NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal ��-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal ��-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4+ T lymphocytes, i.e., CD4+Ki67+ T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8+Ki67+ T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4+Ki67+ T lymphocytes increased over time and was associated with an increase of both naive and memory CD4+ T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4+ T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived���peptide

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T���cells

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

CD8+ T-cell-derived soluble factor(s), but not ��-chemokines RANTES, MIP-1��, and MIP-1��, suppress HIV-1 replication in���monocyte/macrophages

It has been demonstrated that CD8+ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+ T-cell supernatants or ��-chemokines. We demonstrate that: (i) CD8+ T-cell supernatants did, but ��-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1�� (MIP-1��) and MIP-1�� did not, whereas antibodies to interleukin 10, interleukin 13, interferon ��, or interferon �� modestly reduced anti-HIV activity of the CD8+ T-cell supernatants; and (iii) the CD8+ T-cell supernatants did, but ��-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+ T cells is a multifactorial phenomenon, and that RANTES, MIP-1��, and MIP-1�� do not account for the entire scope of CD8+ T-cell-derived HIV-suppressor factors.

The dual functions of Fas ligand in the regulation of peripheral CD8+ and CD4+ T cells

Although Fas ligand (FasL) is well characterized for its capacity to deliver a death signal through its receptor Fas, recent work demonstrates that FasL also can receive signals facilitating antigen (Ag)-specific proliferation of CD8+ T cells. The fact that the gld mutation differentially influences the proliferative capacity of CD8+ and CD4+ T cells presented the intriguing possibility that a single molecule may play opposing roles in these two subpopulations. The present study focuses on how these positive and negative regulatory roles are balanced. We show that naive CD4+ T cells are responsive to FasL-mediated costimulation on encounter with Ag when Fas-mediated death is prevented. Thus, the machinery responsible for transducing the FasL positive reverse signal operates in both CD4+ and CD8+ T cells. Instead, differential control of FasL expression distinguishes the role of FasL in these two T cell subpopulations. FasL costimulation occurs immediately on T cell receptor ligation and correlates with the up-regulation of FasL expression on CD8+ and naive CD4+ T cells, both of which are sensitive to the FasL costimulatory signal. Conversely, FasL-initiated death occurs late in an immune response when high levels of FasL expression are maintained on CD4+ T cells that are sensitive to Fas-mediated death, but not on CD8+ T cells that are relatively insensitive to this signal. This careful orchestration of FasL expression during times of susceptibility to costimulation and conversely, to death, endows FasL with the capacity to both positively and negatively regulate the peripheral T cell compartment.

A mouse CD8 T cell-mediated acute autoimmune diabetes independent of the perforin and Fas cytotoxic pathways: Possible role of membrane TNF

Double transgenic mice [rat insulin promoter (RIP)-tumor necrosis factor (TNF) and RIP-CD80] whose pancreatic �� cells release TNF and bear CD80 all develop an acute early (6 wk) and lethal diabetes mediated by CD8 T cells. The first ultrastructural changes observed in �� cells, so far unreported, are focal lesions of endoplasmic reticulum swelling at the points of contact with islet-infiltrating lymphoblasts, followed by cytoplasmic, but not nuclear, apoptosis. Such double transgenic mice were made defective in either the perforin, Fas, or TNF pathways. Remarkably, diabetes was found to be totally independent of perforin and Fas. Mice lacking TNF receptor (TNFR) II had no or late diabetes, but only a minority had severe insulitis. Mice lacking the TNF-lymphotoxin (LT��) locus (whose sole source of TNF are the �� cells) all had insulitis comparable to that of nondefective mice, but no diabetes or a retarded and milder form, with lesions suggesting different mechanisms of injury. Because both TNFR II and TNF-LT�� mutations have complex effects on the immune system, these data do not formally incriminate membrane TNF as the major T cell mediator of this acute autoimmune diabetes; nevertheless, in the absence of involvement of the perforin or Fas cytotoxic pathways, membrane TNF appears to be the likeliest candidate.

Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.

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.

Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after ���browsing��� their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous ���boiling��� of the cytoplasm and blebbing of the plasma membrane) for 10���20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5���LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity.

Differences in antigen recognition and cytolytic activity of CD8+ and CD8��� T cells that express the same antigen-specific receptor

CD8+ and CD8��� T cell lines expressing the same antigen-specific receptor [the 2C T cell receptor (TCR)] were compared for ability to bind soluble peptide-MHC and to lyse target cells. The 2C TCR on CD8��� cells bound a syngeneic MHC (Kb+)-peptide complex 10���100 times less well than the same TCR on CD8+ cells, and the CD8��� 2C cells lysed target cells presenting this complex very poorly. Surprisingly, however, the CD8��� cells differed little from CD8+ cells in ability to bind an allogeneic MHC (Ld+)-peptide complex and to lyse target cells presenting this complex. The CD8+/CD8��� difference provided an opportunity to estimate how long TCR engagements with peptide-MHC have to persist to initiate the cytolytic T cell response.

Lack of a role for transforming growth factor-�� 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-��1 (TGF-��1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-�� production. Here, we examine the role of TGF-�� in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-�� 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-�� does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-��1���/���, and Smad3���/��� T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-�� by CD4+ T cells. These results indicate that CTLA-4 ligation does not regulate TGF-�� production and that CTLA-4-mediated inhibition can occur independently of TGF-��. Collectively, these data demonstrate that CTLA-4 and TGF-�� represent distinct mechanisms for regulation of T cell responses.

Intralymphatic immunization enhances DNA vaccination

Although DNA vaccines have been shown to elicit potent immune responses in animal models, initial clinical trials in humans have been disappointing, highlighting a need to optimize their immunogenicity. Naked DNA vaccines are usually administered either i.m. or intradermally. The current study shows that immunization with naked DNA by direct injection into a peripheral lymph node enhances immunogenicity by 100- to 1,000-fold, inducing strong and biologically relevant CD8+ cytotoxic T lymphocyte responses. Because injection directly into a lymph node is a rapid and easy procedure in humans, these results have important clinical implications for DNA vaccination.

B lymphocyte involvement in ankylosing spondylitis: the heavy chain variable segment gene repertoire of B lymphocytes from germinal center-like foci in the synovial membrane indicates antigen selection

The synovial membrane (SM) of affected joints in ankylosing spondylitis (AS) is infiltrated by germinal center-like aggregates (foci) of lymphocytes similar to rheumatoid arthritis (RA). We characterized the rearranged heavy chain variable segment (VH) genes in the SM for gene usage and the mutational pattern to elucidate the B lymphocyte involvement in AS.