo upon modification of ALL and CML-BP cells into leukemic antigen presenting cells (APC). Leukemic-APC may be particularly relevant for efficient generation of GvL immune responses after HLA matched DLI, since T cells recognizing allo-antigens in matched HLA molecules are known to reside in the naïve T cell compartment. In contrast, T cells recognizing allo-antigens in mismatched HLA molecules reside in the memory T cell compartment as well. Since memory T cells can also be activated by non-professional APC, HLA mismatched alloSCT and DLI may particularly be considered as a treatment modality for induction of GvL reactivity against poorly immunogenic malignancies. However, T cell responses across HLA barriers can induce severe Graft-versus-Host Disease (GvHD). Mismatched HLA class I molecules, which are broadly expressed on all nucleated cells, are frequent targets of alloreactive T cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, HLA class II mismatched alloSCT and DLI may more selectively induce GvL reactivity without inducing severe GvHD. In this study, we investigated the in vivo immunogenicity of established B-ALL or CML-BP by comparing the anti-tumor responses after fully HLA matched versus HLA class II mismatched DLI in a NOD/scid mouse model. Mice engrafted with primary B-ALL and CML-BP were treated with DLI from HLA matched (12/12 match) or HLA class II mismatched, but HLA class I matched donors. In mice engrafted with B-ALL or CML-BP, treatment with HLA matched DLI induced expansion of human CD4+ and CD8+ T cells in peripheral blood, but leukemic cells were only delayed in growth, and not eliminated. In contrast, after HLA class II mismatched DLI, leukemic cells rapidly disappeared upon emergence of human CD4+ and CD8+ T cells in peripheral blood. To analyze the specificity of the T cells, we clonally isolated CD4+ and CD8+ T cells from bone marrow and spleens of mice after treatment with DLI. All T cell clones were tested for recognition of patient leukemic cells, donor EBV transformed B cells (EBV-LCL) and murine bone marrow derived dendritic cells in IFNg ELISA. Isolated CD8+ and CD4+ T cell clones recognized either patient leukemic cells or murine cells, indicating that the T cell clones were either leukemia-reactive or xeno-reactive. After HLA matched DLI, only 2 of the 106 CD4+ T cell clones, and none of the 183 CD8+ T cell clones, recognized patient leukemic cells. The majority of isolated CD4+ and CD8+ T cell clones were xeno-reactive, as demonstrated by specific recognition of murine bone marrow derived dendritic cells, or non-reactive against any of the tested target cells. In contrast, after HLA class II mismatched DLI, 95 of the 322 CD4+ T cell clones specifically recognized patient leukemic cells. These leukemia-reactive CD4+ T cell clones were shown to be restricted by the mismatched HLA-DRB3, -DQB1 and –DPB1 alleles of the patient. None of the 49 CD8+ T cell clones were leukemia-reactive, but a significant number of CD8+ T cell clones and remaining CD4+ were xeno-reactive. In conclusion, our data show that HLA class II mismatched, but HLA class I matched, DLI is far more effective in inducing anti-tumor reactivity as compared to HLA matched DLI, whereas the in vivo capacity of both DLI's to induce allo-immune reactivity based on the induction of xeno-reactive T cells was similar. Our study emphasizes the necessity of HLA class II disparity for efficient in vivo induction of HLA class II mediated anti-tumor immunity against poorly immunogenic B-ALL and CML-BP in NOD/scid mice. We therefore hypothesize that use of HLA class II mismatched as compared to HLA matched alloSCT and DLI, despite an increased risk for GvHD, may improve the outcome for patients with HLA class II positive high risk acute lymphoblastic leukemia. Disclosures: No relevant conflicts of interest to declare.

HLA Class II Upregulation During An Ongoing Viral Infection Can Lead to HLA-DP Directed Graft-Versus-Host Disease After HLA-DPB1 Mismatched CD4+ Donor Lymphocyte Infusion

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3062-3062 ◽  
Author(s):  
Sanja Stevanovic ◽  
Cornelis A.M. van Bergen ◽  
Simone A.P. van Luxemburg-Heijs ◽  
Jessica C. Harskamp ◽  
C.J.M. Halkes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Hematopoietic Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
T Cell Clones ◽  
Cell Clones

Abstract Abstract 3062 T cell depletion of the graft in allogeneic hematopoietic stem cell transplantation (alloSCT) prevents the occurrence of severe acute Graft-versus-Host Disease (GvHD), but also impairs post-transplant anti-tumor and anti-viral immunity. Early intervention with donor lymphocyte infusion (DLI) after alloSCT may prevent relapse of the malignancy and improve immune reconstitution, but can be associated with reintroduction of GvHD. Since under non-inflammatory conditions HLA class II molecules are predominantly expressed on hematopoietic cells, DLI consisting of only CD4+ T cells can selectively target residual patient (pt) HLA class II + hematopoietic cells without inducing severe GvHD. However, recently in two pts with acute myeloid leukemia we observed severe GvHD after prophylactic CD4+ DLI following a 10/10 HLA allele matched, but HLA-DPB1 mismatched unrelated donor alloSCT. Both pts received a T cell depleted SCT after a non-myeloablative conditioning regimen, resulting in mixed chimerism (>97 % donor) at 3 months after alloSCT, and no GvHD. A single infusion of 0.5*106 purified CD4+ T cells/kg was administered 3.5 months after alloSCT, resulting in a decreasing pt chimerism coinciding with grade 1 skin GvHD, followed by grade 3–4 colonic GvHD 3–8 weeks later. Both pts were successfully treated with immune suppression and are in complete remission (CR) more than one year later. During the clinical immune responses high percentages of activated CD4+ (30–74 %) and CD8+ T cells (9–56 %) were demonstrated in peripheral blood (PB). Using cell sorting, we clonally isolated 777 and 289 CD4+, and 204 and 34 CD8+ T cell clones from pts 1 and 2, respectively, and tested these clones for recognition of multiple pt and donor derived target cells using IFNg ELISA. None of the CD8+ clones were alloreactive. In contrast, 3 and 8 % of the CD4+ T cell clones from pts 1 and 2, respectively, recognized various pt hematopoietic cells, but not donor cells, indicating alloreactivity. Retroviral transduction of donor EBV-LCL with pt HLA-DPB1 alleles identified specific recognition of the mismatched alleles for 2 and 7 % of all CD4+ T cell clones isolated, respectively. The remaining alloreactive CD4+ T cell clones showed a hematopoiesis-restricted minor histocompatibility antigen recognition pattern, since they failed to recognize pt skin fibroblasts pretreated with IFNg to upregulate HLA class II expression. In contrast, the majority of HLA-DPB1 specific CD4+ T cell clones recognized pt IFNg treated skin fibroblasts, indicating a direct role as mediators of GvHD after HLA-DPB1 mismatched CD4+ DLI. Since both pts were in CR, but mixed chimeric at the time of CD4+ DLI, we hypothesized that residual pt HLA-DP+ hematopoietic cells after alloSCT may have served as antigen presenting cells (APC) to induce the HLA-DPB1 specific CD4+ T cell response. Lineage specific chimerism analysis of PB samples prior to CD4+ DLI showed complete donor chimerism in the B cell and myeloid compartments, whereas predominantly pt chimerism (89–100% pt) was demonstrated in the T cell compartment. Flowcytometric analysis showed that 5–25 % of the pt CD4+ and CD8+ T cells were activated and expressed HLA-DP. CMV tetramer analysis demonstrated that 31 % of CD8+ T cells from pt 1 and 10 % from pt 2 were CMV specific, which had expanded as a consequence of CMV reactivation. We hypothesize that the HLA-DPB1 specific CD4+ T cell response has been induced by upregulated HLA-DP expression on activated pt T cells due to preexisting CMV infection, and/or by residual pt derived skin-resident APC, resulting in limited skin GvHD. We demonstrated CMV infection in a colon biopsy at the time of colonic GvHD, suggesting that local production of cytokines by pt derived CMV specific T cells may have upregulated HLA class II expression on non-hematopoietic cells and enhanced the HLA-DPB1 specific CD4+ T cell response, resulting in exacerbation of GvHD. In conclusion, we show in two pts that GvHD after prophylactic CD4+ DLI administered early after HLA-DPB1 mismatched T cell depleted alloSCT was caused by alloreactive CD4+ T cells directed against pt mismatched HLA-DPB1 alleles. Our results suggest that the presence of active viral infections inducing immune responses by residual pt T cells at the time of prophylactic HLA class II mismatched CD4+ DLI increases the likelihood of development of GvHD by influencing HLA class II expression on pt hematopoietic and non-hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

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.

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.

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.

Combining positional scanning peptide libraries, HLA-DR transfectants and bioinformatics to dissect the epitope spectrum of HLA class II cross-restricted CD4+ T cell clones

2010 ◽  
Vol 353 (1-2) ◽  
pp. 93-101 ◽  
Author(s):  
Mireia Sospedra ◽  
Yingdong Zhao ◽  
Marc Giulianotti ◽  
Richard Simon ◽  
Clemencia Pinilla ◽  
...  
Keyword(s):  
T Cell ◽  
Class Ii ◽  
Hla Class Ii ◽  
Peptide Libraries ◽  
Cd4 T Cell ◽  
T Cell Clones ◽  
Hla Dr ◽  
Cell Clones ◽  
Positional Scanning

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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