Efficient induction of HIV-1 replication in latently infected cells through contact with CD4+ T cells: involvement of NF-kappaB activation

Reservoir cells latently infected with HIV-1 pose one of the major obstacles that hamper ultimate eradication of HIV-1 from infected patients. In this report, we showed that direct contact with MOLT-4 T cells induced HIV-1 replication in J(22)-HL-60 latently infected cells without any additional stimulus. Neutralization experiments revealed that pro-inflammatory cytokines, whose production was increased following cell-cell contact, were unlikely to be primarily involved in the induced HIV-1 replication. Cell-cell contact, but not soluble components in the culture supernatant, caused a rapid phosphorylation and degradation of IkappaBalpha, which led to elevated NF-kappaB DNA binding activity in J(22)-HL-60 cells. Furthermore, forced expression of a super-repressor form of IkappaBalpha or pretreatment with ritonavir efficiently blocked the activation of NF-kappaB and HIV-1 replication in J(22)-HL-60 cells co-cultured with MOLT-4 T cells. Moreover, either resting or PHA stimulated primary CD4(+) T cells induced HIV-1 replication in J(22)-HL-60 cells in a similar way with that of MOLT-4 cells. These results indicated that direct contact with CD4(+) T cells induced HIV-1 replication in latently infected cells and provide insight into the molecular mechanism of virus release from myeloid progenitor cells latently infected with HIV-1.     

The mitogenic response of T cells to interleukin-2 requires Raf-1

The product of the c-raf-1 proto-oncogene, Raf-1, is known to encode a 74-kDa ubiquitously expressed cytoplasmic serine/threonine kinase. Various growth factors such as epidermal growth factor, acidic fibroblast growth factor, platelet-derived growth factor, insulin, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-2, IL-3 and erythropoietin have been shown to induce phosphorylation of Raf-1, thereby activating Raf-1 kinase. Raf-1 is, thus, believed to play a role in coupling growth factor receptors to proliferation. We have examined the role of Raf-1 in the mitogenic response of human peripheral blood-derived IL-2 receptor expressing T cells to human recombinant IL-2 employing c-raf antisense (AS) oligodeoxyribonucleotide. Uptake studies of oligonucleotides indicated that incorporation of oligomers was maximal at 4 h and oligdeoxynucleotides remained stable in these cells for up to 24 h. Treatment of T cells with the AS oligodeoxyribonucleotide in intracellular duplex formation followed by efficient translation blockade of c-raf-1. In contrast, sense (S) and nonsense (NS) oligodeoxynucleotides failed to form intracellular duplexes and did not interfere with translation of c-raf-1, suggesting specific elimination of c-raf-1 by the AS oligomer. Proliferation of T cells ([³H]thymidine incorporation) following exposure to IL-2 was substantially reduced when the c-raf-1 AS oligodeoxyribonucleotide was added to cultures, while the mitogenic response to this factor remained almost unaffected in the presence of S and NS oligodeoxy-ribonucleotides.     

Metabolic reprogramming and metabolic dependency in T cells

Upon activation, quiescent naive T cells undergo a growth phase followed by massive clonal expansion and differentiation that are essential for appropriate immune defense and regulation. Accumulation of cell biomass during the initial growth and rapid proliferation during the expansion phase is associated with dramatically increased bioenergetic and biosynthetic demands. This not only requires a metabolic rewiring during the transition between resting and activation but also 'addicts' active T cells to certain metabolic pathways in ways that naive and memory T cells are not. We consider such addiction in terms of the biological effects of deprivation of metabolic substrates or inhibition of specific pathways in T cells. In this review, we illustrate the relevant metabolic pathways revealed by recent metabolic flux analysis and discuss the consequences of metabolic intervention on specific metabolic pathways in T lymphocytes.     

The role of accessory cells in polyclonal T cell activation II. Induction of interleukin 2 responsiveness requires cell-cell contact

It has previously been reported (Hünig, T. et al., Eur. J. Immunol. 1983. 13:1) that highly purified peripheral T cells do not respond to concanavalin A (Con A) even in the presence of Con A-induced spleen cell supernatant as a source of interleukin 2 (IL 2). In the present report, the hypothesis was tested whether this unresponsiveness correlates with the observed inability of Con A to mediate cell-cell contact between highly purified T cells. It was found that T cells, pretreated with neuraminidase to reduce their net negative charge, were both aggregated and rendered IL 2-reactive by Con A. In addition, leukoagglutinin (LA) was found to be able to both agglutinate untreated T cells and to make them IL 2-reactive. Neuraminidase treatment reduced the concentration of LA required to mediate both effects. Neuraminidase treatment did not alter the overall Con A-binding capacity of T cells, nor did it induce reactivity to IL 2 in the absence of lectin. Furthermore, irradiated, neuraminidase-treated T cells served as accessory cells (AC) in the induction of responsiveness to IL 2, but not for production of IL2, which depends on Ia+ AC. Finally, Lyt-2- neuraminidase-treated peripheral T cells responded in the same fashion as whole T cell populations, indicating that at least some T cells of the helper phenotype need not interact with Ia+AC for the induction of IL 2 responsiveness.     

Abnormal expression pattern of Tac and T9 antigens onin vitro activation of leukemic T cells

Mitogen-induced T-lymphocyte proliferation is dependent on the presence of both interleukin 2 (IL-2) and transferrin, even though resting lymphocytes do not have receptors for either. Recently, it has been reported that IL-2 stimulates T-lymphocyte proliferation via IL-2 receptor by induction of transferrin receptor on these cells. Using leukemic T cells as a model of the monoclonal mature T cell, we examined those sequential steps of T-cell activation. Our studies revealed that (i) transferrin receptors do not always appear after IL-2 receptor expression, (ii) IL-2 up-regulates the expression of IL-2 receptor but not of transferrin receptor, and (iii) IL-2 can initiate DNA synthesis without altering transferrin receptor expression. Thus, the sequential activation steps reported for normal T cells were not observed in the present study on leukemic T cells. These data suggest that there are other pathways to DNA synthesis in leukemic T cells and even in normal T cells.     

Colonic epithelial response to injury requires Myd88 signaling in myeloid cells

Proper colonic injury response requires myeloid-derived cells and Toll-like receptor/Myd88 signaling. However, the precise role of Myd88 signaling specifically in myeloid-derived cells that occurs during tissue damage is unclear. Therefore, we created a mouse line with Myd88 expression restricted to myeloid lineages (Myd88(-/-); LysM(Cre/+); ROSA26(Myd88/+); herein Mlcr). In these mice, Myd88 was appropriately expressed and mediated responses to bacterial ligand exposure in targeted cells. Importantly, the severe colonic epithelial phenotype observed in dextran sodium sulfate-injured Myd88(-/-) mice was rescued by the genetic modification of Mlcr mice. During injury, myeloid cell activation and enrichment of Ptsg2-expressing stromal cells occurred within the mesenchyme that surrounded the crypt bases of Mlcr and Myd88(+/-) mice but not Myd88(-/-) mice. Interestingly, these cellular changes to the crypt base mesenchyme also occurred, but to a lesser extent in uninjured Mlcr mice. These results show that Myd88 expression in myeloid cells was sufficient to rescue intestinal injury responses, and surprisingly, these cells appear to require an additional Myd88-dependent signal from a non-myeloid cell type during homeostasis.     

Ultrastructural study of cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro

Many biochemical reports support cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro, however there have been few morphological studies supporting this. Details of cell-cell interaction between osteoclasts and osteoblasts/stroma cells remain unclear. The present study examined cell-cell interaction between osteoclasts and osteoblasts/stroma cells by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Osteoclasts, osteoblasts/stroma cells, and bone marrow cells obtained from 10-day-old ddY mice were cultured on dentin slices for 72 hr. Specimens were fixed, and some were examined by SEM. Specimens were decalcified, embedded in Epon after determination of the tartrate-resistant acid phosphatase activity (TRAP), and TRAP-positive cells for investigation were serially sectioned by alternating semithin and ultrathin sections, and then examined by TEM. By SEM, many cellular contacts were seen between the cells cultured on the dentin, but by TEM there were few special structures on the cell membranes between osteoclasts and osteoblasts/stroma cells, or between osteoclasts and bone marrow cells. A special structure on the cell membranes of osteoclasts was observed between an osteoclast and a cytoplasmic process of osteoblast/stroma cells, and this cell membrane was coated with electron dense or bristle-like structures. These bristle-like structures were very similar to those of coated pits. The present results show that the coated pit-like structure plays an important role in cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro, and suggest that macromolecules binding to the osteoclast-surface receptor via ligands, accumulate in the coated pits, and enter the osteoclast as receptor-macromolecule complexes in endocytic vesicles.     

Gammadelta T cells assist alphabeta T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine

Para-phenylenediamine (PPD) is known to be a common sensitizer of allergic contact dermatitis and contact urticaria. To clarify the mechanism of contact hypersensitivity (CHS) to PPD, we established a mouse model of PPD-induced CHS. BALB/c mice were immunized for 3 consecutive days by painting topically a 2.5% PPD solution on their shaved abdominal skin. On days 5, 7 or 9 after the initial application, the mice were challenged by applications of a 2.5% PPD solution. Maximal ear swelling was determined at 24 h but another statistically significant and smaller ear swelling was observed 1 h after challenge with PPD in a hapten-specific manner. Adoptive cell transfer experiments demonstrated that the ear swelling of the adoptive cell transferred mice displayed an early response at 6 h and a late response from 12 h to 24 h when the recipient mice were challenged immediately after transfer. Both MoAbs and complement treatment of the transferred cells demonstrated that the phenotype of the early response cells which elicited a response at 6 h after challenge was Thy1(+), B220(+), alphabeta TCR(-), gammadelta TCR(-), CD3(-), CD4(-), CD5(+) and CD8(-). The in vitro treatment of effector cells with MoAbs against not only alphabeta TCR but also gammadelta TCR, together with complement, was found to diminish substantially the late response, elicited 12-24 h after challenge. Gammadelta T cells reconstituted the ability of alphabeta T cells to transfer 24 h CHS responsiveness. The phenotype of the gammadelta T cells that assist CHS effector alphabeta T cells was CD3(+), CD4(-) and CD8(+) and these regulatory gammadelta T cells were neither Ag-specific nor MHC-restricted. Furthermore, gammadelta T cells from normal spleen could also assist alphabeta T cells in adoptive transfer of the 24 h CHS response in a non-MHC-restricted manner. RT-PCR demonstrated that alphabeta T cells strongly expressed mRNA IFN-gamma, whereas gammadelta T cells expressed not only IFN-gamma but also IL-4 and IL-10. These data indicate that not only early response cells and alphabeta T cells but also Th2 type gammadelta T cells may play an important role in the elicitation of CHS to PPD.     

Protective T cell response against intracellular pathogens in the absence of Toll-like receptor signaling via myeloid differentiation factor 88

Toll-like receptors (TLR) have been indicated as germline-encoded receptors for sensing a variety of pathogens. Although the role of TLR in innate immunity is beyond question, their function in acquired immunity, in particular in T cell immunity, is less clear. Here, we used experimental Listeria monocytogenes infection of mice to analyze requirements for TLR2, TLR4 and the central TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the generation of specific T cell responses. We demonstrate that following L. monocytogenes infection, mice deficient in TLR2, TLR4 and MyD88 can generate Listeria-specific CD8+ and CD4+ Th1 responses. These T cell responses are sufficient to control secondary infection with a high dose of L. monocytogenes even in the absence of TLR signaling via MyD88. Thus, TLR2-, TLR4- and MyD88-dependent signals are not essential for the generation of CD4+ Th1 and CD8+ T cells, and T cells can protect mice against infection in the absence of these signals.     

Analysis of signaling via surface immunoglobulin receptors on B cells from CBA/N mice

CBA/N mice, which carry the xid immunodeficiency, lack a mature subpopulation of B cells. The residual B cells in these mice do not make antibodies to type-2 T-independent antigens, nor do they synthesize DNA in response to mitogenic forms of anti-Ig antibodies. It is therefore an attractive hypothesis that the surface immunoglobulin receptors (sIgR) on xid B cells signal abnormally following cross-linking. We show here that anti-Ig antibodies do cause inositol phospholipid hydrolysis and Ca2+ mobilization in xid B cells. However, the response of these cells are only 40%-50% of those of normal B cells. Studies with permeabilized cells demonstrated that the hyporesponsiveness is not due to ineffective coupling of sIgR to their associated G-protein. Rather it is apparently due to a quantitative and/or qualitative deficiency in the polyphosphoinositide-specific phosphodiesterase which mediates sIgR-induced inositol phospholipid hydrolysis. These observations may provide a biochemical explanation for the immunological abnormalities resulting from the xid mutation.     

白介素12对T淋巴细胞作用与表面分子表达的关系

目的：探讨IL-12对T细胞作用效应与表面分子（CD25、FasL和TNFR1）变化的关系。方法：dUTP缺口末端标记和Annexin V法检测T细胞凋亡，FITC标记CD25和TNFR1的抗体、生物素标记FasL抗体，用流式细胞仪检测HTB176、TIB152和正常人T细胞表面分子表达的变化。结果：HTB176和TIB152的细胞在IL-12处理1 h，CD25和FasL的表达开始增加，24 h达到高峰，但正常T细胞在24 h后才有反应；正常T细胞经IL-12处理1 h内FasL的表达开始增加，在以后的试验期内始终保持恒定水平；但IL-12不促进三种T细胞表面TNFR1的表达。结论：IL-12对T细胞作用过程有多个表面分子参与，形成不同的效应。     

The tetramer model: a new view of class II MHC molecules in antigenic presentation to T cells

Crystallographic studies suggest a plausible divalent interaction between T-cell receptor (TCR) and MHC class II molecules. In addition, biochemical data suggest that these divalent MHC molecules are preformed at the membrane of the antigen-presenting cell. The tetramer model is based on these preformed tetrameric class II molecules that can be loaded with identical or different peptides in their two grooves. This enables divalent class II molecules to deliver two different messages to T cell: 1) a two-peptide message, in which the tetramer with two identical peptides is able to cross-link two TCRs triggering full activation of a T cell. At the thymic level we propose that this message induces negative selection; or 2) a one-peptide message: only one of the peptides loaded in the class II tetramer is able to interact with that TCR. This message would be involved in triggering partial activation phenomena in mature lymphocytes, whereas in thymocytes this message would mediate positive selection. Since high concentrations of a peptide would favor the load of tetramers with identical peptides, the tetramer could therefore be viewed as a quantitative-qualitative transducer that would trigger different responses depending on the concentration of antigenic peptides.     

Activation of thymic T cells by MHC alloantigen requires syngeneic, activated CD4+ T cells and B cells as APC

We examine here the in vitro requirements to activate immunocompetent T cells, present among thymocytes, to give rise to CTL, CD4+ T cells producing IL-2 and CD8+ T cells producing IFN-gamma. These thymocytes are naive in not having received antigen-dependent signals characteristic of the periphery. Their activation, upon stimulation with allogeneic spleen cells depleted of T cells, referred to here as allogeneic antigen-presenting cells (APCs), to produce allo-MHC-specific effector T cells, requires activated (radiation resistant) CD4+ T cells, syngeneic with the responding thymocytes. We refer here to these T cells as 'help'. Furthermore, optimal T cell activation requires an Ig+ B220+ cell in the allogeneic APC population, most probably a B cell. The allogeneic APCs cannot be replaced by conventional bone marrow (BM)-derived dendritic cells (DCs) activated by CD40 ligation or exposure to LPS. The requirements for both help and allogeneic B cells in the activation of thymocytes contrast with the requirements to generate substantial responses from splenic T cell populations. Activated, BM-derived DCs stimulate substantial splenic responses without help. These different requirements for activation could reflect the fact that thymocytes have not received an exit-thymus signal and/or that splenic T cells are heterogeneous, containing naive, memory and partially-activated T cells.     

The expansion of human gammadelta T cells in response to Daudi cells requires the participation of CD4+ T cells.

The Burkitt's lymphoma cell line Daudi is a potent inducer of human gammadelta T-cell expansion. Using an in vitro culture system comprised of irradiated Daudi cells as stimulators and normal human lymphocytes as responders, the cellular determinants of this response were investigated. Three of four monoclonal antibodies (mAbs 1-1C4, L243, and 9.3F10) directed against disparate epitopes of human major histocompatibility complex (MHC) class II, as well as a mAb with specificity for CD4 (OKT4), inhibited the expansion of gammadelta T cells in response to Daudi cell stimulators. mAbs with a specificity for CD74 and CD8 were non-inhibitory. Lymphocyte depletion experiments demonstrated a critical role for the CD4+ T-cell subset in the expansion of gammadelta T cells. Other data pointed towards requirements for direct cell contact in this system, and the addition of exogenous recombinant interleukin (IL)-2, IL-4, and IL-12 failed to reconstitute gammadelta T-cell expansion in CD4+ lymphocyte-depleted cultures. These results complement previous findings in murine infectious disease and mycobacterial systems, providing a direct demonstration that CD4+ T cells play a role in gammadelta T-cell expansion through an interaction with human leucocyte antigen (HLA) class II on Daudi cells. The data point towards important functional links between the acquired and natural immune systems.     

Mast cell derived heparin activates the contact system: a link to kinin generation in allergic reactions

Contact activation occurs when plasma comes in contact with negatively charged man-made surfaces but no substance that initiates contact activation in vivo has been identified. We have isolated a mast cell heparin proteoglycan (MC-HepPG) from a Furth mouse mastocytoma-derived cell line that is analogous to human tissue-type mast cell HepPG. This material and other glycosaminoglycans (GAGs) were tested for their ability to accelerate the reciprocal activation of factor XII and prekallikrein and the autoactivation of factor XII. Quantitative analysis showed the MC-HepPG to be as active as dextran sulfate on a weight basis; hog intestine heparin, dermatan sulfate. keratan polysulfate and chondroitin sulfate C were less active, other sulfated polysaccharides were essentially inactive. Incubation of MC-HepPG in 1:4 diluted plasma resulted in complete cleavage of high molecular weight kininogen in a factor Xll-dependent reaction. All of the MC-HepPG dependent reactions described above were inhibited by preincubation of MC-HepPG with heparinase I and II but not by pretreatment with heparitinase. chondroitinase ABC or the serine protease inhibitor aPMSF thus indicating that heparin proteoglycan is indeed acting as an initiating ‘surface’. We analysed the proteoglycan preparation by HPLC gel filtration. Fractions spanning a molecular weight range of > 400000–8000 were active initiators. Comparison of the chromatograms obtained before and after cleavage of GAG side chains from the protein core suggested that dissociated GAGs in the MW range 69000–17000 are the most active species rather than the complete proteoglycan. MC-HepPG GAGs therefore represent a physiologic macromolecule with activity comparable to non-physiological surfaces in a purified system and with the capability to induce activation of the contact system in diluted plasma. Its ability to promote kinin generation links cellular and humoral inflammatory responses in the perivasculature and provides a possible explanation for the elevated kinin levels observed after allergen exposure.