Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection

HIV entry into human cells is mediated by CD4 acting in concert with one of several members of the chemokine receptor superfamily. The resistance to HIV infection observed in individuals with defective CCR5 alleles indicated that this particular chemokine receptor plays a crucial role in the initiation of in vivo HIV infection. Expression of human CD4 transgene does not render mice susceptible to HIV infection because of structural differences between human and mouse CCR5. To ascertain whether expression of human CD4 and CCR5 is sufficient to make murine T lymphocytes susceptible to HIV infection, the lck promoter was used to direct the T cell-specific expression of human CD4 and CCR5 in transgenic mice. Peripheral blood mononuclear cells and splenocytes isolated from these mice expressed human CD4 and CCR5 and were infectible with selected M-tropic HIV isolates. After in vivo inoculation, HIV-infected cells were detected by DNA PCR in the spleen and lymph nodes of these transgenic mice, but HIV could not be cultured from these cells. This indicated that although transgenic expression of human CD4 and CCR5 permitted entry of HIV into the mouse cells, significant HIV infection was prevented by other blocks to HIV replication present in mouse cells. In addition to providing in vivo verification for the important role of CCR5 in T lymphocyte HIV infection, these transgenic mice represent a new in vivo model for understanding HIV pathogenesis by delineating species-specific cellular factors required for productive in vivo HIV infection. These mice should also prove useful for the assessment of potential therapeutic and preventative modalities, particularly vaccines.

Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56lck, undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56lck, we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

Fas-dependent CD4+ cytotoxic T-cell-mediated pathogenesis during virus infection

β2-Microglobulin-deficient (β2m−) mice generate a CD4+ major histocompatibility complex class II-restricted cytotoxic T-lymphocyte (CTL) response following infection with lymphocytic choriomeningitis (LCM) virus (LCMV). We have determined the cytotoxic mechanism used by these CD4+ CTLs and have examined the role of this cytotoxic activity in pathogenesis of LCM disease in β2m− mice. Lysis of LCMV-infected target cells by CTLs from β2m− mice is inhibited by addition of soluble Fas-Ig fusion proteins or by pretreatment of the CTLs with the protein synthesis inhibitor emetine. In addition, LCMV-infected cell lines that are resistant to anti-Fas-induced apoptosis are refractory to lysis by these virus-specific CD4+ CTLs. These data indicate that LCMV-specific CD4+ CTLs from β2m− mice use a Fas-dependent lytic mechanism. Intracranial (i.c.) infection of β2m− mice with LCMV results in loss of body weight. Fas-deficient β2m−.lpr mice develop a similar wasting disease following i.c. infection. This suggests that Fas-dependent cytotoxicity is not required for LCMV-induced weight loss. A potential mediator of this chronic wasting disease is tumor necrosis factor (TNF)-α, which is produced by LCMV-specific CD4+ CTLs. In contrast to LCMV-induced weight loss, lethal LCM disease in β2m− mice is dependent on Fas-mediated cytotoxicity. Transfer of immune splenocytes from LCMV-infected β2m− mice into irradiated infected β2m− mice results in death of recipient animals. In contrast, transfer of these splenocytes into irradiated infected β2m−.lpr mice does not cause death. Thus a role for CD4+ T-cell-mediated cytotoxicity in virus-induced immunopathology has now been demonstrated.

Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4+ CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

Virus-specific CD8+ T cell numbers are maintained during γ-herpesvirus reactivation in CD4-deficient mice

The murine γ-herpesvirus 68 replicates in epithelial sites after intranasal challenge, then persists in various cell types, including B lymphocytes. Mice that lack CD4+ T cells (I-Ab−/−) control the acute infection, but suffer an ultimately lethal recrudescence of lytic viral replication in the respiratory tract. The consequences of CD4+ T cell deficiency for the generation and maintenance of murine γ-herpesvirus 68-specific CD8+ set now have been analyzed by direct staining with viral peptides bound to major histocompatibility complex class I tetramers and by a spectrum of functional assays. Both acutely and during viral reactivation, the CD8+ T cell responses in the I-Ab−/− group were no less substantial than in the I-Ab+/+ controls. Indeed, virus-specific CD8+ T cell numbers were increased in the lymphoid tissue of clinically compromised I-Ab−/− mice, although relatively few of the potential cytotoxic T lymphocyte effectors were recruited back to the site of pathology in the lung. Thus the viral reactivation that occurs in the absence of CD4+ T cells was not associated with any exhaustion of the virus-specific cytotoxic T lymphocyte response. It seems that CD8+ T cells alone are insufficient to maintain long-term control of this persistent γ-herpesvirus.

Lack of a role for transforming growth factor-β in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-β1 (TGF-β1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-β production. Here, we examine the role of TGF-β in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-β production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-β does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-β1−/−, and Smad3−/− T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-β by CD4+ T cells. These results indicate that CTLA-4 ligation does not regulate TGF-β production and that CTLA-4-mediated inhibition can occur independently of TGF-β. Collectively, these data demonstrate that CTLA-4 and TGF-β represent distinct mechanisms for regulation of T cell responses.

Activated Ras signals differentiation and expansion of CD4+8+ thymocytes.

We describe a novel approach to assay the ability of particular gene products to signal transitions in lymphocyte differentiation in vivo. The method involves transfection of test expression constructs into RAG-1-deficient embryonic stem cells, which are subsequently assayed by the RAG-2-deficient blastocyst complementation approach. We have used this method to demonstrate that expression of activated Ras in CD4-8- (double negative, DN) prothymocytes in vivo induces their differentiation into small CD4+8+ (double positive, DP) cortical thymocytes with accompanying expansion to normal thymocyte numbers. However, activated Ras expression in DP cells does not cause proliferation or maturation to CD4+8- or CD4-8+ (single positive) thymocytes. Therefore, signaling through Ras is sufficient for promoting differentiation of DN to DP cells, but further differentiation requires the activity of additional signaling pathways.

In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.

Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets.

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.