Immunopathogenesis of human T cell lymphotropic virus type I-associated neurologic disease

This review focuses on current approaches to understanding the immunopathogenesis of human T cell lymphotropic virus (HTLV) type I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) based on newly developed molecular and immunologic techniques that have been adapted to studies of HTLV-I proviral load, HTLV-I mRNA, and HTLV-I tax-specific CD8 T cells. These methods enable researchers to study previously inaccessible aspects of this disease and allow a more detailed analysis of virus/host immune responses as they relate to disease specificity in this disorder. The role of HTLV-I-specific CD8 T cell immune responses is highlighted. The elucidation of the immunopathology of HAM/TSP will enhance our understanding of other HTLV-I-associated disorders plus other neurologic, hematologic, and inflammatory diseases for which viral etiologies have been suggested.     

CD8(+) T cells are an in vivo reservoir for human T-cell lymphotropic virus type I

It is thought that human T-cell lymphotropic virus type I (HTLV-I) preferentially infects CD4(+) T cells in vivo. However, observations of high HTLV-I proviral load in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggest that HTLV-I may infect other cell types in addition to CD4(+) T cells. To identify in vivo T-cell tropisms of HTLV-I, real-time quantitative polymerase chain reaction (PCR) and intracellular protein staining were used. A high amount of HTLV-I proviral DNA was detected from purified CD8(+) T cells by quantitative PCR (between 1.64 and 62.83 copies of HTLV-I provirus per 100 isolated CD8(+) T cells). CD8(+) T cells expressed HTLV-I-related antigens (HTLV-I Tax and p19 protein) after a short time in cultivation. These results demonstrate that CD8(+) T cells are also infected with HTLV-I and express HTLV-I antigens at levels that are comparable to HTLV-I-infected CD4(+) cells. Therefore, CD8(+) cells are an additional viral reservoir in vivo for HTLV-I and may contribute to the pathogenesis of HTLV-I-mediated disorders.     

Impaired function of human T-lymphotropic virus type 1 (HTLV-1)-specific CD8+ T cells in HTLV-1-associated neurologic disease

Despite abundant activated virus-specific cytotoxic T lymphocytes (CTLs), patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) showed a significantly higher frequency of infected T cells than did healthy virus carriers (HVCs). Here, we demonstrate that at a given proviral load, the frequency of CD8(+) T cells that are negative for specific costimulatory molecules was significantly higher in HAM/TSP than in age-matched HVCs and uninfected healthy controls (HCs), whereas the frequency of intracellular perforin-positive CD8(+) T cells was significantly lower in both HAM/TSP and HVCs than in HCs. An inverse correlation between HTLV-1 proviral load (PVL) and percent perforin-positive CD8(+) T cells were observed only in disease-protective allele HLA-A*02-positive HVCs, but not in HAM/TSP patients, whether HLA-A*02 positive or negative, nor in HLA-A*02-negative HVCs. Significantly lower perforin expression was observed in HTLV-1-specific than in cytomegalovirus-specific CD8(+) T cells. Majority of HTLV-1-specific CD8(+) T cells in HVCs showed a CD28(-)CD27(+) phenotype, whereas HAM/TSP showed a CD28(-)CD27(-) phenotype. HTLV-1-specific CD8(+) T cells from HAM/TSP patients showed significantly lower degranulation than HVCs by CD107a mobilization assay. These findings suggest that an impaired function of HTLV-1-specific CTLs is associated with failing antiviral control and disease HAM/TSP.     

In vitro human memory CD8 T cell expansion in response to cytomegalovirus requires CD4+ T cell help

Requirements for human memory CD8(+) T cell expansion are incompletely understood. We found that human cytomegalovirus (HCMV) induced expansion of memory CD8(+) T cells in vitro without requiring intracellular viral peptide synthesis. Peptide-major histocompatibility complex class I tetramer binding confirmed expansion of cells with HCMV-peptide specificity. Expansion of memory CD8(+) T cells was completely dependent on the presence and function of CD4(+) T cells, whose "help" also could be induced by exposure to irrelevant antigen. Recombinant interleukin (IL)-2 or IL-15 could substitute for help provided by CD4(+) T cells, whereas CD8(+) T cell expansion was blocked by anti-IL-2 but not anti-IL-15 antibody. Human memory CD8(+) T cells expand dramatically in vitro in response to cross-presentation of HCMV antigens, and, in contrast to observations made in murine systems, this proliferation was critically dependent on CD4(+) T cells that provide essential IL-2. Thus, in humans, cross-presentation and expansion of memory CD8(+) T cells may be compromised in disease states that result in deficits in CD4(+) T cell numbers or function, such as may be seen in human immunodeficiency virus type 1 infection.     

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.     

Identification and in vitro expansion of CD4+ and CD8+ T cells specific for human neutrophil elastase

Human neutrophil elastase (HNE) and proteinase 3 (PRO3) are myeloid tissue-restricted serine proteases, aberrantly expressed by myeloid leukemia cells. PRO3 and HNE share the PR1 peptide sequence that induces HLA-A*0201-restricted cytotoxic T cells (CTLs) with antileukemia reactivity. We studied the entire HNE protein for its ability to induce CTLs. In an 18-hour culture, HNE-loaded monocytes stimulated significant intracellular interferon gamma (IFN-gamma) production by CD4+ and CD8+ T cells in 12 of 20 and 8 of 20 healthy individuals, respectively. Lymphocytes from 2 HNE responders were pulsed weekly for 4 weeks to generate HNE-specific CTLs. One of 2 HLA-A*0201-negative individuals inhibited the colony formation of HLA-identical chronic myelogenous leukemia progenitor cells (73% inhibition at 50:1 effector-target [E/T] ratio), indicating that peptides other than PR1 can induce leukemia-reactive CTLs. Repetitive stimulations with HNE in 2 of 5 HLA-A*0201+ individuals increased PR1 tetramer-positive CD8+ T-cell frequencies from 0.1% to 0.29% and 0.02% to 0.55%, respectively. These CTLs recognized PR1 peptide or killed HNE-loaded targets. These results indicate that exogenously processed HNE is a source of PR1 peptide as well as other peptide sequences capable of inducing leukemia-specific CD8+ and CD4+ T cells. HNE could, therefore, be used in an HLA-unrestricted manner to induce leukemia-reactive CTLs for adoptive immunotherapy.     

In vitro expansion of human peripheral blood CD34+ cells

To elucidate the role of recombinant human colony-stimulating factors (CSFs) for expanding peripheral blood (PB) CD34+ cells, these cells were purified up to 94.5% +/- 1.3% and the effects of individual and combined CSFs on the proliferation and differentiation of these cells were studied in a 7-day suspension culture. The majority of CD34+ cells coexpressed CD38 (81.8% +/- 5.1%), but was negative for CD33 (88.5% +/- 3.4%). Among the individual CSFs examined, recombinant interleukin-3 (rIL-3) was identified as the most potent factor for expanding PB progenitor cells and increased nonerythroid progenitor cells 13- +/- 4- fold (P < .01). Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant granulocyte-CSF (rG-CSF), recombinant macrophage-CSF (rM-CSF), rIL-6, rIL-11, and recombinant stem cell factor (rSCF) did not alone expand nonerythroid progenitor cells. A combination of 5 CSFs, ie, rIL-3, rIL-6, rGM-CSF, rG-CSF, and rSCF, was identified as the most potent combination of those tested and increased nonerythroid progenitor cells 57- +/- 11-fold. After a 7-day suspension culture of CD34+ cells with these 5 CSFs, CD34+ cells expanded 14.5- fold, and CD34+/CD33- cells and CD34+/CD33+ cells were also expanded 2.9-fold and 307-fold, respectively. Most secondary colonies derived from expanded cells were small; however, the absolute number of large- sized colonies expanded 5.9- +/- 3.3-fold. Thus, the combination of CSFs can achieve a degree of amplification of PB CD34+ cells. The capability of in vitro expansion of PB CD34+ cells as an adjunct to PB stem cell transplantation is worthy of consideration.     

IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro

Recent studies in mice have shown that although interleukin 15 (IL-15) plays an important role in regulating homeostasis of memory CD8+ T cells, it has no apparent function in controlling homeostatic proliferation of naive T cells. We here assessed the influence of IL-15 on antigen-independent expansion and differentiation of human CD8+ T cells. Both naive and primed human T cells divided in response to IL-15. In this process, naive CD8+ T cells successively down-regulated CD45RA and CD28 but maintained CD27 expression. Concomitant with these phenotypic changes, naive cells acquired the ability to produce interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), expressed perforin and granzyme B, and acquired cytotoxic properties. Primed CD8+ T cells, from both noncytotoxic (CD45RA-CD27+) and cytotoxic (CD45RA+CD27-) subsets, responded to IL-15 and yielded ample numbers of cytokine-secreting and cytotoxic effector cells. In summary, all human CD8+ T-cell subsets had the ability to respond to IL-15, which suggests a generic influence of this cytokine on CD8+ T-cell homeostasis in man.     

In vivo expansion coincident with excessive in vitro cell death within the memory subset of CD8+ T cells in HIV type 1 infection

In HIV-1-infected individuals the CD8+ T cell subset is considerably expanded. This has been shown to be caused predominantly by an increase in the number of CD8+CD28- T cells. To characterize further the subsets of CD8+ T cells, we have performed analyses of cell surface phenotype, T cell receptor Vbeta usage, and ability to survive in unstimulated cultures. CD8+CD28- T cells frequently expressed CD45RA. Nonetheless, coincident expression of CD95 (Fas) and high levels of integrins suggested that these cells were immunologically experienced. Contrary to what has been observed in CD8+CD28- T cells from uninfected individuals, a common hierarchy of Vbeta usage was found in CD8+CD28- T cells from HIV-1-infected individuals, which was adhered to by all the study participants, and which was shown to be similar to that observed within CD8+CD28+ T cells. Cells from the memory subsets of CD8+ T cells showed a high incidence of spontaneous death in unstimulated cultures, indicating that these cells have received signals for death by apoptosis in vivo. In contrast, most naive CD8+CD28+CD45RA+ cells survived. The CD8+CD28- memory subset is expanded in vivo despite evidence for coincident excessive cell death in vitro. Our results are consistent with the notion that frequent transitions occur from the memory CD8+CD28+CD45RA- T cell subset to the end-stage CD8+CD28- subset during HIV-1 infection.     

CD4+CD8+ human small intestinal T cells are decreased in coeliac patients, with CD8 expression downregulated on intra-epithelial T cells in the active disease

BACKGROUND/OBJECTIVE: The intestinal lesion of coeliac disease is thought to be initiated and exacerbated by dysregulation of local T-lymphocyte sub-populations. This study examines changes in intestinal T cells from coeliac patients, with a particular focus on CD4CD8 T cells, immunoregulatory cells normally found in relatively high proportions in the small intestine. METHODS: Cells were obtained from duodenal biopsies from active and treated coeliac patients using chelating and reducing agents (epithelial layer) followed by collagenase treatment (lamina propria). Cell yield and viability were assessed and flow cytometric analysis was used to examine CD4CD8 T cells and to quantify CD8 expression. RESULTS: Surprisingly, total T-cell yields in the epithelial layer did not increase in active coeliac disease although enterocyte counts decreased significantly, giving an appearance of infiltration. In active coeliac patients, CD4CD8 T cell percentages were significantly decreased in both the epithelial layer and lamina propria. Levels of CD8 expression by CD4CD8 T cells in the epithelial layer were decreased significantly in patients with active coeliac disease. CD4CD8 T cell proportions did not return to normal in treated coeliac patients whose villous architecture had responded to gluten withdrawal. CONCLUSIONS: No increase of intra-epithelial lymphocytes in the coeliac lesion may require us to reconsider the definition of coeliac disease as an inflammatory condition. Low CD4CD8 populations in treated as well as untreated coeliac patients indicate that these T cells are inherently absent in individuals genetically predisposed to coeliac disease.     

Remarkable increase in CD26-positive T cells in patients with human T lymphotropic virus type I (HTLV-I) associated myelopathy.

We used two-color flow cytometric analysis to investigate CD26+ (Ta1+) cells in peripheral blood T lymphocytes from patients with human T-lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM). The percentage of CD26+ cells among CD3+ cells was markedly increased in patients with HAM, compared with anti-HTLV-I seropositive carriers (p < 0.001) and seronegative controls (p < 0.01). Within the subpopulation of T cells, a significantly high percentage of CD26+ cells was detected in both CD4+ and CD8+ cell populations. Furthermore, analysis of HLA-DR+ T cells revealed similar results. In contrast CD4+CD45RA+ cells were significantly decreased in comparison with controls. These results suggest that immunologically activated or memory T cells found in peripheral blood may be etiologically relevant to HAM.