Regulation of lymphoid homeostasis by IL-2 receptor signals in vivo

High-affinity IL-2R signals are required for peripheral lymphoid homeostasis in vivo. We found that CD25 was required for regulation of peripheral T cells in mice bearing either the DO11.10 MHC class II-restricted TCR transgene or an Iabeta-null mutation, suggesting that MHC class I- and class II-dependent T cell subsets are regulated independently by IL-2R signals. In contrast, deregulation of serum IgG1 levels in CD25-/- mice was dependent on CD4+ T cells. T cell expansion in DO11.10 CD25-/- mice was not preferential for cells escaping allelic exclusion by the TCR transgene, but was suppressed by a Rag-2-null mutation. Together, these findings suggest that endogenous TCR are required to trigger T cell expansion, but that CD25 regulates T cells activated by low-specificity signals. Expansion of DO11.10 T cells in response to cognate Ag was modestly reduced in CD25-/- T cells transferred into the normal lymphoid compartments of BALB/c mice. Moreover, activation-induced clonal contraction and apoptosis in vivo were intact in the absence of CD25. These data indicate that the regulatory role of high-affinity IL-2R signals extends beyond the control of Ag-specific responses and suggest a role for these signals in control of bystander T cell activation.     

Continuous exposure of mice to superantigenic toxins induces a high-level protracted expansion and an immunological memory in the toxin-reactive CD4+ T cells

We analyzed the responses of several T cell fractions reactive with superantigenic toxins (SAGTs), staphylococcal enterotoxin A (SEA), or Yersinia pseudotuberculosis-derived mitogen (YPM) in mice implanted with mini-osmotic pumps filled with SEA or YPM. In mice implanted with the SEA pump, SEA-reactive Vbeta3(+)CD4(+) T cells exhibited a high-level protracted expansion for 30 days, and SEA-reactive Vbeta11(+)CD4(+) T cells exhibited a low-level protracted expansion. SEA-reactive CD8(+) counterparts exhibited only a transient expansion. A similar difference in T cell expansion was also observed in YPM-reactive T cell fractions in mice implanted with the YPM pump. Vbeta3(+)CD4(+) and Vbeta11(+)CD4(+) T cells from mice implanted with the SEA pump exhibited cell divisions upon in vitro restimulation with SEA and expressed surface phenotypes as memory T cells. CD4(+) T cells from mice implanted with the SEA pump exhibited high IL-4 production upon in vitro restimulation with SEA, which was due to the enhanced capacity of the SEA-reactive CD4(+) T cells to produce IL-4. The findings in the present study indicate that, in mice implanted with a specific SAGT, the level of expansion of the SAGT-reactive CD4(+) T cell fractions varies widely depending on the TCR Vbeta elements expressed and that the reactive CD4(+) T cells acquire a capacity to raise a memory response. CD8(+) T cells are low responders to SAGTs.     

Rapid and selective expansion of nonclonotypic T cells in regulatory T cell-deficient, foreign antigen-specific TCR-transgenic scurfy mice: antigen-dependent expansion and TCR analysis

Foreign Ag-specific TCR-transgenic (Tg) mice contain a small fraction of T cells bearing the endogenous Vbeta and Valpha chains as well as a population expressing an intermediate level of Tg TCR. Importantly, these minor nonclonotypic populations contain > or = 99% of the CD4(+)Foxp3(+) regulatory T cells (Treg) and, despite low overall Treg expression, peripheral tolerance is maintained. In the OT-II TCR (OVA-specific, Vbeta5(high)Valpha2(high)) Tg scurfy (Sf) mice (OT-II Sf) that lack Treg, nonclonotypic T cells markedly expanded in the periphery but not in the thymus. Expanded T cells expressed memory/effector phenotype and were enriched in blood and inflamed lungs. In contrast, Vbeta5(high)Valpha2(high) clonotypic T cells were not expanded, displayed the naive phenotype, and found mainly in the lymph nodes. Importantly, Vbeta5(neg) T cells were able to transfer multiorgan inflammation in Rag1(-/-) recipients. T cells bearing dual TCR (dual Vbeta or dual Valpha) were demonstrated frequently in the Vbeta5(int) and Valpha2(int) populations. Our study demonstrated that in the absence of Treg, the lack of peripheral expansion of clonotypic T cells is due to the absence of its high-affinity Ag OVA. Thus, the rapid expansion of nonclonotypic T cells in OT-II Sf mice must require Ag (self and foreign) with sufficient affinity. Our study has implications with respect to the roles of Ag and dual TCR in the selection and regulation of Treg and Treg-controlled Ag-dependent T cell expansion in TCR Tg and TCR Tg Sf mice, respectively.     

Alteration of V beta usage and cytokine production of CD4+ TCR beta beta homodimer T cells by elimination of Bacteroides vulgatus prevents colitis in TCR alpha-chain-deficient mice

A major pathogenic factor for the development of inflammatory bowel disease (IBD) is the breakdown of the intestinal homeostasis between the host immune system and the luminal microenvironment. To assess the potential influence of luminal Ags on the development of IBD, we fed TCR alpha(-/-) mice an elemental diet (ED). ED-fed TCR alpha(-/-) mice showed no pathologic features of IBD, and their aberrant mucosal B cell responses were suppressed. Similar numbers of CD4(+), TCR betabeta homodimer T cells (betabeta T cells) were developed in the colonic mucosa of ED-fed mice; however, Th2-type cytokine productions were lower than those seen in diseased regular diet (RD)-fed mice. The higher cytokine production in diseased RD-fed mice could be attributed to the high incidence of Bacteroides vulgatus (recovered in 80% of these mice), which can induce Th2-type responses of colonic CD4(+), betabeta T cells. In contrast, ED-fed TCR alpha(-/-) mice exhibited a diversification of Vbeta usage of betabetaT cell populations from the dominant Vbeta8 one associated with B. vulgatus in cecal flora to Vbeta6, Vbeta11, and Vbeta14. Rectal administration of disease-free ED-fed mice with B. vulgatus resulted in the development of Th2-type CD4(+), betabeta T cell-induced colitis. These findings suggest that the ED-induced alteration of intestinal microenvironments such as the enteric flora prevented the development of IBD in TCR alpha(-/-) mice via the immunologic quiescence of CD4(+), betabeta T cells.     

Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25- TGF-beta 1+ adaptive regulatory T cells

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.     

Role of the complementarity-determining region 3 (CDR3) of the TCR-beta chains associated with the V alpha 14 semi-invariant TCR alpha-chain in the selection of CD4+ NK T Cells

The NK1.1(+)TCRalphabeta(int) CD4(+), or double negative T cells (NK T cells) consist of a mixture of CD1d-restricted and CD1d-unrestricted cells. The relationships between CD4(+)NK1.1(+) T cells and conventional T cells are not understood. To compare their respective TCR repertoires, NK1.1(+)TCRalphabeta(int), CD4(+) T cells have been sorted out of the thymus, liver, spleen, and bone marrow of C57BL/6 mice. Molecular analysis showed that thymus and liver used predominantly the Valpha14-Jalpha281 and Vbeta 2, 7, and 8 segments. These cells are CD1d restricted and obey the original definition of NK T cells. The complementarity-determining region 3 (CDR3) sequences of the TCR Vbeta8.2-Jbeta2.5 chain of liver and thymus CD4(+) NK T cells were determined and compared with those of the same rearrangements of conventional CD4(+) T cells. No amino acid sequence or usage characteristic of NK T cells could be evidenced: the Vbeta8.2-Jbeta2.5 diversity regions being primarily the same in NK T and in T cells. No clonal expansion of the beta-chains was observed in thymus and liver CD1d-restricted CD4(+)NK T cells, suggesting the absence of acute or chronic Ag-driven stimulation. Molecular analysis of the TCR used by Valpha14-Jalpha281 transgenic mice on a Calpha(-/-) background showed that the alpha-chain can associate with beta-chains using any Vbeta segment, except in NK T cells in which it paired predominately with Vbeta 2, 7, and 8(+) beta-chains. The structure of the TCR of NK T cells thus reflects the affinity for the CD1d molecule rather than a structural constraint leading to the association of the invariant alpha-chain with a distinctive subset of Vbeta segment.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

Antigen-driven interactions with dendritic cells and expansion of Foxp3+ regulatory T cells occur in the absence of inflammatory signals

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.     

Administration of an antigen at a high dose generates regulatory CD4+ T cells expressing CD95 ligand and secreting IL-4 in the liver

Ags administered orally at a high dose are absorbed in immunogenic forms and perfuse the liver, which raises a question regarding the relevance of hepatic lymphocyte activation to the systemic hyporesponsiveness against the ingested Ag. Oral administration of 100 mg of OVA to the mice led to massive cell death of OVA-specific (KJ1-26+)CD4+ T cells by Fas-Fas ligand (FasL)-mediated apoptosis in the liver, which was associated with the emergence of hepatic KJ1-26+CD4+ T cells expressing FasL. Hepatic CD4+ T cells in OVA-fed mice secreted large amounts of IL-4, IL-10, and TGF-beta(1) upon restimulation in vitro and inhibited T cell proliferation. Adoptive transfer of these hepatic CD4+ T cells to naive mice and subsequent antigenic challenge led to suppression of T cell proliferation as well as IgG Ab responses to OVA; this effect was mostly abrogated by a blocking Ab to FasL. i.p. administration of an Ag at a high dose also generated hepatic CD4+FasL+ T cells with similar cytokine profile as T cells activated by oral administration of Ags at a high dose. Finally, we did not see an increase in FasL+ cells in the hepatic CD4+Vbeta8+ T cell subset of MRL/lpr/lpr mice given staphylococcal enterotoxin B, indicating the requirement for Fas-mediated signals. These hepatic CD4+FasL+ regulatory cells may explain the tolerogenic property of the liver and play roles in systemic hyporesponsiveness induced by an Ag administered at a high dose.     

Valpha24-JalphaQ-independent,CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid alpha-galactosylceramide (alphaGC), to a highly conserved NKT cell subset expressing an invariant TCR Valpha24-JalphaQ paired with Vbeta11 chain (Valpha24(+)Vbeta11(+) invariant NK T cell (NKT(inv))). The developmental pathway of Valpha24(+)Vbeta11(+)NKT(inv) is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-alphaGC-tetramers, we demonstrate that in humans, TCR variable domains other than Valpha24 and Vbeta11 can mediate specific recognition of CD1d-alphaGC. In contrast to Valpha24(+)Vbeta11(+)NKT(inv) cells, Valpha24(-)/CD1d-alphaGC-specific T cells express either CD8alphabeta or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8alphabeta(+)Valpha24(-)/CD1d-alphaGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Valpha24(+)/CD1d-alphaGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-alphaGC complex in humans is not uniformly restricted to the Valpha24-JalphaQ/Vbeta11 NKT cell subset, but can be mediated by a diverse range of Valpha and Vbeta domains. The existence of a diverse repertoire of CD1d-alphaGC-specific T cells in humans strongly supports their Ag-driven selection.     

CD28 is required for induction and maintenance of immunological memory in toxin-reactive CD4+ T cells in vivo

We previously reported that Vbeta3+ CD4+ T cells maintained a protracted expansion, with the phenotypes of memory Th2 cells, for 30 days in C57BL/6 (B6) mice implanted with SEA-containing mini-osmotic pumps. In the present study, we followed the fate of Vbeta3+ CD4+ T cells in CD28-/- mice. Vbeta3+ CD4+ T cells increased to a degree similar to that of B6 Vbeta3+ CD4+ T cells until day 10 after implantation, then declined rapidly reaching the control level by 28 days. Remaining Vbeta3+ CD4+ T cells at that time did not exhibit memory phenotypes nor Th2-deviated responses. The rapid drop in Vbeta3+ CD4+ T cells in CD28-/- mice was attributable to upregulated induction of apoptosis owing to marginal inductions of Bcl-2 and Bcl-xL. Collectively, these data indicate CD28 to play critical roles in the generation and maintenance of SEA-reactive CD4+ T cells in vivo.     

Th3 cells in peripheral tolerance. I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-beta T cell-transgenic mice

TGF-beta has been shown to be critical in the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Because Th3 cells produce large amounts of TGF-beta, we asked whether induction of Th3 cells in the periphery was a mechanism by which CD4(+)CD25(+) Tregs were induced in the peripheral immune compartment. To address this issue, we generated a TGF-beta1-transgenic (Tg) mouse in which TGF-beta is linked to the IL-2 promoter and T cells transiently overexpress TGF-beta upon TCR stimulation but produce little or no IL-2, IL-4, IL-10, IL-13, or IFN-gamma. Naive TGF-beta-Tg mice are phenotypically normal with comparable numbers of lymphocytes and thymic-derived Tregs. We found that repeated antigenic stimulation of pathogenic myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+)CD25(-) T cells from TGF-beta Tg mice crossed to MOG TCR-Tg mice induced Foxp3 expression in both CD25(+) and CD25(-) populations. Both CD25 subsets were anergic and had potent suppressive properties in vitro and in vivo. Furthermore, adoptive transfer of these induced regulatory CD25(+/-) T cells suppressed experimental autoimmune encephalomyelitis when administrated before disease induction or during ongoing experimental autoimmune encephalomyelitis. The suppressive effect of TGF-beta on T cell responses was due to the induction of Tregs and not to the direct inhibition of cell proliferation. The differentiation of Th3 cells in vitro was TGF-beta dependent as anti-TGF-beta abrogated their development. Thus, Ag-specific TGF-beta-producing Th3 cells play a crucial role in inducing and maintaining peripheral tolerance by driving the differentiation of Ag-specific Foxp3(+) regulatory cells in the periphery.     

Superantigen enhancement of specific immunity: antibody production and signaling pathways

Superantigens are microbial proteins that induce massive activation, proliferation, and cytokine production by CD4+ T cells via specific Vbeta elements on the TCR. In this study we examine superantigen enhancement of Ag-specific CD4+ T cell activity for humoral B cell responses to T-dependent Ags BSA and HIV gp120 envelope, type I T-independent Ag LPS, and type II T-independent Ag pneumococcal polysaccharides. Injection of BSA followed by a combination of superantigens staphylococcal enterotoxin A and staphylococcal enterotoxin B (SEB) 7 days later enhanced the anti-BSA Ab response in mice approximately 4-fold as compared with mice given BSA alone. The anti-gp120 response was enhanced approximately 3-fold by superantigens. The type II T-independent Ag pneumococcal polysaccharide response was enhanced approximately 2.3-fold by superantigens, whereas no effect was observed on the response to the type I T-independent Ag LPS. The superantigen effect was completely blocked by the CD4+ T cell inhibitory cytokine IL-10. SEB-stimulated human CD4+ T cells were examined to determine the role of the mitogen-activated protein (MAP) kinase signal transduction pathway in superantigen activation of T cells. Inhibitors of the mitogen pathway of MAP kinase blocked SEB-induced proliferation and IFN-gamma production, while an inhibitor of the p38 stress pathway had no effect. Consistent with this, SEB activated extracellular signal-regulated kinase/MAP kinase as well as MAP kinase-interacting kinase, a kinase that phosphorylates eIF4E, which is an important component of the eukaryotic protein synthesis initiation complex. Both kinases were inhibited by IL-10. Thus, superantigens enhance humoral immunity via Ag-specific CD4+ T cells involving the stress-independent pathway of MAP kinase.     

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells

A major group of murine NK T (NKT) cells express an invariant Valpha14Jalpha18 TCR alpha-chain specific for glycolipid Ags presented by CD1d. Murine Valpha14Jalpha18(+) account for 30-50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Valpha24Vbeta11(+) NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3(+) cells) and blood (0.02%). In contrast to those in blood, most hepatic Valpha24(+) NKT cells express the Vbeta11 chain. They include CD4(+), CD8(+), and CD4(-)CD8(-) cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Valpha24(+) T cells are potent producers of IFN-gamma and TNF-alpha, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, alpha-galactosylceramide. Valpha24(+)Vbeta11(+) cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-gamma in response to alpha-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.     

Cutting edge: two distinct mechanisms lead to impaired T cell homeostasis in Janus kinase 3- and CTLA-4-deficient mice

Cytokine receptor signaling and costimulatory receptor signaling play distinct roles in T cell activation. Nonetheless, deficiencies in either of these pathways lead to seemingly similar phenotypes of impaired T cell homeostasis. A dramatic expansion of CD4(+) peripheral T cells with an activated phenotype has been observed in both Janus kinase (Jak) 3-deficient and CTLA-4-deficient mice. Despite these similarities, the mechanisms driving T cell expansion may be distinct. To address this possibility, we examined the TCR repertoire of peripheral T cells in Jak3(-/-) and CTLA-4(-/-) mice using complementarity-determining region 3 spectratype analysis. Interestingly, a restricted and highly biased TCR repertoire was observed in the Jak3(-/-) T cells, strongly supporting a role for foreign Ag in the activation and expansion of these cells. In contrast, CTLA-4(-/-) T cells had a diverse and unbiased TCR repertoire, suggestive of a universal, Ag-independent mechanism of activation and expansion. These findings provide insight into the diverse mechanisms controlling T cell homeostasis.     

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.     

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.     

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.