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　Communication Kitasato Med J 2017; 47: 87-91　

Received 17 November 2016, accepted 19 December 2016Correspondence to: Koichi Kubota, Department of Microbiology, Kitasato University School of Medicine1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, JapanE-mail: koichi-tn@jcom.home.ne.jp

Suppression of the cytopathic effect of herpes simplex virus by thefunctional T cell hybridoma B6HO3: a subset of innate lymphocytesmay have versatile functions for combating early microbial infection

Koichi Kubota

Department of Microbiology, Kitasato University School of Medicine

In a previous study, we found that functional T cell hybridoma B6HO3 cells respond to cell death inbacterial-infected macrophages with the production of IFN-γ that activates the bactericidal functionof macrophages. In the present study, we show that B6HO3 cells also have the ability to respond toherpes simplex virus type 1 infection in J774 macrophage cells and Hepa1-6 cells by suppressing thecytopathic effect of this virus. The functional T cell hybridoma system has been reported to preservethe effector functions of parental lymphocytes, and the parental cell of the B6HO3 hybridoma belongsto innate-like T cells. The findings presented here, therefore, lead us to the hypothesis that a subset ofinnate lymphocytes may be equipped with versatile functions for combating both bacterial and viralinfections.

Key words: arginase, arginine, innate immunity, type 1 interferon

Abbreviations: CPE, cytopathic effect; HSV, herpes simplex virus; NO, nitric oxide; L-NMMA,NG-monomethyl-L-arginine; L-NAME, NG-nitro-L-arginine methyl ester

Findings and Discussion

e previously established a functional T cellhybridoma cell line termed B6HO3 from growth-

arrested hybrids between YACUT lymphoma cells andC3H/He anti-DBA mixed lymphocyte culture cells.1 Thishybridoma responded to cell death in bacterial-infectedmacrophages with the production of IFN-γ, and thisproperty was considered to reflect the effector functionof an innate-like T cell, the parental T cell of thishybridoma.2,3 In the present study, we investigatedwhether or not cell death caused by viral infection alsoinduces B6HO3 cells to produce IFN-γ. To this end,J774 mouse macrophage cells were infected with herpessimplex virus type 1 (HSV-1) and cocultured with B6HO3cells for 48 hours; then, the culture supernatants wereexamined for IFN-γ production. Unlike the resultsobtained with bacterial infection,1 IFN-γ was not detectedin the culture supernatants (data not shown). During thecourse of this study, we observed that, while all of theHSV-1-infected J774 cells underwent cell death in theabsence of B6HO3 cells, many J774 cells remained alivein the coculture wells, suggesting that B6HO3 cells

inhibited HSV-1 replication.To further examine this suppressive effect of B6HO3

cells on HSV-1 replication, J774 cells were inoculatedwith HSV-1 and cocultured with B6HO3 cells in aTranswell to separate both cells with a permeablemembrane. As shown in Figure 1B, HSV-1 causedcytopathic effect (CPE) on J774 cells when HSV-1-inoculated J774 cells were cultured alone for 72 hours,but when HSV-1-inoculated J774 cells were coculturedfor 72 hours with B6HO3 cells in a Transwell, the CPEof HSV-1 was not observed (Figure 1C). This resultindicated that B6HO3 cells produced some soluble factorthat suppressed the CPE of HSV-1. However, becausethe suppression of the CPE did not occur when HSV-1-inoculated cells were cultured for 72 hours together withculture supernatants obtained from 48-hour cocultivationof HSV-1-inoculated J774 cells with B6HO3 cells (Figure1D), the inhibitory factor in the culture supernatantsseemed to be quite unstable. Furthermore, the number ofJ774 cells in the coculture was much lower than that ofcontrol J774 cells that were cultured alone (Figure 1Aand 1C), suggesting that the growth of J774 cells wasalso suppressed in the presence of B6HO3 cells. HSV-1

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itself has the ability to inhibit cell cycle progression.4

However, it is unlikely that the growth inhibition of J774cells was attributable to viral infection, because only asubpopulation of the J774 cells was infected with HSV-1 due to low multiplicity of infection. Thus, the putativeCPE-inhibitory factor produced by B6HO3 cells seemedto also have cell growth-inhibitory activity. This wasalso supported by the fact that the proliferation of B6HO3cells in the upper well of the Transwell decreased in thecoculture (data not shown).

The same results were obtained with the CPE of HSV-1 on mouse hepatoma Hepa1-6 cells (Figure 2B).Confluent Hepa1-6 cells were inoculated with HSV-1and they were separately cocultured with B6HO3 cellsfor 72 hours in a Transwell. Control Hepa1-6 cells areshown in Figure 2A. B6HO3 cells suppressed the CPEof HSV-1 (Figure 2C), but culture supernatants obtainedfrom 48-hour cocultivation of B6HO3 cells with HSV-1-infected Hepa1-6 cells did not have suppressive activity

Figure 1. B6HO3 cells suppress CPE caused by HSV-1 in J774 macrophage cells.

J774 cells (3 × 105/well) were plated in a bottom well of a 24-well Transwell plate and cultured for 16 hours. After culture medium wasremoved, 0.5 ml of HSV-1 in PBS (104.5 TCID50/ml HSV-1) was added to the well, and the plate was incubated at room temperature for1 hour. Then, PBS was removed and 1 ml of culture medium was added to the well. B6HO3 cells (3 × 105) in 0.5ml of culture mediumwere added to an upper well of the Transwell. Subsequently, the plate was incubated in a CO2 incubator at 37℃ for 72 hours (C).(A), Control J774 cells without HSV-1 and B6HO3 cells. (B), HSV-1-inoculated J774 cells without B6HO3 cells. (D), HSV-1-inoculated J774 cells cultured in the mixture of 0.5 ml of fresh culture medium and 0.5 ml of culture supernatants (sup) obtained from 48-hour cocultivation of HSV-infected J774 cells with B6HO3 cells. Cells were observed under a phase contrast microscope at 72 hours afterHSV-1 inoculation (100×). Scale bar = 50μm

(Figure 2D). Furthermore, the B6HO3-mediatedsuppression of the CPE of HSV-1 still occurred evenwhen B6HO3 cells in the upper well were removed fromthe Transwell at 24 hours after coculture (data not shown).This result indicated that B6HO3 cells responding toHSV-1-infected cells completed the inhibition of HSV-1replication and propagation within the first 24 hours ofcoculture.

Nitric oxide (NO), which has been shown to inhibitHSV-1 replication and cell proliferation,5-8 is a candidatefor the unstable factor produced by B6HO3 cells.Therefore, we next examined the involvement of NO inthe B6HO3-mediated suppression of the CPE of HSV-1by adding the arginine analogs, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester(L-NAME) as inhibitors of NO synthase9 to the cocultureof HSV-1-inoculated Hepa1-6 cells with B6HO3 cells ina Transwell (Figure 3). Figure 3D and E show that neitherinhibitor reversed the suppressive activity of B6HO3 cells,

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indicating that NO is not involved in this phenomenon.Noninvolvement of NO was also confirmed by the factthat nitrite formation was not detected with Griess reagent10

in the culture supernatants from those cocultures (datanot shown). Interestingly, addition of excess L-arginineto the coculture completely reversed the suppressiveactivity of B6HO3 cells (Figure 3F). Thus, these resultsunexpectedly revealed that L-arginine concentrations inculture medium play a crucial role in the B6HO3-mediated suppression of the CPE of HSV-1.

L-Arginine has long been known to be required forthe replication of DNA viruses like HSV-111-14 and forcell proliferation.15 It has been reported that activatedmacrophages produce arginase, thereby depletingarginine in culture medium and causing the suppressionof HSV-1 replication when cocultured with HSV-1-infected cells.16,17 Accordingly, instead of envisaging aninhibitory soluble factor that is directly interfering withHSV-1 replication, one may well hypothesize that B6HO3cells promptly produce arginase in response to HSV-1-infected cells, thereby consuming arginine in culturemedium, resulting in suppression of the CPE of HSV-1.

Figure 2. B6HO3 cells suppress CPE caused by HSV-1 in Hepa1-6 cells.

Hepa1-6 cells (2.5 × 105/well) were plated in a 24-well Transwell plate and cultured for 16 hours. The procedure of coculture withB6HO3 was the same as described in Figure 1. (A), Control Hepa1-6 cells. (B), HSV-1-inoculated Hepa1-6 cells without B6H03 cells.(C), HSV-1-inoculated Hepa1-6 cells cocultured with B6HO3 cells. (D), HSV-1-inoculated Hepa1-6 cells cultured in the mixture of 0.5ml of fresh culture medium and 0.5 ml of culture supernatants (sup) obtained from 48-hour cocultivation of HSV-1-infected Hepa1-6 cellswith B6HO3 cells. Cells were observed under a phase contrast microscope at 72 hours after HSV-1 inoculation (100×). Scale bar = 50μm

Since arginase is not an unstable enzyme, it is necessaryto explain why the coculture supernatants do not havethe CPE-suppressive activity. For this, one can furthersuppose that arginine consumption in culture mediumtakes place without B6HO3 cells secreting arginase intoculture medium; i.e., B6HO3 cells may produce cytosolicarginase and cationic amino acid transporter simultaneously,resulting in rapid reduction of extracellular levels of L-arginine;18,19 alternatively, B6HO3 cells may producemembrane-bound arginase.20,21 It is well established thatmyeloid cells such as macrophages produce arginase uponinduction by a variety of stimuli including T-helper type-2 cytokines, thus being associated with a various aspectsof inflammation and immune suppression.19,22,23 However,there is a dearth of information regarding whetherlymphocytes produce arginase or not.19,24 Recently it hasbeen reported that type 2 innate lymphocytes have theability to produce arginase,25 which lends support to ourhypothesis. Verification of our hypothesis awaits furtherinvestigation of the precise mechanism underlying theB6HO3-mediated suppression of the CPE of HSV-1.

Our previous studies showed that B6HO3 hybridoma

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cells produce IFN-γ in response to interleukin-18 releasedto the interface of the cell conjugates between B6HO3cells and dying bacterial-infected macrophages.1,2 B6HO3hybridoma cells were thought to exhibit the phenotypeof an innate-like T lymphocyte, the parental cell of thishybridoma.3 This notion led us to the findings that subsetsof innate lymphocytes respond to dying bacterial-infectedmacrophages with the production of innate IFN-γ, whichplays a crucial role in the bactericidal activation ofmacrophages, and that this IFN-γ production pathway

Figure 3. Effects of inhibitors of nitric oxide synthase and arginine on the CPE-suppressive activity of B6HO3

The procedure for coculture of HSV-1-inoculated Hepa1-6 cells with B6HO3 cells was the same as that in Figure 2. (A), Control Hepa1-6 cells without HSV-1 inoculation. (B), HSV-1-inoculated Hepa1-6 cells. (C), HSV-1-inoculated Hepa1-6 cells cocultured with B6HO3cells. (D), The same coculture as in (C) in the presence of L-NMMA (NG-monomethyl-L-arginine) (1 mM). (E), The same coculture asin (C) in the presence of L-NAME (NG-nitro-L-arginine methyl ester) (1 mM). (F), The same coculture as in (C) in the presence of arginine(10 mM). Cells were observed under a phase contrast microscope at 72 hours after HSV-1 inoculation (100×). Scale bar = 50μm

plays an important role in the early innate defense againstbacterial infection.26,27 The present study shows that, inaddition to the ability to respond to bacterial infection,B6HO3 cells can respond to HSV-1 infection and suppressthe CPE of HSV-1 without producing IFN-γ. Thus,innate-like T lymphocytes that became the parental Tcell of this hybridoma appear to be capable of dealingwith both viral infection and bacterial infection throughdifferent mechanisms. Further detailed analyses of thephenomenon reported here will pave the way for our

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23. Burrack KS, Morrison TE. The role of myeloid cellactivation and arginine metabolism in thepathogenesis of virus-induced diseases. FrontImmunol 2014; 5: 428.

24. Yu H, Yoo PK, Aguirre CC, et al. Widespreadexpression of arginase 1 in mouse tissues: Biochemicaland physiological implications. J HistochemCytochem 2003; 51: 1151-60.

25. Bando JK, Nussbaum JC, Liang H-E, et al. Type 2innate lymphoid cells constitutively express arginase-1 in the naïve and inflamed lung. J Leukoc Biol2013; 94: 877-84.

26. Kubota K. Innate IFN-γ production by subsets ofnatural killer cells, natural killer T cells and γδ Tcells in response to dying bacterial-infectedmacrophages. Scan J Immunol 2010; 71: 199-209.

27. Kubota K, Kadoya Y. Innate IFN-γ-producing cellsin the spleen of mice early after Listeria monocytogenesinfection: importance of microenvironment of thecells involved in the production of innate IFN-γ.Front Immunol 2011; 2: 26.

functional-hybridoma-based prediction that a subset ofinnate lymphocytes is equipped with versatile functionsfor combating both bacterial and viral infections.

Conflict of interests

The author has no conflicts of interest to declare.

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