T-cell receptor Vβ8 for detection of biologically active streptococcal pyrogenic exotoxin type C

Streptococcus pyogenes is an important human patho-gen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPE). The current method for detection cannot distinguish between the biologically active form of SPE that has been reported to cause foodborne outbreaks and the inactivated toxin that poses no health risk. To measure the biological activity of SPE type C (SPE-C), one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vβ8. With this finding, we used a T-cell line natively expressing Vβ8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element in combination with a B-cell line to present the recombinant SPE-C (rSPE-C) toxin via major histocompatibility complex (MHC) class II to the Vβ8 T-cell receptor (TCR) in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross-reactivity with SPE-B and significant loss of SPE-C biological activity in spiked phosphate-buffered saline while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.


INTRODUCTION
Streptococcus pyogenes is gram-positive, cocci, facultative anaerobic bacterial pathogen that causes over 600 million infections annually worldwide, resulting in over 500,000 deaths per year (Carapetis et al., 2005).These include 10,000 severe infections in the United States (Nelson et al., 2016).This bacterium is commonly spread through airborne droplets produced by infected people but can also be spread in food, and there have even been reports of transmission to cattle and back to humans, causing human illness (Henningsen and Ernst, 1938;Spickler, 2020).Streptococcal bovine mastitis is mainly caused by Streptococcus agalactiae, Streptococcus dysgalactiae, or Streptococcus uberis (Kabelitz et al., 2021).In addition, human Strep.pyogenes phage virulence genes, including those for streptococcal pyrogenic exotoxin (SPE) type C (SPE-C) have been located on mobile genetic elements in Strep.dysgalactiae isolates from bovine mastitis, and it is suggested that these could have an important role in zoonosis (Rato et al., 2010(Rato et al., , 2011;;Alves-Barroco et al., 2019).Consumption of foods such as milk, salads, eggs, mayonnaise, tuna, potatoes, cheese, potato salad, and chicken salad contaminated with Strep.pyogenes, which secretes an array of toxins comprising proteases, hemolysins, and superantigens, have been associated with foodborne outbreaks (Hill et al., 1969;Baiter et al., 2000;Katzenell et al., 2001;Kuusi et al., 2006;Wong and Yuen, 2012).These foodborne illness outbreaks usually occur in confined settings, examples include an army unit, an industrial plant, and a prison (Hill et al., 1969;Lossos et al., 1992;Bar-Dayan et al., 1996;Levy et al., 2003;Kaluski et al., 2006), or after attending a social event, for example, a school banquet, wedding, or private party (Horan and Cournoyer, 1986;Gallo et al., 1992;Farley et al., 1993;Levy et al., 2003;Kaluski et al., 2006).It has been shown that bacterial toxin itself (without the bacterium) causes histopathological changes in kidney, spleen, and thymus tissues and mortality (Rasooly et al., 2010).Streptococcus pyogenes produces a group of 13 distinct types of SPE that overstimulate the immune system causing conditions such as sepsis, scarlet fever, guttate psoriasis, and streptococcal toxic shock syndrome (STSS; Hauser et al., 1991;Leung et al., 1995;Spaulding et al., 2013;Commons et al., 2014).The SPE-C are commonly isolated from STSS patients.The SPE-C interacts with the major histocompatibility complex class II molecules on antigen presenting cells, which then present the toxin to T-cells expressing Vβ2.The activated T-cells then release enormous amounts of inflammatory cytokines, such as IFN-γ and TNF-α, leading to fever and shock.There are existing assays for detection of streptococcal exotoxins using ELISA (Cook et al., 2007;Garber et al., 2010), enzyme-linked fluorescence assay (Vernozy-Rozand et al., 2004), and flow cytometry (Miyamoto et al., 2003).In addition, PCR has been used to verify the presence of SPE-C genes while not predicting SPE-C expression level (Fosheim et al., 2011;Berman et al., 2014;Chung et al., 2014).In food processing, heat treatment methods that kill bacteria also inactivate some toxins, but none of these detection methods can discern between biologically active SPE, which pose a foodborne outbreak health risk and the inactive forms of exotoxins.An in vivo rabbit bioassay has been used to measure toxin biological activity through the development of rashes resembling scarlet fever rashes when the animals were challenged with SPE-C, but these methods rely upon expensive procedures using live animals and raise ethical concerns.To give the food industry a way to test if inactivation methods used are effective and to ensure the safety of their products, as well as to help public health officials trace the source of food poisoning outbreaks in which streptococcal pyrogenic exotoxin are suspect and to stop similar outbreaks in the future, we developed a cell-based assay that can discern between biologically active and inactive SPE-C.

Cell Culture
Cells of a Jurkat T-cell line engineered to express luciferase under control of a promoter containing multiple copies of nuclear factor of activated T-cells (NFAT) response elements, GloResponse NFAT-RE-luc2 Jurkat, were obtained from Promega (Madison, WI).Cells of Raji B-cell line (ATCC CCL-86) were obtained from the American Type Culture Collection (ATCC, Rockville, MD).Cell lines were cultured in RPMI 1640 medium from Gibco/Thermo Fisher (Waltham, MA) supplemented with 10% fetal bovine serum from HyClone (Logan, UT), 1× minimum essential medium (MEM) NEAA (Gibco) and 100 mM sodium pyruvate (Gibco).For maintenance of Jurkat cells, 200 μg/mL Hygromy-cin B (Gibco/Thermo Fisher) was additionally added to the cell culture medium.For maintenance of Raji cells, 100 units/mL penicillin (Gibco/Thermo Fisher) and 100 μg/mL streptomycin (Gibco/Thermo Fisher) were added to cell culture medium.Murine splenocytes were obtained by syringe and needle disruption of tissue from spleens aseptically removed from 8-wk-old female C57BL/6 mice.Disruption of tissue was performed in Russ-10 cell culture medium prepared from RPMI 1640 medium without glutamine (Gibco) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 1× MEM NEAA, 1× antibiotic-antimycotic (Gibco) and 50 mM β-mercaptoethanol (Sigma).Harvested splenocytes were centrifuged at 200 × g for 10 min at 4°C, red blood cells were removed in lysis buffer, cells were centrifuged a second time and finally resuspended in Russ-10 medium.Viable cell counts were made on a hemacytometer on the basis of exclusion of trypan blue.All cell lines and splenocytes were incubated at 37°C with a humidified 5% CO 2 atmosphere for maintenance.

Toxins
The staphylococcal enterotoxins SEA and SEE and SPE type B (SPE-B) and recombinant SPE type C (rSPE-C) were obtained from Toxin Technology (Sarasota, FL).The rSPE-C toxin produced by expression in Escherichia coli is comparable to the wild type (Li et al., 1997).

Recombinant SPE-C Splenocyte Proliferation Assay
The proliferation of splenocytes in response to SEA, SEE, and rSPE-C was quantified using the 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay from Calbiochem (San Diego, CA).In this assay, the thymidine analog BrdU is incorporated into DNA as it is newly synthesized in proliferating cells.The amount of BrdU taken up by cells is measured spectroscopically using a horseradish peroxidase-conjugated secondary antibody.Splenocytes in Russ-10 medium at a concentration of 1 × 10 6 cells/mL were aliquoted 200 mL per well in 96-well plates and were exposed to SEA, SEE, and rSPE-C with 37°C static incubation for 72 h under 5% CO 2 .After this time the cells were fixed and washed according to the manufacturer's protocol.A BioTek (Santa Clara, CA) microplate reader was used to measure the absorbance of the samples at 450 and 620 nm.
All experimental animal procedures were performed in accordance with institutional guidelines for husbandry, which have been approved by the Institutional Animal Care and Use Committee of the USDA, West-

Measurement of IL-2 Secretion by ELISA
The IL-2 concentration in cellular supernatant was measured using BD OptEIA mouse IL-2 ELISA Set (BD Biosciences), following the manufacturer's protocol.

Recombinant SPE-C Biochemiluminescence Cell-Based Assay
The mixed culture assay was performed by combining 50 μL each of GloResponse Jurkat cells and Raji cells at a concentration of 2 × 10 6 cells/mL in cell culture medium without Hygromycin B per well of a clear 96well plate followed by addition of 25 μL of an rSPE-C solution to achieve the desired target toxin concentration after dilution in the cell suspension.The plated samples were incubated at 37°C for 5 h and then equilibrated to room temperature for 12 min.Appropriate controls comprised samples into which no rSPE-C was added for negative control and any sample containing toxin as a positive control.Luciferace expression was quantified according to the manufacturer's methods for the BioGlo luciferase assay system (Promega).Luminescence of the products of the reaction were recorded by employing a BioTek microplate reader.The assay is graphically illustrated in Figure 1.

Flow Cytometry
The expression of Vβ8 by GloResponse Jurkat cells was demonstrated by flow cytometry using a FACSAria Fusion instrument manufactured by BD Biosciences (San Jose, CA) running FACSDiva software.Phycoerythrin (PE) labeled anti-Vβ2 mAb and fluorescein isothiocyanate (FITC) conjugated anti-Vβ8 antibody from BD Pharmingen (San Jose, CA) were used to label cells expressing Vβ8 and Vβ2.Data were subsequently analyzed using FlowJo software from BD Biosciences.

Statistical Analysis
SigmaStat 3.5 for Windows from Systat Software (San Jose, CA) was used to perform statistical analysis of data.The proliferation of splenocytes in response to SEA, SEE, and rSPE-C, measurement of active rSPE-C by biochemiluminescence and IL-2 secretion were performed in triplicate.A value of P < 0.05 was used as the basis of statistical significance.A t-test analysis was used to determine whether there were any significant differences between thermal treatments and unheated controls on rSPE-C activity.

Ex Vivo Mouse Cell-Based Activity Assay
To evaluate the ability of an ex vivo assay to quantify the biologically active SPE-C, we tested rSPE-C and utilized the immune response and measured the superantigen activity of rSPE-C at a concentration of 1 μg/mL on mouse splenocyte cells.As positive controls for the assay we used Staphylococcal enterotoxins type A and E, superantigens that are known to induce Tcell activation in splenocytes and are known sources of foodborne disease (Rasooly et al., 2017).After 72 h of incubation BrdU stain was added to cell cultures and cytokine secretion and T-cell proliferation were spectroscopically measured.Our results, presented in

Jurkat T-Cell Line Expresses Vβ8
Prior work reported, based on proliferation and binding studies, that SPE-C specifically binds and activates

IL-2 Secretion by T-Cell Line Natively Expressing TCR Vβ8 for Detection of Active rSPE-C
We applied the genetically engineered human Jurkat T-cell line expressing Vβ8 TCR and the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the rSPE-C-MHC (major histocompatibility complex) class II to the Vβ8 TCR of the engineered T-cell line, forming a bridge between the B-cell and T-cells, leading to T-cell activation and cytokine secretion.
As shown in Figure 4, rSPE-C induced a significant amount of IL-2 secretion over 72 h, with no cross detection of SPE-B.This demonstrates that T-cells carrying the TCR Vβ8 combined with a Raji B-cell can be used for specific detection of active SPE-C.

Light Emission Intensity for Measuring Biologically Active rSPE-C
To examine if rSPE-C can be detected more rapidly than by cytokine secretion, the above mixed cell cultures were exposed to rSPE-C or to SPE-B for 5, 24, and 48 h and luciferase expression was measured by emission of light.As shown in Figure 5, exposure of the above mixed cells cultures to SPE-B did not result in the production of visible light, while rSPE-C induced significant production of visible light after 5 h and this light emission was doubled by 24 h.Therefore, this sig-nificant production of light emission demonstrates that the genetically engineered Jurkat T-cell line expressing the luciferase reporter gene combined with B-cell can be used to detect active rSPE-C rapidly and specifically.

T-Cell Line Natively Expressing TCR Vβ8 for Detection of Active SPE-C in Milk
Because milk and milk products were previously implicated in SPE-C caused outbreaks, we examined whether this in vitro cell-based assay can detect rSPE-C in milk and distinguish between the biologically active form that has been reported to cause foodborne outbreaks and the inactivated toxin that poses no health risk.Milk samples and PBS were spiked with 1 μg/mL of rSPE-C and then were pasteurized at 63°C for 30 min, 72°C for 15 s, or 89°C for 1 s, or heated at 100°C for 5 min.As shown in Figure 6, pasteurization at 63°C for 30 min or 72°C for 15 s, or heat treatment at 100°C for 5 min significantly decreased but did not completely abolish SPE-C biological activity in PBS compared with the unspiked control.However, SPE-C in milk is heat stable.Pasteurization or heat treatment at 100°C for 5 min did not reduce the biological activity of rSPE-C in milk.

DISCUSSION
Reverse PCR dot-blot analysis studies have shown that rSPE-C binds with very high binding affinity to human TCR Vβ2 (Li et al., 1997).Ohara-Nemotol and M. Kaneko, stimulated human peripheral blood   (Abe et al., 1992).The ability to rapidly detect active rSPE-C is very important epidemiologically to identify the causes of Streptococcal outbreaks and to stop similar outbreaks in the future.We demonstrated a novel in vitro cell-based assay combining a Raji B-cell line and a genetically modified luciferase expressing Jurkat T-cell line for detection of rSPE-C activity by measuring IL-2 secretion or more rapidly and practically by measuring light emission in only 5 h after exposure to rSPE-C.Such ability to discern active rSPE-C is important for food safety and for development of processing methods for toxin inactivation in milk or other foodstuffs contaminated with SPE-C.It was suggested that food matrix components that influence Ca ++ influx can falsely affect the NFAT-RE-luciferase assay, but our results in Figure 6 show that milk did not falsely affect the NFAT-RE-luciferase assay.This activity assay can be used for rapid detection of active rSPE-C without cross-reactivity to SPE-B.We cannot rule out nonspecific response to other toxin variants or molecules.However, if this were to become an issue or if it were necessary to increase the sensitivity of the assay, immunomagnetic beads could be utilized to eliminate the confounding components by extracting and concentrating SPE-C from food as described previously (Rasooly andDo, 2008, 2009).Heat treatments are used to destroy spoilage microorganisms and reduce the number of viable pathogens such as Strep.pyogenes.However, despite the inactivation of the causative microorganisms, the risk of heat stable toxin remains.We utilized our in vitro cell-based assay method to assess the effectiveness of pasteurization and heat treatment at 100°C for 5 min on rSPE-C spiked milk or PBS.We observed that rSPE-C stability is dependent on the medium in which the toxin is present.We observed significant loss of rSPE-C activity after heat treatment in PBS but not in milk.Milk protected rSPE-C activity and made it more thermostable in that medium.This was true over the range of conditions of pasteurization we applied.Even heat treatment at 100°C for 5 min reduced rSPE-C activity in PBS but not in milk.The difficulty in inactivating rSPE-C demonstrates the need to prevent early contamination of milk by Strep.pyogenes.Once rSPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.Heat stable rSPE-C will remain long after the Strep.pyogenes has been killed by pasteurization.
Rasooly et al.:  Vβ8 FOR DETECTION OF STREPTOCOCCAL PYROGENIC EXOTOXIN-C ern Regional Research Center.Mice were euthanized by rapid cervical dislocation to minimize suffering, following protocol no.21-1 approved on February 2, 2021.

Figure 1 .
Figure 1.Graphical illustration of cell-based assay.B-cells carrying major histocompatibility complex (MHC) class II on their surface bind streptococcal pyrogenic exotoxin type C (SPE-C) and serve as antigen presenting cells (APC) to genetically modified T-cells via their T-cell receptors.Activation of the genetically modified T-cells leads to luciferase expression.The assay was performed on a microtiter plate, and photon emission was detected using a luminometer.
Figure 2, show that unlike SEA and SEE, we are unable to detect any rSPE-C superantigen activity as measured by murine T-cell proliferation and IFN-γ secretion.
T-cells expressing Vβ2(Ohara-Nemoto and Kaneko, 1996;Li et al., 1997).The present study demonstrates the previously unreported SPE-C activation of T-cells expressing Vβ8.The Jurkat cell line used in this study was confirmed to natively express Vβ8 T-cell receptor (TCR) but not express TCR Vβ2 by staining with FITC labeled anti-Vβ8 mAb or PE labeled anti-Vβ2 mAb and subsequent flow cytometric analysis.The histograms in Figure 3, representing the FITC and PE fluorescence channels, show the binding of FITC conjugated anti-Vβ8 mAb to Vβ8 expressed by this cell line and absence of binding of PE conjugated anti-Vβ2 mAb.The overlap of the histograms in the Vβ2 case confirms the absence of nonspecific binding of the antibody to the cells.

Figure 2 .
Figure 2. Murine splenocyte proliferation and cytokine secretion.After incubation for 3 d, the ex vivo methods were unable to detect recombinant streptococcal pyrogenic exotoxin type C (rSPE-C) at a concentration of 1 μg/mL either by activation of mouse splenocyte T-cell proliferation response as measured by newly synthesized DNA (a) or by IFN-γ secretion (b).OD = optical density.Error bars represent SE.

Figure 3 .
Figure 3. Vβ expression on Jurkat T-cells.Flow cytometric histograms of the fluorescence of live human Jurkat T-cells demonstrate expression of Vβ8 TCR but lack of expression of Vβ2 TCR.Jurkat T-cells were stained with FITC conjugated anti-Vβ8 mAb and PE conjugated anti-Vβ2 mAb.PE = phycoerythrin, FITC = fluorescein isothiocyanate, TCR = T-cell receptor.

Figure 4 .
Figure 4. Detection of recombinant streptococcal pyrogenic exotoxin type C (rSPE-C).Jurkat T-cell and Raji B-cell mixed culture were incubated for 72 h with rSPE-C, streptococcal pyrogenic exotoxin type B (SPE-B), and cell culture media as control.IL-2 secretion was measured by ELISA.Error bars represent SE.

Figure 5 .
Figure 5. Bioassay for measuring biologically active recombinant streptococcal pyrogenic exotoxin type C (rSPE-C).Genetically engineered Jurkat T-cell line expressing the luciferase reporter gene was plated with Raji cells in a 96-well plate.The mixed cultures were co-incubated with 1 μg of rSPE-C or control without toxin.The photon emission was detected by a luminometer and reported as relative luminescence units (RLU).SPE-B = streptococcal pyrogenic exotoxin type B. Error bars represent SE.