The Nef-mediated AIDS-like disease of CD4C/human immunodeficiency virus transgenic mice is associated with increased Fas/FasL expression on T cells and T-cell death but is not prevented in Fas-, FasL-, tumor necrosis factor receptor 1-, or interleukin-1beta-converting enzyme-deficient or Bcl2-expressing transgenic mice

CD4(+)- and CD8(+)-T-cell death is a frequent immunological dysfunction associated with the development of human AIDS. We studied a murine model of AIDS, the CD4C/HIV transgenic (Tg) mouse model, to assess the importance of the apoptotic pathway in human immunodeficiency virus type 1 (HIV-1) pathogenesis. In these Tg mice, Nef is the major determinant of the disease and is expressed in immature and mature CD4(+) T cells and in cells of the macrophage/myeloid lineage. We report here a novel AIDS-like phenotype: enhanced death, most likely by apoptosis (as assessed by 7-aminoactinomycin D and annexin V/propidium iodide staining), of Tg thymic and peripheral CD4(+) and CD8(+) T cells. The Tg CD4(+) and CD8(+) T cells were also more susceptible to cell death after activation in vitro in mixed lymph node (LN) cultures. However, activation-induced cell death was not higher in Tg than in non-Tg-purified CD4(+) T cells. In addition, expression of Fas and FasL, assessed by flow cytometry, was increased in CD4(+) and CD8(+) T cells from Tg mice compared to that of non-Tg littermates. Despite the enhanced expression of Fas and FasL on Tg CD4(+) and CD8(+) T cells, Fas (lpr/lpr) and FasL (gld/gld) mutant CD4C/HIV Tg mice developed an AIDS-like disease indistinguishable from lpr/+ and gld/+ CD4C/HIV Tg mice, including loss of CD4(+) T cells. Similarly, CD4C/HIV Tg mice homozygous for mutations of two other genes implicated in cell death (interleukin-1beta-converting enzyme [ICE], tumor necrosis factor receptor 1 [TNFR-1]) developed similar AIDS-like disease as their respective heterozygous controls. Moreover, the double-Tg mice from a cross between the Bcl2/Wehi25 and CD4C/HIV Tg mice showed no major protection against disease. These results represent genetic evidence for the dispensable role of Fas, FasL, ICE, and TNFR-1 on the development of both T-cell loss and organ disease of these Tg mice. They also provide compelling evidence on the lack of protection by Bcl2 against Tg CD4(+)-T-cell death. In view of the high resemblance between numerous phenotypes observed in the CD4C/HIV Tg mice and in human AIDS, our findings are likely to be relevant for the human disease.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

Primary human immunodeficiency virus type 1 nef alleles show major differences in pathogenicity in transgenic mice

We previously reported that the human immunodeficiency virus type 1 NL4-3 Nef is necessary and sufficient to induce a severe AIDS-like disease in transgenic (Tg) mice when the protein is expressed under the regulatory sequences of the human CD4 gene. We have now assayed additional Nef alleles (SF2, JR-CSF, YU10x, and NL4-3 [T71R] Nef alleles), including some from long-term nonprogressors (AD-93, 032an, and 039nm alleles) in the same Tg system and compared their pathogenicities. All these Nef alleles downregulated cell surface CD4 in human cells in vitro and also, with the exception of Nef(YU10x), in Tg CD4(+) T cells. Depletion of double-positive and single-positive thymocytes occurred with all alleles but was less pronounced in Nef(YU10x) Tg mice. A loss of peripheral CD4(+) T cells was observed with all alleles but was minimal in Nef(YU10x) Tg mice. In Nef(032an) and Nef(SF2) Tg mice, T-cell loss was severe despite lower levels of Tg expression, suggesting a higher virulence of these alleles. All Nef alleles except the Nef(YU10x) and Nef(NL4-3(T71R)) alleles induced an enhanced activated memory (CD25(+) CD69(+) CD44(high) CD45RB(low) CD62L(low)) and apoptotic phenotype. Also, all could interact with and/or activate PAK2 except the Nef(JR-CSF) allele. Organ (lung and kidney) diseases were present in Nef(NL4-3(T71R)), Nef(032an), Nef(039nm), and Nef(SF2) Tg mice, despite very low levels of Tg expression for the last strain. However, no organ disease or minimal organ disease developed in Nef(YU10x) and Nef(AD-93) Tg mice and Nef(JR-CSF) Tg mice, respectively, despite high levels of Tg expression. Our data show that important differences in the pathogenicities of various Nef alleles can be scored in Tg mice. Interestingly, our results also revealed that some phenotypes can segregate independently, such as CD4(+) T-cell depletion and activation, as well as severe depletion of thymic CD4(+) T cells and peripheral CD4(+) T cells. Therefore, expression of Nef alleles in Tg mice under the CD4C regulatory elements represents a novel assay for measuring their pathogenicity. Because of the very high similarity of this murine AIDS-like disease to human AIDS, this assay may have a predictive value regarding the behavior of Nef in infected humans.     

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.     

CD4-mediated signals induce T cell dysfunction in vivo.

Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.     

Naive CD4+ T cells from lupus-prone Fas-intact MRL mice display TCR-mediated hyperproliferation due to intrinsic threshold defects in activation

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.     

The differentiated state of intestinal lamina propria CD4+ T cells results in altered cytokine production, activation threshold, and costimulatory requirements

Intestinal lamina propria (LP) CD4+ T cells are memory-like effector cells that proliferate at relatively low levels and require high levels of TCR signaling and costimulation for full activation in vitro. To study LP CD4+ T cell functional potential we used DO11.10 TCR transgenic (Tg) mice specific for the class II MHC-restricted OVA323-339 peptide and nontransgenic BALB/c mice. Activation of LP Tg+ T cells with Ag using mucosal explants induced high levels of IL-2, IL-4, and IFN-gamma. Culturing isolated LP cells with IL-12 enhanced IFN-gamma production and down-regulated IL-4 and IL-2, whereas addition of IL-4 maintained IL-4 production without inhibiting IFN-gamma production. Systemic administration of relatively high dose (HD; 100 nM) OVA323-339 peptide induced similar levels of bromodeoxyuridine (BrdU) incorporation by LP and splenic Tg+ T cells in vivo, whereas low dose (LD; 4.5 nM) peptide injections induced 4-fold greater levels of BrdU incorporation for LP compared with splenic Tg+ T cells. Coadministration of CTLA-4Ig reduced BrdU incorporation for splenic cells by 70% with HD and LD stimulation, but had little effect on LP responses to HD stimulation. Results of in vivo studies were confirmed in nontransgenic BALB/c mice using HD (200 microg) and LD (10 microg) anti-CD3 mAb+/- CTLA-4Ig. These results suggest that LP T cells are differentiated effector cells that respond at high levels when activated with relatively low levels of Ag- and B7-mediated costimulation in vivo. The reduced activation threshold of LP T cells may facilitate responses to low levels of Ag derived from mucosal pathogens.     

Altered CD4+ T cell phenotype and function determine the susceptibility to mucosal candidiasis in transgenic mice expressing HIV-1

The impairments of protective mucosal immunity which cause susceptibility to oropharyngeal candidiasis (OPC) in HIV infection remain undefined. This study used a model of OPC in CD4C/HIV MutA transgenic (Tg) mice expressing Rev, Env, and Nef of HIV-1 to investigate the role of transgene expressing dendritic cells (DCs) and CD4+ T cells in maintenance of chronic oral carriage of Candida albicans. DCs were depleted in the Tg mice and had an immature phenotype, with low expression of MHC class II and IL-12. CD4+ T cells were quantitatively reduced in the oral mucosa, cervical lymph nodes (CLNs) and peripheral blood of the Tg mice, and displayed a polarization toward a nonprotective Th2 response. Proliferation of CLN CD4+ T cells from infected Tg mice in response to C. albicans Ag in vitro was abrogated and the cells failed to acquire an effector phenotype. Coculture of C. albicans-pulsed DCs with CD4+ T cells in vitro showed that Tg expression in either or both of these cell populations sharply reduced the proliferation of CD4+ T cells and their production of IL-2. Finally, transfer of naive non-Tg CD4+ T cells into these Tg mice restored proliferation to C. albicans Ag and sharply reduced oral burdens of C. albicans. Overall, these results indicate that defective CD4+ T cells primarily determine the susceptibility to chronic carriage of C. albicans in these Tg mice.     

Activation of p21-activated kinase 2 and its association with Nef are conserved in murine cells but are not sufficient to induce an AIDS-like disease in CD4C/HIV transgenic mice

A well conserved feature of human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus (SIV) Nef is the interaction with and activation of the human p21-activated kinase 2 (PAK2). The conservation of this interaction in other species and its significance for Nef pathogenesis in vivo are poorly documented. In the present study, we measured these parameters in Nef-expressing thymocytes, macrophages, and dendritic cells of a transgenic (Tg) mouse model of AIDS (CD4C/HIV). We found that Nef binds to and activates PAK2, but not PAK1 and -3, in these three cell subsets. Nef associates with only a small fraction of PAK2. The Nef-PAK2 complex also comprises beta-PIX-COOL. The impact of the Nef-PAK2 association on disease development was also analyzed in Tg mice expressing 10 different Nef mutant alleles. CD4C/HIV Tg mice expressing Nef alleles defective in Nef-PAK2 association (P69A, P72A/P75A, R105A/R106A, Delta56-66, or G2A (myristoylation site)) failed to develop disease of the non-lymphoid organs (kidneys and lungs). Among these, only Tg mice expressing Nef(P69A) and Nef(G2A) showed some depletion of CD4(+) T cells, although a down-regulation of the CD4 surface protein was documented in all these Tg lines, except those expressing Nef(Delta56-66). Among other Tg mice expressing Nef mutants having conserved the Nef-PAK2 association (RD35AA, D174K, P147A/P150A, Delta8-17, and Delta25-65), only Tg mice expressing Nef(Delta8-17) develop kidney and lung diseases, but all showed partial CD4(+) T cell depletion despite some being defective for CD4 down-regulation (RD35AA and D174K). Therefore, Nef can activate murine PAK2 and associate with a small fraction of it, as in human cells. Such activation and binding of PAK2 is clearly not sufficient but may be required to induce a multiorgan AIDS-like disease in Tg mice.     

The role of CD4+CD25+ immunoregulatory T cells in the induction of autoimmune gastritis

A number of experimental models of organ-specific autoimmunity involve a period of peripheral lymphopenia prior to disease onset. There is now considerable evidence that the development of autoimmune disease in these models is due to the absence of CD4+CD25+ regulatory T cells. However, the role of CD4+CD25+ regulatory T cells in the prevention of autoimmune disease in normal individuals has not been defined. Here we have assessed the affect of depletion of CD4+CD25+ regulatory T cells in BALB/c mice on the induction of autoimmune gastritis. The CD4+CD25+ T cell population was reduced to 95% of the original population in adult thymectomized mice by treatment with anti-CD25 mAb. By 48 days after the anti-CD25 treatment, the CD4+CD25+ regulatory T cell population had returned to a normal level. Treatment of thymectomized adult mice for up to 4 weeks with anti-CD25 mAb did not result in the development of autoimmune gastritis. Furthermore, we have demonstrated that depletion of CD4+CD25+ regulatory T cells, together with transient CD4+ T lymphopenia, also did not result in the development of autoimmune gastritis, indicating that peripheral expansion of the CD4+ T cell population, per se, does not result in autoimmunity in adult mice. On the other hand, depletion of CD4+CD25+ T cells in 10-day-old euthymic mice resulted in a 30% incidence of autoimmune gastritis. These data suggest that CD4+CD25+ regulatory T cells may be important in protection against autoimmunity while the immune system is being established in young animals, but subsequently other factors are required to initiate autoimmunity.     

Lymphoproliferative disorder in CTLA-4 knockout mice is characterized by CD28-regulated activation of Th2 responses

Mice lacking CTLA-4 die at an age of 2-3 wk due to massive lymphoproliferation, leading to lymphocytic infiltration and destruction of major organs. The onset of the lymphoproliferative disease can be delayed by treatment with murine CTLA4Ig (mCTLA4Ig), starting day 12 after birth. In this study, we have characterized the T cells present in CTLA-4-deficient mice before and after mCTLA4Ig treatment. The T cells present in CTLA-4-deficient mice express the activation markers, CD69 and IL-2R; down-regulate the lymphoid homing receptor, CD62L; proliferate spontaneously in vitro and cannot be costimulated with anti-CD28 mAb consistent with a hyperactivated state. The T cells from CTLA-4-deficient mice survive longer in culture correlating with higher expression of the survival factor, Bcl-xL, in these cells. Most significantly, the CD4+ T cell subset present in CTLA-4-deficient mice secretes high levels of IL-4 and IL-5 upon TCR activation. Treatment of CTLA-4-deficient mice treated with mCTLA4Ig reverses the activation and hyperproliferative phenotype of the CTLA-4-deficient T cells and restores the costimulatory activity of anti-CD28 mAb. Furthermore, T cells from mCTLA4Ig-treated mice are not skewed toward a Th2 cytokine phenotype. Thus, CTLA-4 regulates CD28-dependent peripheral activation of CD4+ T cells. This process results in apoptosis-resistant, CD4+ T cells with a predominantly Th2 phenotype that may be involved in the lethal phenotype in these animals.     

CD8+ T cells can be primed in vitro to produce IL-4.

IL-4 production by T lymphocytes from naive mice in response to stimulation by plate-bound anti-CD3 is concentrated among CD4+ T cells. In vitro stimulation of lymph node T cells with anti-CD3 plus IL-2 and IL-4 strikingly increases the frequency of cells that produce IL-4 in response to subsequent stimulation with anti-CD3 plus IL-2. Separation of these primed cell populations into CD4+ and CD8+ T cell by cell sorting reveals that the frequency of IL-4-producing cells in both population is similar. Verification that CD8+ T cells produce IL-4 is provided by the capacity of anti-IL-4 mAb to inhibit the response of the indicator cell line to the growth factor produced by the primed cells and by detection of IL-4 by an IL-4-specific ELISA. The in vitro "priming" of CD8+ T cells to produce IL-4 is not dependent on the presence of CD4+ T cells because highly purified CD8+ T cells can be stimulated to develop into cells capable of producing IL-4 by culture with plate-bound anti-CD3 plus IL-2 and IL-4.     

Adult AIDS-Like Disease in a Novel Inducible Human Immunodeficiency Virus Type 1 Nef Transgenic Mouse Model: CD4+ T-Cell Activation Is Nef Dependent and Can Occur in the Absence of Lymphophenia

CD4C/HIV^nef transgenic (Tg) mice express Nef in CD4⁺ T cells and in the cells of the macrophage/monocyte/dendritic lineage, and they develop an AIDS-like disease similar to human AIDS. In these mice, Nef is constitutively expressed throughout life. To rule out the contribution of any developmental defects caused by early expression of Nef, we generated inducible human immunodeficiency virus type 1 (HIV-1) Nef Tg mice by using the tetracycline-inducible system. Faithful expression of the Nef transgene was induced in (CD4C/rtTA × TRE/HIV^Nef) or (CD4C/rtTA2^S-M2 × TRE/HIV^Nef) double-Tg mice upon doxycycline (DOX) treatment in drinking water. Long-term treatment of these mice with DOX also led to loss, apoptosis, and activation of CD4⁺ T cells, this latter phenotype being observed even with low levels of Nef. These phenotypes could be transferred by bone marrow (BM) transplantation, indicating a hematopoietic cell autonomous effect. In addition, in mixed Tg:non-Tg BM chimeras, only Tg and not non-Tg CD4⁺ T cells exhibited an effector/memory phenotype in the absence of lymphopenia. Finally, the DOX-induced double-Tg mice developed nonlymphoid organ diseases similar to those of CD4C/HIV^Nef Tg mice and of humans infected with HIV-1. These results show for the first time that adult mice are susceptible to the detrimental action of Nef and that Nef-mediated T-cell activation can be independent of lymphopenia. These Tg mice represent a unique model which is likely to be instrumental for understanding the cellular and molecular pathways of Nef action as well as the main characteristics of immune reconstitution following DOX withdrawal.Small animal models able to express the entire human immunodeficiency virus (HIV) genome or selected HIV genes have provided useful information on the pathogenesis of AIDS and still represent important research tools toward this goal. Among these models, transgenic (Tg) mice containing intact copies of HIV DNA, defective provirus with the gag and pol genes deleted, or individual HIV-1 genes have been reported to develop various pathologies, some of which resemble those found in human AIDS (2, 3, 8, 9, 16, 17, 18, 24, 27, 29, 30, 38, 44, 45, 46, 49, 51, 52). The cell type context in which the HIV-1 transgene is expressed in these Tg mice appears to play an important role in determining the type of pathological lesions. Tg mice generated in our laboratory and expressing the entire coding sequence of HIV-1 (CD4C/HIV^WT) or HIV-1 Nef alone (CD4C/HIV^Nef) in the relevant target cells of HIV-1, namely, CD4⁺ T cells, macrophages, and dendritic cells, develop pathologies very similar to those in human AIDS (17, 18). The AIDS-like disease of CD4C/HIV^Nef Tg mice is characterized by immunodeficiency, loss of CD4⁺ T cells, thymic atrophy, activation of T cells and pathologies in heart, lungs, and kidneys (18, 53). Similarly, expression of simian immunodeficiency virus (SIV) Nef in Tg mice under the control of the same promoter sequences (CD4C) results in an AIDS-like disease (42). These studies demonstrated that Nef plays an important role in the development of the AIDS-like disease induced by HIV-1 or SIV in Tg mice.Among the AIDS-like phenotypes of these models, the T-cell activation observed by a number of groups in Tg mice expressing Nef (3, 33, 44, 53) may be of special interest for its resemblance to that of humans or macaques infected with HIV-1 or SIV, respectively. HIV infection results in a state of chronic immune activation which correlates very closely with disease progression in humans (11, 14, 23). Similarly, SIV-infected macaques which develop AIDS show aberrant immune activation (35), while SIV-infected sooty mangabey monkeys, natural hosts of SIV, do not develop immunopathologies and do not show immune activation either (41). Various factors may contribute to this immune activation, including increased plasma lipopolysaccharide levels due to microbial translocation from the gut (4), impaired regulatory T cell function (32), or the action of the HIV-1 gene products themselves, such as Env gp120 and Nef (10, 12, 43). Consistent with this latter scenario, we reported that in CD4C/HIV^Nef Tg mice the extent of T-cell activation correlates with levels of Nef expression in CD4⁺ T cells, thus suggesting a direct involvement of Nef in this activation (53). In contrast, Koenen and coworkers reported that T-cell activation in CD2/Nef Tg mice is induced indirectly by lymphophenia (26). In that study, chimeric mice, which were generated from a mixture of non-Tg and Nef Tg bone marrow (BM) cells, were not lymphopenic, and the donor-derived Nef-expressing Tg T cells did not show an activated phenotype. However, the donor Nef Tg T cells constituted only 1 to 2% of peripheral T cells of these chimeric mice (26). Clearly, alternative experimental approaches are needed to study this phenotype in a more physiological context.In the previously described CD4C/HIV^Nef Tg mice (18), Nef expression begins early in life and is constitutively expressed throughout the life of the animal. The AIDS-like disease caused by this early expression of Nef best represents a model for pediatric AIDS. However, in these Tg mice, Nef may interfere with normal developmental processes and these latter defects may contribute to some of the phenotypes observed. To assess the effects of Nef in fully mature adult animals, and thus develop a model of adult AIDS, temporal regulation of Nef expression in adult mice using an inducible system is required.In the present study, we chose the tet-On (rtTA and rtTA2^S-M2) system (13, 15, 25, 48) to induce expression of HIV-1 Nef in CD4⁺ T cells and cells of the macrophage/dendritic lineage of mice using the CD4C tissue-specific regulatory elements. These CD4C sequences were previously used to generate the constitutively Nef-expressing CD4C/HIV^Nef Tg mice (18). These inducible adult (TRE/HIV^Nef × CD4C/rtTA) and (TRE/HIV^Nef × CD4C/rtTA2^S-M2) double-Tg (DTg) mice express Nef when treated with doxycycline (DOX) and develop an AIDS-like disease very similar to that seen in constitutively Nef-expressing CD4C/HIV^Nef Tg mice. We took advantage of this novel biological system to reassess the role of Nef in T-cell activation. Using a mixed chimera made with BM cells from these inducible Nef Tg mice and from non-Tg mice, we could document CD4⁺ T-cell activation only in donor-derived Nef-expressing Tg cells, but not in non-Tg cells, in the absence of lymphopenia. This result strongly suggests that this CD4⁺ T-cell activation phenotype is most likely driven by expression of Nef in these cells.     

Expression of simian immunodeficiency virus nef in immune cells of transgenic mice leads to a severe AIDS-like disease

In order to study the functions of simian immunodeficiency virus (SIV) Nef in vivo in a small-animal model, we constructed transgenic (Tg) mice expressing the SIV(mac)239 nef gene in the natural target cells of the virus under the control of the human CD4 gene promoter (CD4C). These CD4C/SHIV-nef(SIV) Tg mice develop a severe AIDS-like disease, with manifestations including premature death, failure to thrive or weight loss, wasting, thymic atrophy, an especially low number of peripheral CD8+ T cells as well as a low number of peripheral CD4+ T cells, diarrhea, splenomegaly, and kidney (interstitial nephritis, segmental glomerulosclerosis), lung (lymphocytic interstitial pneumonitis), and heart disease. In addition, these Tg mice fail to mount a class-switched antibody response after immunization with ovalbumin, they produce anti-DNA autoantibodies, and some of them develop Pneumocystis carinii lung infections. All these results suggest a generalized Nef-induced immunodeficiency. The low numbers of peripheral CD8+ and CD4+ T cells are likely to reflect a thymic defect and may be similar to the DiGeorge-like "thymic defect" immunophenotype described for a subgroup of human immunodeficiency virus type 1-infected children. Therefore, it appears that SIV Nef alone expressed in mice, in appropriate cell types and at sufficient levels, can elicit many of the phenotypes of simian and human AIDS. These Tg mice should be instrumental in studying the pathogenesis of SIV Nef-induced phenotypes.     

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.     

Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.     

Persistent TNF-alpha exposure impairs store operated calcium influx in CD4+ T lymphocytes

Persistent tumour necrosis factor alpha (TNF-alpha) exposure uncouples proximal T-cell receptor (TCR)-signalling events. Here, we demonstrate that chronic TNF-alpha exposure also attenuates signalling distal to the TCR, by specifically inhibiting Ca2+ influx evoked by thapsigargin in CD4+ T-cells. Mitogen-induced Ca2+ responses were impaired in a dose dependent manner, and TCR-induced Ca2+ responses were also significantly reduced. The impairment of Ca2+ influx strongly correlated with poor function as proliferative responses to both mitogen and anti-CD3/CD28 stimulation were suppressed. Our findings show that persistent TNF-alpha exposure of T-cells specifically inhibits store operated Ca2+ influx. This may affect gene activation and contribute to the poor T-cell function in chronic inflammatory disease.     

CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.     

Comparative contribution of CD1 on the development of CD4+ and CD8+ T cell compartments

CD1 molecules are MHC class I-like glycoproteins whose expression is essential for the development of a unique subset of T cells, the NK T cells. To evaluate to what extent CD1 contributes to the development of CD4+ and CD8+ T cells, we generated CD1oIIo and CD1oTAPo mice and compared the generation of T cells in these double-mutant mice and IIo or TAPo mice. FACS analysis showed that the number of CD4+ T cells in CD1oIIo mice was reduced significantly compared with the corresponding population in IIo mice. Both CD4+ NK1.1+ and the CD4+ NK1.1- population were reduced in CD1oIIo mice, suggesting that CD1 can select not only CD4+ NK1.1+ T cells but also some NK1.1- CD4+ T cells. Functional analysis showed that the residual CD4+ cells in CD1oIIo can secrete large amounts of IFN-gamma and a significant amount of IL-4 during primary stimulation with anti-CD3, suggesting that this population may be enriched for NK T cells restricted by other class I molecules. In contrast to the CD4+ population, no significant differences in the CD8+ T cell compartment can be detected between TAPo and CD1oTAPo mice in all lymphoid tissues tested, including intestinal intraepithelial lymphocytes. Our data suggest that, unlike other MHC class I molecules, CD1 does not contribute in a major way to the development of CD8+ T cells.