A clonal view of αβ T cell responses

Quantitative analyses of antigen (Ag)-specific αβ T cell populations have provided a large body of information on the natural course of T cell immune responses. New tools are now available to determine the clonal composition of Ag-specific pools in individual responders, an approach which offers direct insights into the generation of T cell immune responses and establishment of protective immunity. The present review discusses the parameters that determine the composition of Ag-specific T cell responses. Emphasis is placed on the role of the naive αβ T cell repertoire and on the dynamics of individual Ag-specific T cell clones during the successive phases of an immune response.     

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17⁺cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c⁺/langerin⁺ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.     

Identification of a homolog of the Cα3′/hs3 enhancer and of an allelic variant of the 3′IgH/hs1,2 enhancer downstream the human immunoglobulin α1 gene

Although four regulatory elements are known downstream the mouse IgH α gene, a single enhancer homologous to hs1,2 has been thus far described downstream each human α gene (Chen, C. and Birshtein, B. K., J. Immunol. 1997. 159: 1310). We characterized a 10-kb region downstream the human α1 gene. Two B cell-specific regulatory elements homologous to the murine Cα3′/hs3 and hs1,2 3′ enhancers were found, which are duplicated downstream α2. The hs1,2 element is in inverted orientation by comparison with a recently reported α1 hs1,2 element: it appears as a common allelic variant carrying an internal tandem repeat insertion and its prevalence in the human population is 60%. As in the mouse, the human hs1,2 enhancer is flanked with long inverted repeats which may have promoted inversion events through homologous recombination. Although the palindromic organization of the region is maintained in human, sequence identity with rodents focuses on core enhancer elements rather than on flanking repeats. Concerted divergence of both sides of the dyad symmetry suggests that inverted repeats are not just evolutionary remnants but rather play an architectural role in the LCR function.     

Deep sequencing of the TCR‐β repertoire of human forkhead box protein 3 (FoxP3)+ and FoxP3– T cells suggests that they are completely distinct and non‐overlapping

Maintenance of peripheral tolerance requires a balance between autoreactive conventional T cells (T_conv) and thymically derived forkhead box protein 3 (FoxP3)⁺ regulatory T cells (tT_regs). Considerable controversy exists regarding the similarities/differences in T cell receptor (TCR) repertoires expressed by T_conv and tT_regs. We generated highly purified populations of human adult and cord blood T_conv and tT_regs based on the differential expression of CD25 and CD127. The purity of the sorted populations was validated by intracellular staining for FoxP3 and Helios. We also purified an overlap group of CD4 T cells from adult donors to ensure that considerable numbers of shared clonotypes could be detected when present. We used deep sequencing of entire TCR‐β CDR3 sequences to analyse the TCR repertoire of T_conv and tT_regs. Our studies suggest that both neonatal and adult human T_conv and tT_reg cells are, in fact, entirely distinct CD4 T cell lineages.     

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.     

Functional and molecular characterization of B cell-responsive Vδ1+ γδ T cells

Cells expressing the Vδ1⁺ gene segment are a minor γδ T cell population in human peripheral blood but predominate in epithelia and (inflamed) tissues. The characteristic dendritic-like morphology of these γδ T cells is consistent with their putative immune surveillance role in epithelia. Their function, however, remains unknown. We and others previously reported that a subset of Vδ1⁺ γδ T cells proliferates after stimulation with Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCL), but not with fresh peripheral blood-derived B cells. These responses were independent of the type of T cell receptor (TcR) γ chain co-expressed with the Vδ1 chain. The in vivo relevance of this LCL-mediated activation as well as the nature of the stimulatory ligand on the LCL is not well established. In this study, we tested the proliferative response of Vδ1⁺ LCL-responsive T cells against non-EBV-transformed B cells, activated through CD40 by murine EL4 B5 cells, and to a panel of B cell lines differing in the expression of EBV nuclear antigen proteins and adhesion/co-stimulatory molecules. The role of the Epstein-Barr virus-derived antigen in the induction of this response could be excluded as the activated (non-EBV-transformed) peripheral blood B cells were also able to induce a proliferative response in the LCL-responsive Vδ1⁺ T cells. Therefore, the stimulatory ligand on B cells is of cellular rather than of viral origin, and its expression is up-regulated upon activation of B cells. The expression of B7 and CD39 molecules on the surface of activated B cells appeared to be crucial since antibodies to these structures could block the induction of proliferation of the Vδ1⁺ T cells. Finally, we investigated the diversity of the responding Vδ1⁺ γδ T cell clones by sequence analysis of the TcRδ junctional regions. No restricted V-D-J sequences were found among the LCL-responsive Vδ1⁺ T cell clones, arguing strongly against a mono- or oligoclonal Vδ1⁺ γδ T cell response to LCL. These findings may explain the presence of polyclonally activated Vδ1⁺ T cells in inflamed tissues where activated B cells are often present.     

Changes in thymic export of γδ and αβ T cells during fetal and postnatal development

The thymus plays an essential role in the generation and selection of T cells and exports approximately 0.5–1% of thymocytes per day in young animals and considerably fewer in older animals. To date there have been no studies directly examining fetal thymic export in any species. Using the technique of intrathymic injection of fluorescein isothiocyanate, followed by an assay for green fluorescent cells in the periphery and for the expression of cell surface antigens on these cells, we have compared directly the export of T cells from the fetal and postnatal ovine thymus. While the thymus exports both αβ and γδ T cells, our results demonstrate that the proportion of thymic γδ T cells that are exported per day is much higher than that of thymic αβ T cells. Moreover, the export rate of γδ T cells increased from approximately 1 in every 60 γδ thymocytes per day emigrating from the fetal thymus to 1 in every 20 from the postnatal thymus. In addition, we identify a population of CD5⁺CD4^?CD8^?γδ^? T cells emigrating from the fetal thymus but greatly reduced among thymic emigrants after birth. These findings have several implications regarding the mechanisms and control of selection of both γδ and αβ T cells.     

DL4‐mediated Notch signaling is required for the development of fetal αβ and γδ T cells

T‐cell development depends upon interactions between thymocytes and thymic epithelial cells (TECs). The engagement of delta‐like 4 (DL4) on TECs by Notch1 expressed by blood‐borne BM‐derived precursors is essential for T‐cell commitment in the adult thymus. In contrast to the adult, the earliest T‐cell progenitors in the embryo originate in the fetal liver and migrate to the nonvascularized fetal thymus via chemokine signals. Within the fetal thymus, some T‐cell precursors undergo programmed TCRγ and TCRδ rearrangement and selection, giving rise to unique γδ T cells. Despite these fundamental differences between fetal and adult T‐cell lymphopoiesis, we show here that DL4‐mediated Notch signaling is essential for the development of both αβ and γδ T‐cell lineages in the embryo. Deletion of the DL4 gene in fetal TECs results in an early block in αβ T‐cell development and a dramatic reduction of all γδ T‐cell subsets in the fetal thymus. In contrast to the adult, no dramatic deviation of T‐cell precursors to alternative fates was observed in the fetal thymus in the absence of Notch signaling. Taken together, our data reveal a common requirement for DL4‐mediated Notch signaling in fetal and adult thymopoiesis.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

T helper 2 (Th2) but not Th1 clones co-stimulate resting T cells in the presence of anti-CD3 monoclonal antibody

We have investigated the effects of monoclonal antibody (mAb) to the CD3 epsilon protein on interactions between small, resting T cells and antigen-specific T helper clones. Highly purified, splenic T cells lacking identifiable accessory cells do not proliferate in a thymidine uptake assay to anti-CD3 mAb, Con A, rIL-2, rIL-4, or irradiated T helper clones (both Th1 and Th2). However, the responding T cells proliferate significantly to the combined stimulus of Th2 clones and anti-CD3 antibody. Only the Th2, not the Th1, subpopulation of T helper cells has the ability to induce a T cell response. The Th2 cell-dependent activation of small resting T cells does not require the external cross-linkage of the anti-CD3 mAb via Fc receptor expressing cells or the secretion of lymphokines from the Th2 helper clones, but it is inhibitable by anti-LFA 1 antibody. Thus, Th2 clones provide a co-stimulatory signal which in conjunction with anti-CD3 mAb causes resting T cell proliferation in the absence of conventional accessory cells.     

Fixing an irrelevant TCRα chain reveals the importance of TCRβ diversity for optimal TCRαβ pairing and function of virus‐specific CD8+ T cells

TCR repertoire diversity can influence the efficacy of CD8⁺ T‐cell populations, with greater breadth eliciting better protection. We analyzed TCRβ diversity and functional capacity for influenza‐specific CD8⁺ T cells expressing a single TCRα chain. Mice (A7) transgenic for the H2K^bOVA_257–264‐specific Vα2.7 TCR were challenged with influenza to determine how fixing this “irrelevant” TCRα affects the “public” and restricted D^bNP ${}_{\text{366}}^{\text{+}}$CD8⁺ versus the “private” and diverse D^bPA ${}_{\text{224}}^{\text{+}}$CD8⁺ responses. Though both D^bNP ${}_{\text{366}}^{\text{+}}$CD8⁺ and D^bPA ${}_{\text{224}}^{\text{+}}$CD8⁺ sets are generated in virus‐primed A7 mice, the constrained D^bNP ${}_{\text{366}}^{\text{+}}$CD8⁺ population lacked the characteristic, public TCRVβ8.3, and consequently was reduced in magnitude and pMHC‐I avidity. For the more diverse D^bPA ${}_{\text{224}}^{\text{+}}$CD8⁺ T cells, this particular forcing led to a narrowing and higher TCRβ conservation of the dominant Vβ7, though the responses were of comparable magnitude to C57BL/6J controls. Interestingly, although both the TCRβ diversity and the cytokine profiles were reduced for the D^bNP ${}_{\text{366}}^{\text{+}}$CD8⁺ and D^bPA ${}_{\text{224}}^{\text{+}}$CD8⁺ sets in spleen, the latter measure of polyfunctionality was comparable for T cells recovered from the infected lungs of A7 and control mice. Even “sub‐optimal” TCRαβ pairs can operate effectively when exposed in a milieu of high virus load. Thus, TCRβ diversity is important for optimal TCRαβ pairing and function when TCRα is limiting.     

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

Human γδ T Cells Express a Higher TCR/CD3 Complex Density Than αβ T Cells

The aim of our study was to compare CD3 expression on γδ T cells and αβ T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human γδ T cells constitutively express approximately twofold more of the TCR/CD3 complex than αβ T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of γδ T cells compared to αβ T cells. These clinical laboratory results confirm the fundamental data described elsewhere. γδ T cells deserve further clinical investigations to understand their precise role in human immunity.     

Activation of murine epidermal γδ T cells through surface 2B4

Dendritic epidermal T cells (DETC) are γδ T cells that normally reside in murine skin. They express on their surface the 2B4 molecule, a 66-kDa glycoprotein of the immunoglobulin gene superfamily thought to be associated with anti-tumor cytotoxicity by natural killer and lymphokine-activated killer cells. Here, we show that ligation of surface 2B4 transduces cell activation signals in DETC. Treatment with anti-2B4 monoclonal antibodies triggers the secretion of interferon-γ and interleukin-2 by DETC lines, induces proliferation of resting DETC lines, amplifies anti-CD3-dependent proliferation of DETC freshly isolated from mouse skin; and up-regulates egr-1 and c-fos mRNA expression. These results indicate a unique pathway for DETC activation.     

Changes in thymic export of L-selectin+ γδ and αβ T cells during fetal and postnatal development

We have used the technique of in situ intrathymic injection of fluorescein isothiocyanate to examine L-selectin expression on γδ and αβ T cells immediately after emigrating from the thymus of fetal and postnatal animals. We found that the percentage of L-selectin⁺ thymocytes exported per day decreased by half after birth and that the export of T cells from the thymus does not rely on expression of the peripheral lymph node homing receptor, L-selectin. Analysis of L-selectin on emigrant and mature T cell subsets revealed a remarkable heterogeneity of expression, both in terms of the numbers of cells expressing this molecule as well as the level of expression. γδ T cells, reportedly not having a propensity for homing to lymph nodes, not only contained the highest proportion of L-selectin⁺ cells, but also expressed far more of this molecule than either CD4⁺CD8^? or CD4^?CD8⁺ αβ T cells. Furthermore, those emigrant T cells expressing L-selectin are somewhat immature in their expression of this molecule. Subsequent maturation resulted in up-regulation of L-selectin on mature peripheral blood T cells, maturation that was clearly independent of extrinsic antigen. This antigen-independent post-thymic maturation appeared to occur as part of the normal progression from immature thymocyte to mature peripheral T cell in both fetal and postnatal animals.     

Administration of anti-interleukin-2 receptor α antibody in vivo induces localized autoimmune disease

Neonatal thymectomy (Tx) of mice at day 3 after birth (Tx-3), but not day 7 (Tx-7), induces organ-localized autoimmune diseases such as oophoritis and gastritis. Lesions in Tx-3 mice can be prevented by injection of splenic CD4⁺ cells from syngeneic normal mice, and this CD4⁺ population with suppressor activity is activated extrathymically by self antigens. Since it is speculated that these CD4⁺ T suppressor cells (Ts) express the interleukin-2 receptor (IL-2R) as an activated T cell population, an attempt was made to eliminate these Ts from the developing immune system of Tx-7 mice and normal mice by i.p. injection of anti-IL-2Rα monoclonal antibodies. Interestingly, organ-localized autoimmune disease with quite similar characteristics to those observed after neonatal Tx developed in not only Tx-7 mice, but also normal mice. The results thus indicate that CD4⁺ cells expressing IL-2Rα play an important role, as Ts in the periphery, in maintaining immune tolerance.