scholarly journals The T cell response of HLA-DR transgenic mice to human myelin basic protein and other antigens in the presence and absence of human CD4.

1995 ◽  
Vol 181 (3) ◽  
pp. 867-875 ◽  
Author(s):  
D M Altmann ◽  
D C Douek ◽  
A J Frater ◽  
C M Hetherington ◽  
H Inoko ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Class Ii ◽  
Hla Class Ii ◽  
T Cell Response ◽  
Disease Models ◽  
Single Chain ◽  
Hla Dr ◽  
Multiple Sclerosis Patients

Analysis of HLA class II transgenic mice has progressed in recent years from analysis of single chain HLA class II transgenes with expression of mixed mouse/human heterodimers to double transgenic mice expressing normal human heterodimers. Previous studies have used either HLA transgenic mice in which there is a species-matched interaction with CD4 or mice which lack this interaction. Since both systems are reported to generate HLA-restricted responses, the matter of the requirement for species-matched CD4 remains unclear. We have generated triple transgenic mice expressing three human transgenes, DRA, DRB, and CD4, and compared HLA-restricted responses to peptide between human-CD4+ (Hu-CD4+) and Hu-CD4- littermates. We saw no difference between Hu-CD4+ and Hu-CD4- groups, supporting the notion that for some responses at least the requirement for species-matched CD4 may not be absolute. Evidence for positive selection of mouse T cell receptors in HLA-DR transgenic mice came both from the acquisition of new, HLA-restricted responses to various peptides and from an increased frequency of T cells using the TCR V beta 4 gene segment. An important goal with respect to the analysis of function in HLA transgenic mice is the clarification of mechanisms which underpin the recognition of self-antigens in human autoimmune disease. As a first step towards 'humanized' disease models in HLA transgenic mice, we analyzed the responses of HLA-DR transgenic mice to the human MPB 139-154 peptide which has been implicated as an epitope recognized by T cells of multiple sclerosis patients. We obtained T cell responses to this epitope in transgenic mice but not in nontransgenic controls. This study suggests that HLA transgenic mice will be valuable in the analysis of HLA-restricted T cell epitopes implicated in human disease and possibly in the design of new disease models.

scholarly journals Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II

2022 ◽  
Vol 12 ◽  
Author(s):  
You-Seok Hyun ◽  
Yong-Hun Lee ◽  
Hyeong-A Jo ◽  
In-Cheol Baek ◽  
Sun-Mi Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
Class Ii ◽  
Hla Class Ii ◽  
T Cell Response ◽  
Hla Dr ◽  
Hla Dq ◽  
Single Allele ◽  
Human Coronaviruses

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4+ T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016–2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance.

scholarly journals Functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecule in antigen recognition by T cells in HLA-DR and DQ transgenic mice.

1994 ◽  
Vol 180 (1) ◽  
pp. 165-171 ◽  
Author(s):  
K Yamamoto ◽  
Y Fukui ◽  
Y Esaki ◽  
T Inamitsu ◽  
T Sudo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Responses ◽  
Class Ii ◽  
Hla Class Ii ◽  
Functional Interaction ◽  
Leukocyte Antigen ◽  
Cell Responses ◽  
Hla Class Ii Molecules

Studies in vitro have suggested that a species barrier exists in functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecules. However, whether mouse CD4+ T cells restricted by HLA class II molecules are generated in HLA class II transgenic mice and respond to peptide antigens across this barrier has remained unclear. In an analysis of T cell responses to synthetic peptides in mice transgenic for HLA-DR51 and -DQ6, we found that DR51 and DQ6 transgenic mice acquired significant T cell response to influenza hemagglutinin-derived peptide 307-319 (HA 307) and Streptococcus pyogenes M12 protein-derived peptide 347-397 (M6C2), respectively. Inhibition studies with several monoclonal antibodies showed that transgenic HLA class II molecules presented these peptides to mouse CD4+ T cells. Furthermore, T cell lines specific for HA 307 or M6C2 obtained from the transgenic mice could respond to the peptide in the context of relevant HLA class II molecules expressed on mouse L cell transfectants that lack the expression of mouse MHC class II. These findings indicate that interaction between HLA class II and mouse CD4 molecules is sufficient for provoking peptide-specific HLA class II-restricted T cell responses in HLA class II transgenic mice.

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.

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.

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

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

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

scholarly journals In vitro inhibition of normal human hematopoiesis by marrow CD3+, CD8+, HLA-DR+, HNK1+ lymphocytes

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1616-1621 ◽  
Author(s):  
G Vinci ◽  
JP Vernant ◽  
M Nakazawa ◽  
M Zohair ◽  
A Katz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Inhibitory Activity ◽  
Colony Growth ◽  
Class Ii ◽  
Hla Class Ii ◽  
Hla Dr ◽  
In Vitro Inhibition

Abstract We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

scholarly journals Direct Ex Vivo Analyses of HLA-DR1 Transgenic Mice Reveal an Exceptionally Broad Pattern of Immunodominance in the Primary HLA-DR1-Restricted CD4 T-Cell Response to Influenza Virus Hemagglutinin

2007 ◽  
Vol 81 (14) ◽  
pp. 7608-7619 ◽  
Author(s):  
Katherine A. Richards ◽  
Francisco A. Chaves ◽  
Frederick R. Krafcik ◽  
David J. Topham ◽  
Christopher A. Lazarski ◽  
...  
Keyword(s):  
T Cells ◽  
Influenza Virus ◽  
Transgenic Mice ◽  
Cd4 T Cells ◽  
Protective Immunity ◽  
Influenza Pandemic ◽  
Class Ii ◽  
T Cell Response ◽  
Human Populations ◽  
Hla Dr

ABSTRACT The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans.

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

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

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

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

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

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

scholarly journals In vitro inhibition of normal human hematopoiesis by marrow CD3+, CD8+, HLA-DR+, HNK1+ lymphocytes

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1616-1621
Author(s):  
G Vinci ◽  
JP Vernant ◽  
M Nakazawa ◽  
M Zohair ◽  
A Katz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Inhibitory Activity ◽  
Colony Growth ◽  
Class Ii ◽  
Hla Class Ii ◽  
Hla Dr ◽  
In Vitro Inhibition

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

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