Abstract
The idiotypes of B cell lymphomas represent tumor-specific antigens. T cell responses induced by idiotype vaccination in vivo are directed predominantly against CDR peptides, whereas in vitro T cells also recognize framework-derived epitopes. To investigate the mechanisms regulating the specificity of idiotype-specific T cells, BALB/c or B10.D2 mice were immunized with mature dendritic cells loaded with H-2Kd-restricted peptides from influenza hemagglutinin, or from shared (J region) or unique (CDR3) structures of the A20 lymphoma idiotype. Antigen-specific T cells were induced in vivo by the CDR3 and influenza epitopes, but not by the J peptide. Gene expression profiling of splenic regulatory T cells revealed vaccination-induced Treg activation and proliferation. Treg activity involved J epitope-dependent IL-10 secretion and functional suppression of peptide-specific effector T cells. Vaccination-induced in vivo proliferation of transgenic hemagglutinin-specific T cells was suppressed by co-immunization with the J peptide and was restored in CD25-depleted animals. In conclusion, Treg induced by a shared idiotype epitope can systemically suppress T cell responses against idiotype-derived and immunodominant foreign epitopes in vivo. The results imply that tumor vaccines should avoid epitopes expressed by normal cells in the draining lymph node to achieve optimal anti-tumor efficacy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00262-010-0918-x) contains supplementary material, which is available to authorized users.
Keywords: Idiotype, Lymphoma, Regulatory T cell, FoxP3
Introduction
Immunoglobulins contain unique primary sequences that are created by Ig gene rearrangement and may be diversified further by somatic hypermutation. The individual composition of immunogenic epitopes within the variable region of a given immunoglobulin is designated as “idiotype” (Id). Since the Id is an unique feature of the respective B cell clone, it represents an individual tumor-specific antigen of malignant B cell lymphomas [1]. Immunization of patients with indolent B cell lymphomas against their lymphoma-derived Id induces specific immune responses [2–6]. In patients immunized during clinical remission, induction of anti-Id antibodies is correlated with a favorable outcome [7, 8]. The role of Id-specific T cells for efficacious anti-lymphoma immunity is less clear.
In animal models, MHC class II-restricted T cells with specificity for hypermutated Id-derived epitopes exert immunosurveillance against the corresponding malignant B cell clone [9]. Augmentation of cellular anti-Id immunity by active immunization may also prevent tumor progression in experimental lymphoma models [9–11]. In contrast, the murine T cell repertoire is generally tolerant to germ line-encoded immunoglobulin sequences and may also be actively tolerized to individual Id epitopes [12].
We have previously described Id-specific, MHC class I-restricted cytotoxic T cells in lymphoma-bearing patients [13]. More recently, a reverse immunology bioinformatics study has indicated MHC class I-dependent immunosurveillance against individual CDRs of the Ids in human follicular lymphoma patients [14]. In accordance with the murine models, Id vaccination of patients induces in vivo preferentially T cells with specificity for CDR peptides or hypermutated epitopes [3, 15, 16]. In order to investigate the mechanisms governing the specificity of vaccination-induced T cell immunity to Id in a wild-type immune repertoire, we performed comparative immunizations with a highly individual CDR3 epitope and a conserved FR epitope in a murine lymphoma model. A highly immunogenic influenza HA peptide [17] served as a reference standard for the analysis of immunization-induced T effector populations.
Materials and methods
Immunization
1 × 106 DC matured and activated under serum-free conditions [18] were loaded with H-2kd-restricted peptides (Proimmune, Oxford, UK) and injected into the hind paw of BALB/c or B10.D2 mice. For in vivo depletion of Treg, 500 μg of anti-CD25 Ab PC61 (ATCC, Manassas, VA, USA) was injected i.p. 3 days prior to immunization. All in vivo experiments were performed at least three times.
In vivo assays
Ag-specific T cells were analyzed 1 week after immunization by flow cytometry with fluorochrome-labeled Ab (BD Biosciences or eBioscience, both in San Diego, CA, USA) and peptide-H-2Kd multimers (Proimmune).
For in vivo detection of specific cytotoxicity, splenocytes were pulsed with 10 μg/ml antigenic or control peptide for 1 h at 37°C and labeled for 10 min separately with 0.5 or 5 μM CFSE, respectively. 1 × 107 cells of each population were mixed and injected i.v. into recipient mice. After 24 h, peripheral blood was analyzed by flow cytometry.
To measure vaccination-induced T cell proliferation, 1 × 106 CD8+ (CD8 isolation kit, Miltenyi Biotech, Bergisch-Gladbach, Germany), CFSE-labeled (5 μM) clone 4 cells were injected i.v. into congenic CD45.2+ mice 3 days after immunization. Clone 4 T cells (kindly provided by D.J. Morgan, University of Bristol) express a transgenic TCR with specificity for an immunodominant HA peptide [19]. 4 days after adoptive transfer, CD8+CD45.1+CD45.2−, H-2Kd-multimer-positive splenocytes were analyzed for CFSE dilution.
Intracellular staining for IFN-γ
Effector cells were incubated for 5–16 h in the presence of 10 μM peptide and 1 μl/ml GolgiStop (BD Biosciences). In restimulation experiments, H-2Kd T2 cells (kind gift of S. Stevanovic, Tübingen, Germany) pulsed for 24 h with 10 μM peptide were added as stimulators. After staining of cellular surface antigens, cells were fixed with 2% formaldehyde and stained in 0.5% saponin with XMG2 anti-IFN-γ Ab (BD Biosciences).
In vitro restimulation of Ag-specific T cells
Splenocytes were cultured in FCS-supplemented RPMI medium with 10 μg/ml peptide. After 2 days, viable cells were recovered by Ficoll gradient centrifugation and cultured in medium supplemented with 50 U/ml rIL-2 (Peprotech, Rocky Hill, NJ, USA). Cells were analyzed after 7–9 days of restimulation.
In vitro cytotoxicity assay
T2 cells were pulsed with 10 μg/ml peptide overnight and stained with 5 μM CFSE. 104 target cells were incubated with effector T cells in different E:T ratios for 4 h. Cytotoxicity was determined by flow cytometry as the CFSE+/propidium iodide+ cell fraction.
Isolation and IL-10 production of CD4+CD25+ Treg
CD4+CD25+ T cells were isolated from LN or spleen by depletion of non-CD4+ cells and subsequent positive selection of CD25+ cells (Regulatory T Cell Isolation Kit; Miltenyi). After 2 days of coculture of 1 × 106 cells of the CD4− fraction with isolated CD4+CD25+ cells, the IL-10 concentration in the culture supernatant was determined by ELISA (BD Biosciences).
Gene expression profiling of Treg
10 μg of biotin-labeled and fragmented cRNA (MessageA-mpTM II-Biotin Enhanced kit, Ambion AM1791) of splenic CD4+CD25+IL7R− Treg [20] were hybridized to GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA, USA) at 45°C for 16 h. The arrays were washed (FS450_0004 protocol, Fluidics Station FS450, Affymetrix), scanned (GeneChip 3000 7G Scanner, Affymetrix), and converted into CEL files (GeneChip Command Console Software Version 1.0, Affymetrix). CEL files were imported into the Refiner module of Expressionist software 5.1.2 (Genedata, Basel, Switzerland), where RMA background subtraction, quantile normalization, and probe summarization with the median polish activity were performed using the Bioconductor RMA condensing algorithm [21]. Data were then imported into the Analyst module of Expressionist and further normalized by median scaling to an expression value of 200 over all probe sets except the bacterial spike probes. Differentially expressed genes were identified with an unpaired Bayes T test (CyberT) with Bayes Confidence Estimate Value set to 10 and a window size of 101 genes [22]. False-discovery rates were estimated by the method of Benjamini and Hochberg [23]. Median expression ratios between Treg and Tconv were calculated for individual genes by the ‘N-fold regulation’-activity of Analyst. Over- or underrepresentation of certain gene ontologies within selected gene lists in Analyst was identified by Fisher’s exact test with a p value threshold of 0.001 and a property size threshold of 10.
Analysis of TCR repertoires
cDNA was synthesized (Superscript II reverse transcriptase; Invitrogen, Carlsbad, CA, USA) from RNA isolated from CD4+CD25+ splenocytes 1 week after vaccination (RNeasy Kit; Qiagen, Hilden, Germany). 2 μl of cDNA were amplified by PCR with 1 μM V and C primers and 0.1 U/μl of Taq polymerase (Qiagen) with an annealing temperature of 60°C [24]. PCR products were labeled with 6-FAM-labeled 3′ C primer and PFU polymerase (Stratagene) for three cycles using an annealing temperature of 60°C. Denatured labeled PCR products (1.3 μl) were analyzed on an Prism 3110 XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with GeneScan 500 LIZ size standard and Genemapper 4.0 software 4.0 (Applied Biosystems).
Results
CDR3- but not J region-specific T cells are induced in vivo by DC immunization
We identified a H-2Kd-restricted CDR3 peptide (YYCSISGDY) from the published A20 IgH sequence [25] by the BIMAS algorithm (http://www-bimas.cit.nih.gov/molbio/hla_bind/). This peptide has no significant protein homologies as determined by BLAST search. The A20 J peptide DYWGQGTEL [26] contains two amino acids that are assigned to the CDR3 region. However, these residues are non-polymorphic, and a BLAST search of the J peptide yielded hundreds of matches with murine IgH sequences (data not shown).
Single immunization of mice with DC loaded with the HA peptide IYSTVASSL [17] efficiently induced specific effector T cells (Fig. 1a). Immunization with a heteroclitic version [27] of the CDR3 peptide (CDR3het, YYCSISGDL) induced Ag-specific T cells that also recognized the native CDR3 peptide (Fig. 1b). Immunization with the J peptide failed to induce a detectable T cell response (Fig. 1a). Since the J peptide effected the highest H-2Kd stabilization in a T2 binding assay, the differential immunogenicity of the peptides was not attributable to MHC binding (Fig. 1c). Peptide-pulsed target cells were eliminated in vivo in HA- and CDR3-immunized but not in J-immunized animals (Fig. 1d). In contrast, in vitro restimulation expanded Ag-specific T cells with strong specific cytotoxicity from all three immunization groups (Fig. 1a, e).
To explore how dependent the lack of J peptide in vivo immunogenicity was on its precise amino acid sequence, we immunized mice with a mutated J peptide (Jmut; DYWEQTEL). This minor modification resulted in high frequencies of peptide-specific splenocytes (Fig. 1f) and moderate but robust in vivo cytotoxicity of Jmut-induced effectors (Fig. 1g).
DC-based peptide J immunization induces Treg activity in vivo
Since the discrepancy between in vivo and in vitro cytotoxicity after J immunization suggested Treg as a potential suppressor mechanism of J-specific T cells in vivo, we performed gene expression profiling of splenic CD4+CD25+IL7R− Treg [20] (Fig. 2a). The Treg phenotype was validated by the expression of Il2ra (CD25), FoxP3, Tnfrsf18 (GITR), and Ctla4 (Supplemental Table 1). Expression of the 603 genes of the so-called “common Treg signature” [28] correlated with published data in naive mice (r = 0.68, p < 0.0001; Supplemental Table 2). This signature was also stable after DC-based immunization (r = 0.82, p < 10−15; blue dots in Fig. 2b). The Treg-specific expression of genes linked to Treg function (annotated in Fig. 2b) was better preserved after DC-based immunization in vivo than published for direct TCR-mediated Treg stimulation in vitro [28].
A reaction of splenic Treg to DC-based immunization was indicated by significant (q < 0.01; p = 2.2 × 10−7–2.4 × 10−14), at least twofold transcriptional change in 1,266 genes (1,098 up- and 168 downregulated). Immunization-induced transcriptional regulation of 300 of these 1,266 genes was restricted to Treg as indicated by an expression ratio of 0.75–1.25 between splenic conventional T cells (Tconv) from immunized and naive mice (red dots in Fig. 2b). Only 6 of these 300 genes (2%) were members of the common Treg signature, and only two genes (Dock10, Wnk1) were known FoxP3 target genes [29]. However, these 300 genes included the upregulated Treg effector cytokine TGF-beta [30], and testing for enriched gene ontology groups identified post-translational protein modification by phosphatase activity (Ppp1cb, Sbf1, Ptp4a3, Ppp5c), gene silencing by methylation (Dnmt3a, Eif2c2, Eif2c3, Adar, Dnmt1), and inhibition of translation initiation (Eif4ebp2, Eif4ebp3) as processes that were significantly enhanced (Supplemental Table 3).
The transcriptional upregulation of Ly6a in splenic Treg was verified in comparison to the Treg surface markers CD25 and folate receptor 4 [31] by flow cytometry for Tconv and FoxP3+ Treg from various anatomical locations (Fig. 2c, d). Vaccination-induced changes of these markers were most pronounced in the draining LN. Intracellular expression of Ki67, which was upregulated 2.05-fold in splenic Treg after vaccination (p = 3.12 × 10−6, q = 0.13), indicated a proliferative response of Treg to DC-based immunization that was higher in the draining LN than in the spleen (Fig. 2c, d). However, even in the draining LN, we were unable to detect any significant differences between J- or CDR3-immunized mice with respect to phenotype (not shown) or total numbers of CD4+CD25+FoxP3+ Treg (Fig. 3a).
Induction of Treg suppressive function by J peptide immunization
After J immunization, FoxP3+ Treg from the draining LN proliferated spontaneously in vitro (Fig. 3b). In cocultures of CD4+CD25+ T cells with CD4− T cells, substantial amounts of IL-10 were detected after J immunization depending on the cellular proportion (Fig. 3c) and independent of the effector T cell specificity (Fig. 3d). CD4+CD25+ Treg from J-immunized animals suppressed peptide-induced in vitro expansion of CDR3-specific CD8+ splenocytes stronger than CDR3-induced cells (Fig. 3e).
J peptide-induced Treg suppress T cell responses to HA at different stages
HA peptide stimulation induced vigorous in vitro proliferation of naive clone 4 T cells in the presence of CD4− splenocytes from HA- or J-immunized animals (Fig. 4a). CD4+ splenocytes from J-immunized mice suppressed peptide-induced clone 4 T cell proliferation more efficiently than CD4+ splenocytes from HA-immunized mice.
To investigate the effects of co-immunization of the J peptide with the highly immunogenic HA peptide on HA-specific T cell responses in vivo, mice were immunized once with DC co-loaded with both the HA peptide and either the J or CDR3 peptide. HA-specific transgenic clone 4 T cells were adoptively transferred 3 days later. HA-specific T cells proliferated vigorously in mice co-immunized with HA and CDR3 peptide (Fig. 4b). Co-immunization with HA and J resulted in markedly reduced proliferation of HA-specific T cells. Depletion of Treg prior to immunization prevented this inhibition.
Finally, we examined the influence of J peptide co-immunization on the initiation of non-TCR-transgenic HA-specific T cells in wild-type mice in vivo. Mice were either immunized with DC loaded with HA and J peptide, or simultaneously with HA peptide-loaded DC into one leg and J peptide-loaded DC into the contralateral leg. The frequency of HA-specific effector T cells in the draining LN was increased after co-immunization with both peptides at the same location (Fig. 4c). However, HA-specific T cells circulated only when the HA immunization was anatomically separated from the J peptide. These findings raise the possibility that co-immunization with the J peptide did not affect the induction and initial local expansion of HA-specific T cells, but possibly reduce their egress from the LN despite the downregulation of CD62L.
The Treg TCR repertoire after DC-based peptide immunization
The differential activity of Treg upon CDR3- and J-immunization prompted us to search for vaccination-dependent changes of the Treg TCR repertoire. However, there was no detectable difference in the distribution of TCR Vβ families in the draining lymph node (Fig. 5a). In addition, no skewing of the CDR length within Vβ transcripts of splenic Treg became apparent after DC-based immunization (Fig. 5b).
Discussion
Natural Treg have the primary function to control autoreactive T cells in the periphery [32]. Treg may be induced by organ-specific self Ag and recognize such Ag through their TCR [33, 34]. When A20 cells expressing HA as an artificial tumor Ag are transferred into mice with HA-specific, MHC class II-restricted transgenic T cells, immunization against HA expands both HA-specific effector cells and suppressive cells including Treg [35]. A dominance of suppression over effector function prevents therapeutic vaccination efficacy in this model.
In contrast to a predetermined specificity of Treg through a transgenic TCR, our experiments are performed by immunizations with MHC class I-restricted peptides and in a natural T cell repertoire. The development of a serum-free protocol to generate “fully licensed” DC in vitro [18] and the identification of an immunogenic CDR3 peptide of the A20 Id enabled us to interrogate the possible extremes in Id vaccination, i.e. to analyze responses to a unique CDR3 peptide versus a FR4/J peptide expressed by many normal B cells. We identified transcriptional changes induced in Treg by an MHC class I-restricted Ag in wild-type mice and compared these transcriptomes to direct Treg stimulation enforced through a transgenic TCR [28]. Since the common Treg signature was considerably more stable after in vivo Treg activation by DC, our experimental system may recapitulate the behavior of Treg in a natural immune response more accurately. We identified upregulation of known Treg effector and cellular interaction molecules, e.g. granzymes, IL-10, TGF-beta [30], neuropilin [36], CXCR6, CD44, and CD18 [37, 38]. Transcriptional induction of protein dephosphorylation, DNA methylation, and inhibition of translation initiation may also be required for suppressive Treg function in an immunostimulatory environment induced by activated and licensed DC [18].
Only the J peptide effected a proliferation of Treg isolated from the draining LN despite similar phenotypic and transcriptional changes in splenic Treg after both immunizations. In addition, TCR repertoire and transcriptome of Treg were practically indistinguishable after immunization with immunogenic and nonimmunogenic peptides. Therefore, the magnitude rather than a particular quality of the Treg response may determine the outcome of peptide immunization. Consistent with this hypothesis, the vaccine formulation may have a critical impact on the immunization efficacy, since the Treg-inducing J epitope was originally identified as a protective Ag after adenoviral Id vaccination [26].
The peptides used in this study cannot be recognized by the MHC class II-restricted TCR of Treg. Therefore, the J-induced Treg response must involve an indirect mechanism. We assume that the immigrating DCs induce Ag-specific T cells in the draining LN. In the case of the CDR3 and HA peptides, these T cells do not encounter their antigen on other cells and are able to establish systemic immunity. In contrast, J-specific T cells can encounter their cognate antigen on other resident B cells immediately after activation, and the resulting release of self Ags from these B cells could lead to efficient activation of resident Treg.
Treg may suppress specific T cell responses by inhibiting their amplification through proliferation [39] and acting directly at the site of T cell activation [40]. Both levels of inhibition may be operational in our experimental system: J peptide-activated Treg exerted systemic inhibition of Ag-specific T cell proliferation. However, we also find preliminary evidence for an impaired exit of activated CD8+ cells from the draining LN after co-immunization with the J peptide.
In summary, our data identify Treg-mediated peripheral tolerance as a mechanism controlling the specificity of vaccination-induced anti-Id cellular immunity. In this model, a peptide shared between neoplastic and normal cells can suppress effector T cell activity against unique tumor-derived and microbial epitopes. The absence of Treg in in vitro stimulation cultures of isolated CD8+ T cells may also permit recognition of FR peptides [41]; however, this T cell activity would be expected to be suppressed in vivo by Treg. Treg activity may also explain the observed specificity of vaccination-induced T cell responses for CDR and hypermutated epitopes in lymphoma patients [3, 15, 16] and the dominant role of CDR epitopes in Id-directed immunosurveillance despite better HLA-binding capacity of FR peptides [14]. An individual idiotype’s net immunogenicity is presumably dependent on the balance between individual immunogenic epitopes and the suppressive effects of conserved peptides. This critical balance could offer a plausible, albeit difficult to prove, explanation for lack of cellular and perhaps humoral immune responses as observed in individual patients in clinical Id vaccination trials [2–7, 16].
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgments
This work was supported by a grant from Wilhelm Sander-Foundation, Munich (2006.049.1), and by the Federal Ministry of Education and Research (BMBF) for the Freiburg Initiative for Systems Biology FRISYS (0313921). We thank H. Pircher for critical discussions.
Conflict of interest
The authors declare that they have no conflict of interest.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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