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DOI: 10.1002/eji.201444819

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Monocyte-derived dendritic cells can induce autoreactive CD4+ T cells showing myeloid lineage directed reactivity in healthy individuals Tin Sing Lam, Marian van de Meent, JH Frederik Falkenburg and Inge Jedema Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands T cells against self-antigens can be detected in peripheral blood of healthy individuals, although intrathymic negative selection removes most high-avidity T cells specific for self-antigens from the peripheral repertoire. Moreover, spontaneous T-cell proliferation following stimulation with autologous monocyte-derived dendritic cells (autoDCs) has been observed in vitro. In this study, we characterized the nature and immunological basis of the autoDC reactivity in the T-cell repertoire of healthy donors. We show that a minority of naive and memory CD4+ T cells within the healthy human T-cell repertoire mediates HLA-restricted reactivity against autoDCs, which behave like a normal antigen-specific immune response. This reactivity appeared to be primarily directed against myeloid lineage cells. Although cytokine production by the reactive T cells was observed, this did not coincide with overt cytotoxic activity against autoDCs. AutoDC reactivity was also observed in the CD8+ T-cell compartment, but this appeared to be mainly cytokine-induced rather than antigen-driven. In conclusion, we show that the presence of autoreactive T cells harboring the potential to react against autologous and HLA-matched allogeneic myeloid cells is a common phenomenon in healthy individuals. These autoDC-reactive T cells may help the induction of primary T-cell responses at the DC priming site.

Keywords: Autologous responses r CD4+ T cells



r

Dendritic cells

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction Autologous or allogeneic T-cell responses against antigens that are either selectively expressed or overexpressed on malignant cells compared to normal cellular counterparts (tumor-associated antigens (TAAs)) are generally assumed to play an important role in immune surveillance against cancer and in cellular immunotherapy strategies [1–5]. In autologous and HLA-matched

Correspondence: Dr. Inge Jedema e-mail: [email protected]  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

allogeneic stem cell transplantation settings, these antigens will be presented and recognized in the context of self-HLA molecules on the cell membrane. When self-antigens and TAAs are expressed in the thymus, potentially harmful T cells expressing a highaffinity T-cell receptor (TCR) specific for these self-antigens or TAAs in the context of self-HLA are deleted from the Tcell repertoire during negative selection [6, 7]. This impedes not only induction of autoimmunity but also antitumor immunity, thereby weakening the concept of functional immune surveillance against malignant cells in the autologous and HLAmatched setting. However, immune responses against autologous malignant cells have frequently been observed, although www.eji-journal.eu

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the functional avidity of these T cells may often be questionable [8–13]. In line with the observations of autologous tumor-reactive T-cell responses, proliferation of (naive) T cells in response to autologous monocyte-derived dendritic cells (autoDCs) has also been demonstrated in in vitro studies [14–17]. The immunological basis and the biological relevance of this autoDC reactivity, seen both in human and in mouse models, have not been clarified so far [15, 16, 18, 19]. These autoDC-reactive T cells may play a role as helper cells in the induction of primary T-cell responses at the DC priming site, but they may also harbor the potential to elicit deleterious autoimmune reactions, and are therefore likely to be controlled under normal circumstances by peripheral tolerance mechanisms, such as naturally occurring regulatory T (Treg) cells [20]. Recently, we demonstrated the ability of naturally occurring CD4+ CD25high Treg cells to actively suppress in vitro priming and expansion of antigen-specific T cells by stimulation of naive donor T cells with peptide-loaded autoDCs. To allow efficient in vitro priming of naive antigen-specific precursor T cells, Treg-cell depletion of the responder T-cell populations has been applied [21]. However, this Treg-cell depletion in combination with priming with autoDCs as professional APCs may allow the outgrowth of autoreactive T cells that are under normal circumstances kept silent by Treg cells [20, 22–28]. This potentially hampers the enrichment of T cells recognizing tumor or pathogen-specific peptides exogenously loaded on these autoDCs, because these autoreactive T cells may even outnumber these antigen-specific precursor T cells that are present in very low frequencies within the naive T-cell repertoire. In this study, we characterized the nature and immunological basis of the autoDC reactivity mediated by naive T cells of healthy donors at bulk and clonal level. We elucidated that a minority of polyclonal naive and memory CD4+ T cells harbored the capacity to mediate HLA-restricted reactivity against autoDCs. This reactivity appeared to be mainly directed against cells of the myeloid lineage. Although profound cytokine production by the reactive T cells was observed, this did not coincide with overt cytotoxic activity against the DCs. This autoDC-reactive T cells may play a physiological role as helper T cells in the induction of primary T-cell responses at the DC activation site.

Results A proportion of naive T cells proliferates in response to stimulation with unpulsed autoDCs in vitro To study whether autoDC reactivity was apparent within purified T-cell populations in the presence or absence of natural regulation of the immune system, we stimulated purified T-cell populations without (total CD3+ cells isolated from peripheral blood mononuclear cells (PBMCs)) or with preceding CD25/Treg depletion (CD25− CD3+ , CD25− CD45RO+ CD3+ (memory), and CD25− CD45RO− CD27+ CD3+ (naive)) with autoDCs for 7 days  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cellular immune response

at a responder to stimulator (R/S; T cell/autoDC) ratio of 1/3. Stimulation was performed in medium supplemented with human serum, 5 ng/mL IL-7, and 0.1 ng/mL IL-15 that was similar to the culture medium used for in vitro priming of antigen-specific T-cell responses from the naive donor T-cell repertoire [21]. As shown in Figure 1A, a profound proliferative response of a proportion of T cells against unpulsed autoDCs was observed, which was not increased by preceding Treg/CD25 depletion of the responding T-cell population. To study whether these autoDCreactive T cells resided in the naive and/or memory T-cell compartment, we stimulated CD25− CD45RO+ CD3+ (memory) and CD25− CD45RO− CD27+ CD3+ (naive) T cells (purities ࣙ 95%) with autoDCs. AutoDC reactivity was visible both in the memory (CD25− CD45RO+ CD3+ ) and naive (CD25− CD45RO− CD3+ ) T-cell compartments. This illustrates that some autoDC-reactive T cells may have been previously activated in vivo. Since gamma chain (γc) cytokines are described to be the driving force behind homeostatic proliferation of naive T cells, we investigated whether the observed autoDC reactivity was in fact caused by (increased) cytokine-driven homeostatic proliferation. Therefore, PKH26-labeled naive T cells were cultured in medium supplemented with 5 ng/mL IL-7 and increasing concentrations of IL-15 in the absence or presence of autoDCs for 7 days. As shown in Figure 1B, the addition of γc cytokines alone caused only limited basal proliferation of the total population of naive T cells, as indicated by slight dilution of the PKH26 staining. However, upon stimulation with autoDCs, a fraction of naive T cells showed a vigorous additional proliferative response reflected by profound PKH26 dilution. Although the total number of cells was increased at higher IL-15 concentration, the fraction of naive T cells mediating the additional autoDC reactivity was similar in all conditions (p > 0.05). At the highest IL-15 concentrations (1 and 5 ng/mL IL-15), the number of T cells in the PKH26-bright quadrant was slightly increased, but did not differ significantly compared to the cultures with low IL-15 (0.1 ng/mL; p > 0.05), indicating minimal cytokine-driven basal proliferation and/or increased survival of naive T cells in the presence of autoDCs and exogenously added IL-15. Stimulation with autoDC without exogenously added IL-7 and IL-15 did not induce significant proliferation indicating that a minimal amount of cytokines is needed in the priming phase of na¨ıve antigen-specific T cells. To limit the additional effect of cytokine-driven proliferation, further experiments were performed at 5 ng/mL IL-7 and 0.1 ng/mL IL-15. We reproducibly confirmed the presence of autoDC reactivity using naive T cells from eight other healthy adult donors. By assessment of the number of cell divisions undergone by the autoDC reactive T cells based on PKH dye dilution, we estimated the frequency of autoDC-reactive T cells to be 1–5% within the naive T-cell repertoire. To investigate whether the observed autoDC reactivity was induced by serum factors taken up and presented in the HLA molecules on the surface of autoDCs, naive T cells were exposed under serum-free conditions to autoDCs that were generated under completely serum-free conditions. Serum-free DCs expressed similar levels of CD80, CD86, HLA-DR, CD11c, and CD83 (data not shown) and induced similar autoDC www.eji-journal.eu

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Figure 1. Proliferation of a minority of (naive) T cells upon stimulation with autologous DCs (autoDCs). (A) Total CD3+ T cells isolated from PBMCs (top) or T-cell populations, after preceding CD25/Treg depletion (CD25− CD3+ , CD25− CD45RO+ CD3+ (memory), CD25− CD45RO+ CD27+ CD3+ (naive), middle and bottom), were stimulated for 7 days with autologous monocyte-derived DCs (autoDCs) at a 1/3 responder (T cell)/stimulator (autoDC) (R/S) ratio in the presence of 5 ng/mL IL-7 and 0.1 ng/mL IL-15. Cells were labeled with PKH26 and proliferation was measured by flow cytometry. Representative plots from one of two experiments (n = 2 donors) are shown. (B) The effect of gamma chain (γc) cytokine concentration on the frequency of naive T cells mediating autoDC reactivity. PKH26-labeled naive T cells were stimulated with irradiated autoDCs at a 1/1 R/S ratio for 7 days in the presence of 5 ng/mL IL-7 and 0.1, 1, or 5 ng/mL IL-15. Proliferation at day 7 was analyzed after CD3 staining by flow cytometry. Representative proliferation dot plots of nonstimulated control T cells (no stim, at day 0 and day 7) and naive T cells stimulated with autoDCs (autoDC, at day 7) are shown from one example of triplicate measurements (top). The absolute numbers of autoDC-reactive/PKH26dim (white bars) and nonreactive/PKH26bright CD3 T cells (black bars) for unstimulated control and stimulation with autoDCs are also shown (bottom). Data represent mean + SD from one experiment performed in triplicate, and were analyzed for statistical difference using Mann–Whitney U test. (C) PKH26-labeled naive T cells were stimulated with serum-free autoDCs and human serum supplemented autoDCs in human serum free or serum supplemented medium, respectively. Proliferation at day 7 was analyzed after CD3 staining by flow cytometry. Representative proliferation dot plots of nonstimulated control (no stim) and naive T cells stimulated with autoDCs (autoDC) are shown from one of the two donors tested independently. (D) PKH-26-labeled naive T cells were stimulated with autoDCs at a 1/3 R/S (naive T cell/autoDC) ratio (5 ng/mL IL-7 and 0.1 ng/mL IL-15) in the presence or absence of autologous CD4+ CD25high CD127low Treg cells or autologous CD4+ CD25low as control. The ratio between naive T cells and Treg cells/control cells was 1 to 2. Proliferation at day 7 was analyzed by flow cytometry. Dot plots are shown from one representative experiment of the three experiments performed.

reactivity under serum-free conditions compared to the condition with human serum, indicating that serum factors were not the target of the autoDC reactivity (Fig. 1C). To study whether naturally occurring Treg cells could inhibit autoDC reactivity in this  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

system when they are outnumbering the precursor T cells mediating autoDC reactivity, we stimulated naive T cells with autoDCs in the presence or absence of autologous purified CD4+ , CD127low , CD25high Treg cells or autologous CD4+ , CD25low control T cells

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Figure 2. Profound autoDC reactivity is particularly found within the naive CD4+ compartment, whereas CD8+ T-cell proliferation is mainly driven by DC-derived cytokines. (A) PKH26-labeled naive T cells (CD4+ alone, CD8+ alone, or combined CD4+ and CD8+ ) were stimulated with irradiated autoDCs at a 1/1 R/S ratio for 7 days (5 ng/mL IL-7 and 0.1 ng/mL IL-15). Cells were gated on CD3+ cells and proliferation at day 7 was analyzed after CD8 staining by flow cytometry. Dot plots are shown from one representative experiment of the two independent experiments, each with one donor. (B) Phenotypic changes of CD4+ (left) and CD8+ (right) T cells. Combined CD4+ and CD8+ naive T cells (PKH-26 labeled) were in vitro stimulated with autoDCs at a 1/3 R/S ratio for 10 days (5 ng/mL IL-7 and 0.1 ng/mL IL-15). Cells were gated on CD3+ cells and stained for CD45RA or CD45RO. Representative proliferation dot plots of one of the three independent experiments, each with one donor, are shown. (C) CD4+ and CD8+ T cells that showed a proliferative response upon stimulation with autoDCs were separated by flow cytometric cell sorting as described. After PKH26 relabeling, autoDC reactivity of these autoDC-reactive CD4+ and CD8+ populations was separately tested by secondary stimulation with autoDCs at a 1/3 R/S ratio for 7 days (5 ng/mL IL-7 and 0.1 ng/mL IL-15). Cells were gated on CD3+ cells and stained for CD4 or CD8 on day 0 directly after PKH26 relabeling and on day 7. Representative proliferation dot plots are shown for one of the three donors, each tested independently. (D) The involvement of cytokines in the proliferation of the responding population was assessed in a transwell culture system. PKH26-labeled, combined CD4+ and CD8+ naive T cells plated in the bottom compartment were exposed to medium (no stim), naive T-cells alone, autoDCs alone, or naive T cells together with autoDCs (at a 1/5 R/S ratio) in the upper compartment for 10 days (5 ng/mL IL-7 and 0.1 ng/mL IL-15). As a positive control, coculture of naive T cells and autoDCs (at a 1/3 R/S ratio) in the bottom chamber was used with medium in the upper compartment. Cells were stained with PKH26 and proliferation was measured by flow cytometry. Histograms for CD4+ and CD8+ cells are shown (black line histogram) with the gray-filled histogram as nonstimulated control from one experiment. One representative histogram out of the three different donors is shown.

(2/1 Treg/naive responder T-cell ratio). As shown in Figure 1D, addition of autologous Treg cells could prevent the induction of autoDC reactivity, whereas the autologous CD4+ control population did not. This indicated that naturally occurring Treg cells could prevent the outgrowth of autoDC-reactive T cells, when present at sufficient numbers at the DC priming site. These data illustrated that naive T cells showed a slow universal in vitro proliferative response to γc cytokines, especially at high concentrations, mimicking homeostatic proliferation. Stimulation of a purified naive T-cell population with autoDCs in the absence of Treg cells in medium supplemented with 5 ng/mL IL-7 and 0.1 ng/mL IL-15 induced specific outgrowth of autoDC-reactive T cells.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

AutoDC reactivity is predominantly seen in the naive CD4+ population To study whether naive CD4+ and CD8+ T cells could both mediate autoDC reactivity independent of each other, naive CD4+ and CD8+ T cells were cultured either alone or together with autoDCs. Figure 2A shows that profound autoDC-reactive proliferation was prominent in the CD4+ population. The additional proliferative response induced in the CD8 population upon autoDC stimulation was less pronounced and appeared to mimic the increased cytokine-mediated proliferative response demonstrated in Figure 1B. The slightly enhanced proliferation observed in the coculture of CD4+ and CD8+ T cells suggests that cytokines produced by www.eji-journal.eu

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Figure 3. AutoDC reactivity is restricted to a subpopulation of naive CD4+ T cells. (A) PKH26-labeled naive T cells were stimulated with autoDCs at different R/S ratios for 7 days. The absolute numbers of autoDC-reactive (white bars) and nonreactive CD4+ T cells (black bars) were quantified by flow cytometry. Data represent mean + SD of four independent experiments, each with a different donor, and p-values (*p < 0.05) are shown for two-tailed paired Student’s t-test. (B) PKH26-labeled naive T cells were stimulated with autoDCs zero to ten times, repetitively, with 24-h intervals for 12 days. The absolute numbers of nonreactive TCR-α/β cells were quantified by flow cytometry. Naive T cells that received single stimulation with autoDCs at a 1/1 R/S ratio were used as a control (white bar). Data are mean + SD of three sample replicates from one experiment.

the autoDC-reactive CD4+ T cells may have further enhanced the CD8 proliferation. We next studied whether naive CD4+ and CD8+ T cells mediating autoDC-induced proliferation retained their naive phenotype or acquired an antigen-experienced phenotype. As shown in Figure 2B, CD4+ T cells mediating clear autoDCinduced proliferation showed a switch in phenotype from CD45RA to CD45RO expression, whereas the non-autoDC-directed (PKH26 bright ) CD4+ T cells mediating only limited cytokine-induced proliferation retained their naive phenotype. A similar trend was seen within the CD8 compartment. To further investigate whether the T cells showing the profound proliferative response upon coculture with autoDCs mediate predominantly antigen-specific autoDC reactivity and/or cytokinemediated proliferation without TCR-mediated antigen recognition, we selected the CD4+ and CD8+ T cells that showed a profound proliferative response upon stimulation with autoDCs and tested their response to secondary stimulation with autoDCs. As shown in Figure 2C, all selected CD4+ T cells showed an additional proliferative response to secondary stimulation with autoDCs, whereas the selected CD8+ T cells only showed cytokine-induced proliferation, similarly both even in the presence and absence of DC stimulation. This suggested that the autoDC-directed proliferation of the CD4+ T cells was mainly caused by antigen-specific recognition, whereas the autoDC-directed proliferation observed in the CD8 compartment was mainly cytokine-driven. To further investigate whether the proliferation of the CD8+ T cells was mediated mainly by soluble factors secreted by autoDCs rather than antigen-specific recognition, we performed transwell experiments using naive T cells in the bottom chamber and culture medium, naive T cells, or autoDCs with or without naive T cells in the upper chamber. In these experiments, minor cytokine-mediated proliferation of both CD8+ and CD4+ T cells was demonstrated when DCs were present in the upper chamber of the transwell system, whereas clear additional autoDC-reactive proliferation was  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

observed in the CD4+ , but not the CD8+ population, when naive T cells and DCs were cocultured in the same chamber (Fig. 2D). These data demonstrated that a fraction of naive CD4+ T cells mediating autoDC reactivity acquired CD45RO expression. This autoDC reactivity required direct cell–cell contact with the DCs and the specific reactivity of these autoDC-reactive CD4+ T cells could be retained upon selection and secondary stimulation. In addition, a low proliferative response could be induced in both CD4+ and CD8+ T cells mediated by cytokines produced by the DCs and autoDC-reactive CD4+ T cells. This cytokine-driven proliferative response could not be blocked by the addition of anti-HLA blocking antibodies (data not shown).

AutoDC reactivity is mediated by a restricted subpopulation of naive CD4+ T cells To investigate whether increment of the numbers of autoDCs used for stimulation or repetitive autoDC stimulations resulted in increased frequencies of responding naive CD4+ T cells mediating autoDC reactivity and whether nonreacting CD4+ T cells remained nonresponsive to autoDC stimulation, we performed single stimulation of naive CD4+ T cells with increasing amounts of autoDCs, or performed daily stimulations with autoDCs at a 1/1 R/S ratio, and quantified the number of autoDC-reactive and nonreacting T cells using quantitative flow cytometric analysis. As shown in Figure 3A, only the absolute numbers of CD4+ T cells that had proliferated increased with addition of increasing numbers of stimulatory autoDCs (white bars), while the absolute numbers of nonreacting cells (black bars) remained constant at all R/S ratios. This illustrated that increasing the numbers of autoDCs only enhanced the proliferation rate of the autoDC-reactive CD4+ T cells, but did not drive more naive T cells into proliferation. To investigate whether all T cells from the naive repertoire could harbor the www.eji-journal.eu

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Cellular immune response

Table 1. Frequencies of autoDC-reactive CD4+ clones that produce IFN-γ, IL4, or both cytokines in response to autoDCs

Donor 1 Donor 2 Donor 3 Donor 4 Mean

Percentage of IFN-γ only

Percentage of IL-4 only

Percentage of both IFN-γ and IL-4

0 100 46.4 8.3 38.7 ± 45.6

12.5 0 21.4 41.7 18.9 ± 17.6

87.5 0 32.1 50 42.4 ± 36.5

Only the autoDC-reactive CD4+ clones (n = 78) were divided into groups that produced either IFN-γ, IL-4, or both cytokines after overnight autoDC stimulation. IFN-γ and IL-4 release was determined by ELISA. Data are shown as mean ± SD of the four donors.

intrinsic potential to respond to stimulation with autoDCs if stimulated at the right moment, we stimulated the naive CD4+ T cells with autoDCs consecutively (one to ten times) with 24-h intervals for 12 days and quantified the number of autoDC-reactive and nonreacting T cells at day 12. Figure 3B shows that irrespective of the number of repeated stimulations with autoDCs, the absolute number of nonreacting T cells remained constant and was similar to the control that had only been stimulated with autoDCs once (white bar). These findings illustrated that only a restricted subpopulation of naive CD4+ T cells harbored the intrinsic potential to mediate autoDC reactivity.

CD4+ T-cell-mediated autoDC recognition is TCR/CD3 dependent and generally triggers cytokine production To further investigate the nature of the autoDC reactivity, we clonally isolated and expanded CD4+ T cells mediating profound autoDC reactivity after primary stimulation by flow cytometric cell sorting based on PKH26 staining. As a control, we also isolated CD8+ T cells showing a proliferative response upon autoDC stimulation. The obtained CD4+ and CD8+ T-cell clones were screened for reactivity against autoDCs by measuring IFN-γ and IL-4 production. In conjunction with previous analyses in the bulk sorted CD8+ populations after secondary stimulation, none of the 175 growing CD8+ clones was reactive against autoDCs. In contrast, 78 of 280 CD4+ T-cell clones, which corresponded to a frequency of 31 ± 27% (n = 4 donors), showed autoDC reactivity upon secondary stimulation, resulting in IFN-γ and/or IL-4 production. In Figure 4A, the total number of CD4+ clones screened, the frequency, and the reactivity patterns of the reactive CD4+ clones are shown for each donor. Table 1 shows that the percentages of reactive CD4+ T cell clones producing IFN-γ alone, IL-4 alone, or both cytokines differed between the donors. Although the majority of clones (77 out of 78 tested clones) produced cytokines in response to secondary stimulation with autoDCs, only one of the clones exerted cytotoxic activity against autoDCs (Fig. 4B). To further investigate whether the autoDC reactivity was induced via classical TCR-mediated recognition of peptide/HLA complexes, we analyzed whether it coincided with downregulation of the TCRαβ/CD3 complex. As a positive control, a classical alloreactive  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

HLA-DPB1*03-restricted CD4+ T-cell clone was used. As shown in Figure 4C, the autoDC-reactive clones showed equivalent levels of downregulation of the TCR-αβ/CD3 complex as the control clone upon stimulation with autoDCs. The level of downregulation of the TCR-αβ/CD3 complex differed significantly from the nonstimulated control (p < 0.05). Thus, CD4+ clones mediating autoDC reactivity upon secondary stimulation with autoDCs showed a specific cytokine response and downregulation of the TCR-αβ/CD3 complex, but the vast majority (77 out of 78 tested clones) did not exert cytotoxic activity against autoDCs.

AutoDC-reactive CD4+ T cells show myeloid lineage directed recognition in an HLA-restricted manner To investigate whether the autoDC-reactive CD4+ T-cell clones recognized other autologous cell populations, we stimulated these clones with autologous cells from various cell lineages at different R/S ratios and measured the cytokine production. Representative data of 13 clones from two donors are shown in Figure 5A. No or minimal reactivity as measured by cytokine production, T-cell activation, and TCR downregulation was detected in response to stimulation with (activated) B cells, PHA-blasts, and Epstein–Barr virus transformed B-cell lines (EBV-LCLs), whereas some T-cell clones showed reactivity against autologous monocytes and immature DCs, with the highest reactivity against the most mature cells (Fig. 5A and data not shown). To exclude that this reactivity was directed against soluble factors that were introduced during maturation and may have been processed and presented in HLA molecules of the autoDCs, the clones were tested against autologous EBV-LCLs pretreated with GM-CSF and IL-4 or the complete DC maturation cocktail. No reactivity was observed against auto-EBV-LCLs with or without cytokine pretreatment, thereby excluding the possibility of recognition of DC maturation cytokines (Fig. 5B). To investigate whether autoDC recognition was HLA-restricted, autoDC-reactive clones were tested against autoDCs in the presence or absence of different HLA class II blocking mAbs. For donor 4, autologous immature DCs were used because of insufficient amounts of monocytes to generate mature DCs. Representative data for three clones are shown in Figure 5C. Cytokine production in response to autoDC stimulation was clearly blocked with anti-HLA class II blocking mAb for all clones. Furthermore, representative examples of specific blocking with mAbs against HLA-DP, -DQ, or -DR are shown in Figure 5C. In total, 9.0, 6.4, and 36% of the reactive CD4+ clones (n = 78) were found to be HLA-DP, -DQ, and -DR restricted, respectively. For the remaining 49% of clones whose response could be blocked with pan-class II, the HLA restriction could not clearly be determined. To confirm the HLA restriction, we tested the autoDC-reactive clones for recognition of a panel of allogeneic DCs (alloDCs), which were matched with the autoDCs for one or more HLA class II molecules. Representative data of three HLA-DP, -DQ, or www.eji-journal.eu

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Figure 4. CD4+ T-cell-mediated autoDC recognition is TCR/CD3-dependent and generally induces cytokine production. (A) CD4+ clones that produced measurable amounts of IFN-γ and/or IL-4 in response to overnight autoDC stimulation (at a 1/5 R/S ratio) were scored as autoDCreactive. IFN-γ and IL-4 secretion by autoDC-reactive CD4+ clones was measured by ELISA. Cumulative IFN-γ (closed circle) and IL-4 (open triangle) release (pg/mL) is shown, and the total numbers of CD4+ clones tested and frequencies of reactive CD4+ clones are listed (bottom). Clones from four donors were tested independently. (B) Cytotoxic potential of reactive CD4+ clones was determined by exposing a constant number of PKH26labeled autoDCs to the clones at different effector/target (E/T) ratios for 24 h. Percentages of lysed target cells are shown. A total of 14 CD4+ clones from two donors are shown, of the 78 clones tested independently. The cytotoxic autoDC-reactive CD4+ clone 1 from donor 2 is shown with open squares. (C) Three different autoDC-reactive CD4+ clones (clone 9 from donor 1, clone 1 from donor 2, and clone 3 from donor 2) were stimulated with autoDCs at a 1/4 R/S ratio for 5 h. TCR-αβ and CD3 expression was analyzed by flow cytometry. As a positive control, a classical cytotoxic HLA-DPB1*03-restricted CD4+ T-cell clone was used. The percentage (mean + SD) of TCR-αβ and CD3 downregulation, calculated as mentioned in the Materials and methods, is shown. Results are pooled from two independent experiments using three different autoDC-reactive CD4+ clones. Statistical differences relative to the nonstimulated control are shown for two-tailed paired Student’s t-test. *p < 0.05.

-DR-restricted clones of the 40 reactive CD4+ clones tested are shown in Figure 5D, showing HLA-DPB1*03:01, DQB1*06:02, and HLA-DRB1*01:01-restricted reactivity, respectively. Table 2 shows that HLA-DP, -DQ, and -DR-restricted autoDC-reactive clones could be isolated from the same individual. The relatively diverse TCR variable β (TCR Vβ) usage of these clones indicated polyclonality, which was in conjunction with the TCR Vβ usage analyses in the bulk sorted autoDC-reactive cells (data not shown). These findings demonstrated that autoDC reactivity was a common phenomenon mediated predominantly by a polyclonal CD4+ T-cell population mediating an HLA-restricted and myeloid lineage directed response.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Discussion In this study, we elucidated the immunological basis and nature of T-cell reactivity against autoDCs that appears to be a common phenomenon. We reproducibly observed that a small population of polyclonal naive and memory CD4+ T cells harbored the potential to mediate a proliferative response upon stimulation with monocyte-derived autoDCs. These autoDC-reactive CD4+ T cells exerted clear HLA-restricted reactivity against autoDCs and this reactivity appeared to be selectively directed against cells of the myeloid lineage. Although profound cytokine production by autoDC-reactive CD4+ T cells was observed, this did not coincide with overt cytotoxicity against autoDCs. In contrast to the www.eji-journal.eu

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Cellular immune response

Figure 5. HLA-restricted recognition of autologous and allogeneic HLA-matched myeloid APCs by autoDC-reactive CD4+ clones. (A) Specificity of reactive CD4+ clones was determined by stimulation with various autologous myeloid (mature DC, immature DC, and monocytes) and nonmyeloid (B cells and EBV-LCL) cells at different R/S ratios. IFN-γ and IL-4 release was measured by ELISA. IFN-γ (closed circle) and IL-4 (open triangle) release in pg/mL is shown for 1 clone from donor 1 and 12 clones from donor 3 from one experiment. (B) Reactive CD4+ clones were stimulated with different amounts of autoEBV-LCLs with or without pretreatment with GM-CSF and IL-4 or DC maturation cytokine mixture for 2 days. Reactivity was assessed by measurement of IFN-γ (left) and IL-4 (right) release by ELISA. One representative experiment of the two experiments is shown for seven clones from donor 3. (C) To determine HLA restriction of reactive CD4+ clones, clones were stimulated with autoDCs (at a 1/5 R/S ratio) in the presence or absence of blocking mAbs against HLA class II, HLA-DP, -DQ, or -DR. Cumulative IFN-γ production (pg/mL) was measured by ELISA. Representative data are shown for 3 of the 78 clones. (D) Reactive CD4+ clones were stimulated with alloDCs matched for one or more HLA class II molecules (at a 1/5 R/S ratio) for 24 h. Cumulative IFN-γ production (pg/mL) was measured by ELISA. Representative data are shown for 3 of the 40 tested clones.

responses seen in the CD4 compartment, the autoDC-induced proliferation of CD8+ T cells appeared to be mainly cytokine-driven rather than induced by TCR-mediated recognition of autoDCs. Similar results were obtained with autoDCs derived from CD34+ progenitor cells (data not shown).  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In agreement with previous studies on autologous mixed lymphocyte reaction, autoDC reactivity was mainly mediated by CD4+ T cells [14–16, 29]. We demonstrated that the initial autoDCinduced proliferation of most CD8+ and few CD4+ T cells was in fact driven by γc- or DC-derived cytokines or cytokines released www.eji-journal.eu

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Table 2. Characteristics of autoDC-reactive CD4 + clones

Block with mAb

Number of clones

Clone

HLAa)

TCR Vβ

IFN-γ

IL-4

DP and pdV5.2

7

9 (donor 1) 24 (donor 1) p13 (donor 3) 12 (donor 4) 13 (donor 4) 65 (donor 4) 95 (donor 4)

DPB1*03:01 DPB1*03:01 DPB1*03:02 DPB1*04:02 DP DPB1*04:02 DPB1*04:02

7.2 4 5.2 ndc) 2 nd 5.1

+b) +

+ + + + + + +

1 (donor 2) 3 (donor 2) 9 (donor 3) 5 (donor 4) 98 (donor 4)

DQ DQB1*06:02 DQB1*05:01 DQ DQB1*06:02

nd nd nd nd 5.1

1 (donor 3) 2 (donor 3) 3 (donor 3) 6 (donor 3) 9 (donor 3) 12 (donor 3) 22 (donor 3) 33 (donor 3) 56 (donor 3) p9 (donor 3) p33 (donor 3) p37 (donor 3) p72 (donor 3) p78 (donor 3) p110 (donor 3) 20 (donor 4) 24 (donor 4) 36 (donor 4) 38 (donor 4) 39 (donor 4) 43 (donor 4) 45 (donor 4) 59 (donor 4) 68 (donor 4) 70 (donor 4) 87 (donor 4) 97 (donor 4) 101 (donor 4)

DRB1*04:01 DRB1*04:01 DRB1*01:01 DRB1*01:01 DRB4*01:03 DRB1*01:01 DRB4*01:03 DRB1*01:01 DRB4*01:03 DRB1*01:01 DRB1*04:01 DRB1*04:01 DRB1*01:01 DRB1*01:01 DRB1*04:01 DR DR DR DR DR DRB1*15:01 DR DR DR DR DR DR DR

5.3 5.3 2 13.1 5.3 1 3 20 13.1 5.1 5.1 16 1 1 3 3 nd 3 nd 11 13.1 12 5.2 1 nd 17 12 nd

DQ and pdV5.2

DR and pdV5.2

pdV5.2

5

28

+ + + + + + +

+ + + + + +

+ + + +

+ + + + + + + + + + + + + + + + + + + + + + +

+

+ +

+

+ + + + + + + +

38

a)

HLA restriction was determined as described in the Material and methods. + indicates the cytokine released by CD4+ clone after overnight autoDC stimulation. c) nd: not detected with the TCR Vβ kit. b)

as result of interactions between T cells and autoDCs, thereby mimicking the homeostatic naive T-cell proliferation [19, 30–32]. This explains why a majority of T cells that have been selected based on autoDC-induced proliferation at bulk or clonal level did not show reactivity against autoDCs upon secondary stimulation. We demonstrated the occurrence of autoDC reactivity even under completely serum-free conditions, thereby ruling out the possi-

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

bility that xeno- and allogeneic antigens or serum factors were the stimulating antigens driving the autoDC reactivity observed in our in vitro study. It is plausible that endogenous antigens of myeloid origin are the targets of the observed autoDC reactivity, as DCs abundantly express endogenous antigens in their HLA molecules and as autoDC reactivity was restricted to cells of the myeloid lineage [33]. Alternatively, it may be possible that

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Eur. J. Immunol. 2015. 45: 1030–1042

autologous apoptotic cells taken up by autoDCs were a potential source of self-antigens for triggering autoDC reactivity, as proposed by earlier studies [29, 34]. Although we were not able to investigate T-cell reactivity against primary, naturally occurring, in vivo matured DCs, the presence of autoDC reactivity within the memory T-cell compartment suggests that some autoDC-reactive T cells may have encountered autoDCs and become antigen-experienced in vivo. The observations that autoDC-reactive CD4+ T cells acquire a memory phenotype upon autoDC stimulation and exert HLAdependent recognition of autoDCs suggest that the basis of autoDC reactivity is in fact similar to a regular antigen-specific immune response recognizing a foreign peptide in the context of self-HLA. Because the fine specificities of autoDC-reactive T cells are not yet deduced, detailed analysis of the TCR affinity by peptide titration experiments was not possible. The autoDC-reactive CD4+ T cells do not appear to be of low functional avidity, even though most T cells bearing high-affinity TCR for self-antigens (including TAAs) are likely to be removed within the thymus [7]. Despite their limited cytotoxic activity, autoDC-reactive CD4+ T cells did display sufficient functional avidity to recognize endogenously processed antigen(s) in the context of self-HLA, and can therefore not be called low functional avidity T cells. Therefore, we postulate that these autoDC-reactive T cells could be of intermediate avidity and may have bypassed the negative thymic selection due to limited or lack of presentation of the target antigen within the thymus [35]. In contrast to the harmful autoreactivity, autoDC reactivity was not functionally absent in the presence of the natural regulation of the immune system. This reactivity was inhibited only when Treg cells were numerically superior to the autoDC-reactive CD4+ T cells. This may suggest that autoDC reactivity is not controlled by peripheral tolerance mechanisms under normal conditions and may be a regular process during the initiation of primary T-cell responses at the DC priming site. We hypothesize that the local ratio between DCs, T cells, and Treg cells may determine whether or not autoDC reactivity can be controlled. The functional biological relevance of autoDC reactivity in vivo remains unclear, but is potentially not harmful considering the limited cytotoxic potential of the autoDC-reactive CD4+ T cells. We speculate that autoDC-reactive T cells may serve as helper cells in the induction of primary T-cell responses at the DC priming site. The cytokine production (IFN-γ and/or IL-4) in response to autoDCs may further activate autoDCs or direct the T-cell response toward a Th 1 or Th 2 response. It is also possible that IL-4 production by autoDC-reactive CD4+ T cells stimulates B-cell activation or antibody production. In support of this hypothesis, there are findings reporting that autoreactive T cells generated in autologous mixed lymphocyte reaction can provide help to syngeneic B cells for antibody production [36, 37]. Moreover, it has been demonstrated in murine models that interaction of naive T cells with autoDCs in the absence of cognate antigen could increase the efficiency of immune responses against foreign antigens [38]. However, in the in vitro settings when peptide-loaded autoDCs are used for priming of antigen-specific T-cell responses, these autoDC-reactive CD4+ T cells may complicate specific enrichment of low-frequency  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cellular immune response

antigen-specific responses. These autoDC-reactive CD4+ T cells may outnumber the low-frequency antigen-specific T cells and hamper their specific enrichment. In conclusion, this study shows the existence of CD4+ T cells harboring the potential to exert HLA-restricted and myeloid lineage directed reactivity against autologous and allogeneic HLA-matched cells in the T-cell repertoire of normal individuals. These autoDC-reactive T cells might have biological relevance in vivo by playing a role as helper T cells in the induction of primary T-cell responses at the DC activation site.

Materials and methods Isolation of responder cells Peripheral blood samples were obtained from healthy donors after informed consent. PBMCs were isolated by Ficoll-Isopaque separation and cryopreserved for further use. Cryopreservation was performed with freezing medium made of Iscove’s modified Dulbecco’s medium (IMDM, BioWhittaker, Lonza, Verviers, Belgium), dimethyl sulfoxide (LUMC, Department of clinical pharmacy, The Netherlands), and human serum albumin (HSA, Sanquin Plasma Products, Amsterdam, The Netherlands). Cryopreserved cells were thawed using IMDM supplemented with HSA. CD3-positive T cells were purified from donor PBMCs using pan T isolation kit according to the manufacturer’s instructions (MACS beads, Miltenyi Biotec, Bergisch Gladbach, Germany) using the AutoMACSTM magnetic cell sorter or LS columns (Miltenyi Biotec). Using antiCD45RO microbeads (Miltenyi Biotec), CD45RO-positive memory T cells were separated from CD45RO-negative T cells that were further enriched for CD27-positive naive T cells with anti-CD27 microbeads (Miltenyi Biotec). Depletion of CD25high T cells was performed with MACS CD25 microbeads (Miltenyi Biotec). MACS buffer contained HSA. Purity of the isolated T-cell subsets varied between 95 and 99%.

Generation of stimulator cells Monocytes were isolated using magnetic CD14 CliniMACS beads (Miltenyi Biotec) according to the manufacturer’s instructions, and transformed into immature DCs by culturing for 2 days at a concentration of 106 cells/mL in IMDM containing 10% heat-inactivated human serum supplemented with 100 ng/mL granulocyte-monocyte colony-stimulating factor (GM-CSF; Novartis, Basel, Switzerland) and 500 IU/mL IL-4 (kindly provided by Schering-Plough, Innishammon, Cork, Ireland). To generate mature DCs, immature DCs were further matured for 2 days in IMDM supplemented with a DC maturation cytokine mix containing 100 ng/mL GM-CSF, 10 ng/mL IL-1β (Cellgenix, Freiburg, Germany), 10 ng/mL IL-6 (Cellgenix), 10 ng/mL TNF-α (Boehringer Ingelheim, Alkmaar, The Netherlands), 500 IU/mL IFN-γ (Immukine, Boehringer Ingelheim), and 1 μg/mL prostaglandin E2 (PGE2; Sigma Aldrich, Zwijndrecht, The www.eji-journal.eu

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Netherlands). In specific experiments, DCs were matured under serum-free conditions using Cellgro DC medium (Cellgenix). Stable EBV-LCLs were generated using standard protocols and were cultured in IMDM supplemented with 10% heat-inactivated fetal bovine serum (FBS, Lonza, Basel, Switzerland). Phytohemagglutinin (Murex Biotech Limited, Dartfort, UK) stimulated T cells (PHA blasts) were generated using standard procedures, and were cultured in IMDM supplemented with 5% heat-inactivated human serum, 5% heat-inactivated FBS, and 100 IU/mL IL-2 (Chiron, Amsterdam, The Netherlands).

Proliferation assay Naive T cells were labeled with PKH26 dye according to the manufacturer’s instructions (Sigma Aldrich), seeded at a concentration of 1–5 × 105 cells/mL, and stimulated with irradiated (25 Gy) autoDCs in IMDM containing 10% heat-inactivated human serum, IL-7 (Biosource/Invitrogen, Breda, The Netherlands), and/or IL-15 (Biosource) at various concentrations in 96-wells U-bottom plates. Cells were counterstained with anti-TCR-αβ-fluorescein isothiocyanate (FITC, BD Biosciences, Breda, The Netherlands), anti-CD3-allophycocyanin (BD Biosciences), anti-CD4-FITC (BD Biosciences), anti-CD3-PerCP (BD Biosciences), and/or anti-CD8allophycocyanin (BD Biosciences). For quantitative flow cytometric analysis, a fixed amount of fluorescent microspheres (Flow-Count Fluorospheres; Beckman Coulter, Woerden, The Netherlands) was added to each sample just prior to the analysis as described previously [39]. Samples were acquired with a FACS Calibur (BD), and data were analyzed using Cellquest (BD) and FlowJo software (Tree Star, Ashland, OR, USA). For each sample, the absolute numbers of specific cell populations in the quantitative analyses were calculated with the following formula: ((numbers of beads added/numbers of beads acquired) × numbers of cells acquired). The frequency of autoDC-reactive T cells was calculated using the proliferation wizard in the FlowJo software following the approach described by Tapirdamaz et al. [40]. Serum-free cell culture in X-VIVO15 medium (Lonza) was used in specific experiments to exclude reactivities against serum antigens. For transwell experiments, corning transwell 96-well microplates (Sigma Aldrich) were used.

Phenotype analysis For phenotypic analyses, the following monoclonal antibodies (mAbs) were used: anti-CD62L-FITC (eBioscience, Vienna, Austria), anti-CD27-FITC, anti-CD45RA-allophycocyanin, antiCD45RO-allophycocyanin, anti-CCR7-FITC, anti-TCR-αβ-FITC, anti-CD28-FITC (BD Biosciences), anti-CD3 PerCP, anti-CD4 PerCP (Biolegend, Uithoorn, The Netherlands), and anti-CD8allophycocyanin (BD Biosciences) or anti-CD8-FITC (BD Biosciences). Cell surface staining was performed at 4°C for 20 min. The TCR Vβ kit (Beckman Coulter) was used to determine TCR Vβ chain usage of the isolated T cells.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Immunol. 2015. 45: 1030–1042

Isolation of autoDC-reactive T-cell lines and clones and Treg cells Purified naive T cells were stained with PKH26 and seeded at a concentration of 106 cells/mL in IMDM, supplemented with 10% heat-inactivated human serum, 5 ng/mL IL-7, and 0.1 ng/mL IL15. Irradiated (25 Gy) autoDCs were added at a 1/1 R/S ratio. After 7–11 days of culture, cells were stained with anti-TCRγ/δ-phycoerythrin (PE, BD), anti-CD4-FITC, and/or anti-CD8allophycocyanin to isolate TCR-α/β-positive (TCR-γ/δ negative), CD4+ , or CD8+ T cells showing proliferation (as visualized by PKH26 dye dilution) in response to autoDC stimulation using flow cytometric cell sorting on a FACSAria (BD). Bulk populations were sorted by two-way sorting of autoDC-reactive (PKH26dim) CD4+ or CD8+ , TCR-α/β-positive (TCR-γ/δ-negative) T cells. Directly after cell sorting, these reactive CD4+ and CD8+ populations were relabeled with PKH26 and tested for their autoDC reactivity by secondary stimulation with autoDCs at an R/S ratio of 1/3 for 7 days in IMDM, supplemented with 10% heat-inactivated human serum, 5 ng/mL IL-7, and 0.1 ng/mL IL-15. For the generation of T-cell clones, cells were sorted one cell per well into 96-well U-bottom microtiter plates containing 100 μL allogeneic feeder mixture consisting of IMDM supplemented with 5% heatinactivated FBS, 5% heat-inactivated human serum, 5 × 104 irradiated (50 Gy) allogeneic feeder cells (PBMCs), 5000 irradiated (50 Gy) allogeneic EBV-LCL, 100 IU/mL IL-2, and 800 μg/mL PHA. Medium of the T-cell clones was refreshed twice per week and the T-cell clones were restimulated every 3 weeks using allogeneic feeder mixture. T-cell clones were used for functional analyses at 2–3 weeks after restimulation. To isolate naturally occurring Treg cells and CD4+ CD25low control population, cells were stained with anti-CD4-pacific blue (BD Biosciences), anti-CD127FITC (BD Biosciences), and anti-CD25-PE (BD Biosciences), and isolated by flow cytometric cell sorting using the FACSAria (BD). Purity was confirmed postisolation by intracellular staining with anti-Foxp3-allophycocyanin (eBiosciences) as described previously [21].

Analysis of T-cell reactivity For analysis of cytokine production, 5000 T cells were cocultured with stimulator cells at different R/S ratios in IMDM, supplemented with 10% human serum and 25 IU/mL IL-2. After 24 h, supernatants were harvested and concentrations of IFN-γ and IL-4 were measured by enzyme-linked immunosorbent assay (ELISA, Sanquin Reagents). For analysis of TCR-αβ and CD3 downregulation, PKH26-labeled T cells were stimulated with autoDCs at a 1/4 R/S ratio. After 5 h of stimulation, TCR/CD3 downregulation was determined using flow cytometric analysis. The percentage of downregulation was calculated as follows: ((mean fluorescence intensity (MFI) of nonstimulated cells − MFI of stimulated cells)/MFI of nonstimulated cells) × 100%. To analyze the HLA restriction of the autoDC reactivity of the T-cell clones, blocking assays were performed using anti-HLA www.eji-journal.eu

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blocking antibodies. AutoDCs were preincubated with saturating concentrations of anti-HLA class II (pdV5.2), anti-HLA-DP (B7.21), anti-HLA-DQ (SPV-L3), or anti-HLA-DR (B8.11.2) mAbs (kindly provided by A. Mulder, IHB, LUMC, The Netherlands) for 1 h at room temperature before addition of the T cells.

Cellular immune response

5 De Visser, K. E., Schumacher, T. N. and Kruisbeek, A. M., CD8+ T cell tolerance and cancer immunotherapy. J. Immunother. 2003. 26: 1–11. 6 Sebzda, E., Mariathasan, S., Ohteki, T., Jones, R., Bachmann, M. F. and Ohashi, P. S., Selection of the T cell repertoire. Annu. Rev. Immunol. 1999. 17: 829–874. 7 Starr, T. K., Jameson, S. C. and Hogquist, K. A., Positive and negative selection of T cells. Annu. Rev. Immunol. 2003. 21: 139–176.

Flow cytometry based cytotoxicity assay

8 Brichard, V., Van, P. A., Wolfel, T., Wolfel, C., De, P. E., Lethe, B., Coulie, P. et al., The tyrosinase gene codes for an antigen recognized by autologous

PKH26-labeled autoDCs were exposed to effector cells at various effector/target (E/T) ratios in IMDM supplemented with 10% heat-inactivated human serum and 25 IU/mL IL-2. After 24 h, samples were stained with anti-TCR-αβ-FITC (BD Biosciences) and anti-CD3-allophycocyanin (BD Biosciences) for 20 min at 4ºC and quantitative FACS analysis was performed to allow calculation of the absolute numbers of surviving target cells and the percentage of specific target cell lysis [39]. The percentages of specific target cell lysis were calculated as follows: ((absolute numbers of viable target cells in control − absolute numbers of viable target cells after coincubation with T cells)/absolute numbers of viable target cells in control) × 100%.

cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 1993. 178: 489–495. 9 Visseren, M. J., van, E. A., van der Voort, E. I., Ressing, M. E., Kast, W. M., Schrier, P. I. and Melief, C. J., CTL specific for the tyrosinase autoantigen can be induced from healthy donor blood to lyse melanoma cells. J. Immunol. 1995. 154: 3991–3998. 10 Scheibenbogen, C., Letsch, A., Thiel, E., Schmittel, A., Mailaender, V., Baerwolf, S., Nagorsen, D. et al., CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia. Blood 2002. 100: 2132–2137. 11 Molldrem, J., Dermime, S., Parker, K., Jiang, Y. Z., Mavroudis, D., Hensel, N., Fukushima, P. et al., Targeted T-cell therapy for human leukemia: cytotoxic T lymphocytes specific for a peptide derived from proteinase 3 preferentially lyse human myeloid leukemia cells. Blood 1996. 88: 2450– 2457.

Statistical analysis

12 Quintarelli, C., Dotti, G., De, A. B., Hoyos, V., Mims, M., Luciano, L., Heslop, H. E. et al., Cytotoxic T lymphocytes directed to the preferentially expressed antigen of melanoma (PRAME) target chronic myeloid

Statistical evaluation of the data was performed using the twotailed paired Student’s t-test or Mann–Whitney U test.

leukemia. Blood 2008. 112: 1876–1885. 13 Fujiwara, H., El, O. F., Grube, M., Price, D. A., Rezvani, K., Gostick, E., Sconocchia, G. et al., Identification and in vitro expansion of CD4+ and CD8+ T cells specific for human neutrophil elastase. Blood 2004. 103: 3076–3083. 14 Opelz, G., Kiuchi, M., Takasugi, M. and Terasaki, P. I., Autologous stimulation of human lymphocyte subpopulation. J. Exp. Med. 1975. 142: 1327–

Acknowledgments: We thank Arend Mulder for providing the blocking mAbs against HLA class II molecules and Guido de Roo and Sabrina Veld for flow cytometric cell sorting. This work was financially supported by the “Landkroon Fellowship van het Doelfonds Leukemie van de Bontius stichting.”

1333. 15 Crow, M. K. and Kunkel, H. G., Human dendritic cells: major stimulators of the autologous and allogeneic mixed leucocyte reactions. Clin. Exp. Immunol. 1982. 49: 338–346. 16 Scheinecker, C., Machold, K. P., Majdic, O., Hocker, P., Knapp, W. and Smolen, J. S., Initiation of the autologous mixed lymphocyte reaction requires the expression of costimulatory molecules B7-1 and B7-2 on human peripheral blood dendritic cells. J. Immunol. 1998. 161: 3966–3973.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

17 Moser, J. M., Sassano, E. R., Leistritz, D. C., Eatrides, J. M., Phogat, S., Koff, W. and Drake, D. R., III, Optimization of a dendritic cell-based assay for the in vitro priming of naive human CD4+ T cells. J. Immunol. Methods 2010. 353: 8–19. 18 Nussenzweig, M. C. and Steinman, R. M., Contribution of dendritic cells

References

to stimulation of the murine syngeneic mixed leukocyte reaction. J. Exp. Med. 1980. 151: 1196–1212.

1 Nanda, N. K. and Sercarz, E. E., Induction of anti-self-immunity to cure cancer. Cell 1995. 82: 13–17. 2 Houghton, A. N. and Guevara-Patino, J. A., Immune recognition of self in immunity against cancer. J. Clin. Invest 2004. 114: 468–471. 3 Restifo, N. P., Dudley, M. E. and Rosenberg, S. A., Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 2012. 12: 269–281.

19 Ge, Q., Palliser, D., Eisen, H. N. and Chen, J., Homeostatic T cell proliferation in a T cell-dendritic cell coculture system. Proc. Natl. Acad. Sci. USA 2002. 99: 2983–2988. 20 Danke, N. A., Koelle, D. M., Yee, C., Beheray, S. and Kwok, W. W., Autoreactive T cells in healthy individuals. J. Immunol. 2004. 172: 5967–5972. 21 Jedema, I., van de Meent, M., Pots, J., Kester, M. G., van der Beek, M. T. and Falkenburg, J. H., Successful generation of primary virus-specific

4 Schetelig, J., Kiani, A., Schmitz, M., Ehninger, G. and Bornhauser, M., T

and anti-tumor T-cell responses from the naive donor T-cell repertoire

cell-mediated graft-versus-leukemia reactions after allogeneic stem cell

is determined by the balance between antigen-specific precursor T cells

transplantation. Cancer Immunol. Immunother. 2005. 54: 1043–1058.

and regulatory T cells. Haematologica 2011. 96: 1204–1212.

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eji-journal.eu

1041

1042

Tin S. Lam et al.

Eur. J. Immunol. 2015. 45: 1030–1042

22 Ota, K., Matsui, M., Milford, E. L., Mackin, G. A., Weiner, H. L. and Hafler,

34 Amel Kashipaz, M. R., Huggins, M. L., Powell, R. J. and Todd, I., Human

D. A., T-cell recognition of an immunodominant myelin basic protein

autologous mixed lymphocyte reaction as an in vitro model for autore-

epitope in multiple sclerosis. Nature 1990. 346: 183–187.

activity to apoptotic antigens. Immunology 2002. 107: 358–365.

23 Pette, M., Fujita, K., Kitze, B., Whitaker, J. N., Albert, E., Kappos,

35 Stoeckle, C., Quecke, P., Ruckrich, T., Burster, T., Reich, M., Weber, E.,

L. and Wekerle, H., Myelin basic protein-specific T lymphocyte lines

Kalbacher, H. et al., Cathepsin S dominates autoantigen processing in

from MS patients and healthy individuals. Neurology 1990. 40: 1770– 1776. 24 Filion, M. C., Proulx, C., Bradley, A. J., Devine, D. V., Sekaly, R. P., Decary, F. and Chartrand, P., Presence in peripheral blood of healthy individuals of autoreactive T cells to a membrane antigen present on bone marrowderived cells. Blood 1996. 88: 2144–2150. 25 Engler, J. B., Undeutsch, R., Kloke, L., Rosenberger, S., Backhaus, M., Schneider, U., Egerer, K. et al., Unmasking of autoreactive CD4 T cells by depletion of CD25 regulatory T cells in systemic lupus erythematosus. Ann. Rheum. Dis. 2011. 70: 2176–2183. 26 Inaba, K., Pack, M., Inaba, M., Sakuta, H., Isdell, F. and Steinman, R. M., High levels of a major histocompatibility complex II-self peptide complex on dendritic cells from the T cell areas of lymph nodes. J. Exp. Med. 1997. 186: 665–672. 27 Scheinecker, C., McHugh, R., Shevach, E. M. and Germain, R. N., Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. J. Exp. Med. 2002. 196: 1079–1090.

human thymic dendritic cells. J. Autoimmun. 2012. 38: 332–343. 36 Finnegan, A., Needleman, B. and Hodes, R. J., Activation of B cells by autoreactive T cells: cloned autoreactive T cells activate B cells by two distinct pathways. J. Immunol. 1984. 133: 78–85. 37 Finnegan, A., Needleman, B. W. and Hodes, R. J., Function of autoreactive T cells in immune responses. Immunol. Rev. 1990. 116: 15–31. 38 Stefanova, I., Dorfman, J. R. and Germain, R. N., Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes. Nature 2002. 420: 429–434. 39 Jedema, I., van der Werff, N. M., Barge, R. M., Willemze, R. and Falkenburg, J. H., New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population. Blood 2004. 103: 2677–2682. 40 Tapirdamaz, O., Mancham, S., van der Laan, L. J., Kazemier, G., Thielemans, K., Metselaar, H. J. and Kwekkeboom, J., Detailed kinetics of the direct allo-response in human liver transplant recipients: new insights from an optimized assay. PLoS One 2010. 5: e14452.

28 Yan, J. and Mamula, M. J., Autoreactive T cells revealed in the normal repertoire: escape from negative selection and peripheral tolerance. J. Immunol. 2002. 168: 3188–3194. 29 Chernysheva, A. D., Kirou, K. A. and Crow, M. K., T cell proliferation induced by autologous non-T cells is a response to apoptotic cells processed by dendritic cells. J. Immunol. 2002. 169: 1241–1250. 30 Kieper, W. C. and Jameson, S. C., Homeostatic expansion and phenotypic conversion of naive T cells in response to self peptide/MHC ligands. Proc. Natl. Acad. Sci. USA 1999. 96: 13306–13311. 31 Geginat, J., Sallusto, F. and Lanzavecchia, A., Cytokine-driven proliferation and differentiation of human naive, central memory, and effector

Abbreviations: autoDC: autologous monocyte-derived dendritic cell · EBV-LCL: Epstein–Barr virus transformed B-cell line · γc: gamma chain · HSA: human serum albumin · R/S: responder to stimulator · TAA: tumor-associated antigen · TCR Vβ: T cell receptor variable β chain Full correspondence: Dr. Inge Jedema, Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, C2R-140, P.O. Box 9600, 2300 RC Leiden, The Netherlands Fax: +31-71-5266755 e-mail: [email protected]

memory CD4(+) T cells. J. Exp. Med. 2001. 194: 1711–1719. 32 Alves, N. L., Hooibrink, B., Arosa, F. A. and van Lier, R. A., IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro. Blood 2003. 102: 2541–2546. 33 Rohn, T. A., Boes, M., Wolters, D., Spindeldreher, S., Muller, B., Langen, H., Ploegh, H. et al., Upregulation of the CLIP self peptide on mature dendritic cells antagonizes T helper type 1 polarization. Nat. Immunol. 2004. 5: 909–918.

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 21/5/2014 Revised: 28/11/2014 Accepted: 19/12/2014 Accepted article online: 26/12/2014

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