TSP

JOURNAL

OF THE

NEUROLOGICAL SCIENCES

ELSEVIER

Journal of the Neurological Sciences 130 (1995) 183-189

Characterization of humoral and cellular immunity in the central nervous system of HAM/TSP Makoto Matsui a,*, Fumio Nagumo b, Jutaro Tadano b, Yasuo Kuroda a a Division of Neurology, Department of Internal Medicine, Saga Medical School, Nabeshima, Saga 849, Japan b Department of Laboratory Medicine, Saga Medical School, Saga, Japan

Received 16 September 1994; revised 10 January 1995; accepted 21 January 1995

Abstract In HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP) immunopathological processes in the central nervous system (CNS) have not been clarified. We compared the humoral and cellular immunity within the CNS and in the systemic circulation of 24 patients with HAM/TSP (8 men and 16 women) to 6 asymptomatic HTLV-I carriers, 7 patients with active multiple sclerosis, 6 patients with acute viral encephalitis, and 39 patients with other non-inflammatory neurological diseases. Significant differences were observed between the HAM/TSP patients and one or more of the control groups: HAM/TSP cerebrospinal fluids (CSF) exhibited higher levels of IgG, IgG index, de novo IgG synthesis rate, and P,-microglobulin, and also a predominance of CD8+ cells that expressed CDlla and CD45RO but lacked CD28 antigens. Results in the 6 patients with acute viral encephalitis suggested that the CD8+ population in the CSF which is positive for CD28 and CD45RO is important for the elimination of virus from infected CNS tissues. Therefore, potentially cytotoxic T cells of a unique CD8+CDlla+CD45RO+CD28phenotype may play a key role in the CNS pathogenesis of HAM/TSP. Keywords:

HAM/TSP;

Viral encephalitis;

Cerebrospinal

fluid; Cytotoxic T lymphocyte;

1. Introduction

Tropical spastic paraparesis (TSP) (Gessain et al., 19851, also known as HTLV-I-associated myelopathy (HAM) (Osame et al., 19861, is a disease entity of chronic myelopathy associated with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of this disorder remains unclear, both humoral and cellular immune perturbations are present in the peripheral blood of patients with HAM/TSP. For example, polyclonal B cell activation (Mori et al., 1988) and an increase in activated T lymphocytes that express activation-related antigen CD25 (Itoyama et al., 1989) and/or HLA-DR (Mori et al., 1988; Jacobson et al., 1988) have been observed in the blood of these patients. These findings appear to indicate aberrant immunoregulation in helper T cells that are CDCpositive (Mori et al., 1988; Itoyama et al., 1989). However, in

* Corresponding author. Tel.: (office) 81-952-31-6511(ext. 2372); Fax: (office) 81-952-32-3026or 30-6629. 0022-510X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0022-510X(95)00036-4

CD28; &-Microglobulin

autopsy studies cellular infiltrates in the central nervous system (CNS) of some HAM/TSP patients have been shown to be predominantly CD8-positive (Moore et al., 1989; Bhigjee et al., 1990). Further, we have observed elevated levels of cytokines interferon-gamma (IFN-7) (Kuroda and Matsui, 1993), interleukin-1 (IL11, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Kuroda et al., 1993) in the CSF, but not in the sera of HAM/TSP patients, indicating the presence of immune activation in the CNS of HAM/TSP. Against this background we performed this study to examine humoral and cellular immunity in the CNS of HAM/TSP patients, in which immunity may differ from that observed in the peripheral blood; this difference may be related to the pathogenesis of HAM/TSP. Employing a flow cytometry system for the detection of surface activation- and function-related antigens on CSF lymphocytes and a turbidimetric immunoassay for IgG and P,-microglobulin levels in the CSF, we first compared the results in groups of patients with HAM/TSP, active multiple sclerosis (MS), and other non-inflammatory neurological diseases (OND). We

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then attempted to define the lymphocyte subsets that may be essential for coping with viral infection in the CNS, by investigating CSF cells in a more aggressive viral infection, acute encephalitis. Finally, the findings for peripheral blood lymphocytes were compared in the HAM/TSP, OND, and asymptomatic HTLV-I carrier groups to determine whether the findings in the CSF of HAM/TSP patients merely reflected the immunological state outside the CNS or whether they represented a CNS-specific pathological process occurring in this disorder. 2. Materials

and methods

Subjects

Twenty-four patients with HAM/TSP (8 men and 16 women, aged 33-72 years; mean, 56.0 years) were studied. The duration of the illness was estimated to be between 10 months and 28 years (mean, 9.1 years). No patient was receiving corticosteroid or immunomodulatory therapy at the time of the study. The criteria we used for the diagnosis of HAM/TSP were essentially those proposed previously (Kuroda et al., 1991), with some modifications. The criteria were: (1) slowly progressive spastic paraparesis with symmetrical hyperreflexia and positive Babinski’s sign, (2) at least slight urinary disturbance or functional disturbances of the detrusor muscles determined by urodynamic studies, (3) no radiological evidence of significant spinal cord compression, and (4) the presence of antibodies to HTLV-I antigens in both serum and the CSF, as detected by a Western blot technique. Antibody titers in the sera were between X 1,024 and X 65,536, while those in the CSF were between x32 and ~2,048 (particle agglutination method). The CSF and the blood were studied simultaneously in 16 of the patients with HAM/TSP; only blood was analyzed in the remaining 8 patients. As HTLV-I-seropositive controls, we included six seropositive women, aged 49-71 years (mean, 60.0), who were negative for anti-HTLV-I antibody in the CSF; these women had various diseases, spinocerebellar degeneration, myotonic dystrophy, cervical spondylosis, remote cerebrovascular disease, neuro-sarcoidosis, and uterine cancer. The antibody titers in the sera were between x64 and X2,048. Both blood and CSF were examined in all these patients; however, CSF cells were present sufficiently for analyses in four patients. As HTLV-I-seronegative controls, we studied 39 patients with other non-inflammatory neurological diseases (OND group). This group consisted of 20 men and 19 women, aged 16-76 years (mean 46.1). They had degenerative disorders of the CNS, e.g., Parkinson’s disease and spinocerebellar degeneration (n = S), remote cerebrovascular disease (n = 6), motor

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neuron disease (n = 6), hysteria (n = 4), myopathy/ muscular dystrophy (n = 4), epilepsy (n = 2), noninflammatory peripheral neuropathy (n = 2), neurogenic muscle atrophy of unknown etiology (n = 3), and other neuropsychological diseases, i.e., cervical spondylosis (n = l), cerebrotendinous xanthomatosis (n = l), dural arteriovenous malformation (n = l), and anorexia nervosa (n = 1). Simultaneous analyses of blood and CSF were performed in 22 subjects in this group; either blood or CSF was examined in the remaining 17. To examine inflammatory diseases in the CNS, we studied 7 patients with multiple sclerosis (MS) and 6 patients with acute viral encephalitis. The 7 MS patients (all female, aged 28-51 years; mean 36.7) were categorized as clinically or laboratory-supported definite MS in accordance with Poser’s criteria (Poser et al., 1983). CSF samples obtained by lumbar puncture at 8 acute relapses of the disease (two separate occasions in one patient) were examined. No patient was receiving corticosteroid or immunosuppressive therapy at the time of study. For the 6 patients with acute viral encephalitis (5 men and 1 woman, mean age 25.5 years), CSF cells were examined in both the acute and convalescent stages of the illness. The relevant viruses were determined as cytomegalovirus, herpes simplex, rubella, and measles viruses in four patients, respectively, and the viruses were undetermined in the remaining two patients. Methods Phenotyping

of cells: Mononuclear cells (MNC) in freshly collected CSF were obtained by low-speed spinning at 4” C, as described previously (Matsui et al., 1990). For inpatients, venous blood was drawn into heparinized and non-heparinized syringes on the same day as atraumatic lumbar puncture was carried out. Only blood was obtained from outpatients. Blood MNC were separated by Ficoll-Paque density-gradient centrifugation. The cells were finally resuspended in RPMI-1640 media with 10% newborn calf serum for staining. The MNC were single-stained with fluorescein (FITC)-conjugated CD3 (OKT3, Ortho Diagnostic Systems, Raritan, NJ), CD20 (OISB20, Ortho), or phycoerythrin (PE)-conjugated CD56 (NKH-1, Coulter Immunology, Hialeah, FL) monoclonal antibodies (MoAb). The cells were simultaneously double-stained with combinations of MoAb; FITC-conjugated CD4 (T4, Coulter) with PE-conjugated CD25 (Cantrell and Smith, 1983) (anti-IL-2R, Becton Dickinson Immunocytometry Systems, San Jose, CA) or CD26 (Fox et al., 1984) (Tal, Coulter). Other combinations of MoAb were also used; PE-conjugated CD4 (Leu3a) or CD8 (Leu2a, Becton Dickinson) with FITC-conjugated CD28 (Damle et al., 1983) (Kolt-2, Nichirei, Tokyo) or

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CD45RO (Beverley, 1986/87) (UCHL-1, Nichirei). A commercially available double-staining reagent T8/S6Fl (Coulter) was used to define CDlla (Morimoto et al., 1987) antigens on CD&positive cells. The MNC were stained on ice for 30 minutes, washed, and then analyzed with a single-laser flow cytometry system (Ortho Spectrum III or Cytron Absolute, Ortho). At least 500 cells/MoAb from the CSF and more than 5,000 cells/MoAb from the blood passed through the instrument. Monocytes and macrophages were gated out, and the cells in the lymphocyte compartment were analyzed for positive fluorescence, as described previously (Matsui et al., 1990). Other immunological parameters: IgG and &-microglobulin concentrations in the CSF were measured by turbidimetric immunoassay (Iatroace IgG, Iatron Laboratories, Tokyo) and with a latex agglutination turbidimetric immunoassay system (LPIA-Ace &m, Iatron), respectively. For evaluating the CSF IgG index (given by the formula: IgG,,,/IgG,,,,, divided by Albumin ,,,/Albumin Serum) and de novo IgG synthesis rate within the CNS, which is given by the formula: [IgG,,, - IgG,,,,,(0.00084 + 0.43 x AIb,,/AIb,,,,Jl x 5, CSF albumin level was measured by radioimmunoassay, and serum albumin and IgG levels were measured by conventional methods. Statistical analyses. Statistical differences in the results among more than two groups were tested by one-factor analysis of variance, and then by Fisher’s protected least significant difference test (PLSD) for Table 1 Humoral factors and percentages of activationand function-related MS patients. All values are expressed as means+ SD. Differences suppressor, NK = natural killer, CTL = cytotoxic, T = lymphocyte. HAM/TSP IgG (mg/dl) IgG Index De novo IgG synthesis (mg/day) P,-Microglobulin (mg/l) CD3+ (%) CD4+ (%) CD8+ (%l CD20+ (%o) CD56+ (%I CD4 + CD25 + (o/o) CD4 + CD26 + (%) CD4+CD45RO+ (%) CD4+CD45RO(%o) CD8+CDlla+ (%l CD8 + CD28 + f%) GD8 + CD45RO + (%l CDS+GDlla(%) CD8 + CD28 - (%l CD8 + CD45RO - (o/o) a Significantly sclerosis (MS) b Significantly

different group. different

(p < 0.01) compared

with

the group

(p < 0.01) compared

with

both

of other

MS and OND

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185

comparison of any two groups. Differences in percentages of lymphocyte subsets between acute and convalescent stages of viral encephalitis were analyzed by paired t and Wilcoxon signed-rank tests; those between the CSF and the peripheral blood of HAM/TSP patients were analyzed by the Student t and MannWhitney U tests. Correlation was tested by Pearson’s correlation coefficient and Spearman’s correlation coefficient analyses. 3. Results

As shown in Table 1, CSF IgG, the IgG index, de novo IgG synthesis, and &-microglobulin levels were significantly elevated in patients with HAM/TSP, compared to the group of patients with other non-inflammatory neurological diseases (OND group). Titers of antibodies to HTLV-I in the CSF of HAM/TSP were significantly correlated with all of the above four parameters, but were not so with HTLV-I titers in the sera. The numbers of CSF mononuclear cells (mean k standard deviation) in the HAM/TSP, MS, and OND groups were 4.1 f 4.8, 3.0 f 2.6 (excluding one extreme result of 901, and 1.7 k 1.2/mm3, respectively. There was no correlation between the above four parameters in the CSF and numbers of CSF cells in any group of patients. Of these parameters the IgG index and de novo IgG synthesis showed a significant inverse correlation with duration of the illness (p < 0.051, whereas

antigen-positive between groups (n = 24)

7.57 f 3.69 b 0.953 + 0.613 a 8.81 f 14.49 a 2.98 + 1.45 = 88.8 * 8.8 a 45.9 f 8.6 b 44.4 + 9.3 b 0.9+ 1.2 2.5 + 2.4 l.O+ 1.6 15.1+7.1 a 28.8 f 10.5 15.9* 10.3 43.8 f 7.3 b 7.9 + 9.5 30.6 f 11.8 b 2.5 f 2.0 37.1+ 13.8 b 15.5 * 7.9

T cell Th Ts/CTL I3 cell NK cell activated memory primed Th naive Th CTL CTL CTL

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neurological groups.

lymphocytes in the cerebrospinal fluid of HAM/TSP were analyzed by Fisher’s PLSD. Th = T helper, Multiple sclerosis

(n = 71

4.23 + 1.80 0.677 f 0.306 0.54 f 7.95 1.47 f 0.60 77.4 + 8.9 64.2 f 10.9 17.9 + 5.5 4.1* 4.3 2.8* 1.9 1.0* 1.0 21.8 f 6.9 38.6* 15.5 19.7 + 8.7 13.2+5.1 2.7+ 2.1 6.9 f 2.6 4.8 f 3.5 15.0 + 12.2 12.6 f 6.5 diseases

(OND)

but not different

and Ts = T

Other neurological diseases (n = 391 4.40 f 2.15 0.506 f 0.159 3.04 f 6.98 1.09+0.46 72.8 f 9.8 57.6 f 10.0 24.4 f 5.3 Ok0 3.Ok3.4 0.1+0.2 29.5 f 8.9 29.4 * 20.1 31.7+ 18.2 19.9 + 6.9 3.2 f 3.8 9.1 f 8.2 4.1 f 5.5 20.9 f 8.4 14.4 f 6.8 compared

with the multiple

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Table 2 Surface CDlla, CD28, and CD45RO antigen-positive CSF lymphocytes in 6 patients with acute viral encephalitis. p values were evaluated by the paired t and Wilcoxon signed-rank tests. Other values represent percentages of individual subsets, expressed as means f SD.

CD4+ (%) CD4 + CD26 + CD4+CD28+ CD4+CD45RO+ CD4+CD45ROCD8+ (%I CD8+CDlla+ CD8+CD28+ CD8 + CD45RO GD8+CDllaCD8+CD28CD8+CD45RO-

f%) (%) (%) (%) (%) f%) + (%) (%) (%) (%)

a Significantly different stage of the infection. NS = not significant.

Acute

Convalescent

p value

35.8k11.6 13.7k5.8 30.4 f 10.5 26.4klO.l 9.3k4.4 a 45.3k20.5 37.9 + 30.3 20.6 f 12.0 a 20.9* 16.9 7.2+ 11.3 29.7k23.1 27.3k21.1

51.5 f 15.8 20.0 f 9.9 26.4 f 18.1 22.0+ 12.1 25.7+ 13.4 39.1+ 15.2 25.8 f 9.2 5.3+5.5 7.1+ 5.6 9.3 f 9.0 32.2+ 15.1 34.2 f 13.6

NS NS NS NS 0.0353 NS NS 0.0068 0.0747 NS NS NS

compared

with

findings

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group (15.1% vs. 29.5%, respectively). About two-thirds of the CD8+ cells possessed both CDlla and CD45RO antigens, whereas only a minority ( < 20%) of the CD8+ cells expressed CD28. In contrast, the CD8+CD28population, which has been reported to contain suppressor cell activity (Lum et al., 19821, was significantly increased in the CSF of HAM/TSP compared with the MS and OND groups. These features in the CSF of HAM/TSP did not differ between patients with either early or late disease. The findings in CSF samples obtained from four asymptomatic HTLV-I carriers were comparable with those in the OND group in terms of IgG (mean 3.57 mg/dl), IgG index (mean 0.4451, de novo IgG synthesis (mean - 6.16 mg/day) and &-microglobulin (mean 1.02 mg/l) levels. The percentage of CD4+ cells (mean 57.0%) was also similar to that in the OND group. However, CD8+ CSF lymphocytes in this group (mean 31.3%) showed intermediate values between those of the HAM/TSP and OND groups; the mean percentages for CD8+CDlla+ and CD8+CD45RO+ subsets were 26.8% and 17.7%, respectively. The CD8+CD28cells did not increase in the CSF of HTLV-I carriers (mean 20.7%), whereas they definitely did in the CSF of HAM/TSP (mean 37.1%). It was important to clarify cytotoxic T lymphocyte (CTL) subsets relevant to immunity against viral infection in the CNS parenchyma. The findings in the 6 patients with acute viral encephalitis indicated that at the acute stage there was a significant increase in CD8+CD28+ cells and an upward trend in the percentage of CD8+CD45RO+ cells in association with an outnumbering of CD8+ lymphocytes in the CSF (Table 2). At the convalescent stage, the percentages of these populations returned to the levels seen in the

in the convalescent

elevation in CSF &-microglobulin levels persisted in patients with a disease duration of more than 10 years. A significant increase in CD8+ cells and a significant decrease in CD4+ cells were observed in the CSF of HAM/TSP patients (Table 1). Conversely, there was a significant decrease in CD8+ cells and a slight increase in CD4+ cells in the CSF of active MS patients, compared with the findings in the CSF of the OND group. Interleukin-2 (IL-2)-receptor (CD25)positive helper T cells were not significantly increased in the CSF of either the HAM/TSP or active MS patients. Another activation-related antigen, CD26, on the CSF CD4+ cells in the HAM/TSP group was down-regulated to half the levels seen in the OND

Table 3 Percentages of activation and functional antigen-positive lymphocytes in the peripheral blood of HAM/TSP patients and asymptomatic HTLV-I carriers. Percentages are expressed as means f SD. Differences between groups were analyzed by Fisher’s PLSD. Th = T helper, Ts = T suppressor, NK = natural killer, CTL = cytotoxic, T = lymphocyte. HAM/TSP CD3 + CD4 + CD8 + CD20 + CD56 + CD4 + CD25 + CD4 + CD26 + CD4 + CD45RO CD4 + CD45RO CD8+CDlla+ CD8 + CD28 + CD8 + CD45RO CD8+CDllaCD8 + CD28 CD8 + CD45RO a Significantly

+ -

+

T cell Th Ts/CTL B cell NK cell activated memory primed naive CTL CTL CTL

different

(p < 0.01) compared

HTLV-I (n = 6)

(n = 24)

68.7 f 12.4 45.5 + 7.1 26.2 f 12.4 11.7k8.6 12.9 k 7.0 4.3 + 2.1 = 18.2 f 6.5 26.0 + 7.0 a 19.4 f 5.9 19.3 + 13.8 a 6.8 f 3.0 a 10.3 f 5.8 a 3.7 + 2.5 = 19.9 f 12.8 16.1+9.1 with the group

of patients

carrier

63.0 f 9.8 43.3 f 8.9 23.8 + 6.0 11.5k5.0 17.9 f 7.8 1.3f0.5 16.9 f 7.4 17.5 f 6.9 25.8k9.6 9.7 + 3.8 10.9 + 4.6 5.9+3.3 7.5 55.1 13.9 f 3.9 17.1 f 7.2

69.6 f 4.3 47.3 rt 6.4 22.9 f 6.4 10.3 f 2.0 16.9 k 8.6 4.4 + 2.6 a 21.6+6.8 22.4 f 2.5 25.0 + 9.2 13.3 + 9.2 7.7 f 2.0 7.6 + 4.7 5.8k3.3 13.lk5.5 14.2 + 4.1 with

other

neurological

Other neurological diseases (n = 39)

diseases.

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group of patients with other non-inflammatory neurological diseases (OND). Table 3 shows the characteristics of the peripheral blood lymphocytes in the HAM/TSP, HTLV-I carrier, and OND groups. The CD8+CDlla+ and CD8+CD45RO+ cells, which were markedly increased in the CSF of HAM/TSP, showed a slight increase in the peripheral blood of HAM/TSP patients compared with the OND group. The percentages of both of these subsets in the blood of HAM/TSP patients were significantly lower than those in the CSF. No correlation in percentages of any of the subsets studied was observed between the blood and the CSF of HAM/TSP patients. These findings indicate that the cells appearing in the CSF of HAM/TSP patients were distinct from those in the blood. The HTLV-I carrier group did not show any significant differences compared with the HAM/TSP group.

4. Discussion This study showed that both humoral and cellular immunity in the CNS of patients with HAM/TSP were altered compared with findings in the group of patients with other non-inflammatory neurological diseases (OND); the findings indicated that the immunopathological process occurring in the CNS of HAM/TSP patients may be distinct from that in the systemic circulation, because the functional property and state of activation of lymphocytes differed in the two compartments. We have found that the CSF of HAM/TSP patients was characterized by a marked increase in CD8+ cells, which was not seen in any asymptomatic HTLV-I carriers and was opposite to the finding in the CSF of active MS patients; an increase in CD4+ cells and a decrease in CD8+ cells were observed in the CSF of active MS patients. MS is an inflammatory demyelinating disease of the CNS, in which CD4+ autoreactive T cells are thought to play a pivotal role (Wucherpfennig et al., 1991). In light of the previous finding that alterations in T cell subsets in active MS patients were detected only in CSF and not in blood (Matsui et al., 19881, we postulated that the CD8+ cells predominantly appearing in the CSF of HAM/TSP represented at least some of the effector cells that may play some role in the pathogenesis of this disease. Most of the CDS+ cells in the CSF of HAM/TSP expressed CDlla and CD45RO molecules but lacked CD28 antigen. Not only CDlla (Morimoto et al., 1987) and CD45RO (Merkenschlager and Beverley, 1989; Akbar et al., 1990) but also CD28 (Damle et al., 1983; Azuma et al., 1992) antigens have been shown to be associated with the cytotoxic T lymphocyte (CTL) activity rendered by CD8+ cells; such CTL play an important role in eliminating virus-infected cells (Zinkerna-

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187

gel and Doherty, 1979). Of these CTL subsets those expressing CD28 and CD45RO antigens appeared to be important for recovery from viral encephalitis in the present study. Indeed, CD8+CD28+ cells isolated from the blood of HAM/TSP patients were shown to inhibit HTLV-I specific antigen expression by cultured autologous blood lymphocytes (Eiraku et al., 1992). These findings can be interpreted as follows: (i) The CD8+ cells infiltrating the CNS of HAM/TSP may mainly consist of CTL specific for HTLV-I antigens (Jacobson et al., 1992). (ii) Those CD8+ cells that are not functionally potent CTL due to the lack of CD28 (Azuma et al., 1993) are, hence, outnumbered in the CSF/CNS as compensation. (iii) The CD8+CD28cells belong to the population that down-regulates immune reactions including antibody production by B cells (Lum et al., 1982). However, the last possibility is unlikely because we have observed elevated levels of IgG and IgG production in the CSF of HAM/TSP in the present study. Taking the first two possibilities together, even if the CD8+ cells predominating in the CSF are reactive with HTLV-I antigens, those CTL may fail to effectively remove HTLV-I-infected CNS tissues because of lack of CD28 antigen. This hypothesis accords with the finding that lymphohematopoietic or neural cells in the CNS may be persistently infected with HTLV-I, as detected by polymerase chain reaction (Bhigjee et al., 1990; Iannone et al., 1992; Kira et al., 1992). Meanwhile, CD28 molecule is a functional membrane protein on the cell surface, which is required for CDS+ cells to exert alloantigen-specific cytolysis (Damle et al., 1983). In another aspect, T cell activation requires a costimulatory signal through the CD28 molecule in addition to antigenic stimuli via the T cell receptor (Jenkins et al., 19911, resulting in cell proliferation and exertion of cytotoxicity (Azuma et al., 1992). With respect to activation-related antigens on lymphocytes, CD25 molecule, which is an a-chain of the receptor for interleukin-2, hence marking cells ready to proliferate, was not up-regulated in the CSF cells of HAM/TSP. CD45RO antigen is known to be expressed by the lymphocytes that encountered the relevant antigen, i.e., primed lymphocytes (Beverley, 1986/87), while CD26 molecule defines memory T cells among CD4+ population (Fox et al., 1984): neither primed nor memory CD4+ helper T cells were increased in the CSF of HAM/TSP. These findings indicate that activated CD4+ helper T cells do not expand within the central nervous system (CNS) in HAM/TSP patients, whereas such cells were reported to increase in the blood of these patients (Mori et al., 1988; Itoyama et al., 1989). Nevertheless, active immunopathological processes may occur within the CNS of HAM/TSP, as indicated by the excess production of IgG and the elevated &-microglobulin levels in the CSF of these patients. B

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cell activation, however, did not appear to be involved primarily in the development of the lesion in the CNS of HAM/TSP, since elevated de novo IgG synthesis rate disappeared as the disease progressed in a chronic fashion. On the other hand, persistently increasing p,-microglobulin levels in the CSF may indicate enhanced expression of class I major histocompatibility complex (MHC) molecules in the brain, possibly due to induction by HTLV-I infection of glial cells (Hoffman et al., 1992; Sawada et al., 1990). This mechanism is suggested to be the case in the pathogenesis of dementia (cerebral lesions) caused by infection with human immunodeficiency virus type 1 (HIV-l) (McArthur et al., 1992). Thus, it is conceivable that interaction between brain cells expressing class I MHC and infiltrating CD8+ cells could induce further immunological events, such as the production of cytokines (Kuroda et al., 1993; Kuroda and Matsui, 19931, which, in turn, could induce class I, as well as class II, MHC expression by CNS tissue (Wong et al., 1985). The excess release of inflammatory cytokines by infiltrating T cells (Di Fabio et al., 1993) or by constituent CNS cells such as microglia (Giulian et al., 1986) may result in CNS tissue injury, as is postulated in HIV-l infection of the CNS (Epstein and Gendelman, 1993). In conclusion, it would help our understanding of the pathomechanisms of HAM/TSP to elucidate the functional properties of the subset of CD8+CDllafCD45RO+CD28lymphocytes that predominate in the CSF of HAM/TSP, in terms of determining specificity to HTLV-I antigens, ability to release cytokines, and cytotoxic as well as immuno down-regulatory activities. It would also be helpful to clarify the presence of another costimulatory surface antigen CTLA-4, which is thought to exert a function similar to that of the CD28 molecule (Linsley and Ledbetter, 1993).

Acknowledgements

A part of this work was supported by a grant from the Immunoneurological Research Committee, Ministry of Health and Welfare of Japan and by a grant-inaid from the Ministry of Education, Science, and Culture of Japan (No. 05807054).

References Akbar, A.N., P.L. Amlot, A. Timms, G. Lombardi, R. Lechler and G. Janossy (1990) The development of primed/memory CD8+ lymphocytes in vitro and in rejecting kidneys after transplantation. Clin. Exp. Immunol., 81: 225-231. Azuma, M., M. Cayabyab, D. Buck, J.H. Phillips and L.L. Lanier (1992) CD28 interaction with B7 costimulates primary allogeneic

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