Molecular dynamics studies of the human CD4 protein

A dynamical model for an N-terminal fragment of the human CD4 protein has been determined by computer simulation. The protein has been studied both in vacuo and in solution. Data from both simulations agree moderately well with each other and with the crystal structure. All elements of secondary structure were retained during simulation. Point mutation and sequence replacement studies have shown that a loop in CD4, residues 40–52, is involved in binding with gp120, the human immunodeficiency virus surface glycoprotein.^1,2 Our results show that the gp120-binding loop and a few regions which bind to monoclonal antibodies and class II MHC molecules are the most highly motile areas of the protein. These results are consistent with the suggestion that CD4 binds to target molecules by using induced-fit contacts. © 1994 John Wiley & Sons, Inc.     

Correlation between the expression of CD4 and the level of CD4 mRNA in human B-cell lines

Lines of Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cells (B-LCLs) differ in the expression of surface CD4 glycoproteins. The aim of the present study was to correlate the expression of CD4 molecules on B-LCL cells with the synthesis of CD4 mRNA. RT-PCR assays were performed with oligonucleotide primers designed to detect mRNA corresponding to intracellular, transmembrane, or extracellular portions of the CD4 molecule. RT-PCR assays with all sets of primers were positive in T-cell populations, but were negative in various B-cell lymphoma lines. The majority of the LCLs established by EBV transfection of non-selected B-cells yielded positive results with at least some of the primer sets used for detection of CD4 mRNA. A significant positive correlation was found between the proportion of CD4+ cells in various B-LCLs and the concentration of CD4 mRNA. LCLs established from B-cells which synthesized various antibodies did not express CD4 molecules and either failed to synthesize CD4 mRNA or produced very low concentrations. These findings indicate that the expression of CD4 on B-LCLs is directly correlated with the concentration of CD4 mRNA synthesized and with the differentiation stage in which B-cells were immortalized by EBV infection.     

CD4-like molecules in human sperm

The expression of a molecule recognized by CD4 monoclonal antibodies was investigated on human sperm using immunolabelling, biochemical and immunochemical methods. Flow cytometry detected a significant fluorescence signal. SDS-PAGE analysis and Western blotting identified a molecule of 60 kDa, consistent with a CD4-like structure as confirmed after selective immunoseparation. Additional bands reacting with anti-CD4 were found in sperm extracts (73 kDa) and seminal fluid (90 kDa). These data indicate that sperm express a molecule similar to the receptor for HIV described on mononuclear cells.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

Design and characterization of stabilized derivatives of human CD4D12 and CD4D1

CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors.     

CD4+CD25high regulatory cells in human peripheral blood

Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD(+)CD25(+) T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4(+) population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4(+)CD25(+) regulatory cells isolated in mice. With TCR cross-linking, CD4(+)CD25(high) cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4(+)CD25(-) responder T cells in a contact-dependent manner. The CD4(+)CD25(high) regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RB(low) on murine CD4(+)CD25(+) regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4(+)CD25(high) regulatory cells and CD4(+)CD25(-) responder cells. Whereas higher ratios of CD4(+)CD25(high) T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.     

Apoptotic human lymphocytes have diminished CD4 and CD8 receptor expression

We used quantitative multiparameter flow cytometric assays to simultaneously detect viable, apoptotic, and necrotic human peripheral blood mononuclear cells (PBMC) and immunophenotyped lymphocyte subsets within the PBMC. Apoptosis was induced by a spectrum of treatments, including camptothecin, cisplatin, dexamethasone, hyperthermia, staurosporine, and etoposide in anti-CD3 mAb-stimulated cells and by cyclohexamide in both quiescent and stimulated cells; apoptosis in the latter was augmented by anti-fas mAb. We found that CD4(+) and CD8(+) cells were significantly underrepresented in the apoptotic PBMC and that the percentage of CD4(+) and CD8(+) PBMC each markedly decreased as apoptosis increased. This suggested that surface expression of these receptors was lessened on apoptotic CD4(+) and CD8(+) cells. This was directly confirmed by observation of sorted CD4(+) PBMC. This analysis of a wide variety of apoptotic stimuli demonstrates that diminished CD4 and CD8 surface receptor expression is a common feature of human T lymphocyte apoptosis.     

Cytofluorometric analyses of human T cell CD2/CD4 inter-molecular interactions

Incubation of human T lymphocytes with saturating concentrations of combinations of certain anti-CD2 and -CD4 mAb results in reciprocal down-regulation of the cell surface density expression of the respective CD molecules. Such reciprocal down-regulation occurs at 0 degrees C in the presence of sodium azide and appears selective for CD2 and CD4 molecules because mAb identifying various other CD T cell surface molecules (anti-Leu2a, -OK-CLL, -W6/32, -beta 2-microglobulin, -4B4) do not modulate CD2 or CD4 R density, and because anti-CD2 mAb (anti-OKT11 and -D66 clone-1) do not alter CD8 R density (anti-OKT8, -Leu2a) and vice versa. Down-regulation of CD2 by mAb specific to CD4 is epitope-specific but does not vary on the basis of the antibody isotype used. The anti-CD4 mAb, Leu3a, was the strongest CD2 down-regulator examined followed by OKT4F. mAb specific to other CD4 epitopes (B, C, D, and E) caused only slight down-regulation of CD2 expression whereas anti-OKT4 and -OKT4A mAb had no significant regulatory effect. Also, mAb specific to the 9.6 (anti-OKT11) and D66 (anti-D66 clone 1) epitopes of the CD2 molecule down-regulated CD4 density detectable with Leu3a, OKT4, and OKT4A anti-CD4 mAb. Down-regulation of CD2 by anti-CD4 mAb also occurred with the transformed T cell line, KE-37, which demonstrates that such effects can occur without mononuclear phagocytic accessory cells. From these data it can be concluded that important T cell immunoregulatory signals may be transmitted intramembranally between CD2 and CD4 glycoproteins.     

Anti-human CD4 induces peripheral tolerance in a human CD4+, murine CD4-, HLA-DR+ advanced transgenic mouse model

Selection in vivo of potent mAbs to human CD4 useful for immunotherapy, e.g., for the induction of immunological tolerance, is restricted for ethical reasons. We therefore used multiple transgenic mice that lack murine CD4, but express human CD4 specifically on Th cells, and HLA-DR3 as its natural counterligand (CD4/DR3 mice). The injection of CD4/DR3 mice with anti-human CD4 (mAb Max.16H5) before immunization with tetanus toxoid (TT, day 0) totally blocked the formation of specific Abs. This state of unresponsiveness persisted a subsequent boost again performed in the presence of anti-human CD4. When these mice were left untreated for at least 40 days, and were then re-exposed with TT, but in the absence of anti-human CD4, they consistently failed to induce specific Abs (long-term unresponsiveness). Exposure to second party Ags (hen egg lysozyme, human acetylcholine receptor) induced specific Abs comparable with control mice, demonstrating that the anti-CD4-induced unresponsiveness was Ag specific (immunological tolerance). Importantly, the concurrent injection of TT and anti-human CD4 at day 0, followed by another two anti-CD4 treatments, also led to tolerant animals, indicating that tolerance was inducible at the same day as the Ag exposure is provided. We finally demonstrate a limited ability of spleen cells to respond to TT in vitro, indicating that T cells are essentially involved in the maintenance of TT-specific tolerance. These data show for the first time that the human CD4 coreceptor mediates tolerance-inducing signals when triggered by an appropriate ligand in vivo.     

Costimulation via CD55 on human CD4+ T cells mediated by CD97

Decay-accelerating factor (CD55) is a complement regulatory protein, which is expressed by most cells to protect them from complement-mediated attack. CD55 also binds CD97, an EGF-TM7 receptor constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells upon activation. Early results suggested that CD55 could further enhance T cell proliferation induced by phorbol ester treatment. The present study demonstrates that coengagement of CD55, using either cross-linking mAbs or its natural ligand CD97, and CD3 results in enhanced proliferation of human peripheral blood CD4(+) T cells, expression of the activation markers CD69 and CD25, and secretion of IL-10 and GM-CSF. Recently, an increase in T cell responsiveness in CD55(-/-) mice was shown to be mediated by a lack of complement regulation. In this study, we show that direct stimulation of CD55 on CD4(+) T cells with CD97 can modulate T cell activation but does not interfere with CD55-mediated complement regulation. Our results support a multifaceted role for CD55 in human T cell activation, constituting a further link between innate and adaptive immunity.     

Heterogeneity of human effector CD4+ T cells

For many years the heterogeneity of CD4⁺ T-helper (Th) cells has been limited to Th1 and Th2 cells, which have been considered not only to be responsible for different types of protective responses, but also for the pathogenesis of many disorders. Th1 cells are indeed protective against intracellular microbes and they are thought to play a pathogenic role in organ-specific autoimmune and other chronic inflammatory disorders. Th2 cells provide protection against helminths, but are also responsible for the pathogenesis of allergic diseases. The identification and cloning of new cytokines has allowed one to enlarge the series of functional subsets of CD4⁺ Th effector cells. In particular, CD4⁺ Th cells producing IL-17 and IL-22, named Th17, have been initially implicated in the pathogenesis of many chronic inflammatory disorders instead of Th1 cells. However, the more recent studies in both humans and mice suggest that Th17 cells exhibit a high plasticity toward Th1 cells and that both Th17 and Th1 cells may be pathogenic. More recently, another two subsets of effector CD4⁺ Th cells, named Th9 and Th22 cells, have been described, even if their pathophysiological meaning is still unclear. Despite the heterogeneity of CD4⁺ effector Th cells being higher than previously thought and some of their subsets exhibiting high plasticity, the Th1/Th2 paradigm still maintains a strong validity.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly ¹⁵N and ¹³C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the ¹H, ¹³C, and ¹⁵N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Interleukin 4 induces CD4+/CD8- to CD8+/CD4- transformation of human neonatal T cells by way of a double positive intermediate

We have determined that IL-4 induces the generation of CD4+/CD8+ T cells in cultures of neonatal lymphocytes. Sorting, positive, and negative selection experiments indicate that these cells arise from a subpopulation of CD4+/CD8- cells present in the neonate but not in the adult. We have further determined that these IL-4 generated "double positive" cells further differentiate to express only the CD8 marker. Our findings suggest an undescribed and dramatic role for IL-4 in T cell differentiation.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

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.