Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells.

Accumulating evidences indicate that the cellular and molecular microenvironment of follicular lymphoma (FL) has a key role in both lymphomagenesis and patient outcome. Malignant FL B cells are found admixed to specific stromal and immune cell subsets, in particular CD4(pos) T cells displaying phenotypic features of follicular helper T cells (T(FH)). The goal of our study was to functionally characterize intratumoral CD4(pos) T cells. We showed that CXCR5(hi)ICOS(hi)CD4(pos) T cells sorted from FL biopsies comprise at least two separate cell populations with distinct genetic and functional features: (i) CD25(pos) follicular regulatory T cells (T(FR)), and (ii) CD25(neg) T(FH) displaying a FL-B cell supportive activity without regulatory functions. Furthermore, despite their strong similarities with tonsil-derived T(FH), purified FL-derived T(FH) displayed a specific gene expression profile including an overexpression of several genes potentially involved directly or indirectly in lymphomagenesis, in particular TNF, LTA, IL4 or CD40LG. Interestingly, we further demonstrated that these two last signals efficiently rescued malignant B cells from spontaneous and rituximab-induced apoptosis. Altogether, our study demonstrates that tumor-infiltrating CD4(pos) T cells are more heterogeneous than previously presumed, and underlines for the first time the crucial role of T(FH) in the complex set of cellular interactions within FL microenvironment.

Bone-metastatic prostate carcinoma favors mesenchymal stem cell differentiation toward osteoblasts and reduces their osteoclastogenic potential.

Bone homeostasis is achieved by the balance between osteoclast-dependent bone resorption and osteoblastic events involving differentiation of adult mesenchymal stem cells (MSCs). Prostate carcinoma (PC) cells display the propensity to metastasize to bone marrow where they disrupt bone homeostasis as a result of mixed osteolytic and osteoblastic lesions. The PC-dependent activation of osteoclasts represents the initial step of tumor engraftment into bone, followed by an accelerated osteoblastic activity and exaggerated bone formation. However, the interactions between PC cells and MSCs and their participation in the disease progression remain as yet unclear. In this study, we show that bone metastatic PC-3 carcinoma cells release factors that increase the expression by human (h)MSCs of several known pro-osteoblastic commitment factors, such as α5/ß1 integrins, fibronectin, and osteoprotegerin. As a consequence, as shown in an osteogenesis assay, hMSCs treated with conditioned medium (C(ed) M) derived from PC-3 cells have an enhanced potential to differentiate into osteoblasts, as compared to hMSCs treated with control medium or with C(ed) M from non-metastatic 22RV1 cells. We demonstrate that FGF-9, one of the factors produced by PC-3 cells, is involved in this process. Furthermore, we show that PC-3 C(ed) M decreases the pro-osteoclastic activity of hMSCs. Altogether, these findings allow us to propose clues to understand the mechanisms by which PC favors bone synthesis by regulating MSC outcome and properties.

A role for T cell-derived interleukin 22 in psoriatic skin inflammation.

Interleukin (IL)-22 is a T cell-derived cytokine that has been reported recently to induce cutaneous inflammation in an experimental murine model of psoriasis, and to induce in vitro an inflammatory-like phenotype. In the present study, we assessed the presence of IL-22 and the IL-22 receptor 1 (IL-22R1) in skin lesions, skin-derived T cells, as well as IL-22 levels in sera from patients with psoriasis. IL-22R1 and IL-10R2 transcripts are expressed at a similar level in psoriatic and healthy skin. In contrast, IL-22 mRNA expression was up-regulated in psoriatic skin lesions compared to normal skin, whereas IL-22 mRNA levels in peripheral blood mononuclear cells from psoriatic patients and normal subjects were similar. Circulating IL-22 levels were significantly higher in psoriatic patients than in normal subjects. T cells isolated from psoriatic skin produced higher levels of IL-22 in comparison to peripheral T cells isolated from the same patients. IL-10 was expressed at similar levels in skin biopsies and peripheral blood mononuclear cells of psoriatic patients and normal subjects. Finally, we show here that supernatants of lesional psoriatic skin-infiltrating T cells induce an inflammatory response by normal human epidermal keratinocytes, resembling that observed in psoriatic lesions. Taken together, the results reported in this study indicate that IL-22 is a cytokine produced by skin-infiltrating lymphocytes that is potentially involved in initiation and/or maintenance of the pathogenesis of psoriasis.

Epigenetic inheritance of fetal genes in allergic asthma.

Asthma has been associated with an exaggerated T-helper type 2 (Th2) over Th1 responses to allergic and nonallergic stimuli, which leads to chronic airway inflammation and airway remodeling. In the present article, we propose that many of the genes involved in IgE synthesis and airways (re)modeling in asthma are persistent or reminiscent fetal genes which may not be silenced during early infancy (or late pregnancy). Genes of the embryologic differentiation of ectodermic and endodermic tissues may explain some of the patterns of airway remodeling in asthma. In utero programming leads to gene expression, the persistence of which may be associated with epigenetic inheritance phenomena induced by nonspecific environmental factors. Clear delineation of these issues may yield new information on the mechanisms of asthma and new targets for therapeutic intervention and primary prevention.

Detection of HLA class II-dependent T helper antigen using antigen phage display.

Major histocompatibility complex (MHC) class II-dependent antigens not only activate CD4+ T helper (Th) cells, but also cytolytic T lymphocytes and effector cells of the innate immune system. These antigens therefore are candidate vaccines against cancer and infectious agents. We have developed a novel approach using a model antigen, tetanus toxoid (TT), which provides the basis for the establishment of a novel strategy of cloning Th antigens. In the TT model system, a cDNA library encoding part of the TT light chain which contained a TT-associated Th epitope recognized by TT-specific Th clones was displayed on a phage vector (TT-phage) and presented to TT-specific Th cells by autologous Epstein-Barr virus-transformed B cells (APC). These TT-phages were able to specifically activate two different TT-specific CD4+ Th cell lines as demonstrated both in [3H]thymidine incorporation and cytokine release assays. Th cell stimulation by TT-phages was significant at a ratio of one TT-phage in 50 irrelevant phages. The described approach provides the basis for the development of a novel strategy of cloning MHC class II-dependent Th antigens, using available Th cells. This strategy has several potential advantages over existing antigen cloning methods or biochemical peptide isolation.

HLA-DR-dependent increased mannan-induced lymphoproliferative response in atopic eczema dermatitis syndrome.

BACKGROUND: In yeast-sensitive atopic eczema dermatitis syndrome (AEDS), yeast mannan induces highly elevated specific IgE levels and lymphoproliferative responses. In healthy individuals the involvement of both human leukocyte antigen (HLA)-dependent T-cell activation and non-HLA-dependent activation, e.g. by crosslinking of the cell surface mannose receptors, has been suggested. In the present study the HLA dependence and the role of crosslinking in the lymphoproliferative response to mannan in AEDS has been analyzed. METHODS: Twenty patients with AEDS and 12 controls with no history of allergic diseases were included in the study. Mannan from Candida albicans was prepared according to the Cetavlon method. Following isolation using Ficoll-Hypaque, peripheral blood mononuclear cells (PBMC) were incubated with the mannan preparation in the absence and presence of different concentrations of neutralizing anti-HLA antibodies and alpha-methylmannoside for 6 days and proliferative responses were measured by 3H-thymidine incorporation and scintilloradiography. RESULTS: In AEDS patients with elevated mannan-specific serum IgE, the C. albicans mannan induced lymphoproliferation. Mannan-induced lymphoproliferative responses could be inhibited, dose-dependently, by neutralizing anti-HLA-DR, but not anti-HLA-DQ antibodies in AEDS patients and healthy controls. The addition of alpha-methylmannoside, that blocks binding to mannose receptors, inhibited lymphoproliferative responses in a dose-dependent way by 50% only in healthy controls, but not in AEDS patients. Levels of inhibition of the proliferation by alpha-methylmannoside correlated inversely with the yeast- and mannan-specific IgE levels. CONCLUSIONS: These results show that in healthy subjects yeast mannan activates lymphocytes both in an HLA-DR-dependent manner and as a result of direct crosslinking of the cell surface. However, in AEDS the elevated lymphoproliferative response is HLA-DR-dependent, although only a slight proportion of this response results from direct crosslinking.

Regulatory T cells and allergic asthma.

Because of the characteristic airway inflammation observed in allergic asthma, the pathogenesis of this disease may be due, in part, to a lack of anti-inflammatory and immune suppressive mechanisms. Here, we discuss the possible involvement and therapeutic use of T regulatory cells and their soluble factors in this multifactorial disease.

Phenotypic characterization of human CD4+ regulatory T cells obtained from cutaneous dinitrochlorobenzene-induced delayed type hypersensitivity reactions.

In this study, we describe the generation and characterization of cloned human CD4+ T lymphocyte populations that have infiltrated into cutaneous, 2,4-dinitrochlorobenzene-induced delayed type hypersensitivity reactions in healthy human subjects. It is shown that, in addition to T helper type 1 clones, elevated numbers of regulatory T clones, producing high levels of interleukin-10 and interleukin-5, but no measurable interleukin-4, were isolated from delayed type hypersensitivity reactions in four of six donors. A subsequent challenge with 2,4-dinitrochlorobenzene of two donors from whom only few interleukin-10-producing T cell clones had been generated after primary challenge, resulted in a decrease in the frequency of T helper type 1 clones and a strong increase in the number of interleukin-10-producing T helper type 2 and regulatory T clones. Culture supernatants from the latter cells, activated with anti-CD3 and anti-CD28 monoclonal antibody, inhibited alloantigen-mediated T cell proliferation which was, partly dependent on interleukin-10, and independent of transforming growth factor-beta. In addition, dendritic cells generated in vitro in the presence of these culture supernatants were impaired in their ability to induce alloantigen-induced proliferative responses. Differential expression of transcripts for the T1/ST2 molecule enabled a phenotypic distinction between resting regulatory T cells and T helper type 2 cells, but not between regulatory T cells and T helper type 1 cells. This experimental model provides a useful tool to isolate human inflammatory and anti-inflammatory T cell subpopulations and, furthermore, enables the study of the kinetics of their appearance into delayed type hypersensitivity reactions.

HIV-1 glycoprotein 120 induces the MMP-9 cytopathogenic factor production that is abolished by inhibition of the p38 mitogen-activated protein kinase signaling pathway.

It has been previously shown that the HIV-1 envelope glycoprotein 120 (gp120) activates cell signaling by CXCR4, independently of CD4. The present study examines the involvement of different intracellular signaling pathways and their physiopathologic consequences following the CD4-independent interaction between CXCR4 or CCR5 and gp120 in different cell types: primary T cells, CD4(-)/CXCR4(+)/CCR5(+) T cells, or glioma cells. These interactions were compared with those obtained with natural ligands, stromal cell-derived factor 1 alpha (SDF-1alpha) (CXCL12) and macrophage inflammatory protein 1 beta (MIP-1beta) (CCL4) of their respective coreceptors. Thus, both p38 and SAPK/Jun N-terminal kinase mitogen-activated protein kinases (MAPKs) are activated on stimulation of these cells with either T- or M-tropic gp120, as well as with SDF-1alpha or MIP-1beta. In contrast, extracellular signal-related kinase 1 and 2 MAPKs are only activated by MIP-1beta but not by M-tropic gp120. Importantly, T- and M-tropic gp120 are able to induce the secretion of matrix metalloproteinase 9 (MMP-9), an extracellular metalloproteinase present in cerebrospinal fluid of patients with HIV-1 by T cells or glioma cells. Specific inhibition of MAPK p38 activation resulted in a complete abrogation of the induction of the MMP-9 pathogenic factor expression by gp120 or chemokines in both cell types. Because neurodegenerative features in acquired immune deficiency syndrome dementia may involve demyelinization by MMP-9, the specific targeting of p38 could provide a novel means to control HIV-induced cytopathogenic effects and cell homing to viral replication sites. (Blood. 2001;98:541-547)

Retrovirus-mediated transduction of primary ZAP-70-deficient human T cells results in the selective growth advantage of gene-corrected cells: implications for gene therapy.

Humans lacking the ZAP-70 protein tyrosine kinase present with an absence of CD8+ T cells and defective CD4+ T cells in the periphery. This severe combined immunodeficiency is fatal unless treated by allogeneic bone marrow transplantation. However, in the absence of suitable marrow donors, the development of alternative forms of therapy is desirable. Because lymphocytes are long-lived, it is possible that introduction of the wild-type ZAP-70 gene into CD4+ ZAP-70-deficient T cells will restore their immune function in vivo. Initial investigations evaluating the feasibility of gene therapy for ZAP-70 deficiency were performed using HTL V-I-transformed lymphocytes. Although transformation was useful in circumventing problems associated with the maintenance of ZAP-70-deficient T cells and low gene transfer levels, the presence of HTL V-I precluded any biological studies. Here, we investigated a retrovirus-mediated approach for the correction of primary T cells derived from two ZAP-70-deficient patients. Upon introduction of the wild-type ZAP-70 gene, TCR-induced MAPK activation, IL-2 secretion and proliferation were restored to approximately normal levels. Importantly, this gain-of-function was associated with a selective growth advantage of gene-corrected cells, thereby indicating the feasibility of a gene therapy-based strategy.

Cytokines and cell surface molecules independently induce CXCR4 expression on CD4+ CCR7+ human memory T cells.

In the present study, we show that IL-2, IL-4, IL-7, and IL-15 are able to induce functional CXCR4 surface expression on resting in vitro-generated CD4+ CXCR4- CCR7+ memory T cells. Cytokine-mediated induction of CXCR4 expression was associated with an increase in CXCR4 transcription, enhanced stromal-derived factor-1-induced T cell migration in vitro, and increased susceptibility of these cells to infection with X4 strains of HIV-1. CXCR4 expression could also be induced through an alternative pathway, following coculture of these cells with CD40-activated, autologous, CD34+ progenitor-derived dendritic cells. Although these dendritic cells express transcripts for IL-7 and IL-15, addition of neutralizing anti-IL-7R and IL-15 mAbs did not block induction of CXCR4 expression. Indeed, dendritic cell-mediated up-regulation of CXCR4 expression was found to depend on CD40/CD154 and CD134/CD134L interactions. Whereas activated autologous dendritic cells induced the expression of both CXCR4 and CD25 on a portion of CCR7+ memory T cells, concomitant CD3-mediated activation of these cells further enhanced CD25 expression, but, in contrast, prevented induction of CXCR4 expression. This observation suggests that triggering of the CD134 and CD154 molecules, in contrast to TCR/CD3 complex-mediated stimulation, results in simultaneous T cell activation and CXCR4 expression. Taken together, these results show that common gamma-chain-interacting cytokines as well as signals mediated via noncognate interactions between activated dendritic cells and memory T cells are involved in the up-regulation of CXCR4 expression.

Alternative antigen receptor (TCR) signaling in T cells derived from ZAP-70-deficient patients expressing high levels of Syk.

ZAP-70-deficient patients present with nonfunctional CD4+ T cells in the periphery. We find that a subset of primary ZAP-70-deficient T cells, expressing high levels of the related protein-tyrosine kinase Syk, can proliferate in vitro. These cells (denoted herein as Syk(hi)/ZAP-70(-) T cells) provide a unique model in which the contribution of Syk to TCR-mediated responses can be explored in a nontransformed background. Importantly, CD3-induced responses, such as tyrosine phosphorylation of cellular substrates (LAT, SLP76, and PLC-gamma1), as well as calcium mobilization, which are defective in T cells expressing neither ZAP-70 nor Syk, are observed in Syk(hi)/ZAP-70(-) T cells. However, Syk(hi)/ZAP-70(-) T cells differ from control T cells with respect to the T cell antigen receptor (TCR)-mediated activation of the MAPK cascades: extracellular signal-regulated kinase activity and recruitment of the JNK and p38 stress-related MAPK pathways are diminished. This distinct phenotype of Syk(hi)/ZAP-70(-) T cells is associated with a profound decrease in CD3-mediated interleukin 2 secretion and proliferation relative to control T cells. Thus, ZAP-70 and Syk appear to play distinct roles in transducing a TCR-mediated signal.

Characterization of T cell subpopulations involved in the pathogenesis of asthma and allergic diseases.

Allergic asthma is a complex and heterogeneous disease which is characterized by intermittent reversible airway obstruction, chronic inflammation of the airways, bronchial hyperreactivity and an infiltration of lymphocytes and eosinophils into the airway submucosa. Animal models and clinical studies in humans have indicated an important role for T helper type 2 lymphocytes, producing IL-4, IL-5 and IL-13, in the pathogenesis of this disorder. However, although IL-4 and IL-13 have strong anti-inflammatory properties, the physiologic anti-inflammatory Th2 response does not seem to be operational in allergic asthma. Moreover, the induction of a Th1 response seems to aggravate, rather than ameliorate, its inflammatory character. This article will focus on the involvement of T lymphocyte subpopulations in the pathogenesis of allergic asthma and allergic diseases. In addition, a potential role of the subpopulation(s) of T regulatory cells in the induction and/or maintaince of the disease process will be discussed.

Prospects for a vaccine in allergic diseases and asthma.

Allergen-specific immunotherapy is widely used to treat allergic diseases, and current research is now focusing on the development of therapeutic vaccines acting on the IgE immune response following allergen challenge. The IgE immune response is dependent on genetic and environmental factors; production of IgE results from complex interactions among B cells, T cells, mast cells, basophils,surface and adhesion molecules and various cytokines. New vaccination methods under investigation involve allergen-specific or nonspecific methodology. Allergen-specific methods currently being developed include allergoids, passive saturation of effector cells, plasmid DNA immunisation and antigen-antibody complexes. The mechanisms of immunotherapy using allergen-specific methods differ with the allergens and the route of immunisation used (parenteral, intranasal, sublingual, oral or bronchial). Many vaccines being developed at present comprise synthetic, recombinant or highly purified subunit antigens, which although they have increased safety may also be less immunogenic.It is hoped that the addition of adjuvants will overcome this drawback. Methods of increasing the dose of allergen while reducing the possibility of an anaphylactic reaction include the use of non-anaphylactic isoforms of the allergens, alteration of the tertiary structure of the allergens and construction of minimal allergen-derived T cell peptides. Nonspecific approaches include humanised anti-IgE antibodies,moderation of the T(H)2 cytokine network and antisense oligodeoxynucleotide therapy.

TCR-mediated activation of allergen-specific CD45RO(+) memory T lymphocytes results in down-regulation of cell-surface CXCR4 expression and a strongly reduced capacity to migrate in response to stromal cell-derived factor-1.

The selective migration of functional T(h) lymphocyte subsets with different cytokine production profiles into inflamed tissue is likely to depend on the state of activation of the cells, as well as on the differential expression of various adhesion molecules and chemokine receptors. In this study, we have analyzed the effect of allergen-specific activation on the expression of the chemokine receptor CXCR4 on T lymphocytes. We show that stimulation of peripheral blood mononuclear cells from atopic patients with the allergen Der p results in down-regulation of CXCR4 surface expression on Der p-activated CD25(+)CD45RO(+) antigen-specific memory cells which was caused by a decrease in CXCR4 gene transcription and did not seem to be mediated by endogenous cytokines, such as IFN-gamma. In contrast, however, CXCR4 surface expression was enhanced on naive CD25(-)CD45RO(-) and resting CD25(-)CD45RO(+) memory T cells, as a result of the presence of endogenous IL-4, most likely produced by Der p-activated memory T cells. Antigen-specific CD25(+)CD45RO(+) T lymphocytes, purified 7 days after stimulation with Der p, had a strongly reduced capacity to migrate in response to stimulation with stromal cell-derived factor (SDF)-1, the ligand for CXCR4. Together, these results suggest that differential expression of CXCR4 on activated and resting T cells is due to the counteracting effects of TCR-mediated down-regulation and IL-4-mediated up-regulation of this chemokine receptor respectively, and furthermore indicate that antigen-activated memory T cells are unlikely to migrate into inflamed tissue in response to SDF-1.

Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-gamma-producing CD4+ and CD8+ T cells.

In this study we have investigated the capacity of human fetal thymocytes to differentiate in vitro into subsets of T cells with polarized Th1 or Th2 cytokine profiles. Stimulation of freshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linked onto CD32,CD58,CD80-expressing mouse fibroblasts and subsequent culture in the presence of exogenous rIL-2 for 6 days, induced the production of both IL-4 and IFN-gamma, which was mainly produced by CD4+ single-positive (SP) and CD8+ SP cells respectively. Addition of rIL-4 during priming augmented IL-4 production in cultures of human fetal thymocytes, which was mainly due to an increased production of IL-4 by CD8SP cells. In contrast, addition of IL-4 to the cultures only slightly enhanced IL-4 production and had little effect on frequencies of IL-4-producing CD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10 and IL-13 at comparable levels, following priming in the presence of rIL-4. Priming in the presence of rIL-12 strongly enhanced the production of IFN-gamma in both CD4SP and CD8SP cells. No correlation between expression of CD27, CD30 and CD60, and a particular cytokine profile of differentiated thymocytes could be demonstrated. Together, these results demonstrate the full capacity of fetal human thymocytes to differentiate into cytokine-producing T cells in a priming milieu with appropriate stimulatory molecules and exogenous cytokines. In addition, CD4SP thymocytes rapidly differentiate into polarized Th2 cells following stimulation in vitro in the absence of exogenous rIL-4.

Recombinant adenovirus vectors for cytokine gene therapy in mice.

BACKGROUND: Adenoviruses have several specific features useful for gene therapy. They infect various lineages of cells irrespective of cell cycle status. However, the exact mechanism of their infection and in vivo kinetics as a gene expression vector have not been elucidated. OBJECTIVE: Using adenovirus vectors expressing marker genes, we examined the infectivity of these vectors (including cellular and tissue tropism), the duration and intensity of transgene expression, and the side effects. METHODS: Various cells were infected with adenovirus expressing LacZ gene at various doses, and beta-galactosidase activity was measured and compared in relation with dose, time course, and cellular vitronectin receptor. Mice were injected with adenoviruses expressing LacZ, luciferase and GM-CSF, and in vivo gene expression was examined. RESULTS: Adenovirus infection induced viral dose-dependent transgene expression that persisted for 2 weeks. Adherent cells were infected much more efficiently than nonadherent cells, probably because the former expressed much higher levels of the vitronectin receptor, one of the main receptors for adenovirus. Studies performed in mice with luciferase-expressing adenovirus revealed that the liver was the main target organ after intravenous injection and showed that the intravenous route was superior to other routes with regard to transgene expression. After intravenous injection of adenovirus expressing human GM-CSF, there was a transient and dose-dependent increase in the serum level of this cytokine. Administration of adenovirus expressing mouse GM-CSF enhanced hematopoiesis in the spleen and bone marrow. CONCLUSION: These results indicated that adenoviruses can be used for in vivo cytokine gene therapy but suggested the necessity of taking into consideration the route of administration, the duration of transgene expression, the toxic dose, and host immune reactions.

Identification of a highly promiscuous and an HLA allele-specific T-cell epitope in the birch major allergen Bet v 1: HLA restriction, epitope mapping and TCR sequence comparisons.

BACKGROUND: Allergen-specific CD4+ T cells play an important regulatory role in atopic allergy. OBJECTIVE: To investigate the human leucocyte antigen (HLA) restriction and T-cell receptor (TCR) usage of allergen-specific T-cell clones (TCCs) that react with defined epitopes of Bet v 1, the major birch pollen allergen. METHODS: Five Bet v 1-specific TCCs derived from two birch pollen-allergic individuals and specific for Bet v 1, were epitope-mapped with overlapping synthetic peptides. In addition, HLA-restriction and TCR CDR3 sequences were determined. RESULTS: Three TCCs reacted with a Bet v 1 peptide containing amino acid residues 21-33 (BP21), the other two TCCs reacted with a minimal peptide comprising residues 37-45 (BP37). Studies using neutralizing anti-HLA-monoclonal antibodies and HLA-typed APCs showed that the BP37-specific TCCs were restricted by a HLA-DQA1*0301/DQB1*0603 heterodimer. In contrast, BP21 was recognized in a highly promiscuous manner. TCCs recognizing this sequence were restricted by HLA-DPB1*0201, a HLA-DQA1*0201/DQB1*0201 heterodimer, or HLA-DRB3*0101. Reverse transcription-polymerase chain reaction with primers for all known TCRAV and TCRBV gene segments, followed by CDR3 region sequencing, revealed the usage of five different TCRAV and four different TCRBV gene segments by the TCCs, as well as diversity in the joining region. All BP21-specific TCCs contained a negatively charged residue in their CDR3alpha regions, the CDR3beta regions showed a high concentration of polar and OH-group bearing residues. BP37-specific TCCs shared the amino acid combination LY in the middle of their CDR3alpha regions, the CDR3beta regions showed high concentration of OH-group bearing or charged residues. CONCLUSIONS: This study shows the existence of a highly promiscuous T-cell epitope in Bet v 1. The presence of additional T-cell epitopes in Bet v 1 may, however, hamper the clinical applicability of the epitope. Likewise, the diversity in TCR usage by T cells recognizing the epitope does not support the development of TCR-directed immunotherapy for birch pollen allergy.

A CD4-independent interaction of human immunodeficiency virus-1 gp120 with CXCR4 induces their cointernalization, cell signaling, and T-cell chemotaxis.

The gp120 envelope glycoprotein of human immunodeficiency virus-1 (HIV-1) interacts with the CXCR4 chemokine receptor, but it is not known whether gp120 activates CXCR4-mediated signaling cascades in the same manner as its natural ligand, SDF1alpha. We assessed the effects of wild-type gp120 and a mutant gp120 that interacts with CXCR4 but not CD4 on CD4(-)/CXCR4(+) cells and CD4(+)/CXCR4(+) cells, respectively. Under both experimental conditions, the interaction of CXCR4 and gp120 resulted in their CD4-independent cointernalization. Both molecules were translocated into early endosomes, whereas neither protein could be detected in late endosomes. Binding of gp120 to CXCR4 resulted in a CD4-independent phosphorylation of Pyk2 and an induction of chemotactic activity, demonstrating that this interaction has functional consequences. Interestingly, however, whereas SDF1alpha activated the ERK/MAP kinase pathway, this cascade was not induced by gp120. Together, these results suggest that the pathology of HIV-1 infection may be modulated by the distinct signal transduction pathway mediated by gp120 upon its interaction with CXCR4.