CD4+ CD25+ regulatory T cells control the induction of antigen-specific CD4+ helper T cell responses in cancer patients

AbstractA proportion of cancer patients naturally develop CD4+ T-helper type 1 (Th1) cell responses to NY-ESO-1 that correlate with anti–NY-ESO-1 serum antibodies. To address the role of T-cell regulation in the control of spontaneous tumor immunity, we analyzed NY-ESO-1–specific Th1 cell induction before or after depletion of CD4+CD25+ T cells in vitro. While Th1 cells were generated in the presence of CD25+ T cells in cancer patients seropositive for NY-ESO-1, seronegative cancer patients and healthy donors required CD25+ T-cell depletion for in vitro induction of NY-ESO-1–specific Th1 cells. In vitro, newly generated NY-ESO-1–specific Th1 cells were derived from naive precursors, whereas preexisting memory populations were detectable exclusively in patients with NY-ESO-1 antibody. Memory populations were less sensitive than naive populations to CD4+CD25+ regulatory T cells. We propose that CD4+CD25+ regulatory T cells are involved in the generation and regulation of NY-ESO-1–specific antitumor immunity.

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Maintenance of Telomere Length in Peripheral Blood CD4+CD25+ Regulatory T-Cells of Cancer Patients Despite Active Proliferation.

Blood ◽

10.1182/blood.v106.11.3309.3309 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3309-3309

Author(s):

Dominik Wolf ◽

Holger Rumpold ◽

Christian Koppelstaetter ◽

Guenther Gastl ◽

Eberhard Gunsilius ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Telomere Length ◽

Cancer Patients ◽

Peripheral Blood ◽

Telomerase Activity ◽

Telomere Shortening ◽

Active Proliferation

Abstract CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the up-regulation of telomerase activity, whose regulation also remains unknown for Treg. We therefore isolated Treg and the respective CD4+CD25− control T-cell population from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17). Analysis of their content of T-cell receptor excision circles (TREC) revealed that the observed increase of Treg frequencies in peripheral blood is due to active cycling rather than to redistribution from other compartments (i.e. secondary lymphoid organs or bone-marrow), as Treg from cancer patients are characterized by a significant decrease of TREC content when compared to TREC content of Treg isolated from healthy age-matched controls. Surprisingly, despite their proven in vivo proliferation, telomere length is not further shortened in Treg from peripheral blood of cancer patients as shown by Flow-Fish, Real-Time PCR and Southern Blotting. Accodingly, telomerase activity of Treg was readily inducible in vitro by OKT3 together with IL-2. Notably, sorting of in vitro proliferating Treg using dilution of CFSE revealed a significant telomere shortening in Treg with high proliferative capacity (i.e. CFSElow fraction) under conditions of strong in vitro stimulatory growth conditions despite a high telomerase activity. Thus, under conditions of strong in vitro stimulation induction of telomerase seems to be insufficient to avoid progressive telomere shortening. In contrast, in actively proliferating peripheral blood Treg from patients with epithelial malignancies induction of telomerase activity is likely to compensate for further telomere erosion.

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Suppression of CTL Responses against AAV-Capsid Epitopes by Peptide-Induced Regulatory T Cells.

Blood ◽

10.1182/blood.v114.22.377.377 ◽

2009 ◽

Vol 114 (22) ◽

pp. 377-377 ◽

Cited By ~ 2

Author(s):

Daniel J Hui ◽

Gary C Pien ◽

Etiena Basner-Tschakarjan ◽

Federico Mingozzi ◽

Jonathan D Finn ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cd8 T Cells ◽

Transgene Expression ◽

T Cell Responses ◽

Effector Cells ◽

Cell Responses ◽

Ctl Activity

Abstract Abstract 377 Hemophilia B represents a promising model for the development of adeno-associated viral (AAV) vectors-based gene therapeutics. In the first clinical trial for AAV serotype 2 mediated gene transfer of Factor IX (F.IX) to the liver of severe hemophilia B subjects, transgene expression was short-lived with a gradual decline of F.IX levels. The loss of transgene expression was accompanied by a transient transaminitis, which we hypothesized to be the result of the reactivation of a pool of capsid-specific memory CD8+ T cells originated from a previous exposure to wild-type AAV. These results were unanticipated since previous work in small and large animal models showed that AAV administration is uneventful, allowing prolonged expression of F.IX transgene at therapeutic levels. We developed an in vitro cytotoxicity assay using a human hepatocyte cell line expressing HLA-B*0702, a common MHC class I allele for which the AAV capsid immunodominant epitope VPQYGYLTL was identified. Using this model, we demonstrated that HLA-matched AAV-specific effector CD8+ T cells were able to lyse target hepatocytes transduced with AAV-2. We now use this in vitro model of CTL killing of AAV-transduced hepatocytes to demonstrate the efficacy of a novel strategy to circumvent undesirable immune response through the engagement of regulatory T cells. A recently characterized MHC Class II-restricted T cell epitope (Tregitope) in the Fc fragment of IgG has been shown to induce regulatory T cells in vitro and in vivo (Blood, 2008; 112: 3303-3311). AAV-specific HLA-B*0702 effector cells expanded in the presence of a human Tregitope peptide resulted in 79% to 89% inhibition of cytotoxic activity against peptide-pulsed and AAV-transduced target cells, respectively. These results were confirmed using PBMCs from 5 different donors. A similar degree of inhibition of CTL activity was observed for the HLA allele A*0101, which binds to the AAV-derived epitope SADNNNSEY; co-culture of effector cells with the Tregitope inhibited CTL-mediated killing by 60%. Interestingly, the same Tregitope efficiently mediated suppression of CTL activity in subjects carrying different HLA alleles, indicating a high level of promiscuity of Tregitope binding. Staining for the regulatory T cell markers CD4, CD25, and FoxP3 supported the hypothesis that Tregitopes suppress T cell responses by expanding regulatory T cells; 62.2% of the CD4+ population stained positive for CD25 and FoxP3 in PBMCs expanded against AAV epitopes in the presence of Tregitope, compared with PBMCs expanded against an AAV epitope alone (3.63%), or against an AAV epitope and an irrelevant control peptide (1.94%). Polyfunctional analysis for markers for T cell activation showed that CD8+ T cells incubated in the presence of Tregitope had an approximately 5-fold decrease in production of IL-2 and IFN-γand a 2-fold reduction in TNF-α production, indicating levels of activation close to naïve CD8+ T cells. We further characterized the mechanism of action of Tregitopes by showing that Tregitopes are required at the time of CD8+ T cell priming, as CTL activity of AAV-expanded CD8+ T cells against transduced hepatocytes was not inhibited by the CD4+ T cell fraction of PBMC expanded separately in vitro with Tregitopes only. We conclude that the use of Tregitopes represents a promising strategy for antigen-specific, Treg-mediated modulation of capsid-specific T cell responses. Disclosures: Martin: EpiVax: Employment. De Groot:EpiVax, Inc.: Employment, Equity Ownership.

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High Prevalence of Functional Laa Specific Cytotoxic T Lymphocytes in Healthy Individuals-Implications for Strategies in Adoptive T Cell Therapies of Relapsed Leukemia

Blood ◽

10.1182/blood.v126.23.5428.5428 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5428-5428 ◽

Cited By ~ 1

Author(s):

Sarah Matko ◽

Marcus Odendahl ◽

Martin Bornhaeuser ◽

Torsten Tonn

Keyword(s):

T Cells ◽

T Cell ◽

Healthy Individuals ◽

Cell Responses ◽

Capture Assay ◽

Healthy Donors ◽

High Prevalence ◽

In Vitro Expansion ◽

Ifn Γ

Abstract While adoptive transfer of virus antigen specific T cells has shown to be effective in therapy of resistant recurrent viremia which is frequently associated with the lack of protective immunity following hematopoietic stem cell transplantation, the transfer of leukemia associated antigen specific (LAA) T cells is less implemented and appears to depend on factors that hamper a successful translation into the clinic. Among them are low frequencies and low antigen affinity of LAA specific T cells which currently mandate laborious in vitro expansion protocols. Moreover, screening of healthy individuals with regard to the presence of LAA specific T cells revealed contradictory results. Since we failed to detect LAA specific T cells in healthy donors using single peptide specificities to known LAA epitopes coupled to MHC Streptamers, here we asked if the use of peptide mixes comprising 15mers overlapping by 11 amino acids and spanning the entire LAA protein could elicit in vitro T cell responses in healthy donors, otherwise undetectable by single peptide staining. A cohort of 48 HLA A*0201 healthy individuals was screened using intracellular cytokine staining (ICS) after stimulation with tumor specific peptide mixes representing well known LAAs (WT1, PRAME, NY-ESO, Survivin and p53). While distinct T-helper cell responses were not observed in either of the specimen tested, cytotoxic T lymphocytes could be elicited and measured after incubation with peptide mixes for 5 hours and subsequent CD8+ IFNγ+ staining in 12 out of 48 healthy subjects. Only one individual displayed specifies against multiple antigens (WT1:0,1%; PRAME:0,5%; NY-ESO:0,1%; p53:0.06%), while the remaining responses were directed to one single antigen per individual. Most prevalent and highest T cell frequencies were found against PRAME in 5 out of all screened subjects (mean 0.4±0.3%; max. 0.8%), followed by WT1 in 4 (mean 0.07±0.03%; max. 0.1%) and NY-ESO in 3 individuals (mean 0,07±0,04%; max. 0,1%); one showed CD8 T cells specific against Survivin (0,03%) and 2 individuals had CD8 frequencies specific against p53 (0,05±0,01; max. 0,06%), respectively. The calculated limit of detection (LOD) for the enumeration of LAA specific T cells was 0,02%. In contrary, testing LAA positive individuals with according MHC Streptamers presenting single peptides of previously described epitopes showed no frequencies exceeding LOD. Further analysis showed LAA specific CD8+ IFNγ+ T cells exhibit mainly a less differentiated phenotype (CD45RA+, CCR7+/-, TNFα+, IL-2+/-) and could be immune-magnetically isolated to purities of 94.5±0.7% using a PRAME-specific IFN-γ capture assay yielding 1*104 antigen specific T cells out of 4*107 PBMCs. Simultaneous enrichment of helper T cells to a purity of 73.0±7.6% proofed their existence, despite no CD4+ response could be detected via ICS in the first place. The cytotoxic potential of the cell product was confirmed in an Europium assay using T2 cells loaded with PRAME peptide mix. The specific lysis accounted to 19.3% at an E:T ratio of 1:1 after 90 minutes of co-incubation. In conclusion, using LAA specific peptide mixes in combination with ICS we were able to show a relatively high prevalence of LAA specific T cells, especially for PRAME, in healthy donors. These LAA specific T cells can be enriched without the need of in vitro expansion culturing ex vivo using the IFN-γ capture assay with regard to achieving a functional LAA specific T cell product for adoptive T cell transfer. Furthermore, a less differentiated phenotype exhibited by a large proportion of LAA specific T cells might contribute to their long term survival in a patient after transplantation. Disclosures No relevant conflicts of interest to declare.

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Altered Heme-Mediated Modulation of Dendritic Cell Function in Sickle Cell Alloimmunization

Blood ◽

10.1182/blood.v126.23.655.655 ◽

2015 ◽

Vol 126 (23) ◽

pp. 655-655

Author(s):

Emmanuelle Godefroy ◽

Yunfeng Liu ◽

Patricia A Shi ◽

W. Beau Mitchell ◽

Devin Cohen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Sickle Cell ◽

Heme Oxygenase 1 ◽

Th1 Cell ◽

Transfusion Therapy ◽

Cell Responses ◽

Healthy Donors ◽

History Of ◽

T Cell Priming

Abstract Red blood cell alloimmunization can be a life-threatening complication for patients with sickle cell disease (SCD) receiving therapeutic transfusions. However, it remains unknown why only some (20-60%) SCD patients develop alloantibodies whereas others do not. Because of ongoing hemolysis in SCD, we have investigated the effects of toxic hemin on T cell responses of patients with SCD on chronic transfusion therapy. We found impaired monocyte control of proinflammatory T cells in response to hemin in alloimmunized SCD patients, partly due to defective levels of monocyte heme-oxygenase-1 (HO-1), an anti-inflammatory heme degrading enzyme that catabolizes heme into iron, biliverdin and carbon monoxide (CO). Monocytes are precursors of dendritic cells (DCs), which represent the antigen presenting cells most able to initiate and regulate immune responses. However, little is known about the functional properties of DCs in SCD patients. We therefore hypothesized that DCs of alloimmunized SCD patients would also have impaired response to hemin. Monocyte-derived DCs (moDCs) from healthy donor controls (n=11) and a cohort of SCD patients (aged 15-30 on chronic transfusion therapy every 3-4 weeks for at least two years using C,E,K phenotyped-matched, leukodepleted units) grouped either as "non-alloimmunized" (no history of antibody production, n=6), versus "alloimmunized" (with a history of having produced at least one alloantibody, n=7) were matured in the absence or presence of hemin (5uM and 20uM) with TLR agonists: LPS/IFNg (TLR4 agonist+STAT1 activation) or R848 (TLR7/8 agonist). Hemin-exposed mature and immature (no TLR agonist) moDCs were then assayed for HO-1 expression, cytokine production, co-stimulation, and T cell priming. Interestingly, upon hemin exposure, immature moDCs from healthy donors and non-alloimmunized patients upregulated more HO-1 (1056±206 fold; mean fluorescent intensity (MFI): 12283±1818 at 20uM hemin) than alloimmunized patients (fold increase 494±49; MFI: 7422±959, p<0.03). In addition, there were no differences among healthy donors and either patient groups in the following mature moDC associated cytokine/surface marker expression after hemin exposure: IL-12p70, IL-6, and TNFa production and surface expression of activation (CD80 and CD86) and antigen presentation (HLA-DR) markers, suggesting that these mature moDC-associated molecules are unlikely to be associated with alloimmunization status. As with healthy donors, hemin inhibited (by 35%) the upregulation of DC-specific maturation marker CD83 expression on mature moDCs of non-alloimmunized patients (MFI before hemin 5199±689 after 20uM hemin 3471±401, p=0.003). Furthermore, HO-1 breakdown product CO, but not biliverdin, inhibited CD83 upregulation on mature moDCs to a similar degree as hemin, suggesting that hemin-mediated CD83 inhibition in mature MoDCs involves HO-1. Remarkably, however, hemin did not alter CD83 expression on mature moDC in alloimmunized patients (before hemin 4382±949 after hemin 4825±1051, p=0.5). To determine whether hemin modulation of CD83 expression affects mature moDC driven differentiation of naïve CD4+ T cells (ie. priming), we performed mixed lymphocyte reactions: whereas hemin inhibited (roughly 30%) moDC-mediated priming of pro-inflammatory IFNg secreting TH1 cells in healthy donors and non-alloimmunized patients (%CD4+ IFNg+ without hemin 58%±9% with 20uM hemin 41%±8%, p=0.005), TH1 cell responses were not suppressed in the alloimmunized group (without hemin: 52%±10% with 20uM hemin: 54%±10%, p=0.4). In summary, hemin-exposed moDCs from healthy donors and non-alloimmunized SCD patients suppress CD83 upregulation and inhibit ensuing TH1 cell responses, possibly through HO-1 induction and subsequent production of CO. In contrast, CD83 upregulation and proinflammatory T cell priming are not inhibited by hemin in alloimmunized patients, probably representing a mechanism by which heme-associated inflammation is maintained in alloimmunized SCD, thereby increasing their risk of alloimmunization. Deciphering this mechanism not only offers a potential biomarker for alloimmunization, but also opens the way to designing innovative treatments for these patients. Disclosures No relevant conflicts of interest to declare.

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Induction of regulatory T cell–resistant helper CD4+ T cells by bacterial vector

Blood ◽

10.1182/blood-2007-09-113761 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1404-1412 ◽

Cited By ~ 24

Author(s):

Hiroyoshi Nishikawa ◽

Takemasa Tsuji ◽

Elke Jäger ◽

Gabriel Briones ◽

Gerd Ritter ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Treg Cells ◽

Th1 Cells ◽

High Avidity ◽

T Helper 1 ◽

Bacterial Vector ◽

Healthy Donors

Abstract Salmonella typhimurium engineered to deliver cancer/testis antigen NY-ESO-1 through type III secretion (S typhimurium–NY-ESO-1) was shown to be an efficient cancer vaccine construct in mice and to stimulate NY-ESO-1–specific CD8+/CD4+ T cells in vitro in patients with cancer with NY-ESO-1 spontaneous immunity. We also showed that individuals without spontaneous immunity to NY-ESO-1 had specific CD4+ T-cell precursors with high avidity to NY-ESO-1 under tight control by CD4+CD25+ regulatory T (Treg) cells. We now found that in healthy donors and patients with melanoma without NY-ESO-1 spontaneous immunity, S typhimurium–NY-ESO-1 elicits CD4+ T helper 1 (Th1) cells in vitro recognizing naturally processed antigen from these high-avidity NY-ESO-1–specific naive precursors. In contrast to peptide stimulation, induction of specific Th1 cells with S typhimurium–NY-ESO-1 did not require in vitro depletion of CD4+CD25+ Treg cells, and this prevailing effect was partially blocked by disruption of interleukin-6 or glucocorticoid-induced TNF receptor (GITR) signals. Furthermore, S typhimurium–induced Th1 cells had higher GITR expression than peptide-induced Th1 cells and were resistant to suppression by CD4+CD25+ Treg cells in a GITR-dependent fashion. We propose that S typhimurium–NY-ESO-1 induces antigen-specific T-cell responses that are resistant to suppression by CD4+CD25+ Treg cells.

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Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients

Blood ◽

10.1182/blood-2006-03-011353 ◽

2006 ◽

Vol 108 (8) ◽

pp. 2655-2661 ◽

Cited By ~ 221

Author(s):

Devi K. Banerjee ◽

Madhav V. Dhodapkar ◽

Elyana Matayeva ◽

Ralph M. Steinman ◽

Kavita M. Dhodapkar

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Regulatory T Cells ◽

Interleukin 2 ◽

Cell Contact ◽

Myeloid Dendritic Cells ◽

Healthy Donors

AbstractCD4+CD25+FOXP3+ regulatory T cells (Treg's) play an important role in the maintenance of immune tolerance. The mechanisms controlling the induction and maintenance of Treg's in humans need to be defined. We find that human myeloid dendritic cells (DCs) are superior to other antigen presenting cells for the maintenance of FOXP3+ Treg's in culture. Coculture of DCs with autologous T cells leads to an increase in both the number of Treg's, as well as the expression of FOXP3 protein per cell both in healthy donors and myeloma patients. DC-mediated expansion of FOXP3high Treg's is enhanced by endogenous but not exogenous interleukin-2 (IL-2), and DC-T-cell contact, including the CD80/CD86 membrane costimulatory molecules. DCs also stimulate the formation of Treg's from CD25- T cells. The efficacy of induction of Treg's by DCs depends on the nature of the DC maturation stimulus, with inflammatory cytokine-treated DCs (Cyt-DCs) being the most effective Treg inducers. DC-induced Treg's from both healthy donors and patients with myeloma are functional and effectively suppress T-cell responses. A single injection of cytokine-matured DCs led to rapid enhancement of FOXP3+ Treg's in vivo in 3 of 3 myeloma patients. These data reveal a role for DCs in increasing the number of functional FOXP3high Treg's in humans.

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Mapping of novel peptides of WT-1 and presenting HLA alleles that induce epitope-specific HLA-restricted T cells with cytotoxic activity against WT-1+ leukemias

Blood ◽

10.1182/blood-2011-11-394619 ◽

2012 ◽

Vol 120 (8) ◽

pp. 1633-1646 ◽

Cited By ~ 35

Author(s):

Ekaterina Doubrovina ◽

Taissia Carpenter ◽

Dmitry Pankov ◽

Annamalai Selvakumar ◽

Aisha Hasan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Wilms Tumor ◽

T Cell Responses ◽

Class Ii ◽

Class I ◽

Hla Alleles ◽

Cell Responses ◽

Healthy Donors

Abstract The Wilms tumor protein (WT-1) is widely recognized as a tumor antigen that is expressed differentially by several malignancies. However, WT-1 peptides known to induce tumoricidal T cells are few. In the present study, we evaluated T-cell responses of 56 healthy donors to in vitro sensitization with autologous APCs loaded with a pool of overlapping 15-mer peptides spanning the sequence of WT-1. Thereafter, we mapped the WT-1 peptides eliciting responses in each individual, defined the immunogenic peptides, and identified their presenting HLA alleles. We report 41 previously unreported epitopes of WT-1: 5 presented by class II and 36 by class I alleles, including 10 that could be presented by more than 1 class I allele. IFNγ+ T cells responding to 98% of the class I and 60% of the class II epitopes exhibited HLA-restricted cytotoxicity against peptide-loaded targets. T cells specific for 36 WT-1 peptides were evaluable for leukemocidal activity, of which 27 (75%) lysed WT-1+ leukemic targets sharing their restricting HLA allele. Each epitope identified induced T-cell responses in most donors sharing the epitopes' presenting allele; these responses often exceeded responses to flanking peptides predicted to be more immunogenic. This series of immunogenic epitopes of WT-1 should prove useful for immunotherapies targeting WT-1+ malignancies.

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