CD4+ T-cells in ginbuna crucian carp, Carassius auratus langsdorfii

Fish & Shellfish Immunology 34 (2013) 136e141

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Direct antibacterial activity of CD8þ/CD4þ T-cells in ginbuna crucian carp, Carassius auratus langsdorfii Sukanta K. Nayak a, Teruyuki Nakanishi b, * a b

Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga-751002, Bhubaneswar, Odisha, India Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 August 2012 Received in revised form 10 October 2012 Accepted 11 October 2012 Available online 23 October 2012

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8aþ T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8aþ T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1e2 h at 26  C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8aþ T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8aþ T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4þ T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgMþ cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8aþ, CD4þ T-cells and surface IgMþ cells in fish. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Antibacterial activity Cytotoxic T cells CD8aþ cells CD4þ cells Ginbuna crucian carp

1. Introduction Cytotoxic lymphocytes which are composed of natural killer (NK) cells and CD8þ cytotoxic T lymphocytes (CTLs) [1e3], are the principal contributors to immune protection from microbial infections and cell transformation. CTLs constitute an important component of adaptive immune response for specific effector mechanism in controlling tumours and viral or bacterial infections. Killing mechanism of CTLs against intracellular pathogens involves the MHC-restricted and antigen-specific recognition and binding of infected host cells [4,5]. Upon recognition by CTLs, killing of the target cell is induced through pathways initiated by death receptors (e.g. Fas) or through granule exocytosis [6,7]. CD8aþ T cells recognize antigens processed and presented by antigen in the context of MHC Class I antigens. Recently, cytotoxic nature of CD4þ T cells against alloantigens, viral-infected cells in various mammalian * Corresponding author. Tel.: þ81 466 84 3383; fax: þ81 466 84 3380. E-mail addresses: [email protected] (S.K. Nayak), tnakanis@ brs.nihon-u.ac.jp (T. Nakanishi). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2012.10.016

system has been also reported [8,9]. Although these activities are well documented, the mechanisms of microbicidal activity of CD4þ T cells have been under investigated. Recent studies have suggested that CTLs have direct antimicrobial activity and can kill different types of pathogens belonging to bacteria, parasites, and fungi [10]. In contrast to killing of tumour and microbial infected cells, direct killing of extracellular pathogens by CTLs is an apparent MHC-independent event since microorganisms do not express MHC [10]. Recognition and killing mechanisms in direct microbicidal activity of CTLs are largely unknown even in mammals, although specific recognition of antigen via MHC and killing mechanism of infected host cells by CTLs are well-known. Therefore, direct antimicrobial activity of CD4þ and CD8þ T cells could be vital in promoting the antimicrobial cell-mediated immune response. The presence of alloantigen or virus-specific cytotoxic T cells has been reported in channel catfish [11], ginbuna [12]. Recently, alloantigen specific killing by CD8aþ T cells [13] along with perforindependent cytotoxic mechanism [14] and helper function of CD4þ T cells of ginbuna crucian carps have been also reported [15].

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However, direct microbicidal activity of fish T cell subsets has not been reported yet. Furthermore, recent evidences indicate the phagocytic and microbicidal nature of fish B cells [16e18]. Therefore, we are aiming to evaluate the direct microbicidal activity of T cell subsets (CD8aþ and CD4þ cells) and other cell types such as surface IgMþ cells and adherent cells of ginbuna. In the present study we found significant bacterial killing activity by sensitized T cell subsets (CD8aþ and CD4þ cells) and surface IgMþ cells from immunised ginbuna against target bacterial cells. This is the first report to demonstrate the conservation of direct microbicidal activity of lymphocytes throughout vertebrates and these findings may shed light on the understanding of protection mechanisms against pathogens from phylogenetical point of view. 2. Materials and methods 2.1. Fish Ginbuna crucian carp, Carassius auratus langsdorfii (OB1 clone, collected from the Okushiri island) of 15e20 g was studied in the present investigation. Ginbuna were maintained at 25 (1  C) in 60 l glass tanks with running water and were fed twice with commercial pellets. 2.2. Bacteria One extracellular pathogen viz., Lactococcus garvieae and one facultative intracellular pathogen namely Edwardsiella tarda was used in the present study. E. tarda was kindly supplied by Dr. Mano, Marine Biotechnology Lab., College of Bioresource Sciences, Nihon University, Japan and L. garvieae, was obtained from Kyoritu Seiyaku Co. Ltd., Japan. 2.3. Monoclonal antibodies Monoclonal antibodies (MAbs) against ginbuna CD8a and CD4 were produced as per the method reported by Akashi et al. [19] and the characteristics of the MAbs have been described in our previous papers [13,15]. A MAb against ginbuna IgM was produced in mice by injecting purified ginbuna IgM according to the standard protocol and has been used to separate sIgMþ and sIgM cells [12,20]. 2.4. Preparation of bacterial antigen E. tarda and L. garvieae were separately grown at 26  C in brain heart infusion (BHI) broth (Fluka, Japan) for 24 h, and then cells were harvested by centrifuging the broth at 10,000  g for 10 min at 4  C. The bacterial antigen was prepared by inactivating the live bacterial suspension in phosphate buffered saline (PBS, pH 7.2) with 1% formalin overnight at 4  C. After overnight inactivation, bacterial cells were centrifuged in a similar manner as described above and finally the pellets containing inactivated cells were suspended in PBS (pH 7.2) after three times washing in PBS (pH 7.2). 2.5. Immunisation of effector donors Two sets of immunisation schedule (single and with one booster dose) was evaluated to find out the best immunisation schedule for optimum sensitization of effector donors. Ginbuna were intraperitoneally immunised with inactivated bacterial antigens (E. tarda and L. garvieae @ 108 CFU/fish). In the single set of immunisation schedule, fish were immunised with one dose of antigen followed by sampling at 1, 3 and 7th day of post injection. Similarly, with one booster dose immunisation schedule, fish were given booster dose after 7 and 15th day of primary injection followed by sampling at 3

137

and 7th day of post immunisation. As per the immunisation schedule, trunk kidney from 3 immunised ginbuna on due sampling time was aseptically removed to prepare the effector cells. 2.6. Preparation of effector cells Leucocyte suspension from the trunk kidney of the immunised ginbuna was prepared by aseptically disaggregating the tissue through sterilized 150-gauge mesh stainless steel sieve in OPTIMEM (Gibco) supplemented with 0.5% heat-inactivated foetal bovine serum (FBS). The leucocytes suspension was then layered over the Percoll density gradient (1.08 g/ml) at equal proportion followed by centrifugation at 450  g for 30 min at 4  C. Lymphocytes rich fraction was finally, collected from the interface of cell suspension and percoll. Different cell fractions (CD8aþ/CD4þ/sIgMþ cells) from the lymphocytes rich fraction were then separated by using specific mAb raised against respective types of cells followed by magnetic activated cell sorting (MACS; Mini Macs, Miltenyi Biotec) according to the method described by Toda el al. [13]. In brief, 1.0  107 cells/ml of trunk kidney cells were incubated with 1:104 diluted rat anti-ginbuna CD8a MAb (mouse ascites) for 45 min on ice. The cells were then washed three times with the medium, adjusted to 1  108 cells/ml, incubated for 15 min at 4  C with 1 ml of a 1:5 dilution of magnetic bead-conjugated goat antirat Ig antibody (Miltenyi Biotec GmbH, Germany), and washed three additional times. CD8a positive and negative cells were separated with MACS by applying the cell suspension to a plastic column equipped with an external magnet. The CD8aþ cells were retained in the column, while the CD8a cells were not. CD8a fraction cells were further separated into CD4 positive and negative fractions using rat anti-ginbuna CD4 MAb (mouse ascites) according to the method described above. Furthermore, CD8a and CD4 double negative cells were separated into sIgM positive and negative fractions using mouse anti-ginbuna IgM MAb (mouse ascites). 2.7. Viability and purity of individual cell fractions The viability of MACS sorted cell fractions was confirmed by trypan blue dye exclusion. The purity of individual cell fractions was checked by flow cytometry by incubating a portion of CD8aþ and/or CD4þ cell fraction with FITC conjugated goat anti-rat IgG þ M þ A antibody (Rockland) with anti-ginbuna CD8 and/or CD4 monoclonal antibodies. Similarly, a portion of sIgMþ cells was incubated with FITC conjugated goat anti-mouse IgG þ M antibody (KPL) with an anti-ginbuna IgM monoclonal antibody. 2.8. Preparation of glass adherent leucocytes cells The glass adherent leucocytes were prepared by spreading 1 ml of trunk kidney leucocyte suspension (1.0  107 cells/ml) into sterilized petri-dishes and then the plates were incubated at 30  C for 4 h in a humidified 5% CO2 incubator followed by three washes in OPTI-MEM supplemented with 0.5% heat-inactivated FBS to remove the majority of non-adherent cells. The cells were then removed from the petri-dishes by trypsinisation and washed three times with the medium and then the viability and types of glass adherent cells were observed under microscope. Antibacterial activity of adherent cells was also determined in a similar manner done for other effector cells as described elsewhere in the text. 2.9. Antibacterial activity of different effector cells Colony-forming unit (CFU) assay was conducted to find out the in vitro antibacterial activity of different types of effector cells

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(CD8aþ/CD4þ/sIgMþ cells) as well as whole leucocytes/lymphocytes and adherent leucocytes/macrophages at different effector and target (bacteria) ratios. The CFU assay was conducted as per the following method. First, the individual pathogen was grown overnight at 26  C in BHI broth and then the cell number was determined by measuring the OD as well as total plate count method. A 100 ml aliquot of the individual bacterial suspension was added to 100 ml of an effector cell suspension at effector : target ratio (1:1, 10:1, 100:1) and then incubated at 26  C for 4 h. The concentration of effector cells used in the study was 106 cells per ml (105 cells per well), unless otherwise mentioned. The number of CFUs of individual bacteria was determined at every one hour interval by lysing the effector cells with sterile distilled water (800 ml) and then plated into brain heart infusion agar plates with appropriate 10fold dilutions. All experiments were performed in triplicate and the % of bacterial killing activity was determined. The percentage of killing was calculated as 1[(viable cells in test)/(viable cells in control [bacterial cells alone])]  100. 2.10. Statistical analysis Statistical analysis was performed by using student ‘t’ test to determine the significant difference (p  0.05) in the mean (S.D) viable bacterial count in CFU assay and/or killing percentage of bacterial cells at a given E/T ratio. 3. Results 3.1. MACS

4.5 4 CFU ( × 103 cfu/ml)

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3.5 3 2.5 2 1.5 1 0.5 0 1h

2h

3h

4h

Co-incubation Period Fig. 1. Effect of co-incubation period of effector cells with target bacteria on viable count of target cells. Effector cells (CD8aþ cells) from immunised ginbuna (n ¼ 3) and target bacteria (E. tarda @ 1.08  105 CFU/ml) at an effector and target ratio of 10:1 were co-incubated in wells of 96 microtiter plate. *: Significantly less viable count in the CFU assay as compared to other incubation period.

CD4þ and sIgMþ effector cells least CFU was recorded at E/T ratio of 10:1 within 1e2 h of co-incubation with pathogen (data not shown). 3.3. Effect of immunisation protocol (schedule) on antibacterial activities

3.2. Requirement of sensitization of effector donors for the induction of antibacterial activities Preliminary experiments were conducted to study the antibacterial activity of different effector cells against bacteria. MACS sorted CD8aþ, CD4þ and sIgMþ effector cells from the immunised ginbuna exhibited antibacterial activity against target bacteria, while the killing activity of effector cells from un-immunised ginbuna was very low (Figs. 3e6). CD8aþ cells significantly reduced (p < 0.05) the viable counts with least CFU of target cells when coincubated for 2 h (Fig. 1). Similarly the CFU assay showed variations in viable counts at different E/T ratio. Maximum reduction of viable count was recorded at an E/T ratio of 10:1 and the viable count was highest at E/T ratio of 1:1 followed by 100:1 (Fig. 2). Similarly, for

A single dose immunisation schedule failed to sensitize the effector donors. All types of effector cells obtained from ginbuna after 1, 3 and 7th day of immunisation with E. tarda did not exhibit any significant reduction of target bacteria (Supplemental Fig. 1). No difference in the viable count was recorded as compared to unimmunised ginbuna effector cells. However, CD8aþ cells from ginbuna immunised with one booster dose at 7th day of primary immunisation followed by sampling after 7 days were found to kill 90.8% E. tarda (Fig. 3). Similar reduction of viable count was

1.8 1.6 1.4

CFU ( × 10 cfu/ml)

Trunk kidney leucocytes from immunised and un-immunised control ginbuna were magnetically sorted into CD8aþ, CD4þ and sIgMþ cells. Flow cytometry analysis of MCAS separated cells exhibited high homogeneity and purity with viability more than 95%. The percentage of lymphocytes in MACS sorted CD8aþ, CD4þ and sIgMþ cell populations were very high, while the percentage of neutrophils, macrophages in each effector cell fractions was very low (Table 1).

1.2 1 0.8 0.6 0.4

Table 1 Composition of magnetically separated CD8aþ, CD4þ and sIgMþ cells from the trunk kidney of immunised ginbuna as determined by flow cytometry. Effector cells

CD8aþ T-cells CD4þ T-cells sIgMþ cells

Viability (Percentage)

Composition of MACS sorted cells (%) Lymphocytes

Neutrophils

Other cell types

96.2  2.8 95.1  3.2 95.4  3.6

85.6  3.6 87.3  4.7 79.8  4.1

4.8  0.7 1.4  0.3 2.4  0.6

4.7  0.8 5.2  1.6 9.8  2.4

Numbers indicate the percentage of cell types in each fraction as the mean  S.D (n ¼ 3).

0.2 0 E/T -1:1

E/T-10:1

E/T-100:1

Different Effector (E) and Target (T) Ratio Fig. 2. Effect of different E/T ratio on the viable count of target bacteria. Effector cells (1  106 cells/ml of CD8aþ cells) from immunised ginbuna (n ¼ 3) were co-incubated at 26  C for 2 h with target bacteria, E. tarda @1.08  106 CFU/ml, 1.08  105 CFU/ml and 1.08  104 CFU/ml to make final E/T ratio of 1:1, 10:1 and 100:1, respectively. *: Significantly less viable count in the CFU assay as compared to E/T ratio.

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120 Percentage of bacterial killing

Percentage of E. tarda killing

120

139

100 80 60 40 20 0

100

80

60

40

20

0 L. garvieae

CD4+ cells from immunised ginbuna

Day of booster injection after primary immunisation/ sampling day after booster dose Fig. 3. Killing activity of CD8aþ cells from immunised ginbuna (n ¼ 3) against E. tarda. Figure shows days of booster injection after primary immnisation/Days at harvest of effector cells after the booster injection.

recorded when ginbuna were re-immunised at 15th day followed by a sampling after 3 days. However, the killing activity of CD8aþ cells collected 7 days after booster injection (15th day of primary immunisation) was maximum (98.9  2.6%) for E. tarda (Fig. 3). 3.4. Bacterial killing activity of different effector cells 3.4.1. CD8aþ T cells As mentioned above, CD8aþ T cells exhibited quite high percentage of killing activity against E. tarda when effector cells were harvested from ginbuna 7 days after booster injection which was given at 15th day after primary immunisation (Fig. 3). Similar but slightly less killing activity of CD8aþ T cells against L. garvieae was observed when compared to intracellular pathogen E. tarda. CD8aþ T cells of ginbuna immunised twice with L. garvieae at 15 days after primary immunisation followed by a sampling at 7 day, killed 92.1 (3.1)% of L. garvieae (Fig. 4).

E. tarda

CD4+ cells from un-immunised ginbuna Fig. 5. Killing activity of CD4þ cells from immunised ginbuna (n ¼ 3) against E. tarda and L. garvieae. Ginbuna was given booster injection at 15th day of primary immunisation and effector cells were collected from trunk kidney 7 days after the booster injection.

3.4.2. CD4þ T cells CD4þ T cells from the immunised ginbuna exhibited significant reduction of viable count of target cells as compared to those from un-immunised ginbuna. CD4þ cells from immunised fish were found to kill 95.7 (3.6)% and 88 (4.2)% of E. tarda and L. garvieae, respectively. However, no significant difference (p  0.05) could be recorded in the killing percentage between E. tarda and L. garvieae (Fig. 5). 3.4.3. sIgMþ cells sIgMþ cells from immunised ginibuna also showed similar killing activity to that of CD8aþ and CD4þ T-cells with highest killing activity exhibited when effector cells were derived from one booster dose immunised ginbuna. sIgMþ cells from immunised ginbuna were found to kill 94.2 (4.7)% of E. tarda and 91 (5.4)% of L. garvieae, respectively. No significant difference (p  0.05) could

120

80 Percentage of bacterial killing

Percentage of L. garvie killing

100

60

40

20

0

L.garvieae

Day of booster injection after primary immunisation/ sampling day after booster dose Fig. 4. Killing activity of CD8aþ cells from immunised ginbuna (n ¼ 3) against L. garvieae. Figure shows days of booster injection after primary immnisation/Days at harvest of effector cells after the booster injection.

E.tarda

sIgM+ cells from immunised ginbuna sIgM+ cells from un-immunised ginbuna Fig. 6. Killing activity of sIgMþ cells from immunised ginbuna against E. tarda and L. garvieae. Ginbuna was given booster injection at 15th day of primary immunisation and effector cells were collected from trunk kidney 7 days after the booster injection.

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be recorded in the killing percentage between E. tarda and L. garvieae (Fig. 6). 3.4.4. Bactericidal activity of leucocytes other than lymphocytes The glass adherent leucocytes (mostly macrophages) exhibited lower antibacterial activity against tested pathogens. Glass adherent leucocytes from immunised ginbuna killed 29 (1.1)% of E. tarda, while those from un-immunised ginbuna killed 16 (1.1)% of the bacteria. On the other hand, glass adherent leucocytes from immunised with L. garvieae and un-immunised ginbuna killed 31 (2.0)% and 21 (1.9)% of L. garvieae, respectively (Fig. 7). 3.5. Antigen specificity The antigen specificity study was conducted by using CD8aþ T cells derived from immunised (twice at 15 days interval and sampled at 7 days after the last immunisation) ginbuna. The CD8aþ T cells from un-immunised ginbuna were found to kill lower percentage of E. tarda (14.8  1.3%) and L. garvieae (19.5  1.5%). In contrast, CD8aþ T cells from immunised ginbuna with E. tarda exhibited quite high killing activity against immunogens (98.9  2.6%). However, CD8aþ T cells from immunised ginbuna with E. tarda non-specifically killed 40 (2.3)% of L. garvieae. Similar non-specific killing was observed when CD8aþ T cells from immunised ginbuna with L. garvieae, i.e. 92.1 (3.1)% of L. garvieae and 42.7 (1.9)% of E. tarda (Table 2). 4. Discussion Cell-mediated immunity is an important component of immune system and plays a crucial role in fighting against tumour cell or invasive pathogens. Although typical phagocytic cells like macrophages/monocytes and neutrophils are involved in microbial killing activities, recent evidences suggest the direct recognition and killing of microorganisms by CTLs. Nevertheless, despite their importance in killing of alloantigen, virus or microbe-infected cells in fish, little is known about the CTL-mediated direct microbial killing activity. In the present study we demonstrated the direct

Percentage of bacterial killing

35

30

25

20

15

10

5

0 E. t a r d a

L. garvieae

Glass adherent leucocytes from immunised ginbuna Glass adherent leucocytes from un-immunised ginbuna Fig. 7. Killing activity of adherent leucocytes from immunised and un-immunised ginbuna. Ginbuna was given booster injection at 15th day of primary immunisation and the glass adherent leucocytes were collected from trunk kidney 7 days after the booster injection.

Table 2 Antigen specific killing activity of target cells by CD8aþ cells of control as well as immunised ginbuna. Target bacteria

E. tarda L. garvieae

CD8aþcells obtained from Control fish

E. tarda immunised fish

L. garvieae immunised fish

14.8  1.7 19.5  1.3

98.9  2.6 40.0  2.3

42.7  1.9 92.1  3.1

Numbers indicate the killing percentage of target bacteria by CD8aþ cells obtained from control and immunised ginbuna as the mean  S.D (n ¼ 3).

killing activity of CD8aþ, CD4þ T-cells as well as sIgMþ cells in fish. All these effector cells killed target bacteria irrespective of their intracellular (E. tarda) or extracellular (L. garvieae) nature. Killing activity was significantly higher against bacteria that had been used for sensitization (as immunogen). However, approximately 50% of non-specific cross killing was also observed. In order to induce optimum antimicrobial activity single dose sensitization is not enough and two times sensitization of effector donors at proper time intervals was required, i.e., one booster dose at 15th day of primary immunisation. The present study also ruled out the possible involvement of macrophages in the antibacterial activity as the percentage of macrophages contamination in the MACS sorted effector cells were very low. Furthermore, adherent cells (mostly macrophages) were also exhibited lower percentage of bacterial killing activity. The results of the present study indicate that the killing activity is dependent on the E/T ratio. The optimum E/T ratio was found to be 10:1 since at this ratio the viable count of target bacteria as measured by CFU assay was significantly low as compared to other ratios of 100:1 and 1:1. The possibility of contact/interaction between the effector and target was higher in E/T ratio of 1:1 but the high viable count of the target bacteria in CFU assay indicates less killing of target bacteria by effector cells. This may be due to the interference of metabolic/toxic byproducts released by target bacteria which in turn could have reduced the number and/or activity of effector cells. Earlier, Pirarat et al. [21] also found that tilapia (Oreochromis niloticus) lymphocytes to be the major cell populations to underwent extensive apoptosis when incubated with a higher concentration of E. tarda. In the present study, the initial bacterial count i.e., 106 CFU per ml was higher in E/T ratio of 1:1, hence, possibility of interference/toxicity against effector cells was higher which in turn lead to high viable bacterial count. As for the case in higher E/T ratio of 100:1 the target bacterial number was less i.e., 104 CFU per ml and the number of effector cells (106 cells per ml) was high for which there may be less possibility of target contact/interaction with effector cell and hence the viability count of target bacteria was high. T cells as well as sIgMþ cells require proper activation/sensitisation for executing antimicrobial effects. CTLs in fish often require repeated priming for executing cell-mediated cytotoxicity against alloantigen [13,22]. In order to induce optimum antimicrobial activity single dose sensitization is not enough and two times sensitization of effector donors at proper time intervals was required, i.e., one booster dose at 15th day of primary immunisation. However, this is not the case for adherent cells and there was not much difference in the killing activity of adherent cells from between immunised and un-immunised fish. CD8þT cell responses are of notable importance for protection against intracellular pathogens and the findings of the present investigation also demonstrated the direct killing of bacterial target as earlier reports in mammals [23,24]. We also found the direct microbicidal activity of CD4þ T cells in fish and these results are in accordance with the previous studies in mammals, e.g. direct killing

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of Cryptococcal neoformes [25,26], Mycobacterium tuberculosis [24] by CD4þ T cells. Several reports have emerged in recent years regarding the potency of fish B cells to execute antimicrobial activity. In the current study, apart from T cells, sIgMþ cells also exhibited notable bactericidal activity. However, it is possible that ginbuna sIgMþ cells may not only contain B (mature and immature) cells but also include the NK-like cells, since sIgMþ cells consist of Fc receptorbearing cells such as neutrophils, macrophages, NK cells [27]. For instance, NK-like cells in channel catfish are also found to be prearmed with IgM via a putative FcR for Igm [27]. Nevertheless, ginbuna sIgMþ cells did not show spontaneous bacterial killing activity unlike NK cells which are the only group of cytotoxic cells that can proliferate and spontaneously kill viruses, bacteria or protozoaninfected cells in an MHC unrestricted manner without need for prior activation/sensitization [28e30]. In ginbuna cytotoxic activity of sIgMþ cells (presumably NK cells) was up-regulated after in vivo sensitization of donor [13]. Therefore, B cells and NK cells are likely responsible cells for direct bacterial killing within sIgMþ cells and neutrophils and macrophages should be excluded, since percentages of these cell types was quite low in sIgM fraction (Table 1). Effector donors had to be sensitized prior to the harvest of effector cells such as CD8þ T cells and sIgMþ cells (presumably NK cells) to induce cytotoxic activity against allogeneic target cells [13]. This is in good agreement with the present direct microbicidal activity of T cell and sIgMþ cells. The antibacterial activity of sIgMþ cells of ginbuna against bacteria went in accordance with the earlier reports which indicated the phagocytic and microbicidal nature of B cells [16e18]. However, involvement of phagocytosis in the killing mechanisms was not examined yet in the present study and further study is needed. Non-specific killing activity was recorded not only for CD8aþ cells (Table 2) but also for CD4þ T-cells and sIgMþ cells (data not shown). The non-specific killing activity of these effector cells may be attributed to the ability of pathogens to trigger effector cells which in turn might lead to release of active components. The involvement of granulysin, perforin in the direct antibacterial activity of CTLs are documented in mammals and further work in this line is going on to characterize the exact killing mechanism of lymphocytes including sIgMþ cells as well as to establish the involvement of these substances in antimicrobial activity in fish. The findings of the present investigation on direct bacterial killing activity of T and B-cells in fish are of notable importance for lower vertebrate like fish that relies heavily on innate defence mechanisms for pathogen clearance. Present results are also useful for the development of evaluation method for vaccine potency in various fish species important in aquaculture, since suitable methods to evaluate the cell-mediated cytotoxic activity are not available in most of fish species to date and present method can be applicable to other fish species where inbred or clonal fish with appropriate cell lines are not available. Acknowledgements This research was supported in part by postdoctoral fellowship for Sukanta Kumar Nayak from the Japanese Society for Promotional of Science (JSPS). The authors are thankful to Dr. T. Moritomo, Laboratory of Fish Pathology, Department of Veterinary Medicine, Nihon University for his help and co-operation for carrying out the present investigation. Appendix A. Supplementary material Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2012.10.016.

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