Molecular Localization and Polymorphism of HLA Class II Restriction Determinants Defined By Mycobacterium Leprae Reactive Helper T Cell Clones from Leprosy Patients

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.

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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 ◽

10.1182/blood.v118.21.3062.3062 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3062-3062 ◽

Cited By ~ 1

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.

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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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Identification, at the genomic level, of an HLA-DR restriction element for cloned antigen-specific T4 cells.

Journal of Experimental Medicine ◽

10.1084/jem.161.6.1569 ◽

1985 ◽

Vol 161 (6) ◽

pp. 1569-1574 ◽

Cited By ~ 11

Author(s):

G Paulsen ◽

E Qvigstad ◽

G Gaudernack ◽

L Rask ◽

R Winchester ◽

...

Keyword(s):

T Cell ◽

Class Ii ◽

Hla Class Ii ◽

T Cell Clones ◽

Genomic Level ◽

Hla Dr ◽

Cell Clones ◽

Length Polymorphisms ◽

Restriction Element ◽

Antigen Presenting

Two T4 cell clones (TLC) specific for antigenic epitopes on Chlamydia trachomatis were studied. Using a panel of allogeneic antigen-presenting cells (APC), both TLC were found to be restricted by HLA class II elements closely associated with, but not identical to the DRw5S specificity, as determined by highly selected alloantisera, a monoclonal antibody (mAb), 109d6, and confirmed on the DNA level by determination of restriction fragment length polymorphisms (RFLP) with a DR beta probe. Furthermore, HLA-DR-specific mAb, including 109d6, but not other HLA class II- or class I-specific antibodies inhibited the two TLC, strongly suggesting that the restriction element is expressed by a DR molecule. Using digestion with Hind III restriction enzyme and a DR beta probe, we found a complete concordance between the appearance of a 9.3 kilobase band and the ability of allogeneic APC to restimulate the T cell clones. Thus, the restriction element for these T cell clones appear to be expressed by DR molecules, but can, at present, only be detected at the genomic level.

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