Presentation of the Goodpasture Autoantigen to CD4 T Cells Is Influenced More by Processing Constraints Than by HLA Class II Peptide Binding Preferences

Abstract Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I–restricted mHags have been identified, the first 2 autosomal HLA-class II–restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II–restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

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IL-6 down-regulates HLA class II expression and IL-12 production of human dendritic cells to impair activation of antigen-specific CD4+ T cells

Cancer Immunology Immunotherapy ◽

10.1007/s00262-015-1791-4 ◽

2016 ◽

Vol 65 (2) ◽

pp. 193-204 ◽

Cited By ~ 31

Author(s):

Yosuke Ohno ◽

Hidemitsu Kitamura ◽

Norihiko Takahashi ◽

Junya Ohtake ◽

Shun Kaneumi ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Cd4 T Cells ◽

Class Ii ◽

Hla Class Ii ◽

Human Dendritic Cells

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Identification of Phosphatidylinositol 4-Kinase Type II β as the First HLA Class II Associated Minor Histocompatibility Antigen Involved in Graft Versus Leukemia Reactivity.

Blood ◽

10.1182/blood.v110.11.1800.1800 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1800-1800

Author(s):

Marieke Griffioen ◽

Edith D. van der Meijden ◽

M. Willy Honders ◽

Caroline Rutten ◽

Simone A.P. van Luxemburg-Heijs ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Class Ii ◽

Hla Class Ii ◽

Type Ii ◽

Cell Therapies ◽

Graft Versus Leukemia ◽

Hla Dq ◽

Phosphatidylinositol 4

Abstract Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLI). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, mHag specific CD4+ T cells may selectively mediate Graft-versus-Leukemia (GvL) reactivity without Graft-versus-Host Disease (GvHD). Clinical studies have shown that adoptive transfer of CD4+ donor lymphocytes after HLA-matched alloSCT may lead to clinical remissions with a reduced incidence of GvHD, emphasizing the relevance of CD4+ T cells and HLA class II associated mHags for development of effective anti-tumor T cell therapies after alloSCT with a low risk for GvHD. The aim of this study was to identify the HLA class II associated mHag that is recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLA-matched alloSCT who developed strong GvL reactivity with mild GvHD of the skin after treatment with DLI. We previously developed recombinant bacteria cDNA expression libraries based on delivery of exogenous antigens for identification of HLA class II antigens and used this method for identification of the first autosomal HLA class II (HLA-DQB1*0603) associated mHag LB-PI4K2B-1S. LB-PI4K2B-1S has a population frequency of 40–50% and is encoded by the broadly-expressed phosphatidylinositol 4-kinase type II β gene. In the patient with CML, a polyclonal CD4+ T cell response against LB-PI4K2B-1S and simultaneous mHag specific CD8+ T cells were demonstrated. LB-PI4K2B-1S specific CD4+ T cells were shown to recognize the CD34+ CML cells of the patient as well as other leukemic cells. Recognition and lysis of normal hematopoietic cells by LB-PI4K2B-1S specific CD4+ T cells critically depended on the number of HLA-DQ molecules expressed at the cell surface and was restricted to high HLA-DQ-expressing B cells, mature dendritic cells (DC) and EBV-transformed B cells. HLA-DQ expression on T cells, PHA-stimulated blasts, monocytes and immature DC was absent or low and not sufficient for T cell recognition. We also demonstrated that HLA-DQ expression on normal cells of non-hematopoietic origin after extensive culturing with IFN-γ was moderately upregulated as compared to HLA-DR and -DP and not sufficient for recognition by LB-PI4K2B-1S specific CD4+ T cells. In conclusion, the data suggest that LB-PI4K2B-1S specific CD4+ T cells mediated tumor rejection by directly eliminating the malignant cells of the patient as effector cells and stimulating the induction and maintenance of CD8+ T cell immunity as helper cells, and HLA-DQ associated mHags may be appropriate targets for T cell therapies with the aim to selectively stimulate GvL after HLA-matched alloSCT with a low risk for GvHD.

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A Single Nucleotide Polymorphism in CD19 Defines a Novel Target for Immunotherapy of B Cell Malignancies with CD4+ Cytotoxic T Cells.

Blood ◽

10.1182/blood.v110.11.1798.1798 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1798-1798

Author(s):

Robbert M. Spaapen ◽

Henk M. Lokhorst ◽

Kelly van den Oudenalder ◽

Brith Otterud ◽

Harry Dolstra ◽

...

Keyword(s):

T Cells ◽

Single Nucleotide Polymorphism ◽

B Cell ◽

Cd4 T Cells ◽

Class Ii ◽

Hla Class Ii ◽

Malignant Cells ◽

Nucleotide Polymorphism ◽

B Cell Malignancies ◽

Single Nucleotide

Abstract The Graft-versus-Tumor (GvT) effect of HLA-matched allogeneic stem cell transplantation (allo-SCT) is largely mediated by donor-derived alloreactive CD4+ and CD8+ T cells. Major targets of this curative effect are the minor Histocompatibility antigens (mHags) expressed on the malignant cells. Here we report the first mHag encoded by a hematopoietic gene and recognized by HLA class II (HLA-DQA1*05/B1*02)-restricted CD4+ T cells. This antigen is encoded by a single nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, a highly important target antigen for immunotherapy of almost all B cell malignancies. We identified this antigen using a novel and powerful genetic strategy, in which a phenotype-genotype correlation scanning was the key step for fine-mapping the genetic locus defined by pair-wise linkage analysis. In functional assays, CD4+ T cells specific for the CD19L-encoded mHag mediated effective peptide-dependent maturation of DCs and polarized them to produce significant levels of interleukin-12. In another assay, the CD19L-mHag-specific T cells facilitated the proliferation of a CD8+ mHag-specific T cell clone in an antigen-dependent manner. Even more important, they also lysed CD19L-positive malignant cells, illustrating the therapeutic advantages of targeting this CD19 L -derived, HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.

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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 ◽

10.1182/blood.v112.11.1245.1245 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1245-1245 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v112.11.3247.3247 ◽

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.

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

Blood ◽

10.1182/blood.v120.21.4351.4351 ◽

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.

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Cytokine response of CD4+T cells responding to HLA class II+ melanoma

Melanoma Research ◽

10.1097/00008390-199706001-00474 ◽

1997 ◽

Vol 7 (Supplement 1) ◽

pp. S136

Author(s):

M Brady ◽

F Lee ◽

S Ree ◽

D Eckles ◽

J Lee

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Class Ii ◽

Hla Class Ii ◽

Cytokine Response

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HLA class II upregulation during viral infection leads to HLA-DP–directed graft-versus-host disease after CD4+ donor lymphocyte infusion

Blood ◽

10.1182/blood-2012-12-470872 ◽

2013 ◽

Vol 122 (11) ◽

pp. 1963-1973 ◽

Cited By ~ 48

Author(s):

Sanja Stevanović ◽

Cornelis A. M. van Bergen ◽

Simone A. P. van Luxemburg-Heijs ◽

Boris van der Zouwen ◽

Ekaterina S. Jordanova ◽

...

Keyword(s):

T Cells ◽

Viral Infection ◽

Graft Versus Host Disease ◽

Cd4 T Cells ◽

Viral Infections ◽

Class Ii ◽

Donor Lymphocyte Infusion ◽

Hla Class Ii ◽

Graft Versus Host ◽

Key Points

Key Points GVHD after HLA-DPB1–mismatched CD4+ DLI after TCD-alloSCT is mediated by allo-reactive HLA-DPB1–directed CD4+ T cells. Viral infections after TCD-alloSCT can induce HLA class II on nonhematopoietic tissues, making them targets for CD4+ T cells in GVHD.

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