Characterization of T Cell Receptors of Th1 Cells Infiltrating Inflamed Skin of a Novel Murine Model of Palladium-Induced Metal Allergy

Metal allergy is categorized as a delayed-type hypersensitivity reaction, and is characterized by the recruitment of lymphocytes into sites of allergic inflammation. Because of the unavailability of suitable animal models for metal allergy, the role of T cells in the pathogenesis of metal allergy has not been explored. Thus, we developed a novel mouse model for metal allergy associated with infiltration of T cells by multiple injections of palladium (Pd) plus lipopolysaccharide into the footpad. Using this model, we characterized footpad-infiltrating T cells in terms of phenotypic markers, T cell receptor (TCR) repertoires and cytokine expression. CD3+ CD4+ T cells accumulated in the allergic footpads 7 days after Pd challenge. The expression levels of CD25, interleukin-2, interferon-γ and tumor necrosis factor, but not interleukin-4 and interleukin-5, increased in the footpads after challenge, suggesting CD4+ T helper 1 (Th1) cells locally expanded in response to Pd. Infiltrated T cells in the footpads frequently expressed AV18-1 and BV8-2 T cell receptor (TCR) chains compared with T cells in the lymph nodes and exhibited oligoclonality. T-cell clones identified from Pd-allergic mouse footpads shared identical CDR3 sequences containing AV18-1 and BV8-2. These results suggest that TCR AV18-1 and BV8-2 play dominant and critical parts in the antigen specificity of Pd-specific Th1 cells.     

T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-idiotypic antibody

The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN- production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN- T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.     

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.     

Surprisingly minor influence of TRAV11 (Vα14) polymorphism on NK T-receptor mCD1/α-galactosylceramide binding kinetics

Defects in natural killer T (NK T) cell function and of interleukin-4 -production in SJL and NOD mice have been linked to susceptibility to autoimmune disease. As SJL and NOD mice both carry the T-cell receptor (TCR) alpha-chain locus "c" (Tcra(c)) haplotype, found in few other strains, we have attempted to determine the influence of Tcra polymorphism on NK T-cell recognition of ligand, selection, and immune responses. The majority of NK T cells use an "invariant" TRAV11J15 (previously called AV14J18 or Valpha14 Jalpha281) alpha- chain paired with either TRBV13-2, BV29, or BV1 to recognize ligands presented by mCD1 molecules, including the glycolipid alpha-galactosylceramide (alpha-GalCer). Sequencing of TRAV11 from the mouse strains B10.A (encoding the Tcra(b) haplotype), B10.A- Tcra(c), and NOD (Tcra(c)) shows that Tcra(c) has a single TRAV11 gene (TRAV11*01) and that Tcra(b) has a single expressed gene (TRAV11*02), plus a closely related pseudogene. There is no apparent difference in alpha-chain J-region usage or in the CDR3alpha sequence at the TRAV11-J15 junction between the haplotypes in TRAV11-bearing NK T cells. Using Biacore and tetramer-binding and decay assays, we have determined that the interaction between Tcra(c) TRAV11*01 NK T TCR and the mCD1/alpha-GalCer complex is slightly weaker than that of Tcra(b) (i.e., TRAV11*02) NK T TCR. These differences are minor compared with differences between agonist and antagonist ligands in other TCR systems, suggesting that it is unlikely that TCR polymorphism explains the defect in NK T cells in the autoimmune mouse strains.     

Cross-reactivity of T-cell clones specific for altered peptide ligands of myelin basic protein

We have determined that certain altered peptide ligands (APLs) can induce T-cells specific for the native peptide myelin basic protein (MBP) p85-99 to secrete Th2-type cytokines such as IL-4 and IL-5 in the absence of significant Th1-type cytokines. However, it is not known whether stimulation with APLs will activate autoreactive T cells or a distinct population of cells. In the present study, 18 T-cell clones that reacted with either MBP p85-99 or one of three APLs of the peptide substituted at TCR contact residues were generated. T-cells were tested functionally for their reactivity to the original stimulating peptide as well as to the MBP APLs. In addition, the T-cell receptor (TCR) alpha and beta chains of each of these clones were sequenced. In a series of T-cell clones isolated from a multiple sclerosis patient, stimulation of T-cells with the APL 93A, which has an alanine for lysine substitution at the TCR contact residue 93, did not induce substantial proliferation of MBPp85-99-specific T-cell clones, indicating that a distinct set of T-cell clones was induced. However, this was not the case for another set of T-cell clones from a different individual in which the 93A peptide induced clonal expansion of T-cells highly reactive with the native MBPp85-99 antigen. Thus, the potential beneficial effect of using APLs to induce downregulatory cytokines appears to depend on the specific T-cell repertoire of the individual patient.     

Relationship between CD8-dependent antigen recognition,T cell functional avidity,and tumor cell recognition

Effective immunotherapy using T cell receptor (TCR) gene-modified T cells requires an understanding of the relationship between TCR affinity and functional avidity of T cells. In this study, we evaluate the relative affinity of two TCRs isolated from HLA-A2-restricted, gp100-reactive T cell clones with extremely high functional avidity. Furthermore, one of these T cell clones, was CD4⁻CD8⁻ indicating that antigen recognition by this clone was CD8 independent. However, when these TCRs were expressed in CD8⁻ Jurkat cells, the resulting Jurkat cells recognized gp100:209–217 peptide loaded T2 cells and had high functional avidity, but could not recognize HLA-A2⁺ melanoma cells expressing gp100. Tumor cell recognition by Jurkat cells expressing these TCRs could not be induced by exogenously loading the tumor cells with the native gp100:209–217 peptide. These results indicate that functional avidity of a T cell does not necessarily correlate with TCR affinity and CD8-independent antigen recognition by a T cell does not always mean its TCR will transfer CD8-independence to other effector cells. The implications of these findings are that T cells can modulate their functional avidity independent of the affinity of their TCRs. Companion Paper: “Characterization of MHC class-I restricted TCRαβ⁺ CD4⁻ CD8⁻ double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination” by Simon Voelkl, Tamson V. Moore, Michael Rehli, Michael I. Nishimura, Andreas Mackensen, and Karin Fischer. doi:.     

TCR Peptide Therapy in Human Autoimmune Diseases

Inflammatory Th1 cells reacting to tissue/myelin derived antigens likely contribute to the pathogenesis of diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis. One regulatory mechanism that may be useful for treating autoimmune diseases involves an innate second set of Th2 cells specific for portions of the T cell receptor of clonally expanded pathogenic Th1 cells. These Th2 cells are programmed to respond to internally modified V region peptides from the T cell receptor (TCR) that are expressed on the Th1 cell surface in association with major histocompatibility molecules. Once the regulatory Th2 cells are specifically activated, they may inhibit inflammatory Th1 cells through a non-specific bystander mechanism. A variety of strategies have been used by us to identify candidate disease-associated TCR V genes present on pathogenic Th1 cells, including BV5S2, BV6S5, and BV13S1 in MS, BV3, BV14, and BV17 in RA, and BV3 and BV13S1 in psoriasis. TCR peptides corresponding to the mid region of these BV genes were found to be consistently immunogenic in vivo when administered either i.d. in saline or i.m. in incomplete Freund's adjuvant (IFA). In MS patients, repeated injection of low doses of peptides (100-300 g) significantly boosted the number of TCR-reactive Th2 cells. These activated cells secreted cytokines, including IL-10, that are known to inhibit inflammatory Th1 cells. Cytokine release could also be induced in TCR-reactive Th2 cells by direct cell-cell contact with Th1 cells expressing the target V gene. These findings indicate the potential of regulatory Th2 cells to inhibit not only the target Th1 cells, but also bystander Th1 cells expressing different V genes specific for other autoantigens. TCR peptide vaccines have been used in our studies to treat a total of 171 MS patients (6 trials), 484 RA patients (7 trials), and 177 psoriasis patients (2 trials). Based on this experience in 824 patients with autoimmune diseases, TCR peptide vaccination is safe and well tolerated, and can produce significant clinical improvement in a subset of patients that respond to immunization. TCR peptide vaccination represents a promising approach that is well-suited for treating complex autoimmune diseases.     

Characterization of human immunodeficiency virus type 1 (HIV-1) Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor following in vitro immunization

Substantial evidence argues that human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T cells play an important role in the control of HIV-1 replication in infected individuals. Moreover, it is increasingly clear that an HIV vaccine should elicit potent cytotoxic lymphocyte and antibody responses that will likely require an efficient CD4(+) T-cell response. Therefore, understanding and characterizing HIV-specific CD4(+) T-cell responses is an important aim. Here we describe the generation of HIV-1 Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor by in vitro immunization with HIV-1 Gag peptides. The Gag peptides were able to induce a strong CD4(+) T-cell immune response in peripheral blood mononuclear cells from the HIV-1-seronegative donor. Six Gag peptide-specific CD4(+) T-cell clones were isolated and their epitopes were mapped. The region of p24 between amino acids 201 and 300 of Gag was defined as the immunodominant region of Gag. A new T helper epitope in the p6 protein of Gag was identified. Two clones were shown to recognize Gag peptides and processed Gag protein, while the other four clones reacted only to Gag peptides under the experimental conditions used. Functional analysis of the clones indicated that both Th1 and Th2 types of CD4(+) T cells were obtained. One clone showed direct antigen-specific cytotoxic activity. These clones represent a valuable tool for understanding the cellular immune response to HIV-1, and the study provides new insights into the HIV-1-specific CD4(+) T-cell response and the induction of an anti-Gag and -Gag peptide cellular primary immune response in vitro.     

Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection. TCR V beta 5 transgenic mice lack effector cells able to cause graft rejection.

Lethally irradiated mice reject within 24 h certain marrow grafts, a phenomenon called either allogeneic or hybrid resistance. The cells responsible for this rejection (NK1+ CD3+ cells (TNK) express Ag of NK cells as well as the TCR-associated CD3 complex. This raises the question whether TCR participate in the function of these cells during graft rejection. By using flow cytometry it is shown that the majority of TNK cells expresses the TCR-alpha/beta chains and by using adoptive cell transfer assays evidence is presented that it is the TCR-alpha/beta expressing cells that cause rejection. To explore whether any particular TCR chains have to be expressed on these cells, C57L mice were assayed and found to be responders suggesting that the V beta chains deleted in these mice are not obligatory. However, introduction of a specific TCR V beta 5 chain into C57BL/6 mice as a transgene leads to inability to transfer resistance. TNK cells of V beta 5 transgenic mice express the introduced gene suggesting that it is the transgenic TCR that is responsible for the lack of function. In assessing T cell functions in V beta 5 transgenic mice it is shown that although these mice generate CTL specific for H-2d targets there is a deficiency to recognize H-2Dd, i.e., of determinants presumed to be recognized in the acute rejection mechanism. Thus TNK cells and CTL share the inability to recognize H-2Dd epitopes due to expression of the V beta 5 transgene. The notion that TCR on TNK cells play a role in the acute rejection process makes it necessary to postulate a receptor selection mechanism for these cells.     

Functional and morphologic characterization of human T lymphocytes expressing the TCR gamma /delta

A minor subset of T lymphocytes express a TCR composed of gamma and delta chains. This subset differs from conventional T cells for a number of phenotypic and functional characteristics. TCR gamma/delta+ cells simultaneously lack both CD4 and CD8 antigens. Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which efficiently lyse tumor target cells. Formal proofs have been provided that TCR gamma/delta+ cells are able to recognize antigens. For example, they proliferated in response to allogeneic mixed lymphocyte culture (MLC); in addition, MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The selection of monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets of TCR gamma/delta+ cells. Both of these mABs, termed BB3 and delta TCS-1 respectively, induced specific activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca++ and/or lymphokine production or cytolytic activity). Analysis of the distribution of subsets expressing different forms of TCR gamma/delta, showed that the BB3-reactive form is prevalent in the peripheral blood. In contrast, delta-TCS-1-reactive cells are relatively unfrequent in peripheral blood but represent the majority of TCR gamma/delta+ cells in tissues.     

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.     

Age-associated rapid and Stat6-independent IL-4 production by NK1-CD4+8- thymus T lymphocytes.

The source of IL-4 required for priming naive T cells into IL-4-secreting effectors has not been clearly identified. Here we show that upon TCR stimulation, thymus NK1-CD4+8- T cells produced IL-4, the magnitude of which was inversely correlated with age. This IL-4 production response by Th2-prone BALB/c mice was approximately 9-fold that of Th1-prone C57BL/10 mice. More than 90% of activated NK1-CD4+8- thymocytes did not use the invariant V alpha 14-J alpha 281 chain characteristic of typical CD1-restricted NK1+CD4+ T cells. Stat6-null NK1-CD4+8- thymocytes produced bioactive IL-4, with induction of IL-4 mRNA expression within 1 h of stimulation. Our results support the possibility that TCR repertoire-diverse conventional NK1-CD4+ T cells are a potential IL-4 source for directing naive T cells toward Th2/type 2 CD8+ T cell (Tc2) effector development.     

Chronic soluble antigen sensitization primes a unique memory/effector T cell repertoire associated with th2 phenotype acquisition in vivo.

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vbeta repertoire, different from the TCR Vbeta profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IA(d)-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.     

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restrictions.     

An increased number of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis

Abstract There is evidence that γ/δ TCR + T cells are specialized in recognizing different antigens, but their immunologic role as a second TCR is still unclear. The aim of this study was to investigate the percentage and absolute numbers of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis (CVH) and to compare with HBsAg⁺, HCV healthy carriers and healthy subjects. Forty nine patients with CVH-24 with chronic active (CAH) and 25 with chronic persistent hepatitis (CPH)-, 21 HBsAg⁺, 20 HCV asymptomatic carriers and 20 healthy subjects were enrolled in the study. Lymphocyte subsets were determined after incubation with monoclonal antibodies to T total (CD5) and T γ/δ cells (γ/δ-1) using immunofluorescence microscopy. An increased number of circulating γ/δ TCR + T cells was found in patients with CVH in comparison with asymptomatic carriers and normal controls: this increase was more profound in patients with CAH, compared to CPH patients. These results indicate a correlation between circulating γ/δ TCR + T cells in CVH patients and activity and chronicity of the disease.     

The relationship between predicted peptide-MHC class II affinity and T-cell activation in a HLA-DRbeta1*0401 transgenic mouse model

The HLA-DRB1*0401 MHC class II molecule (DR4) is genetically associated with rheumatoid arthritis. It has been proposed that this MHC class II molecule participates in disease pathogenesis by presenting arthritogenic endogenous or exogenous peptides to CD4+ T cells, leading to their activation and resulting in an inflammatory response within the synovium. In order to better understand DR4 restricted T cell activation, we analyzed the candidate arthritogenic antigens type II collagen, human aggrecan, and the hepatitis B surface antigen for T-cell epitopes using a predictive model for determining peptide-DR4 affinity. We also applied this model to determine whether cross-reactive T-cell epitopes can be predicted based on known MHC-peptide-TCR interactions. Using the HLA-DR4-IE transgenic mouse, we showed that both T-cell proliferation and Th1 cytokine production (IFN-gamma) correlate with the predicted affinity of a peptide for DR4. In addition, we provide evidence that TCR recognition of a peptide-DR4 complex is highly specific in that similar antigenic peptide sequences, containing identical amino acids at TCR contact positions, do not activate the same population of T cells.     

T cell responses in melanoma patients after vaccination with tumor-mRNA transfected dendritic cells

We have developed an individualized melanoma vaccine based on autologous dendritic cells (DCs) transfected with autologous tumor-mRNA. The vaccine targets the unique spectrum of tumor antigens in each patient and may recruit multiple T cell clones. In a recent phase I/II trial, we demonstrated T cell responses against vaccine antigens in 9/19 patients evaluable by T cell assays. Here, we report a follow-up study that was conducted to characterize interesting T cell responses and to investigate the effects of long-term booster vaccination. Two patients were selected for continued vaccine therapy. The clinical follow-up suggested a favorable clinical development in both patients. The immunological data (T cell proliferation/IFNgamma ELISPOT/Bioplex cytokine assays) indicated sustained T cell responses and suggested an enhancing effect of booster vaccinations. Both CD4(+) and CD8(+) T cell responses were demonstrated. From post-vaccination samples, we generated 39 T cell clones that responded specifically to stimulation by mRNA-transfected DCs and 12 clones that responded to mock-transfected DCs. These data clearly indicate a two-component vaccine response, against transfected and non-transfected antigens. T cell receptor (TCR) clonotype mapping, performed on 11 tDC-specific clones, demonstrated that 10/11 clones had different TCRs. The results thus indicate a broad spectrum T cell response against antigens encoded by the transfected tumor-mRNA. We generally observed mixed Th1/Th2 cytokine profiles, even in T cell clones that were confirmed to be derived from a single cell. This finding suggests that cytokine patterns after cancer vaccination may be more complex than indicated by the classic Th1/Th2 dichotomy.     

FcR gamma presence in TCR complex of double-negative T cells is critical for their regulatory function

TCRalphabeta+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells in humans and mice. Our previous study using cDNA microarray analysis of global gene expression showed that FcRgamma was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRgamma-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRgamma-sufficient DN T cells, FcRgamma-deficient DN T cells were unable to prolong donor-specific allograft survival when adoptively transferred to recipient mice. Protein analyses indicate that in addition to FcRgamma, DN Treg cell clones also express higher levels of TCRbeta, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRgamma associates with the TCR complex and that both FcRgamma and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRgamma expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRgamma deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRgamma plays a role in mediating TCR signaling in DN Treg cells.