Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

Environmental influence on T cell receptor alpha gene rearrangement and expression in vitro.

Experiments were designed to test whether T cell progenitors are committed to particular T cell receptor (TcR) gene rearrangement and expression patterns (prior to rearrangement) or whether such patients are molded by the thymic microenvironment in which T cells develop. To this end, day 14 fetal thymocytes were removed from their normal environment and grown in organ culture (FTOC) for 5 to 12 days. RNA was extracted from organ-cultured cells, processed to cDNA, and TcR alpha sequences were amplified by the polymerase chain reaction for cloning and sequencing. By the examination of N-region additions and V-gene usage, and by the comparison of resultant patterns with those of early vs. adult stages of T cell differentiation in vivo, it was demonstrated that thymocytes in FTOC did not maintain early patterns of gene rearrangement. The thymocyte patterns were most dissimilar from those of normal, early ontogeny when interleukin-4 was added to FTOC. Taken together, results demonstrated the flexibility of T cell progenitors and that environment plays a critical role in the molding of TcR alpha rearrangement and expression patterns.     

A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1^dull γδ thymocytes from DBA/2 mice with anti-γδ monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-γ (IFN-γ), IL-4, IL-10, and IL-3. In contrast, only IFN-γ was detected in parallel cultures of Thy-1^bright γδ thymocytes. Virtually all Thy-1^dull γδ thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1^dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1^dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ-Dδ-Jδ junctions, and somewhat more diverse Vγ-Jγ junctions. We conclude that Thy-1^dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

Antigen-Driven Clonal Accumulation of Peritoneal γδ T Cells in vivo

How the clonality of γδ T cells changes in response to exogenous antigens is uncertain. Here we analyzed kinetics of Vγ1.1 and Vγ2 T cell clonality after intraperitoneal injection of purified protein derivatives (PPD) by the heterogeneity of the third complementarity determining region (CDR3) length in Vγ1.1-Jγ4-Cγ4 and Vγ2-Jγ1-Cγ1 junctions. The V-J junctions were analyzed in intrahepatic lymphocytes (IHL), spleen cells, and peritoneal exudate cells (PEC) by polyacrylamide gel electrophoresis. γδ T cells expressing Vγ1.1 and Vγ2 genes were heterogeneous in normal mice. Accumulation of specific Vγ1.1 T cell clones was transiently detected 7 days after the injection in PEC, but no accumulation was observed in IHL and spleen cells. The accumulated clones disappeared by 4 weeks. Transient accumulation of Vγ2 T cell clones was also observed in PEC at the early phase after the injection. These results suggest that γδ T cells with specific TCR respond to PPD and temporary accumulate in the peritoneal cavity, but not in liver and spleen.     

Phosphoantigen activation induces surface translocation of intracellular CD94/NKG2A class I receptor on CD94− peripheral Vγ9 Vδ2 T cells but not on CD94− thymic or mature γ δ T cell clones

Most adult peripheral blood γ δ T cells express Vγ9/Vδ2-encoded TCR that recognize a restricted set of nonpeptidic phosphorylated compounds, referred to as phosphoantigens. They also express various MHC class I-specific inhibitory receptors (IR), in particular CD94/NKG2-A heterodimers, which participate in the fine tuning of their TCR-mediated activation threshold. Most mature Vγ9/Vδ2 T cells express surface CD94 receptors, unlike cord blood or thymus-derived Vγ9/Vδ2 clones, thus suggesting a role for the microenvironment in IR expression. In the present study we show that most CD94⁻ Vγ9Vδ2 PBL ex vivo express an intracellular pool of CD94/NKG2-A receptors that is translocated to the cell surface upon activation by phosphoantigens or IL-2. In stark contrast, intracellular CD94/NKG2-A complexes are undetectable in CD94⁻ thymus or PBL-derived mature Vδ2 T cell clones, and no surface induction is observed following phosphoantigen activation of T cell clones. Altogether these results provide new insights into the regulation of CD94/NKG2-A expression on T lymphocytes and suggest the existence of distinct mechanisms controlling in vivo and in vitro induction of IR on these cells.     

A regulatory cross‐talk between Vγ9Vδ2 T lymphocytes and mesenchymal stem cells

The physiological functions of human TCRVγ9Vδ2⁺ γδ lymphocytes reactive to non‐peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVγ9Vδ2⁺ lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B‐cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of γδ‐cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX‐2‐dependent production of prostaglandin E2 (PGE₂) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vγ9Vδ2 T lymphocytes. COX‐2 expression and PGE₂ production by MSC were not constitutive, but were induced by IFN‐γ and TNF‐α secreted by activated Vγ9Vδ2 T cells. This regulatory cross‐talk between MSC and Vγ9Vδ2 T lymphocytes involving PGE₂ could be of importance for the antitumor and antimicrobial activities of γδ T cells.     

Specific expression of surface interferon-γ on interferon-γ producing T cells from mouse and man

Interferon (IFN)-γ is a potent immunoregulatory protein secreted by CD4⁺ and CD8⁺ T cells and by natural killer cells. Here, we show that IFN-γ is specifically displayed at a low concentration on the cell surface of those activated T cells from mouse and man which express IFN-γ. It is transiently expressed on the cell surface with kinetics similar to those of intracellular IFN-γ expression. Detectable surface IFN-γ is not expressed by activated T helper (Th) cells producing other cytokines but which do not express IFN-γ. Thus, surface IFN-γ is the first available marker for live T lymphocytes expressing IFN-γ, e.g. Th1 cells.     

Characterization of γ/δ T cell clones isolated from human fetal liverand thymus

The origin and development of T cells bearing γ/δ T cell receptors (TcR) has been extensively studied in the mouse. By contrast, little is known about development patterns and diversity of the human γ/δ T cell lineage. To study therepertoire of human γ/δ⁺ T cells during T cell ontogeny, wehave isolated clonal populations of γ/δ⁺ T cells from 14-week fetal thymus and liver and characterized the molecular compositionof their TcR. The technique of in situ hybridization was used to identify cells expressing TcR genes in fetal liver and thymus. A panel of clones representative of developing T cell populations found in vivo was subsequently isolated from both tissues and clones expressing cell surface γ/δ receptors were identified. Although both the liver-derived γ/δ⁺ T cell clone, L2, and the thymus-derived γ/δ⁺ T cell clone, T6, had similar cell surface phenotypes, namely CD3⁺, CD7⁺, CD45⁺ and CD8^?, their reactivity with anti-CD2 and -CD4 antibodies was different. L2 was CD2^high, CD4^? whereas T6 was CD2^low, CD4^low. Both clones possessed effector functions similar to those of adult T cells as demonstrated by the synthesis and secretion of cytokines in response to stimulation through the CD3/TcR complex. Analysis of the TcR composition of the fetal clones showed both clones to possess similar or identical γ chain components, C_γ1, J_γ1/2, V_γ8, and both utilize V_δ gene segments other than V_δ1. This TcR genotype has not been previously reported in the analysis of adult γ/δ⁺ T cells. Our studies have identified a unique population of human γ/δ⁺ T cells that may be derived extrathymically and appear to be preferentially and perhaps transiently expressed during fetal life.     

Preferentially expanding Vγ1+ γδ T cells are associated with protective immunity against Plasmodium infection in mice

γδ T cells play a crucial role in controlling malaria parasites. Dendritic cell (DC) activation via CD40 ligand (CD40L)‐CD40 signaling by γδ T cells induces protective immunity against the blood‐stage Plasmodium berghei XAT (PbXAT) parasites in mice. However, it is unknown which γδ T‐cell subset has an effector role and is required to control the Plasmodium infection. Here, using antibodies to deplete TCR Vγ1⁺ cells, we saw that Vγ1⁺ γδ T cells were important for the control of PbXAT infection. Splenic Vγ1⁺ γδ T cells preferentially expand and express CD40L, and both Vγ1⁺ and Vγ4⁺ γδ T cells produce IFN‐γ during infection. Although expression of CD40L on Vγ1⁺ γδ T cells is maintained during infection, the IFN‐γ positivity of Vγ1⁺ γδ T cells is reduced in late‐phase infection due to γδ T‐cell dysfunction. In Plasmodium‐infected IFN‐γ signaling‐deficient mice, DC activation is reduced, resulting in the suppression of γδ T‐cell dysfunction and the dampening of γδ T‐cell expansion in the late phase of infection. Our data suggest that Vγ1⁺ γδ T cells represent a major subset responding to PbXAT infection and that the Vγ1⁺ γδ T‐cell response is dependent on IFN‐γ‐activated DCs.     

Developmental Expression Profile of the CXCL12γ Isoform: Insights Into its Tissue‐Specific Role

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an highly conserved gene that plays pivotal, non‐redundant roles during development. The interaction of the highly cationic carboxy‐terminal (C‐ter) domain of CXCL12γ with glycosaminoglycans (GAG) critically determines the biological properties of this chemokine. Indeed, CXCL12γ isoform displays sustained in vivo recruitment of leukocytes and endothelial progenitor cells as compared to other CXCL12 isoforms. Despite the important, specific roles of CXCL12γ in vivo, the current knowledge about its distribution in embryo and adult tissues is scarce. In this study, we have characterized by both RT‐PCR and immunohistochemistry the expression profile and tissue distribution of CXCL12γ, which showed a distinct mRNA expression pattern during organogenesis that correlates with the specific expression of the CXCL12 γ protein in several tissues and cell types during development. Our results support the biological relevance of CXCL12 γ in vivo, and shed light on the specific roles that this novel isoform could play in muscle development and vascularization as well as on the regulation of essential homeostatic functions during the embryonic development. Anat Rec, 292:891–901, 2009. © 2009 Wiley‐Liss, Inc.     

Shared amino acid sequences in the NDβN and Nα regions of the T cell receptors of tumor-infiltrating lymphocytes within malignant glioma

The purpose of this study was to assess the V-(D)-J junctional region of the T cell receptor (TCR), the CDR3 region, which is responsible for glioma-specific antigen contact in αβ TCR-mediated recognition. We sequenced the TCR α and β chians of Vα7, and Vβ13.1 cDNA derived from tumor-infiltrating lymphocytes (TIL) of 12 glioma patients and also the corresponding clones from the patients' peripheral blood lymphocytes (PBL). A shared Vβ13.1 DJ sequence of the CDR3 region, NDβN, was demonstrated in 49 of 66 Vβ13.1⁺ clones (74.2 %) from the glioma TIL, whereas only 4 of 33 clones (12.1 %) were observed in the Vβ13.1⁺ clones from the PBL (p < 0.001). A common VDJ sequence, FCASS (Vβ13.1)-YRLPWGTSDS (NDβN)-GELFF(Jβ2.2), was observed not only in the gliomas from each patient, but also among all the patients with a preference for Vβ13.1. In contrast, the amino acid sequences of the Vβ13.1⁺ PBL clones were diverse and random. Next, we sequenced subclones from other Vβ subfamilies randomly selected to compare their VDJ region rearrangements (Vβ3 and Vβ5.1). In contrast to Vβ13.1, the amino acid sequences of these junctional regions were completely different in these subclones. The V-J junctional region of the α chain is dominated by a few clones in some patients, and no shared amino acid sequences were detected in the TCR Vα junctional region. However, in the Nα region of the Vα7-bearing TIL clones, arginine was used in 27 of 44 clones (61.4%) compared to only 3 of 12 clones from the PBL (p < 0.05). These results are consistent with the hypothesis that a clonal expansion/accumulation of glioma lineage-specific T cells occurred in vivo at the tumor site and that these T cells may be recognizing glioma-specific antigens.     

Different cytokine production profiles of γδ T cell clones: Relation to inflammatory arthritis

This study was performed to investigate whether γδ T cells could also be divided into subsets, identified by a cytokine profile, as described for αβ T helper (Th) cell subsets. Cytokine production was studied in 22 γδ T cell clones obtained from the synovial fluid and peripheral blood of one patient with inflammatory arthritis and compared to that of 26 αβ T cell clones of the same and different patients. Interferon-γ (IFN-γ) was produced by 18 (82%) and interleukin-4 (IL-4) by 17 (77%) out of 22 γδ T cell clones, respectively. In contrast, IL-10 was not produced, except at very low level in one case. The mean levels of IL-4 were lower for clones derived from synovial fluid. When considering the production of IFN-γ as an indicator of Th1 and that of IL-4 as an indicator of Th2, respectively, the most common pattern was a γδTh1-like pattern, with the combination of high levels of IFN-γ and low levels of IL-4. This pattern was found in Vδ1⁺ clones, all from synovial fluid. Additional patterns were also observed: a mixed, probably γδ Th0-like pattern with a more balanced production of both IFN-γ and IL-4; a γδTh1 pattern with the production of IFN-γ alone; a γδTh2 pattern with the production of IL-4 alone. These three patterns were also seen in blood γδ T cells which were all Vδ2, indicating that these patterns were independent of the γδ phenotype. γδ T cell clones produced lower levels of IFN-γ (p = 0.001) and higher levels of IL-4 than αβ clones (p < 0.02). These differences in cytokine production between αβ and γδ subsets and within these subsets may contribute to their respective role in chronic inflammation.     

Interferon-γ- and interleukin-4-producing T cells can be primed on dendritic cells in vivo and do not require the presence of B cells

The antigen-presenting cell (APC) requirements for the in vivo induction of Th1-and Th2-type responses were investigated using a severe combined immunodeficiency (SCID)mouse chimera model. SCID mice adoptively transferred with either T cells [SCID(T)] or T + B cells [SCID(T + B)] and immunized with antigen in adjuvant were able to generate antigen-specific T cells which could produce both interferon (IFN)-γ and interleukin (IL)-4 upon in vitro restimulation. This suggests that B cell APC are not necessary for the priming of either IFN-γ- or IL-4-producing T cells in vivo. The ability of different APC to activate Th2-dependent effector mechanisms was also investigated. SCID(T) and SCID(T + B) mice were infected with the nematode parasite Nippostrongylus brasiliensis and analyzed for the development of IL-5-dependent peripheral blood eosinophilia. Following infection both SCID(T) and SCID(T + B) mice generated similar numbers of peripheral blood eosilnophils, suggesting that similar amounts of IL-5 had been produced. Therefore, B cell APC are also not required for the in vivo activation of Th2 cells to lymphokine production. To establish more precisely which APC prime T cells to produce IFN-γ and IL-4, normal mice were immunized by injection of syngeneic splenic dendritic cells which had been pulsed with antigen in vitro. T cells from these immunized mice were able to produce good IFN-γ and IL-4 responses upon in vitro restimulation with specific antigen; therefore, dendritic cells appear to be sufficient APC for the in vivo priming of both IFN-γ- and IL-4-producing T cells.     

Analysis of TCR β chains in Lewis rats with experimental allergic encephalomyelitis. II. Vβ8.2+ T cells with limited CDR3 N region additions derive from the adult thymus

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68 – 86. The TCR β chains of these disease-causing T cells show three distinct features: they are almost exclusively Vβ8.2, they use AspSer as the first two amino acid residues of the third complementarity-determining region (CDR3) and these junctional region sequences show few if any non-germline N-region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68 – 86-reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery – i.e., that such β chains are conformationally the most appropriate for triggering by an epitope of 68 – 86 complexed to class II RT1.B^l MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post-irradiation adult thymus. These disease-causing T cells are heavily Vβ8.2⁺ , CDR3 AspSer⁺ and use few N region additions. We conclude that T cells with these TCR β chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.