Recognition of human T cell leukemia virus type I (HTLV-I) gag and pX gene products by MHC-restricted cytotoxic T lymphocytes induced in rats against syngeneic HTLV-I-infected cells

We established rat T cell lines expressing human T cell leukemia virus type I (HTLV-I) Ag from inbred strains of rats, WKA/H, DA, and F344, to study CTL response against the HTLV-I-infected cells. HTLV-I-specific Ag expressed in these rat cells were HTLV-I gag Ag, p19, p24, and p15, and pX Ag, p40tax and p27rex, but not env Ag, as determined by immunofluorescence and immunoblot assays. By immunization of rats with syngeneic HTLV-I-infected cells, CTL against syngeneic HTLV-I-infected cells and antibodies to HTLV-I Ag were generated in WKA/H and DA rats. The bulk CTL cultures from WKA/H and DA rats lysed specifically syngeneic SV40-transformed kidney cells infected with recombinant vaccinia viruses (RVV) expressing HTLV-I gag and pX Ag, but not those infected with RVV expressing HTLV-I env Ag or a control vaccinia virus. From WKA/H rat CTL cultures, four CTL clones reactive with syngeneic HTLV-I-infected cells were isolated, three of which were specific for p27rex/p21x, but the Ag recognized by the other CTL clone was not defined with any RVV used. These results indicate that HTLV-I gag and pX gene products are recognized by MHC-restricted rat CTL specific for syngeneic HTLV-I-infected cells.     

Functional alterations of herpes simplex virus-specific CD4+ multifunctional T cell clones following infection with human T lymphotropic virus type I

In an attempt to understand the mechanisms of immunodeficiency induced by human T lymphotropic virus type I (HTLV-I), HSV-specific CD4+ human multifunctional T cell clones were infected with HTLV-I in vitro. Early after HTLV-I infection, when their growth was still IL-2-dependent, clones were found to have almost completely lost their cytotoxic activity. At that time, their HSV-Ag-induced proliferative response and helper function for anti-HSV antibody production by B cells were only partially impaired. After this initial phase, the HTLV-I-infected clone became IL-2-independent, and the helper function was also completely lost. IL-2-dependent HTLV-I-infected clones showed degrees of proliferative response and elevation of intracellular free Ca2+ concentration induced by anti-CD3 mAb equivalent to those of HTLV-I-uninfected clones. On the other hand, during the IL-2-independent stage, expression of CD3-TCR complex on the cell surface was markedly decreased, and no significant elevation of intracellular free Ca2+ concentration was detected in response to anti-CD3 mAb. These data indicated that the loss of cytotoxic activity of HSV-specific T cell clones observed early after HTLV-I infection was not the result of impaired antigen recognition via the CD3-TCR complex, but might be due to dysfunction in the effector phase. On the other hand, the dysfunction of helper activity found late after HTLV-I infection might have mainly occurred in the recognition phase due to the decreased expression of CD3-TCR complex. The present data appear to suggest certain aspects of the pathogenesis of the immunodeficiency occurring in HTLV-I infection.     

Infection of human natural killer (NK) cells with replication-defective human T cell leukemia virus type I provirus. Increased proliferative capacity and prolonged survival of functionally competent NK cells.

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of human cell types, but only T lymphocytes are efficiently immortalized after HTLV-I infection. This study reports an attempt to infect and to immortalize NK cells with HTLV-I. Co-cultivation of freshly isolated NK cells with a HTLV-I-producing T cell line did not result in NK cell infection. However, NK cells activated with an anti-CD16 mAb and co-cultivated with a HTLV-I-producing T cell line were reproducibly infected by HTLV-I. HTLV-I infection was documented in NK cell lines and clones by the detection of defective integrated provirus by both Southern blot and polymerase chain reaction analysis. Although HTLV-I-infected NK cells produced viral proteins, they did not produce infectious viral particles. HTLV-I-infected NK cells were phenotypically indistinguishable from their uninfected counterparts (CD16+, CD2+, CD56+, CD3-). They also retained the ability to mediate both natural and antibody-dependent cell cytotoxicity. The IL-2-dependent proliferation of HTLV-I-infected NK cells was significantly greater than that of uninfected NK cells. The doubling time of this infected population was reduced from 9 days to 3 days, and the overall survival of the culture in the absence of restimulation was extended from 5 wk to 18 wk. Unlike T lymphocytes, HTLV-I-infected NK cells were not immortal, implying a fundamental difference between these two lymphocyte populations.     

Production of phagocytosis-inducing factor and expression of 4B4 antigen by cloned human T cells before and after transformation with HTLV-I

The characteristics of 4 T-cell clones, each capable of producing phagocytosis-inducing factor (PIF), were compared before and after transformation with human T-lymphotropic virus Type 1 (HTLV-I). Before transformation, the four clones produced PIF transiently after stimulation with antigen or mitogen and expressed the phenotype T3(CD3)+, T4(CD4)+, T8(CD8)-, 4B4+, and 2H4-; the three clones that could be studied also expressed the OKT17 marker. After transformation, the cells expressed the same phenotypic markers, except for two clones that lost the CD3 antigen. The clones that were available for study before and after transformation also expressed the antigen detected by the monoclonal antibody 5/9. In addition, all clones secreted PIF constitutively after transformation. These characteristics of the four transformed T-cell clones closely resembled those of three long-term HTLV-I-transformed T-cell lines, HUT-102, C5/MJ, and MT-2, which also produced PIF constitutively and expressed the CD4 and 4B4, but not 2H4, markers. In addition, two other HTLV-I-transformed lines generated in the present study produced PIF constitutively. Since all nine HTLV-I transformed cell lines and all four untransformed clones secreted PIF, and since our previous studies have shown that only approximately 20% of CD4+ peripheral blood lymphocytes secrete PIF, these results suggest that HTLV-I may preferentially transform PIF-secreting CD4+ lymphocytes. The predominant 4B4+, 5/9+, 2H4- phenotype (characteristic of antigen-responsive T cells) of the untransformed and transformed clones as well as the long-term HTLV-I-transformed lines also suggests that the subset of CD4+ lymphocytes that proliferates in response to soluble antigen may be especially susceptible to transformation with this virus.     

Human T lymphotropic virus I infection deregulates surface expression of the transferrin receptor

Human T-lymphotropic virus I (HTLV-I) is an etiologic agent in adult T cell leukemia. In an effort to understand the relationship between HTLV-I infection and malignant transformation, we have examined transferrin receptor expression in HTLV-I-infected cells. Transferrin receptor expression in normal T cells is tightly regulated and essential for cell proliferation. We have used matched T cell sets originating from a normal donor, consisting of tetanus toxoid-specific normal T cell clones (TM3 and TM5) and their in vitro HTLV-I-infected counterparts (TM3H and TM5H). Using these matched sets of virus-infected and normal T cells, we have determined that HTLV-I infection leads to hyperexpression of surface transferrin receptors (five- to six-fold higher than normal counterparts). Although the growth rates of the virus-infected cells did not differ significantly from their normal controls, HTLV-I-infected cells constitutively hyperexpressed surface transferrin receptors, whereas the level of surface receptor expression of normal counterpart cells varied during the cycle of antigenic stimulation. Immunoprecipitation of total (surface plus cytoplasmic) transferrin expression showed that the HTLV-I-infected cells did not possess a greater total number of transferrin receptors than their normal counterparts. This data was supported by Northern blot analysis, which showed equivalent transferrin receptor mRNA expression in HTLV-I-infected and uninfected cells. Functional analysis revealed a marked defect in 59Fe-transferrin internalization in the HTLV-I-infected cells. Furthermore, the HTLV-I-infected cells showed markedly decreased transferrin receptor phosphorylation and internalization in response to active phorbol ester. Thus the data demonstrate that in peripheral blood T cells, HTLV-I infection is accompanied by surface transferrin receptor overexpression secondary to subcellular redistribution and defective internalization.     

Model for studying virus attachment. II. Binding of biotinylated human T cell leukemia virus type I to human blood mononuclear cells potential targets for human T cell leukemia virus type I infection.

Purified human T cell leukemia virus type I (HTLV-I) was biotinylated and used to study its attachment to human PBMC. The use of biotinylated HTLV-I (biot-HTLV-I) in conjunction with mouse mAb specific for selected cell-surface molecules and flow cytometric analysis allowed us to positively identify virus-binding cells among a heterogeneous blood mononuclear cell population. Biot-HTLV-I efficiently bound not only to T cells, but also to B cells and monocytes. Preincubation of monocytes with excess of unlabeled HTLV-I significantly reduced the attachment of biot-HTLV-I. HTLV-I not only bound to, but also infected, B cells, as suggested by: i) in situ hybridization of a 35S-labeled full length HTLV-I DNA probe with EBV-transformed B cells, previously cocultured with HTLV-I-producing (G11MJ) T cells, and ii) hybridization of the same nick-translated 32P-labeled DNA probe with blotted DNA from similar HTLV-I-infected EBV-transformed B cells. HTLV-I infection did not affect the ability of B cells to secrete IgG. These findings suggest that HTLV-I cannot only infect cells of the T lineage, but can also infect B cells.     

Human T lymphotrophic virus type I may not be associated with multiple sclerosis in Japan.

To study the possible involvement of human T lymphotrophic virus type I (HTLV-I) or a related retrovirus in Japanese cases of multiple sclerosis (MS), we first performed a Western blot analysis with purified Ag of HTLV-I. Ten out of 31 MS patients (32.2%), 19 of 66 patients (28.8%) with other neurologic diseases, and 2 of 64 healthy blood donors (3.1%) had antibodies reactive with Ag corresponding to the group-specific Ag (gag) proteins (p15, p19, p24) on their sera. There were no significant differences between MS and other neurologic diseases concerning the patterns and the frequency. Second, we tried to establish T cell lines from PBMC of 22 MS patients with crude IL-2 without accessory cells, because HTLV-I-infected T cells can be immortalized in a high ratio under those conditions. Only one T cell line (MS-14C), however, could be maintained in long term culture. MS-14C and cultured T cells for 3 to 5 wk derived from MS patients were examined by Southern blot analysis under both stringent and low stringent conditions with HTLV-I as a probe. No HTLV-I related bands could be detected. By polymerase chain reaction examination, we also could not detect HTLV-I provirus genome in the fresh PBMC from 20 MS patients, although some of them had gag-reactive antibodies. Our data do not favor the hypothesis of HTLV-I or an HTLV-I-related human retrovirus in the etiology of MS.     

In vitro generated human monoclonal trinitrophenyl-specific B cell lines. Evidence that human and murine anti-trinitrophenyl monoclonal antibodies cross-react with Escherichia coli beta-galactosidase

Stable human antigen-specific monoclonal B cell lines were established without prior in vivo immunization. This was accomplished by expanding the anti-trinitrophenyl (TNP) B cells in vitro with the antigen TNP-Brucella abortus and then immortalizing them with Epstein-Barr virus. Five anti-TNP clones were selected by sequential limiting dilution. All five anti-TNP clones secreted IgM kappa antibodies. When tested against a panel of self and environmental antigens, all five anti-TNP clones exhibited cross-reactivity with an Escherichia coli-derived beta-galactosidase. To determine whether this was a more general phenomenon, a panel of murine monoclonals were tested and found to bind to beta-galactosidase. It is therefore possible that human and murine anti-TNP beta cell responses reflect reactivity against an environmental antigen, namely an epitope present on E. coli-derived beta-galactosidase. This approach of expanding human antigen-specific B cells by antigen stimulation in vitro, with a T-independent hapten-carrier conjugate before Epstein-Barr virus transformation, may prove useful in the development of human monoclonals for therapeutic purposes.     

Construction of a full-length human T cell leukemia virus type I genome from MT-2 cells containing multiple defective proviruses using overlapping polymerase chain reaction

Human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia, integrates into the host genome as a provirus. Multiple defective copies of the integrated provirus are often present in the host genome. For this reason it is difficult to clone the intact provirus from HTLV-I-infected cells using conventional techniques. Here, we used overlapping polymerase chain reaction (PCR) to construct a full-length provirus of HTLV-I directly from an HTLV-I-transformed cell line, MT-2, which contains multiple defective proviruses. First, four overlapping proviral HTLV-I fragments (1.4-3.9 kb each) were constructed from genomic MT-2 DNA using PCR. Next, the complete HTLV-I proviral DNA (9 kb) was generated from these fragments using asymmetric PCR and cloned into a plasmid vector. 293 T cells transfected with this plasmid produced virus-like particles, and we show that these particles are capable of infecting a human T cell line. We propose that this cloning technique constitutes a powerful tool for constructing infectious molecular clones from cells of patients infected with HTLV-I or other viruses.     

Human T cell leukemia virus type I induces DNA synthesis and immunoglobulin secretion in human lymphocyte cultures

Viral particles obtained from HTLV-I (human T cell leukemia virus, type I)-transformed T cell lines induced immunoglobulin production by normal peripheral blood lymphocytes. Conversely, no immunoglobulin could be detected in the supernatant medium in purified B cells cultivated with HTLV-I, suggesting that the presence of T cells is mandatory for HTLV-I to induce B cell polyclonal activation. The T cell help was mediated by soluble factors, as indicated in experiments showing that cell-free conditioned medium from T lymphocytes activated by HTLV-I was able to induce B cell proliferation and differentiation. Furthermore, a direct effect of HTLV-I on B cell proliferation was demonstrated when viral particles were added to purified B cells together with suboptimal doses of Staphylococcus aureus Cowan strain I (SAC). These observations show that an immediate early effect of HTLV-I infection was exerted on B cells, mainly in a T cell-dependent manner. Such an effect may account for the hypergammaglobulinemia observed in HTLV-I seropositive individuals, and in patients with HTLV-I-associated neurological disorders.     

Regulation of IgA secretion by T cell clones derived from the human gastrointestinal tract

The role of T cells in Ig isotype regulation is still unclear. To address this question, we generated mitogen-stimulated T cell clones from normal human lymphoid follicles of the gut-associated lymphoid tissue (appendix). Both the T cell clones and clonal supernatants provided preferential help for IgA secretion by PWM-stimulated B cells. Many of these CD3+, CD4+, 4B4+, DR+ helper clones co-expressed Fc-gamma and Fc-alpha R, but there was poor correlation between the expression of Fc-alpha R and IgA help (p = 0.31). Most of the T cell clones helped both IgM+A- and IgM-A+ B cell populations to secrete IgA, suggesting that they mediate switch of isotype-uncommitted B cells as well as post-switch expansion of IgA-committed B cells; however, some of the T cell clones helped IgM+A- B cell populations much more than IgM-A+ B cell populations, suggesting that, in this case, the regulatory effect is predominantly at the level of B cell switch. In all, these results show that the mucosal immune system contains individual T cells which are capable of positively regulating IgA-specific isotype differentiation at two levels of B cell development, thus allowing for efficient generation of IgA-secreting B cells.     

Human T lymphotropic virus type-I (HTLV-I) immortalizes human T cells in vitro--its implication in the pathogenesis of adult T cell leukemia (ATL).

HTLV-I is the first human retrovirus that was isolated from a patient with T-cell malignancy in 1980 in the United States. HTLV-I is detected in most patients with adult T cell leukemia (ATL) and healthy carriers, who are frequently found in the southwestern parts of Kyushu and Shikoku Districts. HTLV-I-infected cells express IL-2 receptors, and HTLV-I-infected T cell lines can be established from most of ATL patients in culture in the presence of IL-2. Furthermore, these IL-2 dependent T cell lines often begin to proliferate in the absence of IL-2 and to not respond to IL-2, despite IL-2 receptors on their cell surface, thus mimicking ATL cells in vivo. These findings suggest that HTLV-I is an etiological agent of ATL. In this mini-review, the T cell immortalizing activity of HTLV-I in vitro, with special reference to the evolution of ATL cells based on our results, is described.     

Expression of parathyroid hormone-related protein in a human T cell lymphotrophic virus type I-infected T cell line

We analyzed human T cell lymphotrophic virus type I (HTLV-I)-infected T cells for the presence of mRNA coding for parathyroid hormone-related protein (PTHrP) by Northern blotting using synthetic DNA probes. We report here that PTHrP mRNAs were detected in a HTLV-I-infected T cell line, MT-2, but not in uninfected T cell or B cell lines, and that PTH-like bioactivity was detected only in the conditioned medium of MT-2 cells. Our study suggests that the pathophysiology of hypercalcemia in patients with adult T cell leukemia/lymphoma may resemble that which occurs with solid tumors.     

Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells

The effect of human T cell leukemia/lymphoma virus type I (HTLV-I) infection on the function and the phenotype of a human proliferating/cytotoxic T cell clone, specific for tetanus toxin, was investigated. During the period after infection, two distinct phases were observed, based on growth properties, phenotype, and functional activity of the infected cells. Phase I HTLV-I infected cells (0 to about 150 days after infection) proliferated in an IL-2-dependent way, but without the requirement for repetitive antigenic stimulation. No differences in expression of the CD2, CD3, CD4, Tp103, and CD28 Ag between these cells and the parental cells could be demonstrated, with the exception of the expression of IL-R p55 and HLA-DR Ag, which were constitutively expressed on the phase I cells. The phase I HTLV-I-infected cells, as well as the parental 827 cells reacted with a mAb specific for an epitope on the variable part of the TCR beta-chain, indicating that the TCR was not altered after HTLV-I infection. Like the parental clone, the phase I cells proliferated in response to tetanus toxin, but the tetanus toxin-specific response of the phase I cells did not require the presence of APC. Results of experiments, in which the levels of intracellular Ca2+ were measured, indicated that HTLV-I cells can acquire the capability to process Ag and present that to themselves. Phase I HTLV-I-infected T cells had lost their cytotoxic activity which was likely to be due to an effect on the lytic machinery rather than on Ag recognition by the TCR, inasmuch as it was found that phase I HTLV-I-infected T cells did no longer contain N-alpha-benzyloxy-L-lysine thiobenzylester-serine esterase activity. Furthermore, it was found that phase I HTLV-I-infected T cells had a diminished capacity to form conjugates with target cells. From a period of about 200 days after HTLV-I infection, phase II cells emerged that proliferated strongly in the absence of IL-2 and that had lost all functional activity. These cells did not express the CD3/T cell receptor complex on their surface. Phase I as well as phase II HTLV-I-infected cells were targets for CTL raised in the autologous donor.