scholarly journals Banked, GMP Grade Third Party T-Cell Lines Specific for CMVpp65 Epitopes Presented By Certain Prevalent HLA Alleles More Consistently Clear CMV Infections in a Genetically Heterogeneous Population of HSCT Recipients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 309-309 ◽  
Author(s):  
Aisha N. Hasan ◽  
Susan E. Prockop ◽  
Guenther Koehne ◽  
Ekaterina Doubrovina ◽  
Richard J. O'Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Adoptive Transfer ◽  
Third Party ◽  
Clinical Activity ◽  
Allelic Variants ◽  
Hla Alleles ◽  
Allogeneic Hsct ◽  
T Cell Lines

Abstract Adoptive transfer of virus-specific T-cells (CTLs) derived from allogeneic HSCT donors or HLA partially matched third party donors can eradicate EBV, CMV or adenovirus infections in a high proportion of allogeneic HSCT recipients. At our center, the response rate to third party CMVpp65-specific T-cells (CMVCTLs) has been about 60%. The specific characteristics of CTLs from third party donors that can predict efficacy after adoptive transfer have not been fully defined. However, current evidence indicates that for third party CTLs to be effective, CTL lines selected for treatment must at least be restricted by an HLA allele shared by the cells infected in the patient and the T-cell donor. We have developed a CMV CTL bank containing 132 CTL lines that was generated using a pool of overlapping peptides spanning the sequence of CMVpp65. Each CMV CTL line in the bank has been characterized as to T-cell memory phenotype, cytokine profile, epitope specificity and the HLA allele restriction of the cytotoxic T-cells. Although our pool of CTL donors has inherited a diverse group of widely prevalent HLA alleles, the cytotoxic activity of the T-cell lines in the bank is restricted by only 11 alleles. Nevertheless, these T-cells provided an appropriately restricted CTL line for 51 of 56 requested cases. In the present study, we examined 52 HSCT recipients who had received CMV CTLs of defined HLA restriction from either their HSCT donor (n=23) or a third party donor (n= 29) to determine whether and to what degree specific immunodominant T-cells restricted by specific HLA alleles exhibited differential clinical activity following adoptive transfer. We did this analysis because in analyzing responses to epitopes presented by these alleles, we noted that epitopes presented by certain alleles elicited quantitatively greater in-vitro responses as measured by the number of IFNγ+ or tetramer+ T-cells/106T-cells administered. For 9 CTL lines used in our series, the epitope eliciting the immunodominant T-cell response was an 11-15 aa peptide that was presented by both an HLA class-I and class-II allele. These class-I and class-II shared epitopes elicited more robust, multifunctional CD8+ and CD4+ T-cells that generated IFNγ, TNFα and IL-2. 8 of 9 patients treated with one of these CTL lines rapidly cleared viremia and end organ disease. Another group of 4 CTL lines responded to 2 epitopes, 1 dominant and 1 subdominant, that were restricted by one HLA A and one B allele; specifically A01 and B08, A 01 and B35 and A24 and B35. Treatment with such dual epitope responsive CTL lines also yielded complete responses in 4/4 cases. Among lines specific for single immunodominant epitopes, HLA restricted CMV CTLs responding to 4 CMVpp65 epitopes also consistently induced rapid clearance of viremia and organ disease including colitis, retinitis and encephalitis; NLVPMVATV (14/16 Complete responses CR), TPRVTGGGAM/RPHERNGFTV (7/7 CR) and HERNGFTV (6/6 CR) presented by HLA A0201, B0702 and allelic variants of B40 or B44 (B4001-06 and/B4401-03 ) respectively. In contrast, none of the 10 patients treated CTL lines responding to epitopes presented by HLA A2601, A2407, A2902, B0705, B5001, or allelic variants of B35 responded to CTL treatment and progressed. These findings suggest a hierarchy of epitopes presented by prevalent HLA alleles that may exhibit more consistent clinical activity in-vivo. Selection strategies utilizing such data may permit the development of a CTL selection algorithm for CMV CTLs derived from third party donors for treatment of CMV infections that would, more consistenly, induce clearance of CMV viremia and disease. These data also offer the potential to focus strategies for generation of CMV CTLs using a limited peptide pool or a panel of AAPCs expressing specific HLA alleles, thereby making this treatment approach more financially and ligisticaly feasible. These data could also be applied toward the development of an effective CMV vaccine for high risk individuals. Disclosures No relevant conflicts of interest to declare.

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 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.

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.

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.

Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3565-3572 ◽  
Author(s):  
Georg Rauser ◽  
Hermann Einsele ◽  
Christian Sinzger ◽  
Dorothee Wernet ◽  
Gabriele Kuntz ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Lines ◽  
Adoptive Transfer ◽  
Cd8 T Cell ◽  
Cell Transplants ◽  
Ifn Γ ◽  
Rapid Generation ◽  
T Cell Lines

Abstract Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4+ and CD8+ CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-γ (IFN-γ) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 × 108 combined CD4+ and CD8+ CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-γ production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4+ and CD8+ T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4+ and CD8+ T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4+ and CD8+ CMV-specific T cells under conditions mimicking good manufacturing practice. (Blood. 2004; 103:3565-3572)

scholarly journals Identification of Hexon-Specific CD4 and CD8 T-Cell Epitopes for Vaccine and Immunotherapy

2007 ◽  
Vol 82 (1) ◽  
pp. 546-554 ◽  
Author(s):  
Ann M. Leen ◽  
Anne Christin ◽  
Mariam Khalil ◽  
Heidi Weiss ◽  
Adrian P. Gee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Adoptive Transfer ◽  
Cytotoxic T Cell ◽  
Cell Immune Response ◽  
T Cell Epitopes ◽  
Human Pathogens ◽  
T Cell Lines ◽  
Cytotoxic T Cell Lines

ABSTRACT Adenoviral infections in the immunocompromised host are associated with significant morbidity and mortality. Although the adoptive transfer of adenovirus-specific T cells may prevent and treat such infections, the T-cell immune response to the multiplicity of adenovirus serotypes and subspecies that infect humans has not been well characterized, impeding the development of such approaches. We have, therefore, analyzed the specificities of T-cell responses to the viral capsid hexon antigen, since this structure is highly conserved in human pathogens. We screened 25 human cytotoxic T-cell lines with adenovirus specificity to extensively characterize their responses to adenoviral hexon and to identify a panel of novel CD4+ and CD8+ T-cell epitopes. Using a peptide library spanning the entire sequence of the hexon protein, we confirmed the responsiveness of these cytotoxic T-cell lines to seven peptides described previously and also identified 33 new CD4- or CD8-restricted hexon epitopes. Importantly, the majority of these epitopes were shared among different adenovirus subspecies, suggesting that T cells with such specificities could recognize and be protective against multiple serotypes, simplifying the task of effective adoptive transfer or vaccine-based immunotherapy for treating infection by this virus.

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.

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.

Anti-Viral Cytotoxic T-Lymphocyte Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-50-SCI-50
Author(s):  
Catherine M. Bollard
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cell Lines ◽  
Adoptive Transfer ◽  
Peripheral Blood ◽  
Cell Subpopulation ◽  
Secondary Resistance ◽  
Allogeneic Hsct

Abstract Abstract SCI-50 Cytomegalovirus (CMV), Epstein Barr virus (EBV) and adenovirus (Ad) are particularly problematic in patients after hematopoietic stem cell transplantation (HSCT) and are associated with significant morbidity and mortality. While antiviral pharmacotherapy may help prevent or treat CMV or Ad, and CD20-specific antibodies may control EBV-associated lymphoproliferation, these drugs are expensive, toxic and often ineffective due to primary or secondary resistance. These deficiencies in conventional therapeutics have increased interest in an immunotherapeutic approach to viral disorders. Adoptive transfer of T-cells in the form of donor lymphocyte infusions (DLI) has been used to treat viral infection after allogeneic HSCT, but has so far proved to be of limited effectiveness for patients with viral infections such as Ad, and often produce GvHD. Adoptive transfer of peripheral blood derived virus-specific cytotoxic T lymphocytes (CTL) directed to CMV, EBV and Ad can rapidly reconstitute anti-viral immunity post-HSCT without causing GvHD. We have shown that peripheral blood-derived T-cell lines enriched in cells recognizing CMV, EBV and Ad can reproducibly control infections due to all three viruses after allogeneic HSCT. This study demonstrated that the multivirus-specific T cells expand in vivo and are active against all three viruses. Furthermore, by restoring immunity to three viruses simultaneously, the need for continued prophylaxis with pharmacotherapy is eliminated, thus, improving the efficiency and cost effectiveness of protecting HSCT recipients from these potentially lethal viruses. In principle, this strategy could be applied with comparable success to recipients of cord blood (CB) transplants; however, certain obstacles to the extension of this approach must be circumvented. These include: (i) the limited numbers of CB T-cells available for manipulation and (ii) the naivety of CB T-cells. Hence, the development of virus-protective T-cell therapy for patients undergoing CBT requires the priming and extensive expansion of naïve T-cells rather than the more limited and simple direct expansion of pre-existing virus-specific memory T-cell populations from virus-experienced donors. Further, CB T-cells have lower cytotoxic activity and higher activation-induced cell death than peripheral blood T-cells. These limitations have prevented the production of virus-specific cord blood-derived CTL in sufficient numbers for clinical use. Recent studies demonstrate that ex vivo priming of naïve T-cells can be achieved in the presence of combinations of soluble cytokines such as IL-7, IL-15, and IL-12 that respectively decrease the antigen concentration threshold, direct T-cells towards a central memory phenotype, and influence the polarization of Th1 cells. We have now shown that Ad5f35pp65-transduced CB-derived APC can be used to generate large numbers of autologous T-cells specific for CMV and Ad, even from the phenotypically naive T-cell subpopulation. Addition of EBV-transformed B-lymphoblastoid cell lines (LCL) to the APCs allows the Ad/CMV specificity of the CB T-cells to be extended to EBV. In addition, the multivirus-specific T-cells recognize an array of epitopes after only 2 weeks expansion in vivo. We have now initiated a clinical trial using CB-derived multivirus-specific T cells to infuse to patients after CB transplant. Moreover, we propose that these virus-specific T cells generated from cord blood or peripheral blood can be genetically modified to (i) redirect specificity to leukemia as well as viral antigens and (ii) overcome tumor immune evasion strategies. Disclosures: No relevant conflicts of interest to declare.

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