Generation of Cytomegalovirus (CMV)-Specific CD4 and CD8 T Cell Lines Using Protein-Spanning Pools of pp65 and IE1 Derived Peptides.

Abstract BACKGROUND: Reactivation of latent CMV in immunocompromised recipients of allogeneic stem cell transplantation remains a major cause of morbidity and mortality. Reconstitution of immunity by CMV specific immunotherapy is an attractive alternative to drugs currently used, which show high toxicity and are sometimes ineffective. It has been demonstrated that CD4 helper T-cell function is crucial for the persistence of in vivo transferred CD8 CMV-specific CTL. Based on this finding, we have explored the feasibility of generating both anti-CMV CD4 and anti-CMV CD8 T-cell lines. METHODS: Dendritic Cells (DC) were generated from donor peripheral blood (PB) monocytes after a 7-day culture in the presence of GM-CSF plus IL-4 and matured with TNF-α, IFN-α, IFN-γ, IL1-β, POLI I:C. Matured-DC were then pulsed with a pool of 50 peptides spanning pp65 and IE1 proteins which are recognised by both CD4 and CD8 T lymphocytes. Donor T cells were stimulated three times at a T cell/DC ratio of 1:6 on day 0, +7 and +14 with mature peptide pulsed-DC. At the end of the culture the specificity of generated T cells was determined as percentage of pentamer-positive cells and intracellular IFN-γ production after incubation with peptide pulsed-DC. Cultured T cells were also analysed for their ability to proliferate in response to peptide pulsed-target cells, to kill them in a standard citotoxicity assay and to migrate in response to inflammatory (CXCL9, CCL3 and CCL5) and constitutive (CXCL12) chemokines. RESULTS: CMV-specific T cell lines were generated from five CMV seropositive donors. In four cases CD4 and CD8 CMV-specific T cell lines were expanded successfully. Cultured T cells expressed CD8 (mean= 70%, range 60–81%) and CD4 (mean= 20%, range 15–28%) and showed a CD45RA- CCR7- Effector Memory phenothype (mean=26%, range 19–30%) or a CD45RA+ CCR7- T Effector Memory RA-Positive phenothype (mean=67%, range 59–77%). An enriched CMV-specific T cell population was observed after staining with pentamers (7–45% pentamer-positive T cells). Furthermore, 90% of CD8+ and 40% of CD4+ T cells expressed high levels of intracytoplasmatic perforin and granzyme. In 4/5 cases tested, cutured T cells showed a cytolitic activity against CD8-peptide pulsed target cells (average lysis=50%, range 40–55%) and to a lesser extent against CD4-peptide pulsed target cells (average lysis=35%, range 30–40%). In addition, cultured T lymphocytes were able to proliferate and to produce intracytoplasmic IFN-γ (average production=50%, range 35–60%) after exposure to peptide-pulsed DC. Finally, Cultured T cells strongly migrated in response to chemokines (CXCL9, CCL3 and CCL5) involved in the recruitment of effector cells during viral infection. DISCUSSION: In conclusion, a great advantage of this method is represented by the possibility to generate anti-CMV CD4+ T cells, which could support in vivo the persistence of re-infused CMV-specific CTL. Moreover, the possibility of generating peptides under GMP conditions would facilitate the translation of this approach into clinical intervention.

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

Blood ◽

10.1182/blood-2003-09-3056 ◽

2004 ◽

Vol 103 (9) ◽

pp. 3565-3572 ◽

Cited By ~ 120

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 ◽

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)

Generation of GMP-Grade CMV pp65-Specific CD8+ and CD4+ Donor T Cell Lines for Treatment of CMV Reactivation after Transplantation.

Blood ◽

10.1182/blood.v108.11.2931.2931 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2931-2931

Author(s):

Maarten L. Zandvliet ◽

J.H. Frederik Falkenburg ◽

Inge Jedema ◽

Roelof Willemze ◽

Henk-Jan Guchelaar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Cd4 T Cells ◽

Target Cells ◽

Specific Cell ◽

Specific Response ◽

Specific Lysis ◽

Abstract Reactivation of CMV remains a major cause of morbidity and mortality in immunocompromised recipients of allogeneic stem cell transplantation. Antiviral pharmacotherapy may not be sufficient due to significant toxicity and moderate efficacy. It has been shown that adoptive transfer of donor-derived CMV-specific T cells may be an effective strategy to control established CMV infection. For a persistent function in vivo the presence of both virus-specific CD8+ and CD4+ T cells is essential. Therefore, we developed an optimized protocol for the generation of CMV pp65-specific CD8+ and CD4+ T cell lines which is fully compliable with Good Manufacturing Practice (GMP) conditions. Enrichment for CMV-specific T cells followed by only a short culture period is likely to retain maximal in vivo potential. PBMCs from 7 CMV seropositive donors were stimulated with recombinant pp65 protein (7–70 μg/ml) and/or HLA-A*0201/HLA-B*0702 restricted immunodominant pp65 peptides (NLV/TPR). Peptides used were clinical grade, and recombinant protein was gamma-irradiated (50 kGy, −80 C°) to eliminate possible microbiological contamination. High dose gamma-irradiation of pp65 protein resulted in partial degradation, but antigenic presentation was maintained. IFNγ producing cells were enriched using the IFNγ secretion assay (Miltenyi Biotec) at day 1 after stimulation, and cultured with autologous feeders (10x) and IL-2 (10 or 50 IU IL-2/ml) with or without CD3/28 expansion beads. Addition of high concentrations of protein during initial stimulation had a negative effect on enrichment probably due to non-specific stimulation of cells. Addition of immunodominant pp65 peptides promoted isolation efficiency and proliferation of epitope-specific CD8+ T cells in some donors. Cell lines were analyzed at different time points (day 4–15) using peptide-MHC tetramer and phenotypic markers. In addition, pp65-specificity was evaluated by intracellular IFNγ staining after restimulation with a pp65 protein-spanning pool of 15-mer peptides. CMV-specific lysis was tested in a 51-Cr release assay on pp65-transduced target cells. Enrichment of IFNγ producing cells after pp65 protein stimulation resulted in pp65-specific cell lines consisting of both CD8+ and CD4+ T cells. The T cell subset distribution directly after enrichment did not change during culture and was reproducible for each donor. Moreover, the composition of T cell lines reflected the pp65-specific response in donor PBMC starting material. The CD8+ compartment contained the known immunodominant tetramer staining cells (range 5–100%). The majority of both CD8+ and CD4+ T cells produced IFNγ upon restimulation with the pp65 peptide-pool, and showed CMV-specific lysis of target cells. The phenotype of pp65-specific T cells was predominant CD28+/CD45RO+ and CD45RA−/CCR7−/CD62L−, although CCR7 and CD62L were transiently expressed at day 4 and 7 after stimulation. Cryopreservation did not affect the composition or functionality of T cell lines. In conclusion, this procedure yields GMP-grade T cell lines comprising both CD8+ and CD4+ CMV-specific T cells. Processing and presentation of CMV protein by donor antigen-presenting cells enables selection of the full pp65-specific donor repertoire, without restrictions related to HLA or known epitopes. The choice for a moderate or more vigorous expansion after enrichment remains arbitrary and needs to be evaluated in clinical trials.

CD8+ T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo.

10.1084/jem.175.6.1531 ◽

1992 ◽

Vol 175 (6) ◽

pp. 1531-1538 ◽

Cited By ~ 170

Author(s):

J T Harty ◽

M J Bevan

Keyword(s):

T Cells ◽

Listeria Monocytogenes ◽

T Cell ◽

Cell Lines ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Significant Protection ◽

Peptide Epitope ◽

Class I major histocompatibility complex (MHC)-restricted CD8+ T cells have been demonstrated to be effective mediators of both acquired and adoptive immunity to the intracellular bacterium Listeria monocytogenes. We have recently determined that L. monocytogenes-infected H-2d mice recognize a nonamer peptide, residues 91-99, of the secreted protein listeriolysin O (LLO), in a H-2Kd-restricted fashion. In this report we have generated CD8+ T cell lines with specificity for LLO 91-99 in the context of H-2Kd by in vitro stimulation with P815 (H-2d) cells transfected with LLO. These CD8+ lines have been generated from immune donors after sublethal infection with L. monocytogenes, or after in vivo immunization with syngeneic spleen cells coated with synthetic LLO 91-99 peptide. LLO-specific CD8+ T cells derived from either protocol were capable of significant protection against L. monocytogenes infection. The in vivo protection by these CD8+ T cell lines has been shown to be solely due to recognition of LLO 91-99 in the context of H-2Kd. These studies demonstrate that CD8+ T cell immunity to a single, naturally produced peptide epitope has the potential for significant protection in a bacterial infection. Thus, the allele-specific motif approach to epitope prediction has identified a naturally produced bacterial epitope with biological relevance.

Selective activation of Fas/Fas ligand-mediated cytotoxicity by a self peptide.

10.1084/jem.183.6.2449 ◽

1996 ◽

Vol 183 (6) ◽

pp. 2449-2458 ◽

Cited By ~ 57

Author(s):

P Brossart ◽

M J Bevan

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Fas Ligand ◽

Cd8 T Cell ◽

The Self ◽

Target Cells ◽

Ifn Gamma ◽

T Cell Lines ◽

Self Antigen

To study how MHC-associated self antigens may regulate the function of T cells in the periphery, we generated CD8+ T cell lines specific for a single residue variant of a self peptide. The self peptide (GAYEFTTL) was isolated from H-2-Kb class I MHC molecules immunopurified from tumor cells. CD8+ CTL lines from H-2b mice were generated against a variant peptide, pE4R, (arginine for glutamic acid at the TCR contact position 4). In short-term 51Cr-release assays, these CTL lysed H-2Kb targets that were pulsed with picomolar levels of pE4R but did not lyse target cells coated with the self peptide at micromolar levels. However, in overnight assays the CTL lysed Fas-positive target cells in the presence of nanomolar levels of the self peptide. This killing was shown to be entirely Fas/Fas ligand mediated by blocking with anti-Fas antibody and Fas-Fc chimeric molecules. While the self peptide was unable to induce serine esterase release from the CTL, it did induce secretion of IFN-gamma. By these criteria then, the unmodified self ligand served as a partial agonist for the CTL raised against a single-residue variant. CD8+ T cell lines raised by in vitro stimulation with the self peptide were likewise unable to kill self peptide-coated targets via the perforin pathway but did lyse targets via Fas. These and similar data from other groups show that self antigens (i.e., MHC/peptide complexes) may be recognized by mature peripheral T cells. The T cell population is tolerant of the self antigen in the sense that they do not respond to physiological levels of the MHC/peptide complex. However, when the level of self antigen is increased (by using synthetic peptide loading) CD8+ T cells may respond by proliferation, IFN-gamma secretion, Fas ligand upregulation, and Fas-mediated cytolysis but are still unable to respond by perforin-mediated cytolysis or granzyme release. The physiological significance of such partial activation in regulation of the immune system remains to be demonstrated.

Generation of Human CD8 T Regulatory Cells by CD40 Ligand–activated Plasmacytoid Dendritic Cells

10.1084/jem.20011603 ◽

2002 ◽

Vol 195 (6) ◽

pp. 695-704 ◽

Cited By ~ 475

Author(s):

Michel Gilliet ◽

Yong-Jun Liu

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cd8 T Cells ◽

Transforming Growth Factor ◽

Cd8 T Cell ◽

Cd40 Ligand ◽

Plasmacytoid Dendritic Cells ◽

Target Cells ◽

Ifn Γ

Although CD8 T cell–mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand–activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand–activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-γ upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-γ, and no IL-4, IL-5, nor transforming growth factor (TGF)-β. The addition of anti–IL-10–neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10–producing anergic CD8 T cells. IL-10–producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-β. IL-10–producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10–producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10–producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell–mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.

Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9

Nature Communications ◽

10.1038/s41467-020-19672-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Lintao Liu ◽

Enguang Bi ◽

Xingzhe Ma ◽

Wei Xiong ◽

Jianfei Qian ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Effector Memory ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Ifn Γ

AbstractCAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells

10.1084/jem.20122006 ◽

2013 ◽

Vol 210 (3) ◽

pp. 491-502 ◽

Cited By ~ 106

Author(s):

Shlomo Z. Ben-Sasson ◽

Alison Hogg ◽

Jane Hu-Li ◽

Paul Wingfield ◽

Xi Chen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Granzyme B ◽

Interleukin 1 ◽

Cd8 T Cell ◽

Cell Receptor ◽

In Vivo Models ◽

Ifn Γ

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.

Generation of Acute Myeloid Leukemia (AML)-Specific Autologous CD4 and CD8 T Cell Lines by Limiting Dilution AML Dendritic Cell Culture.

Blood ◽

10.1182/blood.v108.11.2018.2018 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2018-2018

Author(s):

Rui-kun Zhong ◽

Thomas A. Lane ◽

Edward D. Ball

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Myeloid Leukemia ◽

Cd8 T Cell ◽

Ifn Gamma ◽

T Cell Lines ◽

Limiting Dilution ◽

Acute Myeloid

Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (>90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy. Figure Figure

Minor Histocompatibility Antigen HA-1-Specific Immune Reactions after Hematopoietic Cell Transplantation (HCT).

Blood ◽

10.1182/blood.v108.11.5183.5183 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5183-5183

Author(s):

Michael Hudecek ◽

Haifa Al-Ali ◽

Annette Reinhardt ◽

Dietger Niederwieser ◽

Sabine Tschiedel

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Elispot Assay ◽

Target Cells ◽

Cell Frequency ◽

Immune Reactions ◽

Time Points ◽

Tetramer Staining ◽

Abstract Minor histocompatibility antigens can be responsible for both GvHD and GvT effects after HCT, depending on their tissue distribution. HA-1 is expressed on hematopoietic cells and several types of solid tumors, but not on GvHD target cells. Two alleles have been defined, of which HA-1H is immunogenic in the presence of HLA-A*0201. Here, we investigate HA-1-specific immune responses in HLA-A*0201 HA-1H individuals after related, HLA-matched, HA-1 mismatched HCT. Methods: A total of 9 patients with hematological malignancies received HCT from HLA-matched, HA-1-mismatched donors, in seven cases following reduced intensity conditioning (RIC). The appearance of HA-1-specific T-cells was monitored with HLA-A*0201/HA-1H Tetramers and with the IFN-γ Elispot Assay at different time points before and after HCT (days 0, 28, 56, 84 and monthly thereafter). T-cell lines were generated from 2 patients at different time points after engraftment. Results: HA-1-specific immune reactions were detected in 3 patients in the months following HCT, 2 after RIC and 1 after conventional HCT. Frequencies of HA-1-specific T-cells increased further with time. Both RIC-transplanted patients had secondary AML. The first patient had a complete remission (CR) of his malignancy coincident with a grade I skin GvHD. HA-1-specific T-cells persisted for at least 3 years after engraftment at an average of 0.05% total mononuclear cells. After discontinuation of immunosuppression at day 1000 post HCT, frequencies of up to 0.24% were detected and strong HA-1-specific reactions were observed by Elispot. The second RIC patient required immunosuppression to control acute GvHD. On day 298 after HCT, CD34+ donor chimerism (DC) declined to 15% as an early indication of relapse. After reducing cyclosporine, a reversion to 100% DC occurred simultaneously with an increase of HA-1-specific T-cells from 0.05 to 0.14%. The patient went into remission, but again experienced GvHD. Increased immunosuppression was associated with a decrease in HA-1-specific T-cells. A similar decrease in CD34+ DC occurred at day 591 and 726 after HCT and was treated with low-dose chemotherapy and reduction of immunosuppression. On these occasions, the level of HA-1-specific T-cells increased from 0.08% to 0.21% and from 0.05% to 0.18% respectively, and was accompanied by the restoration of full DC. Interestingly, the fluctuations of HA-1-specific T-cells observed with Tetramer staining were not detected in the Elispot assay. T-cell lines were generated from PBMC of the 2 patients during periods of different HA-1 specific T-cell frequency. They reacted similarly with HA-1 peptide pulsed target cells and HA-1H EBV-LCL in the Elispot and Cr-release assay. Tetramer staining revealed up to 77,2% HA-1-specific CD8+ T-cells after 6 weeks of culture. Of the 2 patients with conventional HCT, 1 patient with CML was in CR with up to 0.17% HA-1 specific T-cells 557 days after tranplant. Conclusions: HA-1-specific immune reactions are observed in vivo after HA-1 mismatched HCT. Reactivity to HA-1 increases during the first 3 months after HCT and after reduction/discontinuation of immunosuppression. In one patient, increases of HA-1 specific T-cell frequency were associated with reductions in tumor cell burden (leukemic host CD34+ cells) and decreases with reappearance of tumor cells. Tetramer staining was clearly more sensitive than the Elispot assay in monitoring the endogenous HA-1-specific immune response. T cell lines can be established from PBMC independent of the frequency of HA-1-specific T cells.

Alloreactivity of Virus Specific T-Cells.

Blood ◽

10.1182/blood.v112.11.3249.3249 ◽

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 ◽

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.