Sensitization of Human T Cells with Overlapping Pentadecapeptides Spanning the Wt1 Protein Induces Expansion of Leukemocidal T Cells Specific for Both Previously Identified and Novel WT1 Epitopes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3873-3873
Author(s):  
Ekaterina Doubrovina ◽  
Jacob Dupont ◽  
Deepa Trivedi ◽  
Elena Kanaeva ◽  
Richard J. O’Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Hla Class I ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
Hla Alleles ◽  
Peptide Epitopes ◽  
Human T Cells ◽  
T Cell Lines

Abstract The oncofetal protein, WT1, is differentially expressed in 60–80% of acute leukemias and CML. Previously, four peptide epitopes presented by HLA-A0201 or HLA-A2402 have been identified. We used a pool of 15-mers with 10 amino acid overlaps spanning the full sequence of WT1, loaded on autologous monocyte-derived dendritic cells or EBV-transformed BLCL to sensitize and raise T cell lines from a series of normal donors expressing or not expressing HLA-A0201 or HLA-A2402. Specific 15-mers eliciting responses were identified by quantitating IFNg+ CD8+ or CD4+ T cell responses to secondary stimulation with 15-mer subpools organized in a mapping grid with single overlaps. Responses against targets loaded with the identified stimulating 15-mer and sharing single HLA class I or II alleles permitted identification of restricting HLA alleles. Patients inheriting HLA-A0201 have generated CD8+ T cells in response to the complete pool which are selectively reactive against single 15-mers containing the known immunogen 252–260RMFPNAPYL. However, in certain donors, responses to 15-mer 28, containing a heretofore unreported epitope 136–144ALLPAVPSL, (which has a high predicted affinity for HLA-A0201) have been dominant. By mapping responses of T cells sensitized with the complete pool, defining the presenting HLA alleles and delineating immunogenic sequences within specific 15-mers, additional novel epitopes presented by HLA- B0801 and DRB10301 have also been defined. T cells generated by this approach also lyse both peptide-loaded targets as well as primary WT1+ leukemic cells expressing the restricting HLA allele. Thus, sensitization with pools of overlapping synthetic 15-mers spanning WT1 may selectively stimulate WT1-specific CD4+ and CD8+ T cell lines restricted by HLA alleles other than HLA- A0201 or A2402 that are leukemocidal and therefore of potential use for adoptive immunotherapy.

Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3531-3540 ◽  
Author(s):  
Angelo A. Cardoso ◽  
J. Pedro Veiga ◽  
Paolo Ghia ◽  
Hernani M. Afonso ◽  
W. Nicholas Haining ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Adoptive T Cell Therapy ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

Generation of Cytomegalovirus-Specific T Lymphocytes Using Protein-Spanning Pools of pp65-Derived Over-Lapping Pentadecapeptides for Adoptive Immunotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2236-2236
Author(s):  
Guenther Koehne ◽  
Deepa Trivedi ◽  
Roxanne Y. Williams ◽  
Richard J. O’Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Alleles ◽  
Hla Genotypes ◽  
T Cell Lines

Abstract Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We utilized a pool of 138 synthetic overlapping pentadecapeptides over-spanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T-cells and in analyses of T-cell lines from 5 separate HLA-A*0201+ individuals demonstrate that this pp65-derived pentadecapeptide-pool selectively induced T-cells specifically reactive against sub-pools of pentadecapeptides which contained the HLA-A*0201 binding epitope NLVPMVATV. The specificity of these T-cells for this immunodominant nonapeptide was confirmed by MHC-tetramer staining and intracellular interferon-γ production, demonstrating that 38 – 60% of the CD8+ cell population were specific for this A*2-restricted peptide after 3 weeks of culture. These T cells also killed both nonapeptide-pulsed and CMV-infected target cells. In subsequent experiments using auotlogous monocyte-derived DC’s pulsed with the pentadecapeptide pool for the stimulation of CMV-specific T-cell lines in individuals other than HLA-A*2, the generated T cells selectively recognized 1–3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide loaded targets sharing single HLA class I or II alleles permitted the identification the restricting HLA alleles. Those T-cell lines from HLA-A*2 neg. donors contained high frequencies of CD4 and/or CD8 T-cells selectively reactive against peptides presented by other HLA alleles including known epitopes such as aa 341–350QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as aa 267–275HERNGFTVL (HLA-B*4001 and B* 4002). In some donors, the peptide-specific IFN-g+ T-cells generated have been predominantly CD4+ T-cells. Like the peptide-specific CD8+ T-cells, we could determine both epitope and HLA-class II restricting element, e.g. aa513–523 FFWDANDIYRI (HLA-DRB1* 1301). These CD4+ T-cells also consistently exhibited cytotoxic activity against infected targets as well as peptide-loaded cells expressing the restricting HLA class II allele. Thus, synthetic overlapping pentadecapeptides spanning the sequence of the immunodominant protein of CMV-pp65, when loaded on DCs can consistently stimulate the in vitro generation of CD8+ and CD4+ T-cell lines from seropositive donors of diverse HLA genotypes. These cell lines are selectively enriched for T-cells specific for a limited number of immunodominant epitopes each presented by a single HLA class I or class II allele. This approach fosters expansion and selection of HLA-restricted CMV-pp65-reactive T-cell lines of high specificity which also lyse CMV-infected targets and may have advantages for generating virus-specific T-cells for adoptive immunotherapy.

Allogeneic Virus-Specific T Cells with HLA Alloreactivity Do Not Produce Graft-Versus-Host Disease In Human Subjects

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1252-1252
Author(s):  
J. Joseph Melenhorst ◽  
Ann M. Leen ◽  
Catherine M. Bollard ◽  
Máire F Quigley ◽  
David A Price ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Graft Versus Host Disease ◽  
Hla Class I ◽  
Class I ◽  
Hla Alleles ◽  
Graft Versus Host ◽  
Specific Ctls ◽  
T Cell Lines

Abstract Abstract 1252 We and others have recently established that T cell reactivity with non-self HLA (HLA alloreactivity) arises not only from naive T cells but also from the antigen-experienced T cell pool, including Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific T cells. Virus-specific T cells could therefore mediate graft-versus-host disease (GvHD) if infused into partially HLA mismatched recipients. We reviewed our clinical experience with adoptive transfer of allogeneic hematopoietic stem cell transplant donor-derived virus-specific T cell lines in 153 recipients who received donor-derived EBV-specific CTLs (N=114), bivirus CTLs specific for adenovirus and EBV (n=14), and trivirus CTLs specific for CMV, adenovirus and EBV (n=25). Seventy three donor-recipient pairs were partially HLA-mismatched, with the degree of HLA mismatching varying from one allele to a full haplotype. De novo GvHD did not develop after infusion of cytotoxic T lymphocytes (CTL), and the incidence of GvHD reactivation was 6.5% and not significantly different between recipients of HLA matched or mismatched CTL. Thus, virus-specific CTL did not mediate GvHD, even in recipients of partially matched CTL. Next we analyzed the HLA alloreactivity of four donor-infused bivirus-specific T cell lines, using activated T cells, that are known to lack CMV and EBV antigen expression, as antigen presenting cells (TAPC). We used a panel of 44 TAPC covering the most frequent HLA class I and II alleles. The CTL lines were labeled with CFSE and stimulated with TAPC for 6 hours, after which production of TNFα and IFNγ/IL-2 by CD4+ and CD8+ T cells in the CFSE-positive fraction was analyzed by flow cytometry. All CTLs responded to a number of TAPC, with some APC being recognized strongly. The majority elicited only weak or no response from the CTLs. We then assessed whether the CTLs recognized TAPC expressing the recipient's HLA alleles. We found moderate reactivity of the CTL with 1–5 TAPC expressing recipient HLA alleles. Taken together, our data indicate that reactivity of virus-specific CTLs with hematopoietic APC does not correlate with the risk of developing GvHD, and that virus-specific CTL can safely be infused into HLA class I and/or II mismatched recipients. Disclosures: No relevant conflicts of interest to declare.

Alloreactivity of Virus Specific T-Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3249-3249
Author(s):  
Avital L. Amir ◽  
Lloyd J.A. D’Orsogna ◽  
Marleen M. van Loenen ◽  
Dave L. Roelen ◽  
Ilias I.N. Doxiadis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Memory T Cells ◽  
Target Cells ◽  
T Cell Clones ◽  
Hla Alleles ◽  
Alloreactive T Cells ◽  
Cell Clones ◽  
T Cell Lines

Abstract Graft versus host disease (GVHD) in allogeneic stem cell transplantation (SCT) and graft rejection is caused by alloreactive T-cells. Alloreactivity can be exerted by naïve as well as by memory T-cells. Persistent latent viral infections, like those with herpes viruses, have a profound impact on the repertoire of memory T-cells. This implies that virus specific memory T-cells are also potentially alloreactive. Previously it has been shown that virus specific T-cell clones can cross react against allo-HLA. We investigated the frequency of alloreactivity mediated by virus specific T-cells. Mixed lymphocyte reactions, previously used to determine precursor frequencies of alloreactive T-cells, give an underestimation of the total frequency of alloreactive T-cells, due to limited number of allo-HLA alleles tested in this system. Therefore, in this study multiple CD8+ virus specific T-cells lines and clones were tested for alloreactivity against almost all frequent HLA class I and II alleles. From different healthy individuals we derived CD8+ virus specific T-cell lines, specific for Epstein Barr virus (EBV), Cytomegalovirus (CMV), Varicella Zoster virus (VZV) and Influenza virus (Flu) which were restricted to different HLA molecules. The generation of the T-cell lines and clones was performed by bulk sorting and single cell sorting, based on staining with viral peptide/MHC complex specific tetramers. The viral specificity of the expanded lines and clones was confirmed by tetramer staining and cytotoxicity and cytokine production assays. Polyclonality of the T-cell lines and monoclonality of the T-cell clones was confirmed by TCR Vβ analysis. Next, the T-cell lines and clones were screened for alloreactivity by testing against a panel of 29 different EBV transformed LCLs, together covering almost all frequent HLA class I and II molecules. 90% of tested virus specific T-cell lines and 40% of virus specific T-cell clones were found to be alloreactive, recognizing at least one of the allo-HLA alleles. For several lines and clones the specific recognized allo-HLA molecule was further identified using a panel of HLA typed target cells in combination with HLA specific blocking antibodies. Additionally, single HLA antigen expressing cell lines were used as target cells. Thus far we found EBV EBNA3A specific, HLA-A3 restricted T-cell clones to recognize HLA-A31. A CMV pp50 specific, HLA-A1 restricted T-cell line recognized HLA-A68. One VZV IE62 specific, HLA-A2 restricted clone showed recognition of HLA-B57, while another clone with the same specificity but with a different TCR Vβ recognized HLA-B55. An EBV BMLF specific, HLA-A2 restricted T-cell line showed recognition of HLA-A11. Finally an EBV BRLF specific, HLA-A3 restricted clone recognized HLA-A2. Our results show that a high percentage of virus specific T-cells can exert alloreactivity against allo- HLA molecules. Previously it was assumed that virus specific T-cells are not alloreactive against foreign HLA, allowing safe application of virus specific T-cell lines derived from HLA disparate donors in patients without the risk of inducing GVHD. Our data indicate that applying virus specific T-cell lines over HLA barriers does give a significant risk of GVHD and suggest that lines should be tested for alloreactivity against patient specific HLA alleles prior to application. A substantial part of the memory T-cell pool consists of virus specific T-cells, which are dominated by a limited repertoire of virus specific T-cell clones, present in high frequencies. Thus, virus specific T-cells recognizing allo-HLA alleles may also play an essential role in graft rejection.

scholarly journals The Scope of Allo-HLA Cross-Reactivity By (Third Party) Virus Specific T Cells Is Surprisingly Affected By HLA Restriction Rather Than Virus Specificity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2048-2048
Author(s):  
Wesley Huisman ◽  
Didier A.T. Leboux ◽  
Lieve E. van der Maarel ◽  
Lois Hageman ◽  
Derk Amsen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
K562 Cells ◽  
Cross Reactivity ◽  
Third Party ◽  
Cell Populations ◽  
Hla Alleles ◽  
Hla Restriction ◽  
T Cell Lines

Abstract Reactivations of cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) are frequently seen in immune compromised patients after allogeneic stem cell transplantation (alloSCT), and are associated with high morbidity and mortality. T cell immunity is essential for anti-viral protection, but a fully competent T cell repertoire generally does not develop until 3-6 months after transplantation. Especially patients transplanted with a virus non- experienced donor are at risk of developing severe complications. Adoptive transfer of partially HLA-matched virus specific T cells from healthy third party donors is a potential strategy to temporarily provide anti-viral immunity to these patients. However, these partially HLA-matched T cells harbor a risk of mediating allo-HLA cross-reactivity. Here, we investigated whether virus specificity and HLA restriction of the virus specific T cells influence the risk of allo-HLA cross-reactivity, and thus the development of GVHD. To determine the occurrence and diversity of allo-HLA cross-reactivity, virus specific CD8 T cells from homozygous HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02 donors were isolated by cell sorting using tetramers for various peptides from CMV, EBV and AdV. Allo-HLA cross-reactivity was tested using an allogeneic EBV-LCL panel covering 116 different HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA alleles of interest. A significant proportion of the virus specific T cell populations (n=174; 20 specificities) isolated from 27 healthy donors exerted allo-HLA cross-reactivity, as measured by recognition of 1 or more HLA mismatched EBV-LCLs from the panel. Similar frequencies were found for the various viral specificities showing 30% of the CMV, 46% of the EBV and 36% of the AdV-specific T cell populations to be allo-HLA cross-reactive. However, for some specificities (e.g. HLA-A*0201-restricted EBV-LMP2-FLY) allo-HLA cross-reactivity was infrequent (n=1/11), whereas for other specificities (e.g. HLA-B*08:01-restricted EBV-BZLF1-RAK) the majority of the T cell populations (n=9/13) was allo-HLA reactive. Surprisingly, a much larger fraction of HLA-B*08:01 restricted virus specific T cell populations showed allo-HLA cross-reactivity (72%, 36 out of 50 T cell lines), compared to the other HLA restricted virus specific T cell populations (29% of HLA-A*01:01, 30% of HLA-A*02:01 and 26% of HLA-B*07:02 restricted virus specific T cell lines). HLA-B*08:01 restricted virus specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 allo EBV-LCLs (range 1-17). In contrast, HLA-A*01:01, HLA-A*02:01 and HLA-B*07:02 restricted virus specific T cells reacted to a median of 1, 2 and 3 (ranges 1-7) allo EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivity using the panel of 40 different single HLA transduced K562 cells further illustrated the extensive allo-HLA cross-reactivity for HLA-B*08:01 restricted T cells isolated from homozygous HLA-A*01/B*08 donors compared to virus specific T cells restricted by other HLA alleles. These data show that allo-HLA cross-reactivity by virus specific T cells is highly influenced by the HLA restriction and not by the viral specificity of the T cell populations. Of the HLA-A*01, A*02, B*07 and B*08-restricted virus specific T cell populations isolated from homozygous donors, HLA-B*08:01 restricted virus specific T cells showed the highest frequency and diversity of allo-HLA cross-reactivity. Our results indicate that selection of virus specific T cells with specific HLA restrictions may decrease the risk of developing GVHD after infusion of third-party virus specific T cells to patients with uncontrolled viral reactivation after alloSCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Failure of regulation of Tac antigen/TCGF receptor on adult T-cell leukemia cells by anti-Tac monoclonal antibody

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 1014-1016
Author(s):  
M Tsudo ◽  
T Uchiyama ◽  
H Uchino ◽  
J Yodoi
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Leukemic Cells ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Down Regulation ◽  
Adult T Cell Leukemia ◽  
T Cell Lines

Anti-Tac monoclonal antibody, which blocks the membrane binding and action of human T-cell growth factor (TCGF), is strongly proposed to recognize TCGF receptor. We have demonstrated that anti-Tac antibody reacted with leukemic cells from patients with adult T-cell leukemia (ATL) and reacted with T-cell lines established from ATL cells. Although antigenic modulation, or down-regulation, of Tac antigen on activated normal T cells was induced by anti-Tac antibody, the expression of Tac antigen on ATL cells or T-cell lines was not affected when examined by the fluorescence-activated cell sorter (FACS) and the radioassay using 125I-staphylococcal protein A. These results indicate that regulation of Tac antigen-TCGF receptor is different between normal and malignant T cells, suggesting that failure of down- regulation of Tac antigen on leukemic cells by anti-Tac antibody may play an important role in the malignant proliferation of ATL cells.

Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2793-2801 ◽  
Author(s):  
Deepa Trivedi ◽  
Roxanne Y. Williams ◽  
Richard J. O'Reilly ◽  
Guenther Koehne
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Adoptive Immunotherapy ◽  
High Specificity ◽  
Hla Class I ◽  
Target Cells ◽  
Cell Mediated Immunity ◽  
Regulatory Perspective ◽  
T Cell Lines

Abstract Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We used a pool of 138 synthetic overlapping pentadecapeptides overspanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T cells that selectively recognized 1-3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide-loaded targets sharing single HLA class I or II alleles identified the restricting HLAalleles. HLA-A*0201+ donors consistently responded to pentadecapeptides containing HLA-A*0201-binding epitopeaa495-503NLVPMVATV. T-cell lines from other donors contained high frequencies of CD4 and/or CD8 T cells selectively reactive against peptides presented by other HLA alleles, including both known epitopes such as aa341-350QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as aa267-275HERNGFTVL (HLA-B*4001 and B*4002) and aa513-523FFWDANDIYRI (HLA-DRB1*1301). These T cells consistently lysed CMV-infected target cells. Thus, this approach fosters expansion and selection of HLA-restricted CMV-pp65–reactive T-cell lines of high specificity that also lyse CMV-infected targets, and from a functional and regulatory perspective, may have advantages for generating virus-specific T cells for adoptive immunotherapy.

scholarly journals Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4+ T Cells of the Mycobacterium lepraeMemory Repertoire

1999 ◽  
Vol 67 (11) ◽  
pp. 5683-5689 ◽  
Author(s):  
Abu S. Mustafa ◽  
Knut E. A. Lundin ◽  
Robert H. Meloen ◽  
Thomas M. Shinnick ◽  
Fredrik Oftung
Keyword(s):  
T Cells ◽  
T Cell ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Cell Lines ◽  
Restriction Analysis ◽  
Cell Repertoire ◽  
Peptide Epitopes ◽  
Hla Dr ◽  
T Cell Lines

ABSTRACT By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4+ T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosisHSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) representedM. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae andM. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4+ T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.

scholarly journals Dual-Sensitized T-Cells Responding to EBV Blcl and Either CMVpp65 or WT-1 Peptide Pools Have Distinct or Shared HLA Restrictions That May Depend on the Presenting HLA Alleles

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4596-4596
Author(s):  
Ekaterina Doubrovina ◽  
Aisha N. Hasan ◽  
Susan Prockop ◽  
Karim Baroudy ◽  
Richard O'Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Research Funding ◽  
Third Party ◽  
Hla Alleles ◽  
Immunogenic Peptides ◽  
Healthy Donors ◽  
T Cell Lines

Abstract Adoptive Immunotherapy with virus-specific T-cells generated from transplant or third party donors can induce durable remissions of severe infections or EBV lymphomas post-transplant. T-cells sensitized with antigens from multiple viruses have also shown promise. However, in any individual donor, immunogenic peptides from different viruses might be expected to elicit T-cell responses restricted by different HLA alleles. In HLA non-identical patients, the efficacy of T-cells reactive against any one virus would be eliminated if the T-cells specific for that virus are restricted by an HLA allele not shared by the patient. To examine this hypothesis, we evaluated the HLA restrictions of T-cells generated from 42 healthy donors after dual sensitization with either autologous EBV-transformed B-cells (EBVBLCL) loaded with a pool of overlapping 15-mer peptides spanning the sequence of CMVpp65 (n=20) or autologous EBVBLCL loaded with a pool of 15-mers spanning the oncofetal protein WT-1 (n=22). The HLA restrictions of the CMVpp65-specific and WT1 specific T cells were assessed by their cytotoxic activity against a panel of Cr51 labeled dendritic cells sharing a single HLA allele with the T cells donor. The EBV restrictions of the dual sensitized EBV CTLs were identified by their cytotoxic activity against EBV BLCLs sharing the same single HLA alleles derived from the same donors. In 13/20 CMVpp65/EBV sensitized T cells (65%) and 17/22 WT1/EBV sensitized T cells (77%) the CMV or WT1 specific T cell lines were restricted by single HLA alleles. In 10 of the 20 (50%) lines sensitized with EBV BLCL and CMVpp65, CMVpp65 specific T cells were restricted by an HLA allele that was also one of the restricting alleles for EBV CTLs in the same line. However, in the other 10(50%) the CMVpp65 T cells were restricted by an HLA allele different from that of the EBV CTLs. In the 22 lines co-sensitized with EBV and WT1, WT1 specific T cells were restricted by an allele different from those of the EBV CTLs in 13 (59%) lines. Comparison of EBVCTLs from dual sensitized T cell lines with EBVCTLs contemporaneously generated from the same donors but sensitized with EBV BLCL alone revealed that in 2/4 CMVpp65/EBV lines and 2/5 WT1/EBV lines in which the HLA restriction of CMVpp65 or WT1 specific T cells differed from that of EBV T cells in the same culture, the HLA allele differentially presenting the CMV or WT1 antigen but not an EBV antigen in the dual sensitized cultures was a prominent restricting allele of T cells sensitized with an autologous EBV BLCL alone. In our bank of 135 CMVpp65-specific T-cells sensitized with autologous APCs loaded with the same pool of overlapping CMVpp65 peptides, T-cells specific for epitopes presented by HLA B0702 were dominant in 33/34 donors inheriting this allele. Furthermore, for T-cell lines generated from 50 donors inheriting HLA A0201, HLA A0201 restricted T-cells specific for the NLV peptide of CMVpp65 were dominant for all lines except those 13 that co-inherited HLA B0702. Disclosures Doubrovina: Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding. Hasan:GlaxoSmithKline: Employment. Prockop:Atara Biotherapeutics: Research Funding; Mesoblast: Research Funding. O'Reilly:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding.

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