Characterization Of The HLA Class II Ligandome In Acute Myeloid Leukemia (AML) Reveals Novel Candidates For Peptide-Based Immunotherapy

Abstract CD4+ T cells are crucial for the induction and maintenance of cytotoxic T cell responses, but can also mediate direct tumor rejection. The therapeutic efficacy of peptide-based cancer vaccines may thus be improved by including HLA class II epitopes to stimulate T helper cells. In contrast to HLA class I ligands, only a small number of class II ligands of TAA has been described so far. We recently reported on the overexpression of HLA class II in AML cells as compared to autologous monocytes and granulocytes as well as on the first HLA class I leukemia associated antigens identified directly on the cell surface of primary AML cells (Stickel et. al. abstract in Blood 2012). In this study we characterized the HLA class II ligandome in AML to identify additional ligands for a peptide-based immunotherapy approach. HLA class II ligands from primary AML cells as well as bone marrow and peripheral blood mononuclear cell (BMNCs/PBMCs) of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. Most abundantly detected peptides were functionally characterized with regard to their ability to induce a specific CD4+ T-cell response in healthy donors and in tumor patients using ELISpot. Samples from 10 AML patients (5 FLT3-ITD mutated) and 18 healthy donors were analyzed. We identified more than 2,100 AML-derived HLA class II ligands representing >1,000 different source proteins, of which 315 were exclusively represented in AML, but not in healthy PBMC/BMNC. Data mining for broadly represented LiTAAs pinpointed 26 HLA class II ligands from 8 source proteins that were presented exclusively on more than 40% of all analyzed AML samples as most promising targets. Amongst them were already described TAAs (e.g., RAB5A) as well as several so far understated proteins (e.g. calsyntenin 1, glycophorin A, mannose-binding lectin 2). Subset analysis revealed 58 LiTAAs presented exclusively on FLT3-ITD mutated AML cells. Additional screening for HLA class II ligands from described leukemia associated antigens showed positive results for NPM1 (1 peptide sequence) and MPO (13 peptide sequences). Peptides from calsyntenin 1 and RAB5A were able to elicit CD4+-T-cell response in 25% of tested AML patients (n=16). Thus, our study identified, for the first time, HLA class II tumor associated antigens directly obtained from the HLA ligandomes of AML patients and thereby represents a further step to our goal of developing a multipeptide vaccine for immunotherapy of AML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text

Comprehensive analysis of human CMV T cell response reveals epitopes common to both HLA class I and class II restriction

Human Immunology ◽

10.1016/j.humimm.2004.07.136 ◽

2004 ◽

Vol 65 (9-10) ◽

pp. S72

Author(s):

Maria P. Bettinotti ◽

Stefanie Slezak ◽

Lorraine Caruccio ◽

David Stroncek

Keyword(s):

T Cell ◽

Cell Response ◽

Hla Class I ◽

Class Ii ◽

Comprehensive Analysis ◽

T Cell Response ◽

Class I ◽

Human Cmv

Download Full-text

Effects of cyclophosphamide and IL-2 on regulatory CD4+ T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response

Cancer Immunology Immunotherapy ◽

10.1007/s00262-013-1397-7 ◽

2013 ◽

Vol 62 (5) ◽

pp. 897-908 ◽

Cited By ~ 20

Author(s):

Chiara Camisaschi ◽

Paola Filipazzi ◽

Marcella Tazzari ◽

Chiara Casati ◽

Valeria Beretta ◽

...

Keyword(s):

T Cell ◽

Cell Response ◽

Hla Class I ◽

T Cell Response ◽

Cd4 T Cell ◽

Class I ◽

Cell Frequency ◽

Melanoma Patients ◽

And Function

Download Full-text

Identification of a Coordinated CD8 and CD4 T Cell Response Directed Against Mismatched HLA Class I Causing Severe Acute Graft-versus-Host Disease

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2011.10.018 ◽

2012 ◽

Vol 18 (2) ◽

pp. 210-219 ◽

Cited By ~ 13

Author(s):

Avital L. Amir ◽

Renate S. Hagedoorn ◽

Simone A.P. van Luxemburg-Heijs ◽

Erik W.A. Marijt ◽

Alwine B. Kruisselbrink ◽

...

Keyword(s):

T Cell ◽

Graft Versus Host Disease ◽

Cell Response ◽

Hla Class I ◽

T Cell Response ◽

Cd4 T Cell ◽

Class I ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Download Full-text

Public T-cell epitopes shared among SARS-CoV-2 variants are presented on prevalent HLA class I alleles

10.1101/2021.10.13.463911 ◽

2021 ◽

Author(s):

Saskia Meyer ◽

Isaac Blaas ◽

Ravi Chand Bollineni ◽

Marina Delic-Sarac ◽

Trung T Tran ◽

...

Keyword(s):

T Cell ◽

Cell Response ◽

T Cell Responses ◽

Hla Class I ◽

Cd8 T Cell ◽

T Cell Response ◽

Class I ◽

T Cell Epitopes ◽

Low Frequencies ◽

Cell Responses

T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. Here, we investigate CD8 T-cell responses to 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identify nine conserved SARS-CoV-2 specific epitopes restricted by four of the most prevalent HLA class I alleles in Caucasians, to which responding CD8 T cells are detected in 70-100% of convalescents expressing the relevant HLA allele, including two novel epitopes. We find a strong correlation between immunoprevalence and immunodominance. Using a new algorithm, we predict that a vaccine including these epitopes would induce a T cell response in 83% of Caucasians. Significance Statement: Vaccines that induce broad T-cell responses may boost immunity as protection from current vaccines against SARS-CoV-2 is waning. From a manufacturing standpoint, and to deliver the highest possible dose of the most immunogenic antigens, it is rational to limit the number of epitopes to those inducing the strongest immune responses in the highest proportion of individuals in a population. Our data show that the CD8 T cell response to SARS-CoV-2 is more focused than previously believed. We identify nine conserved SARS-CoV-2 specific CD8 T cell epitopes restricted by four of the most prevalent HLA class I alleles in Caucasians and demonstrate that seven of these are endogenously presented.

Download Full-text

The CD4+ T-cell response to adenovirus is focused against conserved residues within the hexon protein

Journal of General Virology ◽

10.1099/vir.0.82867-0 ◽

2007 ◽

Vol 88 (9) ◽

pp. 2417-2425 ◽

Cited By ~ 23

Author(s):

David Onion ◽

Laura J. Crompton ◽

Donald W. Milligan ◽

Paul A. H. Moss ◽

Steven P. Lee ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Response ◽

Protein A ◽

T Cell Response ◽

Cd4 T Cell ◽

T Cell Epitopes ◽

Hexon Protein ◽

Healthy Donors ◽

Fiber Proteins

Adenovirus is a significant pathogen in immunocompromised patients and is widely utilized as a gene delivery vector, so a detailed understanding of the human immune response to adenovirus infection is critical. This study characterized the adenovirus-specific CD4+ T-cell response of healthy donors by incubation with whole virus or with individual hexon and fiber proteins. Adenovirus-specific CD4+ T cells averaged 0.26 % of the CD4+ T-cell pool and were detectable in all donors. T cells recognizing the highly conserved hexon protein accounted for 0.09 %, whereas no response was observed against the fiber protein. A panel of hexon-specific CD4+ T-cell clones was generated and shown to lyse targets infected with adenovirus from different serotypes and species. Three CD4 T-cell epitopes are described, which map to highly conserved regions of the hexon protein.

Download Full-text

T cell MHC class II molecules downregulate CD4+ T cell response following LAG-3 binding

Human Immunology ◽

10.1016/0198-8859(96)85066-x ◽

1996 ◽

Vol 47 (1-2) ◽

pp. 69

Author(s):

Frédéric Triebel ◽

Bertrand Huard

Keyword(s):

T Cell ◽

Mhc Class Ii ◽

Cell Response ◽

Class Ii ◽

T Cell Response ◽

Cd4 T Cell ◽

Mhc Class Ii Molecules

Download Full-text

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.

Download Full-text