Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes

T cell receptor (TCR) alpha alleles undergo primary and secondary rearrangement in double-positive (DP) thymocytes. By analyzing TCRalpha rearrangement in orphan nuclear receptor RORgamma-deficient mice, in which the DP lifespan is shorter, and in Bcl-x(L)-transgenic mice, in which the DP lifespan is extended, we show that the progression of secondary V(alpha) to J(alpha) rearrangements is controlled by DP thymocyte survival. In addition, because Bcl-x(L) induces a bias towards 3' J(alpha) usage in peripheral T cells, we conclude that the programmed cell death of DP thymocytes is not simply a consequence of failed positive selection. Rather, it limits the progression of rearrangement along the J(alpha) locus and the opportunities for positive selection, thereby regulating the TCRalpha repertoire.     

4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells

Interactions between 4-1BB and its ligand, 4-1BBL, enhance CD8(+) T cell-mediated antiviral and antitumor immunity in vivo. However, mechanisms regulating the priming of CD8(+) T cell responses by 4-1BB remain unclear, particularly in humans. The 4-1BB receptor was undetectable on naive or resting human CD8(+) T cells and induced in vitro by TCR triggering. Naive cord blood cells were therefore primed in vitro against peptides or cellular antigens and then co-stimulated with 4-1BBL or agonistic antibodies. Co-stimulation enhanced effector function such as IFN-gamma production and cytotoxicity by augmenting numbers of antigen-specific and effector CD8(+) T cells. OKT3 responses also showed reduced cell death and revealed that the proliferation of CD8(+) T cells required two independently regulated events. One, the induction of IL-2 production, could be directly triggered by 4-1BB engagement on CD8(+) T cells in the absence of accessory cells. The other, expression of CD25, was induced with variable efficacy by accessory cells. Thus, suboptimal accessory cells and 4-1BB co-stimulation combined their effects to enhance IL-2 production and proliferation. Reduced apoptosis observed after co-stimulation in the presence of accessory cells correlated with increased levels of Bcl-X(L) in CD8(+) T cells, while Bcl-2 expression remained unchanged. Altogether, 4-1BB enhanced expansion, survival and effector functions of newly primed CD8(+) T cells, acting in part directly on these cells. As 4-1BB triggering could be protracted from the TCR signal, 4-1BB agonists may function through these mechanisms to enhance or rescue suboptimal immune responses.     

Heterogeneity of CD4(+) and CD8(+) T cells

There is extensive plasticity in the T-cell response to antigen. Helper CD4(+) T cells, cytotoxic CD8(+) T cells, the progression from na?ve to effector and memory T cells, and differentiation into Th1, Tc1, Th2 and Tc2 subsets have long been recognized. More recently it has become apparent that T-cell populations display additional diversity in terms of phenotype, anatomical distribution and effector function.     

PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2

Programmed death-1 (PD-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor expressed upon T cell activation. PD-1(-/-) animals develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses. Members of the B7 family, PD-L1 and PD-L2, are ligands for PD-1. This study examines the functional consequences of PD-1:PD-L engagement on murine CD4 and CD8 T cells and shows that these interactions result in inhibition of proliferation and cytokine production. T cells stimulated with anti-CD3/PD-L1.Fc-coated beads display dramatically decreased proliferation and IL-2 production, while CSFE analysis shows fewer cells cycling and a slower division rate. Costimulation with soluble anti-CD28 mAb can overcome PD-1-mediated inhibition by augmenting IL-2 production. However, PD-1:PD-L interactions inhibit IL-2 production even in the presence of costimulation and, thus, after prolonged activation, the PD-1:PD-L inhibitory pathway dominates. Exogenous IL-2 is able to overcome PD-L1-mediated inhibition at all times, indicating that cells maintain IL-2 responsiveness. Experiments using TCR transgenic CD4(+) or CD8(+) T cells stimulated with antigen-presenting cells expressing PD-L1 show that both T cell subsets are susceptible to this inhibitory pathway. However, CD8(+) T cells may be more sensitive to modulation by the PD-1:PD-L pathway because of their intrinsic inability to produce significant levels of IL-2.     

Functions of tetramer-stained HIV-specific CD4(+) and CD8(+) T cells

Significant insight has been gained into constraints on the sensitivity and specificity of staining with class I tetramers. The function of the populations that are defined varies with the clinical situation. Insight has also been gained into the determinants of the CD8(+) T cell response during primary and chronic HIV infection. Human class II tetramers have been synthesised but their role in defining CD4(+) T cell function in HIV infection remains to be determined.     

IL-7 induces proliferation of CD3 /low CD4 CD8 human thymocyte precursors by an IL-2 independent pathway

The proliferation potential of highly purified human CD3^–CD4^–CD8^–(triple negative) and CD3^low(lo)CD4^–CD8^– thymocyteprecursors in response to various cytokines was investigated.High in vitro growth ability was observed in response to recombinanthuman IL-2 (riL-2) and human riL-7, both in the absence of anyco-mitogen and in combination with phorbol 12-myristate 13-acetate(PMA). Furthermore, the proliferation of these thymocyte precursorsin the presence of rlL-7, although accompanied by a significantincrease of IL-2 receptor (IL-2R) p55 expression, appeared independentof that mediated by the autocrine IL-2 pathway, since mAbs toIL-2 and IL-2R p55 did not eliminate responsiveness to rlL-7.Synergism of rlL-7 with rlL-2 was also observed, while no cooperationwas detectable with rlL-4 or rlL-6. Analysis of surface phenotypeand cell cycle status of cells cultured in the presence of rlL-7,both plus and minus PMA, showed that CD3^– as well as CD3¹⁰cells readily proliferated to rlL-7. Upregulation of the levelsof expression of CD3 antigen was also observed in these cultures.These results, together with the previous characterization ofIL-7 as a human pre-B cell and mature T cell growth factor,Identify IL-7 as a cytokine with biologic activities on a varietyof target cells. They also suggest that IL-7, in analogy withthe mouse system, might play a role in human T cell ontogeny.     

Effect of murine thymic epithelial cell line (MTEC1) on the functional expression of CD4(+)CD8(-) thymocyte subgroups

To determine the effect of thymic stromal cells on the functional maturation of CD4 single-positive (SP) thymocytes, the functional status of isolated CD4 SP thymocyte subgroups was investigated by means of cell proliferation and cytokine production in response to concanavalin A (Con A) prior and after co-culturing with a murine thymic epithelial cell line (MTEC1). Mouse medullary CD4 SP thymocytes were phenotypically divided into seven discrete subgroups predicted to reflect the maturation pathway from newly emerging CD4 SP thymocytes to terminally differentiated cells. For functional analysis, six major subgroups (6C10(+)CD69(+), 6C10(-)CD69(+), 6C10(-)CD69(-)3G11(+)Qa-2(-), 6C10(-)CD69(-)3G11(+)Qa-2(+), 6C10(-)CD69(-)3G11(-)Qa-2(-) and 6C10(-)CD69(-)3G11(-)Qa-2(+)) cells were isolated and their functional status in response to Con A stimulation assessed. A functional hierarchy is revealed among these subgroups, consistent with their phenotypic maturation status, which may imply that these cells undergo a functional maturation process within thymic medulla. The function of cytokine production by CD4 SP thymocytes is acquired in a stepwise manner from a low to high level and characterized by T(h)0-type cytokines in the main stream of differentiation pathway. However, a minor subgroup that appeared at the late stage as 3G11(-)6C10(-) cells was biased to produce T(h)2-type cytokines. Nevertheless, the functional capacity of the final two Qa-2(+) subgroups of CD4 SP thymocytes was still significantly lower than that of spleen CD4(+) T cells. After co-cultivation with MTEC1 cells, four subgroups of TCRalphabeta(+)CD4(+)CD8(-) thymocytes exhibited significantly higher levels of proliferation capability and modulation in cytokine production capability. However, co-culturing with MTEC1 cells did not change the pattern of T(h)0- or T(h)2-like cytokine production by respectively medullary CD4 SP thymocyte subgroups nor could MTEC1 induce CD4 SP thymocytes to secrete T(h)1-type cytokines. The results suggest that MTEC1 can regulate the functional status of these thymocyte subgroups.     

Diacylglycerol kinase alpha activity promotes survival of CD4+ 8+ double positive cells during thymocyte development

The diacylglycerol kinases (DGK) form a family of isoenzymes that catalyse the conversion of diacylglycerol (DAG) to phosphatidic acid (PA), both powerful second messengers in the cell. DGKalpha is expressed in brain, peripheral T cells and thymocytes and has been shown to translocate to the nuclear matrix upon T-cell receptor (TCR) engagement. Here, we show that high level expression of DGKalpha is induced following a signal transmitted through the pre-TCR and the protein tyrosine kinase, lck. Activity of DGKalpha contributes to survival in CD4+ 8+ (DP) thymocytes as pharmacological inhibition of DGK activity results in death of this cell population both in cell suspension and thymic explants. DGKalpha promotes survival in these thymocytes through a Bcl-regulated pathway. A consequence of inhibition of DGKalpha is the specific down-regulation of Bcl-xl, whereas in transgenic mice that over-express Bcl-2, death induced by the inhibitor is partially blocked. Thus we report a novel activity of DGKalpha in survival of thymocytes immediately after entry into the DP stage in development.     

Heterogeneity within medullary-type TCRalphabeta(+)CD3(+)CD4(-)CD8(+) thymocytes in normal mouse thymus

The functional maturation process of medullary-type CD4(-)CD8(+) [CD8 single-positive (SP)] thymocytes remains largely uncharacterized. We describe a phenotypic analysis of CD8 SP medullary-type thymocytes and find a remarkable heterogeneity within this thymic cell population. While mature CD8(+) T cells in the periphery are relatively homogeneous (TCRalphabeta(+)CD3(+)Qa-2(+) HSA(-)3G11(-)6C10(-)CD69(-)), CD8 SP medullary-type thymocytes contain discrete subpopulations that can be identified by differential expression of several cell-surface markers. We have identified at least six discrete subpopulations in the subset of TCRalphabeta(+)CD3(+) CD8 SP cells in the thymus. According to the expressed phenotypes, a linear developmental pathway is predicted among these CD8 SP subpopulations as follows: 6C10(+)CD69(+)HSA(hi)3G11(+)Qa-2(-) --> 6C10(-)CD69(+)HSA(hi/int)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(int)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(lo)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(-/lo)3G11(-)Qa-2(-) --> 6C10(-)CD69(-)HSA(-/lo)3G11(-)Qa-2(+). This study provides a framework for understanding CD8 SP T cell maturation in the thymic medulla.     

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge

DNA vaccination offers the advantages of viral gene expressionwithin host cells without the risks of infectious virus. Likeviral vaccines, DNA vaccines encoding internal influenza virusproteins can induce immunity to conserved epitopes and so maydefend the host against a broad range of viral variants. CD8⁺cytotoxic T lymphocytes (CTL) have been described as essentialeffectors in protection by influenza nucleoprotein (NP), althougha lesser role of CD4⁺ cells has been reported. We immunizedmice with plasmids encoding influenza virus NP and matrix (M).NP + M DNA allowed B6 mice to survive otherwise lethal challengeinfection, but did not protect B6-ß₂m(–/–)mice defective in CD8⁺ CTL. However, this does not prove CTLare required, because ß₂m(–/–) mice have multipleimmune abnormalities. We used acute T cell depletion in vivoto identify effectors critical for defense against challengeinfection. Since lung lymphocytes are relevant to virus clearance,surface phenotypes and cytolytic activity of lung lymphocyteswere analyzed in depleted animals, along with lethal challengestudies. Depletion of either CD4⁺ or CD8⁺ T cells in NP + MDNA-immunized BALB/c mice during the challenge period did notsignificantly decrease survival, while simultaneous depletionof CD4⁺ and CD8⁺ cells or depletion of all CD90⁺ cells completelyabrogated survival. We conclude that T cell immunity inducedby NP + M DNA vaccination is responsible for immune defense,but CD8⁺ T cells are not essential in the active response tothis vaccination. Either CD4⁺ or CD8⁺ T cells can promote survivaland recovery in the absence of the other subset.     

Naive CD8(+) but not CD4(+) T cells induce maturation of dendritic cells

We have shown previously that the generation of tumor-reactive CD8(+) cytotoxic T lymphocytes require qualitatively different signals from CD4(+) and CD8(+) T cells that most likely are provided to dendritic cells (DCs). This raises the question of whether the two T cell subsets are equally able to deliver the initial activation signal to DCs. Using ovalbumin as a model antigen we show that naive CD4(+) T cells cannot activate immature DCs and do not become activated, even though they recognize antigen on immature DCs. In contrast, naive CD8(+) T cells rapidly activate DCs and subsequently start to proliferate. This suggests that CD8(+) T cells contribute to DC activation prior to CD4(+) T cells and implies that CD8(+) T cells can provide help to CD4(+) T cells.     

CD4(+) T cell-released exosomes inhibit CD8(+) cytotoxic T-lymphocyte responses and antitumor immunity

T cells secrete bioactive exosomes (EXO), but the potential immunoregulatory effect of T-cell EXO is largely unknown. In this study, we generated activated ovalbumin (OVA)-specific CD4⁺ T cells in vitro via coculture of OVA-pulsed dendritic cells (DC_OVA) with naive CD4⁺ T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTII mice. CD4⁺ T-cell EXO were then purified from the CD4⁺ T-cell culture supernatants by differential ultracentrifugation. CD4⁺ T-cell EXO exhibited the ‘saucer'' shape that is characteristic of EXO with a diameter between 50 and 100 nm, as assessed by electron microscopy, and contained the EXO-associated proteins LAMP-1, TCR and lymphocyte function associated antigen-1 (LFA-1), as determined by western blot. Flow cytometric analysis showed that CD4⁺ T-cell EXO expressed CD4⁺ T-cell markers (CD4, TCR, LFA-1, CD25 and Fas ligand), but to a lesser extent than CD4⁺ T cells. We demonstrated that DC_OVA took up CD4⁺ T-cell EXO via peptide/major histocompatibility complex (pMHC) II/TCR and CD54/LFA-1 interactions. OVA-specific CD4⁺ T-cell EXO from OTII mice, but not ConA-stimulated polyclonal CD4⁺ T-cell EXO from wild-type C57BL/6 mice inhibited DC_OVA-stimulated in vitro CD4⁺ T-cell proliferation and in vivo CD8⁺ cytotoxic T lymphocyte (CTL) responses and antitumor immunity against OVA-expressing B16 melanoma BL6-10_OVA cells. In addition, EXO derived from a T-cell hybridoma cell line, MF72.2D9, expressing an OVA-specific CD4⁺ TCR, had a similar inhibitory effect as OTII CD4⁺ T-cell EXO on CTL-mediated antitumor immunity. Taken together, our data indicate that antigen-specific T-cell EXO may serve as a new type of immunosuppressive reagent for use in transplant rejection and treatment of autoimmune diseases.     

The CD4(+) T cell response to HIV-1

Although virus-specific CD4(+) T cells have proven to be a critical component of the immunologic control of chronic viral infections in a number of models, the role and even the existence of HIV-1-specific CD4(+) T cells in human HIV-1 infection has been controversial. Recent advances in quantifying and functionally characterizing HIV-1-specific T cells may shed light on the participation of these cells in anti-HIV-1 host defense.     

Rac1-mediated Bcl-2 induction is critical in antigen-induced CD4 single-positive differentiation of a CD4+CD8+ immature thymocyte line

Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a "molecular switch" in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single-positive (CD4-SP) T cells from CD4CD8 double-positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4-SP cells upon TCR stimulation in vitro. DPK expressing dominant-negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4-SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR-dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1-expressing DPK. We found that TCR-dependent induction of Bcl-2 was suppressed greatly in dnRac1-expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl-2 inhibited TCR-dependent induction of apoptosis and restored CD4-SP generation in dnRac1-expressing DPK without restoring TCR-induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4-SP from the DP cell line by preventing TCR-induced apoptosis via Bcl-2 up-regulation.     

Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity

Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing.     

Peripheral blood extrathymic CD4(+)CD8(+) T cells with high cytotoxic activity are from the same lineage as CD4(+)CD8(-) T cells in cynomolgus monkeys

We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin-granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-gamma and produced a high level of IFN-gamma when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR V(beta) with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys.     

Rat eosinophils stimulate the expansion of Cryptococcus neoformans-specific CD4(+) and CD8(+) T cells with a T-helper 1 profile

Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, revealing a strong granulomatous response and a low susceptibility to dissemination. Moreover, it has been shown that eosinophils are components of the inflammatory response to C. neoformans infections. In this in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, and that the phenomenon involves the engagement of FcγRII and CD18. Moreover, our results showed that the phagocytosis of opsonized C. neoformans triggers eosinophil activation, as indicated by (i) the up‐regulation of major histocompatibility complex (MHC) class I, MHC class II and costimulatory molecules, and (ii) an increase in interleukin (IL)‐12, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) production. However, nitric oxide (NO) and hydrogen peroxide (H₂O₂) synthesis by eosinophils was down‐regulated after interaction with C. neoformans. Furthermore, this work demonstrated that CD4⁺ and CD8⁺ T lymphocytes isolated from spleens of infected rats and cultured with C. neoformans‐pulsed eosinophils proliferate in an MHC class II‐ and class I‐dependent manner, respectively, and produce important amounts of T‐helper 1 (Th1) type cytokines, such as TNF‐α and IFN‐γ, in the absence of T‐helper 2 (Th2) cytokine synthesis. In summary, the present study demonstrates that eosinophils act as fungal antigen‐presenting cells and suggests that C. neoformans‐loaded eosinophils might participate in the adaptive immune response.     

Ontogeny of a novel CD4+CD8-CD3- thymocyte subpopulation: a comparison with CD4- CD8+ CD3- thymocytes

We have studied the ontogeny of a novel thymocyte subset, CD4+CD8-CD3-. Three-colour flow cytometric analysis demonstrated that these cells constituted approximately 1% of the total thymocyte content in adult CBA mice, and were not present in lymph nodes. They were mainly blastic, cortisone-sensitive, and localized in the outer thymic cortex. During foetal life they were first observed at day 15 and reached a maximum (6%) at day 17, beyond which they decreased to the adult level. This kinetic profile was similar to that of the CD4-CD8+CD3- subpopulation, except that the CD4+CD8-CD3- cells appeared slightly earlier and their percentage was lower. Both these populations appeared after the CD4-CD8-CD3- cells but before the CD4+CD8+CD3- cells. Similar observations were made during thymic reconstitution following dexamethasone treatment. In this case, both CD4+CD8-CD3- and CD4-CD8+CD3- thymocytes disappeared 48 h after the treatment. While their absolute number increased up to 14 days post-treatment, their percentage was maximal at day 7 post-treatment and returned to normal values by day 10 post-treatment. These results argue strongly that not only the CD4-CD8+CD3- population but also the CD4+CD8-CD3- population can be considered an intermediate precursor in CBA thymuses.