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 Off-Target HLA Cross-Reactivity By (Third Party) Virus-Specific T Cells Is Surprisingly Affected By HLA Restriction and HLA Background but Not By Virus Specificity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4440-4440
Author(s):  
Wesley Huisman ◽  
Didier A.T. Leboux ◽  
Lieve E. van der Maarel ◽  
Lois Hageman ◽  
Derk Amsen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
K562 Cells ◽  
Cross Reactivity ◽  
Third Party ◽  
Viral Reactivation ◽  
Cell Populations ◽  
High Morbidity ◽  
Hla Restriction ◽  
Restricted Group

Reactivations of cytomegalovirus (CMV), Epstein Barr virus (EBV) and adenovirus (AdV) occur frequently in immune compromised patients after allogeneic stem cell transplantation (alloSCT) and cause 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 graft from a virus non-experienced donor are at risk. 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, such T cells harbor a risk of mediating off-target toxicity due to allo-HLA cross-reactivity. It is not currently known whether the degree of allo-HLA cross-reactivity is random or whether rules exist that might allow prediction of specific T-cell populations. Here, we investigated whether virus specificity, HLA type of the donor or HLA restriction of the virus-specific T cells influence the risk of allo-HLA cross-reactivity. Through cell sorting using tetramers for various peptides from CMV, EBV and AdV, 164 CD8 T-cell populations (21 specificities) were isolated from peripheral blood of 24 healthy donors, homozygous for HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02. 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. Forty percent of all virus-specific T-cell populations exerted allo-HLA cross-reactivity. Similar frequencies were found for the various viral specificities showing 33% of the CMV, 43% of the EBV and 38% of the AdV-specific T-cell populations to be allo-HLA cross-reactive. Surprisingly, a much larger fraction of the HLA-B*08:01-restricted virus-specific T-cell populations exhibited allo-HLA cross-reactivity (77%) than from those restricted by the other HLAs (32% of HLA-A*01:01, 38% of HLA-A*02:01 and 26% of HLA-B*07:02-restricted virus-specific T-cell populations). HLA-B*08:01-restricted virus-specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 different allogeneic 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 (range 1-7) different allogeneic EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivities using a panel of 40 different single HLA-A, B, or C-transduced K562 cells further illustrated recurrent recognition of a restricted group of allogeneic HLA-B molecules by HLA-B*08:01-restricted T-cell populations, mediated by single T-cell clones. Heterozygosity for recurrently recognized allo-HLA-B molecules led to a significant decrease in the broadness of allo-HLA cross-reactivity by HLA-B*08:01-restricted T-cell populations, presumably due to negative thymic selection. In contrast, heterozygosity HLA-B molecules that were not part of the restricted group of cross-recognized alleles did not significantly decrease allo-HLA cross-reactivity. These data show that allo-HLA cross-reactivity by virus-specific T cells is highly influenced by their HLA restriction and the HLA background of the donors, but not by their virus specificity. 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 with recurrent recognition of groups of specific mismatched allogeneic HLA-B alleles. Our results indicate that selection of virus-specific T cells with specific HLA restrictions and HLA backgrounds may decrease the risk of off-target toxicity 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.

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.

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.

Universal CD137 Expression upon Activation Allows Efficient Isolation of a Broad Repertoire of Virus-Specific CD8+ and CD4+ T Cells for Adoptive Immunotherapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2222-2222
Author(s):  
Maarten L. Zandvliet ◽  
J.H. Frederik Falkenburg ◽  
Inge Jedema ◽  
Roelof Willemze ◽  
Henk-Jan Guchelaar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd8 T Cell ◽  
Cell Populations ◽  
T Cell Lines ◽  
Kinetics Of ◽  
Ifng Production

Abstract Reactivation of adenovirus (ADV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can cause serious morbidity and mortality during the prolonged period of immune deficiency following allogeneic stem cell transplantation. It has been shown that adoptive transfer of donor-derived virus-specific T cells can be a successful strategy to control viral reactivation. To provide safe and effective anti-viral immunotherapy, we aimed to generate combined CD8+ and CD4+ T cell lines with high specificity for a broad range of viral epitopes. Isolation by the IFNg capture assay of virus-specific T cells that produce IFNg upon activation allows the generation of highly specific T cell lines without the need for extensive culture. However, it has been recently shown that specific upregulation of the co-stimulatory molecule CD137 upon antigen-specific activation of CD8+ and CD4+ T cells can also be used for isolation. We therefore analyzed IFNg production and CD137 expression by CD8+ and CD4+ T cells upon incubation of peripheral blood mononuclear cells (PBMC) from seropositive donors with peptides corresponding to 17 defined MHC class I restricted minimal epitopes from 10 different ADV, CMV, EBV and influenza (FLU) proteins, and 15-mer or 30-mer peptides containing MHC class II restricted epitopes from CMV pp65 or ADV hexon. Using tetramer and intracellular IFNg staining we first determined the fraction of CD8+ T cells that produced IFNg upon activation with the minimal epitopes. Specific IFNg production was observed for 58–100% of tetramer+ CD8+ T cells specific for CMV pp65 (n=6), and 83% for FLU (n=1), but only 18–58% for CMV pp50 (n=3) or IE-1 (n=3), 4–91% for EBV latent (n=3) and lytic (n=3) epitopes, and 41–63% for ADV hexon (n=2). In contrast to the variation in the fraction of IFNg-producing cells, we observed homogeneous upregulation of CD137 by the virus-specific tetramer+ T cell populations upon activation. In 2 cases where no CD137 expression by tetramer+ T cells could be detected, no IFNg production was observed either. These data suggest that the majority of CD8+ T cells specific for CMV pp65 or FLU can be isolated on basis of IFNg production, but only part of CD8+ T cell populations specific for other viral proteins, while complete virus-specific CD8+ T cell populations may be isolated on basis of CD137 expression. Activation of CD4+ T cells specific for CMV pp65 or ADV hexon with 15-mer or 30-mer peptides induced both specific IFNg production and CD137 expression. To investigate whether multiple virus-specific T cell populations could be isolated simultaneously, we next determined the kinetics of IFNg production after activation with defined MHC class I epitopes or peptides containing MHC class II epitopes. CMV- and EBV-specific CD8+ T cells and CMV-specific CD4+ T cells showed a rapid induction of IFNg production, which peaked after 4 hours and decreased thereafter. In contrast, ADV- and FLU-specific CD8+ T cells and ADV-specific CD4+ T cells, predominantly having a more early differentiation phenotype (CD27+CD28+) compared to CMV- and EBV-specific T cells, showed peak IFNg production after 8 hours that continued for more than 48 hours. This difference in phenotype and IFNg kinetics may suggest that the persistent and frequent presentation of CMV and EBV epitopes in vivo, in contrast to an intermittent exposure to ADV and FLU epitopes, drives differentiation and shapes the kinetics of the IFNg response of specific T cells. Kinetic analysis of CD137 expression showed uniform upregulation by virus-specific CD8+ T cell populations from day 1 to day 4 after activation, which peaked at day 2, suggesting that this may be the optimal time point for CD137-based isolation. In a limited number of experiments, virus-specific CD8+ and CD4+ T cells could be isolated based on CD137 expression within the same timeframe. These data indicate that virus-specific T cell populations can be more efficiently isolated at one time point on basis of CD137 expression than on basis of IFNg production, due to differences in IFNg kinetics. In conclusion, this study shows that T cell lines generated by CD137 isolation may comprise a significant number of virus-specific T cells which do not produce IFNg, but may have other effector functions. Furthermore, CD137-based enrichment may be more robust and allows the efficient simultaneous isolation of multiple virus-specific T cell populations due to uniform kinetics of CD137 expression.

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.

HLA-DPB1 Mismatching Results in the Generation of a Full Repertoire of HLA-DPB1 Specific T Cell Responses Showing Immunogenicity of All HLA-DPB1 Alleles

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3504-3504
Author(s):  
Caroline E. Rutten ◽  
Simone A.P. van Luxemburg-Heijs ◽  
Edith D. van der Meijden ◽  
Marieke Griffioen ◽  
Roelof Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Hela Cells ◽  
Cd4 T Cells ◽  
Cross Reactivity ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Lines

Abstract In unrelated donor hematopoietic stem cell transplantation (URD-SCT) patients are preferably transplanted with stem cells from a fully HLA matched donor, usually defined as identical for HLA-class I, -DR and -DQ. Since HLA-DPB1 is often not taken into consideration in donor selection, 80–90% of URD-SCTs are mismatched for HLA-DPB1. The role of HLA-DPB1 as transplantation antigen has been unclear, since clinical reports on the impact of matching for HLA-DPB1 on transplant outcome showed conflicting results. HLA-DPB1 mismatching has been associated with an increased risk of graft versus host disease (GVHD). However, we recently demonstrated that HLA-DPB1 specific T cells can mediate a potent graft versus leukemia effect without inducing GVHD. It has been suggested that the controversial effects of matching for HLA-DPB1 in URD-SCT could partly be explained by the assumption that not all HLA-DPB1 differences are immunogenic. This theory was based on the cross-reactive recognition of two HLA-DPB1* 09 specific T cell clones that recognized other HLA-DPB1 alleles sharing amino acids (aa) in position 8–11 of HLA-DPB1 (Zino et al, blood 2004). It was hypothesized that there would be no induction of T cell responses between individuals expressing HLA-DPB1 molecules sharing this aa sequence. This was translated into a classification of permissive and non-permissive HLA-DPB1 mismatches in order to allow a broader donor selection. To investigate whether cross-reactive recognition of other HLA-DPB1 molecules by our previously generated HLA-DPB1*02 or *03 specific CD4+ T cell clones depended on the presence of specific aa sequences we tested recognition of a panel of 14 EBV-LCL expressing 9 different HLA-DPB1 molecules. All HLA-DPB1*02 as well as all *03 specific T cell clones showed cross-reactivity with other HLA-DPB1 alleles and each T cell clone exhibited its own pattern of cross-reactivity. Two HLA-DPB1*0201 specific T cell clones with different TCR-Vβ showed also recognition of EBV-LCL expressing HLA-DPB1*1001 and *1701 or HLA-DPB1*1001, *0901 and *1601 respectively. Five HLA-DPB1*03 reactive T cells clones with different TCR-Vβ showed differential cross-recognition of EBV-LCL expressing HLA-DPB1*0101, *0601, *1101, *1301 and *1401. To identify immunogenic differences the aa sequences of the HLA-DPB1 molecules recognized by the various T cell clones were compared. The HLA-DPB1 molecules recognized by the HLA-DPB1*02 specific T cell clones shared an aa substitution at position 69 compared to the responder cell. However, HLA-DPB1*0601,*0901 and *1901 with the same substitution were not recognized by both T cell clones. This phenomenon was also observed for the HLA-DPB1*03 specific T cell clones, indicating that the cross-reactive recognition of HLA-DPB1 could not be predicted by aa sequences. Next, we analyzed the immunogenicity of various HLA-DPB1 alleles in different stimulator/responder combinations to verify the classification of permissive and non-permissive mismatches. We developed a model to generate allo-HLA-DP responses by transducing HLA-class II negative HELA cells with various HLA-DP molecules and used these cells to stimulate purified CD4+ T cells from HLA-DPB1 homozygous donors. HELA cells transduced with HLA-DPB1*0101, *0201, *0301, *0401, *0402, *0501, *0601, *0901, *1101, *1301, *1401 or *1701 were used as stimulator cells. Responder CD4+ T cells were typed HLA-DPB1* 0201, *0301, *0401 or *0402. 14 days after stimulation, CD4+ T cells were tested for recognition of the stimulator cells and of HELA cells transduced with the responder HLA-DPB1 molecule as a negative control. For these 4 responders, stimulation with 12 different HLA-DP transduced HELA cell lines resulted in specific IFN-γ production in response to the stimulator cells in 47 out of 48 stimulations. 28 CD4+ T cell lines also showed cross-reactive recognition of HELA cells transduced with at least one other HLA-DPB1 molecule. In conclusion, we showed that cross-reactive recognition of various HLA-DPB1 molecules by HLA-DPB1 specific T cells is a common observation. However, we demonstrated that cross-reactivity between HLA-DPB1 molecules by allo-HLA-DPB1 specific T cells does not exclude the generation of immune response between individuals expressing these HLA-DPB1 molecules. By generating multiple allo-HLA-DP specific T cell lines, we showed that all HLA-DPB1 mismatch combinations are immunogenic.

scholarly journals Magnitude of Off-Target Allo-HLA Reactivity by Third-Party Donor-Derived Virus-Specific T Cells Is Dictated by HLA-Restriction

2021 ◽  
Vol 12 ◽  
Author(s):  
Wesley Huisman ◽  
Didier A. T. Leboux ◽  
Lieve E. van der Maarel ◽  
Lois Hageman ◽  
Derk Amsen ◽  
...  
Keyword(s):  
T Cells ◽  
Hla Class I ◽  
K562 Cells ◽  
Cross Reactivity ◽  
Third Party ◽  
Thymic Selection ◽  
Hla Restriction ◽  
Selection For ◽  
Hla Molecules ◽  
Selection Of

T-cell products derived from third-party donors are clinically applied, but harbor the risk of off-target toxicity via induction of allo-HLA cross-reactivity directed against mismatched alleles. We used third-party donor-derived virus-specific T cells as model to investigate whether virus-specificity, HLA restriction and/or HLA background can predict the risk of allo-HLA cross-reactivity. Virus-specific CD8pos T cells were isolated from HLA-A*01:01/B*08:01 or HLA-A*02:01/B*07:02 positive donors. Allo-HLA cross-reactivity was tested using an EBV-LCL panel covering 116 allogeneic HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA-class-I alleles of interest. HLA-B*08:01-restricted T cells showed the highest frequency and diversity of allo-HLA cross-reactivity, regardless of virus-specificity, which was skewed toward multiple recurrent allogeneic HLA-B molecules. Thymic selection for other HLA-B alleles significantly influenced the level of allo-HLA cross-reactivity mediated by HLA-B*08:01-restricted T cells. These results suggest that the degree and specificity of allo-HLA cross-reactivity by T cells follow rules. The risk of off-target toxicity after infusion of incompletely matched third-party donor-derived virus-specific T cells may be reduced by selection of T cells with a specific HLA restriction and background.

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