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.

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.

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.

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.

Human T Cells with Distinct Specificities Mediate Graft-Versus-Leukemia Reactivity and Xenogeneic Graft-Versus-Host Disease in a NOD/Scid Mouse Model for Human Acute Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1330-1330
Author(s):  
Sanja Stevanovic ◽  
Bart Nijmeijer ◽  
Marianke LJ Van Schie ◽  
Roelof Willemze ◽  
Marieke Griffioen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
T Cell Clones ◽  
Cell Clones ◽  
Human T Cells

Abstract Abstract 1330 Poster Board I-352 Immunodeficient mice inoculated with human leukemia can be used as a model to investigate Graft-versus-Leukemia (GvL) effects of donor lymphocyte infusions (DLIs). In addition to GvL reactivity, treatment with DLI induces xenogeneic Graft-versus-Host Disease (GvHD) in mice, characterized by pancytopenia and weight loss. In patients treated with DLI for relapsed or residual leukemia after allogeneic stem cell transplantation, immune responses against non-leukemic cells may also cause GvHD. It has been suggested that GvL reactivity and GvHD, which co-develop in vivo, can be separated and that distinct T cells exist with the specific capacity to mediate GvL reactivity or GvHD. Since adoptive T cell transfer models that allow analysis of separation of GvL and GvHD are rare, we aimed to establish whether GvL reactivity and xenogeneic GvHD could be separated using our model of human leukemia-engrafted NOD/scid mouse after treatment with human donor T cells. In this study, non-conditioned NOD/scid mice engrafted with primary human acute lymphoblastic leukemic cells were treated with CD3+ DLI. Established tumors were effectively eliminated by emerging human T cells, but also induced xenogeneic GvHD. Flowcytometric analysis demonstrated that the majority of emerging CD8+ and CD4+ T cells were activated (HLA-DR+) and expressed an effector memory phenotype (CD45RA-CD45RO+CCR7-). To investigate whether GvL reactivity and xenogeneic GvHD were mediated by the same T cells showing reactivity against both human leukemic and murine cells, or displaying distinct reactivity against human leukemic and murine cells, we clonally isolated and characterized the T cells during the GvL response and xenogeneic GvHD. T cell clones were analyzed for reactivity against primary human leukemic cells and primary NOD/scid hematopoietic (BM and spleen cells) and non-hematopoietic (skin fibroblasts) cells in IFN-g ELISA. Isolated CD8+ and CD4+ T cell clones were shown to recognize either human leukemic or murine cells, indicating that GvL response and xenogeneic GvHD were mediated by different human T cells. Flowcytometric analysis demonstrated that all BM and spleen cells expressed MHC class I, whereas only 1-3 % of the cells were MHC class II +. Primary skin fibroblasts displayed low MHC class I and completely lacked MHC class II expression. Xeno-reactive CD8+ T cell clones were shown to recognize all MHC class I + target cells and xeno-reactive CD4+ T cells clones displayed reactivity only against MHC class II + target cells. To determine the MHC restriction of xeno-reactive T cell clones, NOD/scid bone marrow (BM) derived dendritic cells (DC) expressing high levels of murine MHC class I and class II were tested for T cell recognition in the presence or absence of murine MHC class I and class II monoclonal antibodies in IFN-g ELISA. Xeno-reactive CD8+ T cell clones were shown to be MHC class I (H-2Kd or H-2Db) restricted, whereas xeno-reactive CD4+ T cell clones were MHC class II (I-Ag7) restricted, indicating that xeno-reactivity reflects genuine human T cell response directed against allo-antigens present on murine cells. Despite production of high levels of IFN-gamma, xeno-reactive CD8+ and CD4+ T cell clones failed to exert cytolytic activity against murine DC, as determined in a 51Cr-release cytotoxicity assay. Absence of cytolysis by CD8+ T cell clones, which are generally considered as potent effector cells, may be explained by low avidity interaction between human T cells and murine DC, since flowcytometric analysis revealed sub-optimal activation of T cells as measured by CD137 expression and T cell receptor downregulation upon co-culture with murine DC, and therefore these results indicate that xenogeneic GvHD in this model is likely to be mediated by cytokines. In conclusion, in leukemia-engrafted NOD/scid mice treated with CD3+ DLI, we show that GvL reactivity and xenogeneic GvHD are mediated by separate human T cells with distinct specificities. All xeno-reactive T cell clones showed genuine recognition of MHC class I or class II associated allo-antigens on murine cells similar as GvHD-inducing human T cells. These data suggest that our NOD/scid mouse model of human acute leukemia may be valuable for studying the effectiveness and specificity of selectively enriched or depleted T cells for adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Diversity of HLA Class I and Class II Restricted Minor Histocompatibility Antigens in Graft-Versus-Leukemia Reactivity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4084-4084
Author(s):  
Marieke Griffioen ◽  
M. Willy Honders ◽  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A.M. van Bergen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematopoietic Cells ◽  
Cd4 T Cell ◽  
Cell Recognition ◽  
T Cell Clones ◽  
T Cell Recognition ◽  
Cell Clones ◽  
T Cell Clone ◽  
Ifn Γ

Abstract Abstract 4084 Poster Board III-1019 Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T-cells recognizing minor histocompatibility antigens (mHags) encoded by single nucleotide polymorphisms (SNPs) on malignant cells of the recipient. Donor T-cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues. The aim of this study was to investigate the specificity and diversity of mHags recognized by T-cells in Graft-versus-Leukemia (GvL) reactivity. Activated (HLA-DR+) CD8+ and CD4+ T-cell clones were isolated from a patient successfully treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. GvL reactivity in this patient was accompanied with mild GvHD of the skin. Isolated T-cell clones were shown to recognize 13 different mHags. CD8+ T-cell clones were specific for HA-1 and HA-2 in HLA-A*0201, one unknown mHag in B*0801 and 4 unknown mHags in B*4001. CD4+ T-cell clones were specific for one unknown mHag in HLA-DQ and 5 unknown mHags in DR. By screening plasmid (class I) and bacteria (class II) cDNA libraries, we identified a mHag in HLA-DQ encoded by the PI4K2B gene (Griffioen et al., PNAS 2008), 4 mHags in HLA-DR encoded by the PTK2B, MR-1, LY75 and MTHFD1 genes (Stumpf et al., Blood 2009) and a mHag in B*4001 encoded by the TRIP10 gene. For the 3 T cell clones recognizing unknown mHags in B*4001, we performed Whole Genome Assocation scanning (WGAs). A panel of 60 EBV-LCL was retrovirally-transduced with B*4001 and tested for T-cell recognition. In parallel, genomic DNA was isolated and more than one million single nucleotide polymorphisms (SNPs) were determined by the Illumina beadchip array. Statistical analysis revealed significant association between T-cell recognition of EBV-LCL and the presence of coding SNPs in the SON DNA-binding protein and SWAP-70 genes. To get more insight into the role and potential use of the mHags in GvL reactivity and GvHD, all T-cell clones were analyzed in detail for reactivity against hematopoietic and non-hematopoietic cells. Hematopoietic cells included peripheral blood cells (monocytes, B-cells and T-cells), professional antigen presenting cells (APC) and leukemic cells (CML, ALL and AML). All CD8+ T-cell clones recognized (subsets of) peripheral blood cells as well as CML cells, except for the T-cell clone for TRIP10. Recognition of (subsets of) peripheral blood cells was also observed for all CD4+ T-cell clones, but CML cells were differentially recognized. CML cells were strongly recognized by the T-cell clones for MTHFD1 and the unknown mHag in HLA-DR, whereas no or low reactivity was observed for all other CD4+ T-cell clones. All CD8+ and CD4+ T-cell clones strongly recognized professional APC, including monocyte-derived dendritic cells and in vitro differentiated CML cells with APC phenotype. All T-cell clones were also capable of recognizing AML and ALL, except for the T-cell clone for TRIP10, which showed restricted recognition of AML-M4 and -M5 of monocytic origin. As non-hematopoietic cells, patient-derived fibroblasts were cultured with and without IFN-γ and tested for T-cell recognition. In the absence of IFN-γ, all T-cell clones failed to recognize fibroblasts, except for the T-cell clone for the unknown mHag in B*0801. After treatment with IFN-γ, additional reactivity was observed for the T-cell clones for SON DNA-binding protein and the unknown mHag in B*4001. Our data showed the specificity and diversity of mHags recognized by T-cells induced in a patient successfully treated with DLI for relapsed CML. The T-cell response was directed against 13 different mHags, of which 10 mHags in HLA class I and class II have now been identified by different techniques. Detailed analysis of T-cell recognition of hematopoietic and non-hematopoietic cells provides evidence that the mHags played different roles in the onset and execution of GvL and GvHD. Moreover, only one of the 10 identified mHags was expressed on fibroblasts after treatment with IFN-γ, indicating the characterization of mHags with potential relevance for T-cell based immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Major Histocompatibility Complex–independent Recognition of a Distinctive Pollen Antigen, Most Likely a Carbohydrate, by Human CD8+ α/β T Cells

1997 ◽  
Vol 186 (6) ◽  
pp. 899-908 ◽  
Author(s):  
Silvia Corinti ◽  
Raffaele De Palma ◽  
Angelo Fontana ◽  
Maria Cristina Gagliardi ◽  
Carlo Pini ◽  
...  
Keyword(s):  
T Cells ◽  
Major Histocompatibility Complex ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Response ◽  
Major Histocompatibility ◽  
T Cell Clones ◽  
Histocompatibility Complex ◽  
Cell Clones

We have isolated CD8+ α/β T cells from the blood of atopic and healthy individuals which recognize a nonpeptide antigen present in an allergenic extract from Parietaria judaica pollen. This antigen appears to be a carbohydrate because it is resistant to proteinase K and alkaline digestion, is hydrophilic, and is sensitive to trifluoromethane-sulphonic and periodic acids. In addition, on a reverse-phase high performance liquid chromatography column the antigen recognized by CD8+ T cells separates in a fraction which contains >80% hexoses (glucose and galactose) and undetectable amounts of proteins. Presentation of this putative carbohydrate antigen (PjCHOAg) to CD8+ T cell clones is dependent on live antigen presenting cells (APCs) pulsed for >1 h at 37°C, suggesting that the antigen has to be internalized and possibly processed. Indeed, fixed APCs or APCs pulsed at 15°C were both unable to induce T cell response. Remarkably, PjCHOAg presentation is independent of the expression of classical major histocompatibility complex (MHC) molecules or CD1. CD8+ T cells stimulated by PjCHOAg-pulsed APCs undergo a sustained [Ca2+]i increase and downregulate their T cell antigen receptors (TCRs) in an antigen dose– and time-dependent fashion, similar to T cells stimulated by conventional ligands. Analysis of TCR Vβ transcripts shows that six independent PjCHOAg-specific T cell clones carry the Vβ8 segment with a conserved motif in the CDR3 region, indicating a structural requirement for recognition of this antigen. Finally, after activation, the CD8+ clones from the atopic patient express CD40L and produce high levels of interleukins 4 and 5, suggesting that the clones may have undergone a Th2-like polarization in vivo. These results reveal a new class of antigens which triggers T cells in an MHC-independent way, and these antigens appear to be carbohydrates. We suggest that this type of antigen may play a role in the immune response in vivo.

CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus–transformed B cells

Blood ◽  
2009 ◽  
Vol 114 (4) ◽  
pp. 807-815 ◽  
Author(s):  
Heather M. Long ◽  
Jianmin Zuo ◽  
Alison M. Leese ◽  
Nancy H. Gudgeon ◽  
Hui Jia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Epstein Barr Virus ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
T Cell Clones ◽  
Barr Virus ◽  
Cell Clones ◽  
Epstein Barr

Abstract Epstein-Barr virus (EBV)–specific T-cell preparations, generated by stimulating immune donor lymphocytes with the autologous virus-transformed B-lymphoblastoid cell line (LCL) in vitro, can be used to target EBV-positive malignancies. Although these preparations are enriched for EBV antigen–specific CD8+ T cells, most also contain a CD4+ T-cell population whose specificity is unknown. Here, we show that, although CD4+ T-cell clones derived from such cultures recognize HLA class II–matched LCLs but not mitogen-activated B lymphoblasts, many (1) do not map to any known EBV antigen, (2) can be raised from EBV-naive as well as EBV-immune persons, and (3) can recognize a broad range of human B lymphoma–derived cell lines irrespective of EBV genome status, providing those lines to express the relevant HLA class II–restricting allele. Importantly, such CD4+ clones not only produce IFNγ but are also cytotoxic and can control the outgrowth of HLA-matched lymphoma cells in cocultivation assays. We infer that such CD4+ T cells recognize cellular antigens that are preferentially up-regulated in EBV-transformed but not mitogen-activated B lymphoblasts and that are also expressed in a range of B-cell malignancies. Such antigens are therefore of potential value as targets for CD4+ T cell–based immunotherapy.

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