Characterization of bovine homologues of granulysin and NK-lysin

Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3(+) T cells, CD4(+) T cells, CD8(+) T cells, WC1(+) gammadelta T cells, and PBMC depleted of CD3(+) T cells, but was absent in CD21(+) cells and CD14(+) cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.     

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN- dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.     

Coordinate expression of CC chemokine ligand 5, granulysin, and perforin in CD8+ T cells provides a host defense mechanism against Mycobacterium tuberculosis

The ability of CD8+ T cells to kill intracellular pathogens depends upon their capacity to attract infected cells as well as their secretion of cytolytic and antimicrobial effector molecules. We examined the Ag-induced expression of three immune effector molecules contained within cytoplasmic granules of human CD8+ T cells: the chemokine CCL5, the cytolytic molecule perforin, and the antimicrobial protein granulysin. Macrophages infected with virulent Mycobacterium tuberculosis triggered the expression of CCL5 in CD8+ T cells only in donors with previous exposure to the tuberculosis bacteria, not in naive donors. Functionally, CCL5 efficiently attracted M. tuberculosis-infected macrophages, but failed to exert direct antibacterial activity. Infected macrophages also triggered the expression of granulysin in CD8+ T cells, and granulysin was found to be highly active against drug-susceptible and drug-resistant M. tuberculosis clinical isolates. The vast majority of CCL5-positive cells coexpressed granulysin and perforin. Taken together, this report provides evidence that a subset of CD8+ T cells coordinately expresses CCL5, perforin and granulysin, thereby providing a host mechanism to attract M. tuberculosis-infected macrophages and kill the intracellular pathogen.     

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.     

Human NKT cells express granulysin and exhibit antimycobacterial activity

Human NKT cells are a unique subset of T cells that express an invariant V alpha 24 TCR that recognizes the nonclassical Ag-presenting molecule CD1d. Activation of NKT cells is greatly augmented by the marine sponge-derived glycolipid alpha-galactosylceramide (alpha GalCer). Because human monocyte-derived cells express CD1d and can harbor the intracellular pathogen Mycobacterium tuberculosis, we asked whether the addition of alpha GalCer could be used to induce effector functions of NKT cells against infected monocytes, macrophages, and monocyte-derived dendritic cells. NKT cells secreted IFN-gamma, proliferated, and exerted lytic activity in response to alpha GalCer-pulsed monocyte-derived cells. Importantly, alpha GalCer-activated NKT cells restricted the growth of intracellular M. tuberculosis in a CD1d-dependent manner. NKT cells that exhibited antimycobacterial activity also expressed granulysin, an antimicrobial peptide shown to mediate an antimycobacterial activity through perturbation of the mycobacterial surface. Degranulation of NKT cells resulted in depletion of granulysin and abrogation of antimycobacterial activity. The detection of CD1d in granulomas of tuberculosis patients supports the potential interaction of NKT cells with CD1d-expressing cells at the site of disease activity. These studies provide evidence that alpha Gal Cer-activated CD1d-restricted T cells can participate in human host defense against M. tuberculosis infection.     

A role for IL-12 receptor expression and signal transduction in host defense in leprosy.

The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12Rbeta1/IL-12Rbeta2 heterodimer, with the IL-12Rbeta2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12Rbeta2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12Rbeta1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12Rbeta2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12Rbeta2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12Rbeta2 expression and function in host defense in leprosy.     

CD8 T cell-mediated killing of Cryptococcus neoformans requires granulysin and is dependent on CD4 T cells and IL-15

Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl(2,) or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.     

A lipopeptide facilitate induction of Mycobacterium leprae killing in host cells

Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells.     

Identification of a human HLA-E-restricted CD8+ T cell subset in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine

Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4-CD56- T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-gamma production. Increases in the net frequency of IFN-gamma spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-gamma production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.     

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.     

A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy

The interaction of CD40 ligand (CD40L) expressed by activated T cells with CD40 on macrophages has been shown to be a potent stimulus for the production of IL-12, an obligate signal for generation of Th1 cytokine responses. The expression and interaction of CD40 and CD40L were investigated in human infectious disease using leprosy as a model. CD40 and CD40L mRNA and surface protein expression were predominant in skin lesions of resistant tuberculoid patients compared with the highly susceptible lepromatous group. IL-12 release from PBMC of tuberculoid patients stimulated with Mycobacterium leprae was partially inhibited by mAbs to CD40 or CD40L, correlating with Ag-induced up-regulation of CD40L on T cells. Cognate recognition of M. leprae Ag by a T cell clone derived from a tuberculoid lesion in the context of monocyte APC resulted in CD40L-CD40-dependent production of IL-12. In contrast, M. leprae-induced IL-12 production by PBMC from lepromatous patients was not dependent on CD40L-CD40 ligation, nor was CD40L up-regulated by M. leprae. Furthermore, IL-10, a cytokine predominant in lepromatous lesions, blocked the IFN-gamma up-regulation of CD40 on monocytes. These data suggest that T cell activation in situ by M. leprae in tuberculoid leprosy leads to local up-regulation of CD40L, which stimulates CD40-dependent induction of IL-12 in monocytes. The CD40-CD40L interaction, which is not evident in lepromatous leprosy, probably participates in the cell-mediated immune response to microbial pathogens.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus.

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

Immunopathogenic role of T-cell subsets in Borna disease virus-induced progressive encephalitis.

Borna disease is an immunopathological virus-induced encephalopathy comprising severe inflammation and degenerative brain cell lesions which results in organ atrophy and chronic debility in rats. CD4+ and CD8+ T cells have been reported to be involved in the development of this disease of the central nervous system. A virus-specific homogeneous T-cell line, established in vitro after immunization of rats with the recombinant 24-kDa virus-specific protein, showed antigen-specific proliferation in the presence of the 24-kDa but not the 38-kDa Borna disease virus-specific protein, another major virus-specific antigen. This T-cell line, P205, was found to exhibit characteristics of a T-helper cell: CD4+ CD8- IL-2- IL-4- IFN-gamma+ IL-6+ IL-10+. Furthermore, this T-cell line expressed the alpha/beta T-cell receptor and the alpha 4 integrin (VLA-4). Adoptive transfer of this helper cell resulted in an increase of antibody titers and two different types of disease in virus-infected rats after cyclophosphamide-induced immunosuppression. (i) Rats receiving T cells between 10 and 18 days after treatment with cyclophosphamide showed an acute lymphoproliferative disease in the gut and lungs within 9 days after adoptive transfer and died. (ii) Passive transfer within the first 5 days after immunosuppressive treatment resulted in typical Borna disease associated with neurological symptoms such as ataxia and paresis starting 14 to 16 days after transfer. Immunohistological analysis of the brains of rats with Borna disease uniformly revealed the presence of CD8+ T cells in encephalitic lesions in addition to CD4+ cells that were found in the brains of recipients of the virus-specific CD4+ T-cell line, irrespective of whether neurological symptoms developed or not. However, recipient rats treated with antibodies against CD8+ T cells developed neither encephalitis nor disease. Therefore, CD4+ T cells appear to accumulate in the brain and cause perivascular inflammatory lesions which alone obviously do not cause disease. In contrast, the presence of CD8+ cells apparently directly correlates with the development of neurological symptoms.     

Transformation of T-lymphocyte subsets by Marek''s disease herpesvirus.

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.     

NK cells use perforin rather than granulysin for anticryptococcal activity

Cytotoxic lymphocytes have the capacity to kill microbes directly; however, the mechanisms involved are poorly understood. Using Cryptococcus neoformans, which causes a potentially fatal fungal infection in HIV-infected patients, our previous studies showed that granulysin is necessary, while perforin is dispensable, for CD8 T lymphocyte fungal killing. By contrast, the mechanisms by which NK cells exert their antimicrobial activity are not clear, and in particular, the contribution of granulysin and perforin to NK-mediated antifungal activity is unknown. Primary human NK cells and a human NK cell line YT were found to constitutively express granulysin and perforin, and possessed anticryptococcal activity, in contrast to CD8 T lymphocytes, which required stimulation. When granulysin protein and mRNA were blocked by granulysin small interfering RNA, the NK cell-mediated antifungal effect was not affected in contrast to the abrogated activity observed in CD8 T lymphocytes. However, when perforin was inhibited by concanamycin A, and silenced using hairpin small interfering RNA, the anticryptococcal activities of NK cells were abrogated. Furthermore, when granulysin and perforin were both inhibited, the anticryptococcal activities of the NK cells were not reduced further than by silencing perforin alone. These results indicate that the antifungal activity is constitutively expressed in NK cells in contrast to CD8 T lymphocytes, in which it requires prior activation, and perforin, but not granulysin, plays the dominant role in NK cell anticryptococcal activity, in contrast to CD8 T lymphocytes, in which granulysin, but not perforin, plays the dominant role in anticryptococcal activity.     

Systemic and local characterization of regulatory T cells in a chronic fungal infection in humans

The long-term persistence of pathogens in a host is a hallmark of certain infectious diseases, including schistosomiasis, leishmaniasis, and paracoccidioidomycosis (PCM). Natural regulatory T (Treg) cells are involved in control of the immune responses, including response to pathogens. Because CTLA-4 is constitutively expressed in Treg cells and it acts as a negative regulator of T cell activation in patients with PCM, here we investigated the involvement of Treg cells in the control of systemic and local immune response in patients with PCM. We found that the leukocyte subsets were similar in patients and controls, except for CD11c+CD1a+ cells. However, a higher frequency of CD4+CD25+ T cells expressing CTLA-4, glucorticoid-inducible TNFR, membrane-bound TGF-beta, and forkhead-box 3 were observed in PBMC of patients. In accordance, these cells exhibited stronger suppressive activity when compared with those from controls (94.0 vs 67.5% of inhibition of allogeneic T cell proliferation). In addition, the data showed that CD4+CD25+ T cells expressing CTLA-4+, glucocorticoid-inducible TNFR positive, CD103+, CD45RO+, membrane-bound TGF-beta, forkhead-box 3 positive, and the chemokines receptors CCR4 and CCR5 accumulate in the Paracoccidioides brasiliensis-induced lesions. Indeed, the secreted CCL17 and CCL22, both associated with the migration of Treg cells to peripheral tissues, were also detected in the biopsies. Moreover, the CD4+CD25+ T cell derived from lesions, most of them TGF-beta+, also exhibited functional activity in vitro. Altogether, these data provide the first evidence that Treg cells play a role in controlling local and systemic immune response in patients with a fungal-induced granulomatous disease advancing our understanding about the immune regulation in human chronic diseases.     

Highly enhanced expression of CD70 on human T-lymphotropic virus type 1-carrying T-cell lines and adult T-cell leukemia cells

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4(+) T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4(+) T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.     

Virus and Autoantigen-Specific CD4+ T Cells Are Key Effectors in a SCID Mouse Model of EBV-Associated Post-Transplant Lymphoproliferative Disorders

Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies.     

Heparan sulfate proteoglycans mediate attachment and entry of human T-cell leukemia virus type 1 virions into CD4+ T cells

Heparan sulfate proteoglycans (HSPGs) are used by a number of viruses to facilitate entry into host cells. For the retrovirus human T-cell leukemia virus type 1 (HTLV-1), it has recently been reported that HSPGs are critical for efficient binding of soluble HTLV-1 SU and the entry of HTLV pseudotyped viruses into non-T cells. However, the primary in vivo targets of HTLV-1, CD4(+) T cells, have been reported to express low or undetectable levels of HSPGs. For this study, we reexamined the expression of HSPGs in CD4(+) T cells and examined their role in HTLV-1 attachment and entry. We observed that while quiescent primary CD4(+) T cells do not express detectable levels of HSPGs, HSPGs are expressed on primary CD4(+) T cells following immune activation. Enzymatic modification of HSPGs on the surfaces of either established CD4(+) T-cell lines or primary CD4(+) T cells dramatically reduced the binding of both soluble HTLV-1 SU and HTLV-1 virions. HSPGs also affected the efficiency of HTLV-1 entry, since blocking the interaction with HSPGs markedly reduced both the internalization of HTLV-1 virions and the titer of HTLV-1 pseudotyped viral infection in CD4(+) T cells. Thus, HSPGs play a critical role in the binding and entry of HTLV-1 into CD4(+) T cells.