Expansion of unconventional T cells with natural killer markers in malaria patients

Parasitology International 52 (2003) 61–70

Expansion of unconventional T cells with natural killer markers in malaria patients Hisami Watanabea,1, Anura Weerasingheb, Chikako Miyajia, Hiroho Sekikawaa, Sinichi Toyabea, M. Kaiissar Mannora, Sufi Reza M. Morsheda, Ramesh C. Haldera, Jun Kobayashic, Hiromu Tomac, Yoshiya Satoc, Kuni Iwaid, Hiroki Matsuokad, Toru Aboa,* a

Department of Immunology, Niigata University School of Medicine, Niigata 951-8510, Japan b Medical Research Institute, Colombo, Sri Lanka c Faculty of Medicine, Department of Parasitology, University of Ryukyus, Nishihara, Okinawa 903-0215, Japan d Department of Medical Zoology, Jichi Medical College, Minamikawachi, Tochigi 329-0498, Japan Received 12 July 2002; accepted 8 October 2002

Abstract Immunological states during human malarial infection were examined. In parallel with parasitemia and anemia, granulocytosis was induced in the blood of patients, especially those infected with Plasmodium (P.) falciparum. At that time, the level of lymphocytes remained unchanged or slightly increased in the blood. However, the distribution of lymphocyte subsets was modulated, showing that the proportion of CD56qT cells, CD57q T cells, and gdT cells (i.e. all unconventional T cells) had increased in patients infected with P. falciparum or P. vivax. This phenomenon occurred at the early phase of infection and disappeared in the course of recovery. The data from patients with multiple attacks of P. vivax infection showed that there was no augmentation of these responses. In adult cases, the increase in the proportion of unconventional T cells seemed to closely parallel disease severity. However, all these responses were weak in children, even those infected with P. falciparum. In conjunction with accumulating evidence from mouse malaria experiments, the present results suggest that the immunological state induced by malarial infection might mainly be an event of unconventional T cells and that the immunological memory might not be long-lasting, possibly due to the properties of unconventional T cells. 䊚 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human malaria; CD56qT cells; CD57qT cells; gdT cells; Immunological memory

1. Introduction *Corresponding author. Tel.: q81-25-227-2133; fax: q8125-227-0766. E-mail address: [email protected] (T. Abo). 1 Present address: Division of Cellular and Molecular Immunology, Center of Molecular Bioscience, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan

Malarial parasites invade hepatocytes in the liver stage and erythrocytes in the blood stage after infection w1–3x. We have to consider how the immune system recognizes these intracellular par-

1383-5769/03/$ - see front matter 䊚 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 3 - 5 7 6 9 Ž 0 2 . 0 0 0 8 5 - 5

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asites, namely, whether conventional T and B cells or unconventional lymphocytes recognize the parasites. This question arises from the fact that conventional T and B cells (or immunoglobulins secreted by them) encounter difficulty in recognizing intracellular particles, except specific stages of malarial infection including the stages when disseminated sporozoites and merozoites appear. In recent mouse studies, unconventional T cells, including natural killer T (NKT) cells and NK1.1y extrathymic T cells, were found to be associated with the protection against malaria w4– 6x. Since these unconventional T cells have autoreactivity against abnormal self-cells w7,8x, it is expected that such autoreactivity may be responsible for the elimination of hepatocytes or erythrocytes infected with intracellular parasites. In other words, unconventional T cells may play a major role in innate immunity against intracellular pathogens. In the case of human malaria, Plasmodium (P.) falciparum and P. vivax are the most common species. P. falciparum gives rise to severe diseases including severe anemia, cerebral malaria, acute renal failure, and hypoglycemia and is a significant cause of mortality. On the other hand, although the general clinical features of P. vivax are similar to those of P. falciparum, resulting mortality is not high. In light of these findings, we examined whether human unconventional T cells with properties similar to such murine cells expand in the peripheral blood of malaria patients. In the case of humans, the populations of CD56qT cells and CD57qT cells, namely, T cells with natural killer (NK) markers, are relatively low in the blood but large in the liver (and bone marrow) w9,10x. These T cells are almost completely absent in the thymus. It was demonstrated that these unconventional T cells, including gdT cells, increased in number in the blood of malaria patients, especially adults with severe malaria. We have to consider the possibility that innate immunity mediated by these unconventional T cells, if not completely, might be responsible for the resistance to malarial infection.

2. Materials and methods 2.1. Subjects Sri Lanka Project: Thirty-four adult patients were studied. Doctors in Sri Lanka identified these patients in a field study. As a control group, 11 healthy subjects in the Medical Research Institute, Colombo, Sri Lanka were also studied. Adult P. vivax patients (ns27) consisted 16 males and 11 females and ranged from 12 to 60 years of age (means34.0). Adult P. falciparum patients (ns 7) consisted of 4 males and 3 females and ranged from 25 to 64 years of age (means45.6). Healthy adult controls consisted 7 male and 4 females and ranged from 27 to 47 years of age (means36.2). Lao P.D.R. (Laos) Project: Ten pediatric patients (P. falciparum) living in the Province of Khammouane, Lao P.D.R. were studied. These patients came to hospitals by themselves. As a control group, 21 healthy children in Niigata, Japan were studied. The age of the control and malaria children was ranged from 5 to 15 years. The study was performed with the informed consent of the donors or their families. In addition to these foreign patients, some Japanese malaria patients were also examined. 2.2. Diagnosis of malaria Thin blood films were examined to detect malarial parasites by experienced laboratory technicians in Sri Lanka and Laos. 2.3. Hematological profiles To analyze the hematological parameters, leukocyte counts of fresh whole peripheral blood were determined by hemocytometer and were stained by the Giemsa method. The percentages of hemoglobin (Hb) in the blood were obtained by a routine laboratory technique, the cianometohemoglobin method. 2.4. Cell preparations Peripheral blood lymphocytes (PBL) were isolated from heparinized blood and then obtained by

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Table 1 Hematological profile Subject

n

Granulocyte (=103yml)

Lymphocyte (=103yml)

Hb (gydl)

Healthy controls

11

4.1"1.1

2.1"0.3

14.1"0.8

Malaria patients P. vivax P. falciparum

27 7

4.0"2.0 6.3"1.8*

2.5"1.5 2.7"0.9

12.5"2.1* 12.0"1.1*

*

P-0.05 compared with healthy controls.

Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden) gradient (1.077) centrifugation w11x. In these experiments, PBL were isolated in the field (Sri Lanka and Laos) and transported under refrigeration to our laboratory within 24–48 h by air. 2.5. Flow cytometric analysis The cell surface phenotype of PBL was examined by two-color staining of mAbs w12,13x. The mAbs used here included FITC or PE-conjugated anti-CD3, anti-CD56, anti-CD57, anti-CD4, antiCD8 (BD PharMingen Co., San Diego, CA), antiabTCR, anti-gdTCR (Beckman Coulter Inc., Fullerton, CA), anti-Vg9TCR, and antiVa24mAbs (BD PharMingen Co.). The cells were analyzed by a FACScan (Becton-Dickinson Co., Mountain View, CA). Dead cells were excluded by forward scatter, side scatter, and propidium iodide gating. 2.6. Statistical analysis Data are presented as means"1 S.D. Statistical analysis was performed by the Mann–Whitney Utest. Differences were taken to be significant when P values were -0.05. 3. Results 3.1. General features of adult malaria patients In collaboration with doctors in Sri Lanka, we obtained blood samples from 34 malaria patients, including those infected with P. vivax (ns27) or P. falciparum (ns7). We also obtained blood samples from healthy controls in Sri Lanka. The hematological profiles of the tested subjects are

represented in Table 1. It is interesting that the number of granulocytes was greater in patients with P. falciparum. The number of lymphocytes was almost the same (or slightly increased) in healthy controls. As expected, the contents of Hb decreased (i.e. anemia) in patients infected with both P. vivax and P. falciparum. 3.2. Expansion of CD56qT and CD57qT cells in malaria patients We examined whether there was a change in the distribution of lymphocyte subsets, especially unconventional T cells with NK markers (i.e. NKqT cells) (Fig. 1a). In this experiment, twocolor staining for CD3 and CD56 and that for CD3 and CD57 were conducted for the blood of one patient infected with P. vivax and one patient infected with P. falciparum. The data from one healthy control showed that while there were few unconventional NKqT cells (CD56qT and CD57qT cells), conventional NKyT cells and NK cells were abundant. On the other hand, a considerable proportion of NKqT cells appeared in patients infected with P. vivax and in those infected with P. falciparum. The proportion of CD56qNK cells tended to increase in these patients. We previously reported that unconventional NKqT cells contain both abT and gdT cells and double-negative (DN) CD4y8y cells w9,10x. In contrast, conventional NKyT cells do not contain such unusual T cell populations. We examined whether these unusual T cells appeared in the blood of malaria patients (Fig. 1). In this experiment, two-color staining for CD3 and TCRgd, that for CD3 and a mixture of CD4 and CD8, and that for CD4 and CD8 were conducted. In healthy control subjects, neither TCRgdq cells nor

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H. Watanabe et al. / Parasitology International 52 (2003) 61–70

Fig. 1. Phenotypic characterization of lymphocytes in the blood of healthy controls and malaria patients. (a) Two-color staining for CD3 and CD56 and that for CD3 and CD57; (b) Two-color staining for CD3 and TCRgd (or a mixture of CD4 and CD8) and that for CD4 and CD8. One typical healthy control and one patient infected with P. vivax (case 2) and P. falciparum are represented. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas.

DNCD4y8y cells were detected. On the other hand, considerable proportions of both gdT cells and DNCD4y8y cells appeared in malaria patients. It was also demonstrated that the proportion of CD8q cells, but not that of CD4q cells, tended to increase after malarial infection. To examine how abT and gdT cells expressed CD56 and CD57 antigens, two-color staining for TCRab and CD56 (or CD57) and that for TCRgd and CD56 (or CD57) were conducted (Fig. 2).

One patient infected with P. falciparum (case 1) and one patient infected with P. vivax (case 2) were examined. These patients were different from those in Fig. 1. A considerable proportion of TCRabq cells expressed CD56 or CD57 antigens. This tendency was more striking on TCRgdq cells. The majority (60–90%) of gdT cells expressed CD56 and CD57 antigens. However, Va24q cells were an extremely minor population among TCRabq cells in both patients. All of these experiments were repeated in controls and malaria patients (Table 2). The proportions of both CD56qT and CD57q cells tended to increase with malarial infection. This change was significant in patients infected with P. falciparum (P-0.05). The proportions of gdqT cells and DNCD4y8y cells were also elevated. In other words, the deviation to unconventional T cells was more striking in P. falciparum patients than in P. vivax patients. In the case of mice, NKT cells preferentially use an invariant chain of Va14Ja281 for TCRa w14–16x. Since Va24 is a human counterpart of mouse Va14 w17,18x, we then examined whether the proportion of Va24qT cells increased in malaria patients (Table 3). As already known w17,18x, the population of Va24qT cells is an extremely minor population among whole T cells (-0.5%) in the peripheral blood. However, this population is greater among CD56qT cells and DNCD4y8y cells w18x. We therefore examined the population of Va24qT cells within these populations. Although the proportion of Va24q cells tended to increase slightly in patients with P. falciparum, this level was not statistically significant (P)0.05). 3.3. Time-kinetics after malarial infection and repeated infection of malaria To determine the relationships between the shift of immunoparameters and the time after malarial infection, we classified P. vivax patients into three groups (Table 4A). By this classification, increased proportions of CD57qT cells and DNCD4y8y cells were seen at the earlier stage, i.e. 1–7 days after malarial infection. The proportion of CD56qT cells and gdT cells had the same tenden-

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Fig. 2. The majority of gdT cells expressed CD56 and CD57 antigens. One adult patient infected with P. falciparum (case 1) and one adult patient infected with P. vivax (case 2) are represented. Two-color staining of the indicated combinations were conducted. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas.

cy. The shift of these immunoparameters gradually disappeared at 8–10 days after malarial infection. Some patients had previously experienced multiple attacks (2–4 times) of malarial infection. We classified these patients into two groups (ns6 in each group) and examined the immunoparameters (Table 4B). Although the values of tested immunoparameters varied, there was no statistical difference (P)0.05). 3.4. A time-kinetic study of a patient infected with P. falciparum

immunoparameters during the entire period (Fig. 3). Although malarial therapy was started after the onset of disease, the proportions of CD56qT cells, CD57qT cells, and gdT cells began to increase in the blood, showing peaks around week 4. In parallel with the decrease in parasitemia and symptoms, the proportions of these unconventional T cells decreased. In this case, there was another characteristic: the proportion of gdT cells were extremely high in comparison with those of CD56qT and CD57qT cells. 3.5. Subfamilies of gdT cells in malaria patients

We experienced one patient (a 25-year-old female) infected with P. falciparum from just after the onset of disease to her recovery from the disease in Japan. We were able to survey the

Human gdT cells are classified into two subfamilies, Vg9q and Vg9y cells. The majority of gdT cells are Vg9q while the minority are Vg9y in

Table 2 Distribution of lymphocyte subpopulations during P. vivax and P. falciparum infections Subject

n

% Positive cells CD56qT cells

CD57qT cells

gdT cells

CD4qT cells

CD8qT cells

DNCD4y8y cells

Healthy controls

11

3.6"1.2

7.5"3.8

3.2"1.5

40.1"9.6

23.8"4.5

6.2"3.2

Malaria patients P. vivax P. falciparum

27 7

7.8"6.9 8.7"3.7*

14.8"12.7 16.9"7.6*

6.9"8.2 11.7"12.0

45.1"18.7 33.6"13.1

20.8"10.9 22.0"6.4

7.7"6.9 12.6"5.3*

*

P-0.05 compared with healthy controls.

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66

Table 3 A comparison of the proportion of Va24q cells among T cell subsets between healthy controls and malaria patients Subject

n

% Va24q cells among CD56qT cells

DNCD4y8y cells

Healthy controls

8

2.1"0.9

1.7"1.1

Malaria patients P. vivax P. falciparum

8 3

1.0"0.9 2.7"1.3

1.3"1.3 4.9"0.0

Va24q cells were a minor population among whole T cells (-0.1%). However, they were enriched into CD56qT cells and DNCD4y8y cells.

the peripheral blood of humans w19–21x. The distribution of these subfamilies was identified in controls and malaria patients (Table 5). In this experiment, both Japanese cases and Sri Lankan cases were examined. The Vg9q cells in malaria patients in both countries tended to be elevated. However, since controls in Sri Lanka had elevated levels of Vg9q cells, the statistical difference between controls and malaria patients was not calculated. Although Vg9q cells increased in the patients, the percentages of Vg9q ygdT cells rather decreased due to the parallel expansion of Vg9y cells. 3.6. Immunoparameters in children infected with P. falciparum In Laos, many children suffer from P. falciparum malaria. We thus investigated whether the eleva-

tion of unconventional T cells occurred in the blood of such children (ns7) (Table 6). To determine whether children have a quite low level of unconventional T cells in the peripheral blood, we collected control samples from healthy Japanese children (ns21) and healthy Laos children (ns3). Primarily, the proportions of CD56qT cells, CD57qT cells, and gdT cells were very low in children of both countries. The proportion of CD57qT cells increased slightly, but those of other CD56qT cells and gdT cells remained unchanged even after malarial infection. 4. Discussion In this study, we demonstrated that the proportion of unconventional T cells with NK markers increased in the blood of malaria patients, especially in the adult cases. Since the number of total lymphocytes increased in all these cases, the absolute number of unconventional T cells was estimated to increase. As shown previously w9,10x, unconventional T cells with NK markers include CD56qT and CD57qT cells, the former cells being abundant in the liver and the latter being abundant in the liver and bone marrow. Although these CD56qT and CD57qT cells were few in the blood of healthy controls, both of them increased in malaria patients. In the case of mice, NK1.1qT cells (i.e. NKT cells) or NK1.1y extrathymic T cells are abundant in the liver and increase in number in mice with malaria w4–6x. Therefore, the

Table 4 Lymphocyte subsets during P. vivax infection n

% Positive cells CD56qT cells

CD57qT cells

gdT cells

DNCD4y8y cells 13.9"7.7* 6.8"2.2* 3.1"1.6

(A) Sampling time after infection (days) 1–3 4–7 8–10

5 5 4

8.2"9.0 9.9"6.4 4.6"4.6

16.3"9.9* 20.7"16.6 5.6"5.6

11.7"12.6 5.2"1.5 2.9"3.4

(B) Number of attacks Single Multiple (2–4)

6 6

7.1"8.5 7.6"6.9

10.1"10.4 20.2"11.2

9.8"12.3 4.1"1.9

*

P-0.05 compared to 8–10 days.

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Fig. 3. Time-kinetics of the proportions of CD56qT cells, CD57qT cells, and gdT cells in one patient infected with P. falciparum. Blood samples of one female adult malaria patient were obtained at the indicated weeks, the sampling being started several days after the appearance of malarial fever and parasitemia.

present results indicate that the major expanding T cells might be unconventional T cells rather than regular T cells in both mice and humans with malarial infection. Unconventional T cells comprise both abT and gdT cells, expressing either CD4q, CD8q, or DNCD4y8y w9,10x. Therefore, the present results do not contradict those of previous studies, in

67

which CD4q w22x, CD8q w23x and DNCD4y8y T cells w5x were found to expand in malaria patients. We previously reported that unconventional T cells have a phenotype as memory T cells (e.g. CD45ROq) w24x. This is also true in humans w25x. It has been reported that such memory-like T cells expanded in malaria patients w26,27x. All of these previous data show one of the properties ascribed to unconventional T cells. It is well established that IFNg is associated with the protection from malarial infection w28x or the pathology of malaria w29x. Unconventional T cells as well as NK cells have been reported to be good producers of IFNg w12x. In addition to CD56qT and CD57qT cells, the proportion of gdT cells was found to increase in malaria patients. We have previously reported that gdT cells are present within the populations of CD56qT and CD57qT cells w9,10x. Therefore, these responses, seen after malarial infection are coincidental. It is known that Vg9qgdT cells respond in vitro to P. falciparum-infected erythrocytes w30–32x. However, there was no deviation from Vg9ygdT cells to Vg9qgdT cells in malaria patients in this study. If malarial infection induces a polyclonal activation of T cells, we must also consider the in vivo activation of Vg9y cells. A similar result was reported in African children with P. falciparum infection by Hviid et al. w33x. It is known that Vg9 is coupled with Vd2 while Vg9y is coupled with Vd1 w19–21x. Another possibility may be an endemic difference regarding this matter w34,35x. The proportion of abT cells among CD56qT and CD57qT cells is usually greater than that of gdT cells w9,10x. In the case of mice, NK1.1q and NK1.1y subsets are present within unconventional abT cells with an intermediate density of the

Table 5 Subfamilies of gdT cells during P. vivax infection Subject

Healthy controls Malaria patients *

n

5 8

%Vg9q cells

%Vg9q ygdT cells

Japanese

Sri Lankan

Japanese

Sri Lankan

1.3"0.7 2.4"0.3*

0.9"0.3 1.3"0.2

76.4"5.2 44.0"5.3*

46.7"3.1 32.6"15.1

P-0.05 compared with healthy controls in the same country.

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Table 6 Lymphocytes subpopulations of children with P. falciparum malaria in Laos Subject

Japan (Niigata) Laos Laos *

Age

5–15 8–12 5–14

n

21 3 7

Malaria

y y q

% Positive cells CD56qT cells

CD57qT cells

gdT cells

1.3"1.0 1.1"0.2 0.8"0.4

3.5"1.1 5.2"2.7 6.4"2.7*

4.1"1.7 4.1"2.9 5.3"2.7

P-0.05 compared with Japanese subjects.

TCR–CD3 complex (i.e. TCRint cells) w8,36x. We previously proposed the possibility that CD56qT cells are similar to murine NK1.1qTCRint cells and that CD57qT cells are similar to NK1.1yTCRint cells w10x. This notion arises from their tissue-dependent distribution. However, there is another difference between mice and humans. The majority of murine NKT cells use an invariant chain of Va14 for TCRa and recognize some glycolipids in the context of CD1d (a MHC class I-like molecule) w14–16x. In the case of humans, Va24qT cells (equivalent to murine Va14qT cells) are an extremely minor population among CD56qT cells w17,18x, although Va24qT cells are included within this population w18x. This was also the case in malaria patients as shown in this study. It is speculated that Va24yT cells among CD56qT cells might be regulated by monomorphic (and polymorphic) MHC and MHC-like class I antigens (e.g. HLA-E–G and CD1a–c) other than CD1d. These widespread molecules of MHC and MHC class I-like antigens in humans may reduce the proportion of Va24qT cells among CD56qT cells. The increase in the proportion of CD56qT and CD57qT cells was much more prominent in the P. falciparum cases than in the P. vivax cases. In the former, the increase of unconventional T cells was seen at the early phase of infection. In a mouse malaria study, we reported that extrathymic T cells with or without the NK1.1 marker expanded in the liver and that some of them leaked into the circulation w6x. Concerning these murine data, human unconventional T cells which are found in the blood of malaria patients might have been derived from the liver. Although some of the present evidence overlaps the data from previous studies (e.g. the elevation of gdT cells in malaria

patients) w37,38x, a simultaneous determination of unconventional T cells, including their time-kinetics and origin, in the patients was performed for the first time in this study. We have previously reported that the memory of malarial infection decreased as a function of time in mice and that these mice had completely lost such immunity 1 year after the infection w6x. This phenomenon may be due to the primitive nature of extrathymic T cells, namely, incompleteness of memory. A similar phenomenon is known in humans w39x. Patients who have suffered multiple attacks of P. vivax do not show an elevated level of unconventional T cells, in comparison with patients who have experienced only a single attack. The elevation in the proportion of unconventional T cells in children was less prominent than that in adult patients. Concerning the age-associated increase of unconventional T cells in mice and humans w13x, children may have less ability to induce the expansion of unconventional T cells. As a result, these children with malaria may usually show severe symptoms or a prolonged time-course of malaria. Alternatively, children with malaria may use another mechanism of immunity (e.g. regular T and B cells, including antibodies, against disseminated sporozoites and merozoites). An elevated level of granulocytes was seen in adult patients infected with P. falciparum. Such an increased level of granulocytes is often related to an increased level of sympathetic nerve activation (e.g. tachycardia) w40,41x. In a recent study, we encountered a high level of granulocytosis in cerebral malaria cases (our unpublished observation). Granulocytes are the major source of the production of superoxides and free radicals which sometimes induce tissue destruction w42,43x.

H. Watanabe et al. / Parasitology International 52 (2003) 61–70

Therefore, we must focus attention on granulocytes as well.

w11x

Acknowledgments This paper was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. The authors wish to thank Mrs Yuko Kaneko for preparation of the manuscript.

w12x

w13x

w14x

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