R. A. W. Van Lier

Leukocyte adhesion deficiency type 1 (LAD-1)/variant. A novel immunodeficiency syndrome characterized by dysfunctional beta2 integrins.

Leukocyte adhesion deficiency (LAD) is characterized by the inability of leukocytes, in particular neutrophilic granulocytes, to emigrate from the bloodstream towards sites of inflammation. Infectious foci are nonpurulent and may eventually become necrotic because of abnormal wound healing. LAD-1 is characterized by the absence of the beta2 integrins (CD11/CD18) on leukocytes. When expression is completely absent, patients often die within the first year. However, low levels of beta2 expression may result in a milder clinical picture of recurrent infection, which offers a better prognosis. In this paper, we describe the in vivo and in vitro findings on a patient with clinical features of a mild LAD-1 disorder, i.e., suffering from bacterial infections without apparent pus formation in the presence of a striking granulocytosis, showing no delayed-type hypersensitivity reaction upon skin testing, no specific antibody generation, but normal in vitro T cell proliferation responses after immunization. Expression levels of CD11/CD18 proteins were completely normal, but leukocyte activation did not result in CD11/ CD18 activation and high-avidity ligand-binding. In vitro chemotaxis and endothelial transmigration of the neutrophils as well as leukocyte aggregation responses were almost absent. On the other hand, beta1 and beta3 integrin-mediated adhesion functions were completely normal. During follow-up, a bleeding tendency related to decreased beta3 activation became clinically apparent, different from previously described cellular adhesion molecule variants. Therefore, this is the first well-documented case of a clinical combined immunodeficiency syndrome that results from nonfunctional CD11/CD18 molecules, and thus designated LAD-1/ variant.

Functional and phenotypic evidence for a selective loss of memory T cells in asymptomatic human immunodeficiency virus-infected men.

In addition to a well-documented depletion of CD4+ T helper cells in later stages of human immunodeficiency virus (HIV) infection, evidence has been provided for a specific unresponsiveness to triggering either by specific antigen in the context of autologous major histocompatibility molecules (self + X) or anti-CD3 monoclonal antibodies (MAb) in both CD4 and CD8 cells from asymptomatic HIV-infected individuals. In the present study we analyzed this unresponsiveness using mitogenic antibodies to distinct T cell membrane receptors. T cells from HIV-infected men who had normal numbers of CD4+ T cells responded poorly to activation signals via the CD3 membrane antigen in both accessory cell-dependent as well as accessory cell-independent culture systems. A similar low response was observed in an anti-CD2-driven system. In contrast, proliferation induced by anti-CD3, anti-CD2, or the phorbol ester Phorbol myristate acetate could be normally enhanced by anti-CD28 MAb. We demonstrated that this unresponsiveness is not due to a failure to induce early events required for activation, such as increased intracellular concentration of free calcium and activation of protein kinase C, but is caused by an imbalance between naive and memory T cells. In HIV-infected asymptomatic men, CD29+ memory T cells are selectively depleted which results in a poor responsiveness to self + X. These findings provide new insights that may have implications for our understanding of the immunopathogenesis of AIDS.

Treatment with depleting CD4 monoclonal antibody results in a preferential loss of circulating naive T cells but does not affect IFN-gamma secreting TH1 cells in humans.

CD4(pos) TH1 T cells are considered to play a central role in a number of human autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis. Experimental treatment protocols aimed at selectively eliminating CD4(pos) T cells thus far have yielded disappointing clinical results. Here we analyzed phenotype and function of circulating T cells in multiple sclerosis patients treated with the chimeric CD4 mAb cM-T412 in a randomized, double-blind, placebo-controlled, magnetic resonance imaging-monitored phase II trial. Treatment resulted in a long-lasting depletion of CD4(pos) T cells but did not affect CD8(pos) T cell numbers. Analysis of CD4(pos) subpopulations showed that unprimed, CD45RA(pos)/R0(neg) lymphocytes were approximately three times more sensitive to the mAb than primed, CD45RA(neg)/R0(pos) T cells. Notably, within the CD45RA(pos) subset, T cells with phenotypic evidence of prior activation, i.e., expressing Fas, were relatively insensitive to cM-T412, compared with Fas(neg) cells. Remarkably, while a decrease in the number of IL-4-producing T helper 2 (TH2)-type cells in the anti-CD4 treated group was observed, numbers of IFN-gamma-producing T helper 1 (TH1)-type cells remained stable, resulting in a significant increase in the TH1/TH2 ratio. Our data show that treatment with depleting CD4 mAb does not eliminate the cells most strongly involved in the disease process, i.e., primed, IFN-gamma-producing TH1-type cells, and may therefore give an explanation for the lack of beneficial clinical effects of depleting CD4 mAb in human chronic autoimmune disease.

Immunological and virological markers in individuals progressing from seroconversion to AIDS

Six men were selected from a large cohort of homosexual men participating in a study on HIV infection that was followed from seroconversion to AIDS. The patients were studied retrospectively for immunological functions of T cells, T-cell subset distribution and biological phenotype of HIV. A severe decrease in anti-CD3 monoclonal antibody (MAb)-induced T-cell proliferation at seroconversion was observed in two out of six men. After this acute phase, CD4+ T-cell numbers were in the normal range in the early asymptomatic period; the proliferative response was subnormal, whereas the capacity to generate cytotoxic T cells (CTL) was normal. From seroconversion on, CD4+CD29+ memory T-cell numbers were decreased to approximately 50% of normal values, which may contribute to loss of T-cell reactivity. In the asymptomatic phase only slow-replicating non-syncytium-inducing HIV variants were observed. The T-cell proliferative response further declined with the depletion of naive CD4+ CD45RA+ T cells and CD4+ T-cell numbers started to decline. This second decrease in T-cell function coincided with the emergence of more rapidly replicating, often (four out of six) syncytium-inducing variants. At diagnosis of AIDS, T-cell proliferation and CD4+ T-cell numbers were extremely low in five out of six patients and CTL function had declined in three out of five individuals tested. Circulating CD8+ cells had gradually shifted to an immature CD38+CD28- phenotype. Our findings support the theory that HIV-induced immune dysfunction allows for the emergence of virulent HIV variants associated with CD4+ cell loss and disease.

Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation

We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgVH mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.

Deep Sequencing of Antiviral T-Cell Responses to HCMV and EBV in Humans Reveals a Stable Repertoire That Is Maintained for Many Years

CD8+ T-cell responses against latent viruses can cover considerable portions of the CD8+ T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8+ T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8+ T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8+ T-cell receptor Vβ repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.

Dichotomy in NF-kappaB signaling and chemoresistance in immunoglobulin variable heavy-chain-mutated versus unmutated CLL cells upon CD40/TLR9 triggering.

Chronic lymphocytic leukemia (CLL) cells circulating in peripheral blood (PB) differ from the leukemic fraction in lymph nodes (LNs) with respect to cell division and drug sensitivity. CD40 stimulation of PB CLL cells in vitro results in chemoresistance and provides a partial model for the LN microenvironment. The TLR9 ligand CpG induces proliferation in immunoglobulin variable heavy-chain-unmutated CLL, but apoptosis in immunoglobulin variable heavy-chain-mutated CLL. To juxtapose proliferative with antiapoptotic signals, we investigated the effects of CpG in the context of CD40 ligation in mutated versus unmutated CLL cells in this study. Prolonged CD40 ligation induced classical, followed by alternative nuclear factor-κB (NF-κB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-XL levels, respectively. A dichotomy in NF-κB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-XL levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-XL and remained chemoresistant. The pivotal contribution of Bcl-XL to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-XL levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-κB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-XL levels.

AICL : A NEW ACTIVATION-INDUCED ANTIGEN ENCODED BY THE HUMAN NK GENE COMPLEX

Abstract The NK gene complex on mouse chromosome 6 and its human homologue on chromosome 12 encode type II transmembrane proteins with a C-type lectin domain which trigger or inhibit target cell lysis by NK cells (NKR-P1, Ly49, NKG2, CD94) or function as cellular activators of various hematopoietic cells (CD69). We herein report the cDNA cloning of a new molecule, designated activation-induced C-type lectin (AICL), whose gene maps to the human NK gene complex proximal to the CD69 gene. AICL is a 149-amino acid (aa) polypeptide with a short cytoplasmic part of seven aa and a C-type lectin domain separated from the transmembrane region by only nine aa. The highest sequence similarity is found to the C-type lectin domains of CD69 and the chicken lectin 17.5. The presence of AICL transcripts in different cell types of hematopoietic origin, a rapid increase of gene transcription during lymphocyte activation, and a short half-life of the mRNA characterize AICL as a new, broadly expressed activation antigen.

Antigen-presenting cell-derived signals determine expression levels of CD70 on primed T cells

Interaction between CD27 and its ligand CD70 provides a second signal for T‐cell proliferation and tumour necrosis factor‐α (TNF‐α) production. Whereas CD27 is broadly expressed during T‐cell development, expression of CD70 in vivo is restricted. To determine when CD27–CD70 interactions can occur in immune reactions, we here analysed the regulation of CD70 expression on activated T cells. Mitogenic stimulation of purified T cells with either immobilized CD3 monoclonal antibody (mAb) or a combination of CD2 mAb induces only low levels of CD70 membrane expression. Markedly, expression of the CD27‐ligand is strongly enhanced by antigen‐presenting cells (APC) and APC‐associated signals such as interleukin‐1α (IL‐1α), IL‐12, TNF‐α and CD28‐ligation. In contrast, T‐cell derived cytokines, such as IL‐4, counteract CD70 up‐regulation on activated T cells. Analysis of the small subset of circulating CD70+ T cells revealed that these cells have a primed phenotype as they express CD45RO and HLA‐DR antigens and are in high frequency able to secrete interferon‐γ (IFN‐γ). We conclude that T–T interactions involving CD27 and CD70 are likely to occur relatively early in immune reactions, after productive T‐cell priming by APC and that expression of CD70 on circulating T cells is a reflection of recent priming by antigen.

Intracellular cytokine profile in T-cell subsets of multiple sclerosis patients: different features in primary progressive disease:

Objective: To evaluate the expression of cytokines in both CD4+ and CD8+ T cells derived from peripheral blood of untreated multiple sclerosis (MS) patients with either relapsing-remitting (RR), secondary progressive (SP) or primary progressive (PP) MS and healthy controls (HC). Background: MS is an immune-mediated disease and cytokines have been hypothesized to contribute significantly to disease progression. Compared to the relapse-onset (RR, SP) form of the disease, PPMS patients have different clinical, immunological and pathological features. Surprisingly, the ability of their circulating T cells to produce immunoregulatory cytokines has not been extensively studied so far. Methods: Seventy-two MS patients (24 RR, 26 SP, 22 PP) and 34 HC were studied. Stimulated peripheral blood derived CD4+ and CD8+ T cells were analyzed for IFN-g, IL-2, TNF-a, IL-4, IL-10 and IL-13 production. Results: MS patients express significantly more CD4+ and CD8+ T cells producing IFN-g compared to HC. Compared to the other forms of the disease, PPMS patients display a significant decrease in CD4+ T cells producing IL-2, IL-13 and TNF-a and a significant increase in CD8+ T cells producing IL-4 and IL-10. Conclusions: The data presented here demonstrate that patients with PPMS express less pro- and more anti-inflammatory cytokine producing T cells compared to the relapse-onset form of the disease, confirming the view on PPMS as a distinct disease entity.