Increased IL-4+ CD8+ T cells in peripheral blood and autoreactive CD8+ T cell lines of patients with inflammatory arthritis

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.

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

Journal of Experimental Medicine ◽

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 ◽

T Cell Lines

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.

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In Vitro Expansion of Human Tumor-Reactive CD8+ T Cell Lines Using Superparamagnetic CD3/CD28/CD137 Beads.

Blood ◽

10.1182/blood.v114.22.5118.5118 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5118-5118

Author(s):

Daniel Teschner ◽

Eva Distler ◽

Elke Schnuerer ◽

Gregor Wenzel ◽

Axl Neurauter ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cell Lines ◽

Peripheral Blood ◽

Cd8 T Cell ◽

Strong Increase ◽

In Vitro Expansion ◽

T Cell Lines

Abstract Abstract 5118 Introduction Efficient methods for the reliable in vitro expansion of tumor-reactive T cells will surely broaden the applicability of adoptive T cell therapy in cancer. In this study we investigated the antigen-independent stimulation and expansion of human T cells in peripheral blood mononuclear cells (PBMC) and in long-term cultured tumor-reactive CD8+ T cell lines using superparamagnetic beads coated with antibodies to CD3 and the costimulatory molecules CD28 and CD137. Methods T cell numbers were measured in healthy donor PBMC after in vitro stimulation with Dynabeads® coated with CD3/CD28/CD137 versus Dynabeads® coated with CD3/CD28 (all beads +/- 100 U/mL IL-2) versus IL-2 alone at different bead/cell ratios (3:1, 1:1). Expansion was also analyzed in human renal cell carcinoma-reactive CD8+ T cell lines after restimulation with tumor cells (weekly), CD3/CD28 beads and CD3/CD28/CD137 beads, respectively (bead/cell ratio of 1:5, 100 U/mL IL-2 added). Expanded T cell lines were phenotyped for expression of activation, differentiation and homing molecules (i.e. CD27, CD28, CD45RA, CD45RO, CD57, CD62L, CD137, CCR7) and were also tested for function. Results T cells in PBMC showed an increased expansion rate of up to 17-fold during a 2-week culture period using beads with IL-2 added versus IL-2 alone (p<0.0001 for CD3/CD28/CD137; p<0.0001 for CD3/CD28). The difference between CD3/CD28/CD137 beads and CD3/CD28 beads was not significant (p=0.4). Bead/cell ratios of 1:1 and 3:1 expanded T cells in PBMC with similar efficiency. In addition, IL-2 was essential to obtain maximum T cell proliferation. Peripheral blood CD4+ and CD8+ T cells showed a strong increase of CD137 surface expression starting 12-24 hours upon stimulation, regardless which beads were used. In contrast to PBMC, tumor-reactive CD8+ T cell lines expanded more rapidly using CD3/CD28/CD137 beads versus CD3/CD28 beads (p=0.03). Stimulation with CD3/CD28/CD137 beads was comparably efficient versus the control arm using weekly addition of tumor cells and IL-2. Simultaneous addition of beads and tumor cells did not have a synergistic effect. CD8+ T cell lines analyzed 12 days after bead-induced in vitro expansion versus weekly tumor stimulation showed a comparable level of tumor reactivity in IFN-g ELISPOT assay. Phenotypically, expression of CD137 on CD8+ T cell lines showed maximum up-regulation 24 hours after beads stimulation and persisted for at least 72 hours. In contrast, cultures stimulated solely with tumor cells showed a much shorter and transient CD137 expression with an earlier peak level after 12 hours. Other phenotypic markers were similar on tumor-reactive T cell cultures, except for increased CD62L expression after bead-induced stimulation. Conclusion Antigen-independent in vitro expansion of T cells in PBMC was equally efficient using CD3/CD28 beads or CD3/CD28/CD137 beads, respectively. In contrast, we observed an increased growth rate for tumor-reactive CD8+ T cell lines when activated with CD3/CD28/CD137 beads compared to CD3/CD28 beads. Antitumor reactivity of T cell lines was maintained during the antigen-independent stimulation step. Bead activation was associated with increased expression of the lymph node homing receptor CD62L on antitumor CD8+ T cell lines, which indicates a central memory phenotype. Our data suggest that the conjugation of anti-CD137 antibodies to the traditionally used CD3/CD28 beads improves their expansion capacity for antitumor CD8+ T cell lines. Disclosures No relevant conflicts of interest to declare.

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Expansion of Simian Immunodeficiency Virus (SIV)-Specific CD8 T Cell Lines from SIV-Naive Mauritian Cynomolgus Macaques for Adoptive Transfer

Journal of Virology ◽

10.1128/jvi.00993-15 ◽

2015 ◽

Vol 89 (19) ◽

pp. 9748-9757 ◽

Cited By ~ 8

Author(s):

Mariel S. Mohns ◽

Justin M. Greene ◽

Brian T. Cain ◽

Ngoc H. Pham ◽

Emma Gostick ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Simian Immunodeficiency Virus ◽

Cell Lines ◽

Cd8 T Cells ◽

Adoptive Transfer ◽

Cd8 T Cell ◽

Immunodeficiency Virus ◽

Cynomolgus Macaques ◽

Transfer Strategies

ABSTRACTCD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). However, the specific qualities and characteristics of an effective CD8 T cell response remain unclear. Although targeting breadth, cross-reactivity, polyfunctionality, avidity, and specificity are correlated with HIV control, further investigation is needed to determine the precise contributions of these various attributes to CD8 T cell efficacy. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques (MCM). These cells exhibited an effector memory phenotype, produced cytokines in response to cognate antigen, and suppressed viral replicationin vitro. We further cultured cell lines specific for four SIV-derived epitopes, Nef103–111RM9, Gag389–394GW9, Env338–346RF9, and Nef254–262LT9. These cell lines were up to 94.4% pure, as determined by major histocompatibility complex (MHC) tetramer analysis. After autologous transfer into two MCM recipients, expanded CD8 T cells persisted in peripheral blood and lung tissue for at least 24 weeks and trafficked to multiple extralymphoid tissues. However, these cells did not impact the acute-phase SIV load after challenge compared to historic controls. The expansion and autologous transfer of SIV-specific T cells into naive animals provide a unique model for exploring cellular immunity and the control of SIV infection and facilitate a systematic evaluation of therapeutic adoptive transfer strategies for eradication of the latent reservoir.IMPORTANCECD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Autologous adoptive transfer studies followed by SIV challenge may help define the critical elements of an effective T cell response to HIV and SIV infection. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques. This is an important first step toward the development of autologous transfer strategies to explore cellular immunity and potential therapeutic applications in the SIV model.

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

Blood ◽

10.1182/blood.v104.11.2236.2236 ◽

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.

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T Cell Recognition of HCMV: Pan-Genome Analysis of Immunogenic Open-Reading Frames.

Blood ◽

10.1182/blood.v104.11.606.606 ◽

2004 ◽

Vol 104 (11) ◽

pp. 606-606 ◽

Cited By ~ 1

Author(s):

Louis J. Picker ◽

Andrew W. Sylwester ◽

Bridget L. Mitchell ◽

Cara Taormina ◽

Christian Pelte ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cell Response ◽

Cd8 T Cell ◽

Cross Reactivity ◽

T Cell Response ◽

Cd4 T Cell ◽

Cell Responses

Abstract Human Cytomegalovirus (HCMV) is among the largest and most complex of known viruses with 150–200nm virions enclosing a double stranded 230kb DNA genome capable of coding for >200 proteins. HCMV infection is life-long, and for the vast majority of immune competent individuals clinically benign. Disease occurs almost exclusively in the setting of immune deficiency, suggesting that the stable host-parasite relationship that characterizes these infections is the result of an evolutionarily “negotiated” balance between viral mechanisms of pathogenesis and the host immune response. In keeping with, and perhaps because of this balance, the human CD4+ T cell response to whole HCMV viral lysates is enormous, with median peripheral blood frequencies of HCMV-specific cells ~5–10 fold higher than for analogous preparations of other common viruses. Although certain HCMV ORFs have been identified as targets of either the CD4+ or CD8+ T cell response, the specificities comprising the CD4+ T cell response, and both the total frequencies and component parts of the CD8+ T cell response are unknown. Here, we used cytokine flow cytometry and ~14,000 overlapping 15mer peptides comprising all 213 HCMV ORFs encoding proteins >100 amino acids in length to precisely define the total CD4+ and CD8+ HCMV-specific T cell responses and the HCMV ORFs responsible for these responses in 33 HCMV-seropositive, HLA-disparate donors. An additional 9 HCMV seronegative donors were similarly examined to define the extent to which non-HCMV responses cross-react with HCMV-encoded epitopes. We found that when totaled, the median frequencies of HCMV-specific CD4+ and CD8+ T cells in the peripheral blood of the seropositive subjects were 4.0% and 4.5% for the total CD4+ or CD8+ T cell populations, respectively (which corresponds to 9.1% and 10.5% of the memory populations, respectively). The HCMV-specific CD4+ and CD8+ T cell responses included a median 12 and 7 different ORFs, respectively, and all told, 73 HCMV ORFs were identified as targets for both CD4+ and CD8+ T cells, 26 ORFs as targets for CD8+ T cells alone, and 43 ORFS as targets for CD4+ T cells alone. UL55, UL83, UL86, UL99, and UL122 were the HCMV ORFs most commonly recognized by CD4+ T cells; UL123, UL83, UL48, UL122 and UL28 were the HCMV ORFs most commonly recognized by CD8+ T cells. The relationship between immunogenicity and 1) HLA haplotype and 2) ORF expression and function will be discussed. HCMV-seronegative individuals were non-reactive with the vast majority of HCMV peptides. Only 7 potentially cross-reactive responses were identified (all by CD8+ T cells) to 3 ORFs (US32, US29 and UL116) out of a total of almost 4,000 potential responses, suggesting fortuitous cross-reactivity with HCMV epitopes is uncommon. These data provide the first glimpse of the total human T cell response to a complex infectious agent, and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

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

Blood ◽

10.1182/blood.v106.11.477.477 ◽

2005 ◽

Vol 106 (11) ◽

pp. 477-477

Author(s):

Erica Dander ◽

Giuseppina Li Pira ◽

Ettore Biagi ◽

Fabrizio Manca ◽

Andrea Biondi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Cd8 T Cell ◽

Effector Memory ◽

Target Cells ◽

Pulsed Dc ◽

Ifn Γ ◽

T Cell Lines

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.

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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

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AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Blood ◽

10.1182/blood.v108.11.455.455 ◽

2006 ◽

Vol 108 (11) ◽

pp. 455-455 ◽

Cited By ~ 1

Author(s):

Federico Mingozzi ◽

Marcela V. Maus ◽

Denise E. Sabatino ◽

Daniel J. Hui ◽

John E.J. Rasko ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Cell Population ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Transgene Expression ◽

Cd8 T Cell ◽

Target Cells

Abstract Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes. It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

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Autoproliferation Contributes to Clonal Expansion and Changes In Cellular Topography In T Cell Large Granular Lymphocyte (LGL) Leukemia

Blood ◽

10.1182/blood.v116.21.1373.1373 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1373-1373

Author(s):

JianXiang Zou ◽

Jeffrey S Painter ◽

Fanqi Bai ◽

Lubomir Sokol ◽

Thomas P. Loughran ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd8 T Cell ◽

Effector Memory ◽

Ki67 Expression ◽

Terminal Effector ◽

Lgl Leukemia

Abstract Abstract 1373 Introduction: LGL leukemia is associated with cytopenias and expansion of clonally-derived mature cytotoxic CD8+ lymphocytes. The etiology of LGL leukemia is currently unknown, however, T cell activation, loss of lymph node homing receptor L-selectin (CD62L), and increased accumulation of T cells in the bone marrow may lead to suppressed blood cell production. The broad resistance to Fas (CD95) apoptotic signals has lead to the hypothesis that amplification of clonal cells occurs through apoptosis resistance. However, the proliferative history has not been carefully studied. To define possible mechanism of LGL leukemia expansion, T cell phenotype, proliferative history, and functional-related surface marker expression were analyzed. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 16 LGL leukemia patients that met diagnostic criteria based on the presence of clonal aβ T cells and >300 cells/ml CD3+/CD57+ T cells in the peripheral blood. Samples were obtained from 10 age-matched healthy individuals from the Southwest Florida Blood Services for comparisons. Multi-analyte flow cytometry was conducted for expression of CD3, CD4/8, CD45RA, CD62L, CD27, CD28, CD25, CD127, IL15Ra, IL21a, CCR7 (all antibodies from BD Biosciences). The proliferative index was determined by Ki67 expression in fixed and permeabilized cells (BD Biosciences) and the proliferative history in vivo was assessed by T-cell-receptor excision circle (TREC) measurement using real-time quantitative PCR (qRT-PCR) in sorted CD4+ and CD8+ T cells. TRECs are episomal fragments generated during TCR gene rearrangements that fail to transfer to daughter cells and thus diminish with each population doubling that reflects the in vivo proliferative history. Results: Compared to healthy controls, significantly fewer CD8+ naïve cells (CD45RA+/CD62L+, 8.4 ± 10.8 vs 24.48 ± 11.99, p=0.003) and higher CD8+ terminal effector memory (TEM) T cells (CD45RA+/CD62L-, 67.74 ± 28.75 vs 39.33 ± 11.32, p=0.007) were observed in the peripheral blood. In contrast, the percentage of CD4+ naïve and memory cells (naïve, central memory, effector memory, and terminal effector memory based on CD45RA and CD62L expression) was similar in patients as compared to controls. The expression of CD27 (31.32 ± 34.64 vs 71.73 ± 20.63, p=0.003) and CD28 (31.38 ± 31.91 vs 70.02 ± 22.93, p=0.002) were lower in CD8+ T cell from patients with LGL leukemia and this reduction predominated within the TEM population (17.63±24.5 vs 70.98±22.5 for CD27, p<0.0001 and 13±20.5 vs 69.43± 21.59 for CD28, p<0.0001). Loss of these markers is consistent with prior antigen activation. There was no difference in CD25 (IL2Ra, p=0.2) expression on CD4+ or CD8+ T cells, but CD127 (IL7Ra, p=0.001), IL15Ra, and IL21Ra (p=0.15) were overexpressed in TEM CD8+ T cell in patients vs controls. All of these cytokine receptors belong to the IL2Rβg-common cytokine receptor superfamily that mediates homeostatic proliferation. In CD8+ T cells in patients, the IL-21Ra was also overexpressed in naïve, central and effector memory T cells. The topography of the expanded CD8+ T cell population was therefore consistent with overexpression of activation markers and proliferation-associated cytokine receptors. Therefore, we next analyzed Ki67 expression and TREC DNA copy number to quantify actively dividing cells and determine the proliferative history, respectively. We found that LGL leukemia patients have more actively dividing CD8+ TEM T cells compared to controls (3.2 ± 3.12 in patients vs 0.44 ± 0.44 in controls, p=0.001). Moreover, the TREC copy number in CD8+ T cells was statistically higher in healthy individuals after adjusting for age (177.54 ± 232 in patients vs 1015 ± 951 in controls, p=0.019). These results show that CD8+ cells in the peripheral compartment have undergone more population doublings in vivo compared to healthy donors. In contrast, the TREC copies in CD4+ T-cells were similar between LGL patients and controls (534.4 ± 644 in patients vs 348.78 ± 248.16 in controls, p>0.05) demonstrating selective cellular proliferation within the CD8 compartment. Conclusions: CD8+ T- cells are undergoing robust cellular activation, contraction in repertoire diversity, and enhanced endogenous proliferation in patients with LGL leukemia. Collectively, these results suggest that clonal expansion is at least partially mediated through autoproliferation in T-LGL leukemia. Disclosures: No relevant conflicts of interest to declare.

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