Characterization of T-cell subsets in response to foot-and-mouth disease bivalent inactivated vaccine in Chinese Holstein cows

ABSTRACT Traditional vaccine efficacy evaluation predominantly relies on antibody levels, while the assessment of T-cell responses remains underexplored. In this study, we employed multi-parameter flow cytometry to comprehensively analyze T-cell responses in cows vaccinated against foot-and-mouth disease (FMD). We categorized the cows into high and low vaccine potency groups based on antibody levels and investigated differences in T-cell responses between these groups. Phenotypic analysis revealed a significant reduction in CD4, CD8, and γδ T cells in peripheral blood following FMD vaccine inoculation, concomitant with altered CD44 expression. Intriguingly, FMD vaccination induced a marked increase in the percentage of CD4+CD8+ double-positive (DP) T cells in cow peripheral blood. Notably, cows with high vaccine potency exhibited a significantly higher proportion of CD4+CD8+ DP T cells compared to those with low potency post-vaccination, suggesting their potential involvement in FMD vaccine-induced immune protection, possibly through the regulation of B-cell antibody secretion. Additionally, FMD vaccination led to the generation of central memory CD4 and CD8 T cells but not γδ T cells. Our groundbreaking findings shed light on the latent function of CD4+CD8+ DP T cells in FMD vaccine-induced immune protection and established a novel method for evaluating FMD vaccine efficacy based on T-cell responses. IMPORTANCE Vaccination plays a crucial role in the prevention and control of FMD; however, outbreaks persist occurring worldwide. Assessing the immune response to FMD vaccines is essential for effective prevention of FMD. In this study, a seven-color flow cytometry protocol was developed to systematically evaluate the T-cell response of Chinese Holstein cows vaccinated with FMD bivalent inactivated vaccine. Our findings showed that while most T-cell subsets (%) decreased post-vaccination, a significant increase was observed in CD4+CD8+ DP T cells, which was consistent with the levels of specific foot-and-mouth disease virus (FMDV) antibodies. These findings suggested that CD4+CD8+ DP T cells could serve as a potential biomarker for the evaluation of cellular and humoral responses to FMDV vaccination. Additionally, we should be aware of the potential decline in cellular immunity among cattle during FMD vaccination, as this may increase the risk of other pathogen-related issues.

F oot-and-mouth disease (FMD) is a severe and highly infectious disease caused by the foot-and-mouth disease virus (FMDV).It is characterized by fever, blistering, and ulceration of oral mucosa and hoof (1).There are seven serotypes of foot-and-mouth disease virus, including A, C, O, Southern African Territories SAT 1, SAT 2, SAT 3, and Asia 1 (2).FMD inflicts significant economic losses on dairy farms worldwide, impact ing milk yield and reproductive performance in dairy cows (3,4).Vaccination serves as the primary means of controlling FMD transmission in dairy cows.However, the protection conferred by FMD vaccines is relatively short-lived and may vary, depending on the strain, but it can be improved and extended by optimizing vaccine efficacy and administering booster doses (1).Therefore, it is crucial for veterinarians to comprehend the efficacy of FMD vaccines and fine-tune the dosing and immunization intervals.
Various methods are available for evaluating the effectiveness of FMD vaccines.Conventionally, prior to registering a new vaccine strain, the vaccine's efficacy is assessed through the inoculation of cattle's tongues with the virulent (homologous) FMDV as the vaccine strain, followed by monitoring for 21-28 days.If the challenge virus is prevented from spreading to the feet and causing blisters, cows are considered as protected (1).However, this method is laborious and expensive for routine use.While challenge tests cannot be replaced entirely, there is a need for alternative evaluation methods.The immune response to FMDV is characterized by the induction of lymphocyte stimulation responses and the production of serotype-specific neutralizing antibodies (5).Antibody levels, commonly measured using virus neutralization tests or indirect enzyme-linked immunosorbent assays (ELISA) (6,7), are widely employed as predictors of protection against FMD in vaccinated animals.Nevertheless, few studies have explored the T-cell responses induced by FMD vaccines in cows (8,9), despite the potential of this approach as a novel means of evaluating vaccine effectiveness.
In China, FMD has caused substantial economic losses to dairy farms, with serotypes O and A being endemic in many provinces of northwest and southeast China since 2010 (10).The bivalent FMD vaccine has effectively controlled the spread of FMDV in China.However, The recent outbreak of FMD in developing countries has served as a wake-up call (11,12), reminding us of the mutating nature of viruses and the possibility of a more threatening FMDV subtype emerging in the future.While the neutralizing antibody levels against FMD have been extensively studied and nearly established, animals with low levels or with absence of neutralizing antibodies may still be protected when challenged with FMDV (13).Consequently, a more comprehensive understanding of the animal immune responses induced by FMD vaccines is imperative for developing and reserving additional techniques and methods for evaluation of FMD vaccine.This study evaluates the dynamic changes in T-cell subsets in the peripheral blood of cows following inoculation with bivalent inactivated FMD vaccine (serotypes O and A) and compares the differences in T-cell activation and effects in Chinese Holstein cows that responded to the bivalent vaccine with varying levels of antibody response.Our goal is to establish a method for evaluating bivalent inactivated FMD vaccine efficacy based on T-cell responses.

Animal selection and vaccination
In this experiment, 210 healthy cows were carefully selected from 1,675 lactating Chinese Holstein cows from a well-managed pasture in Jiangsu Province, China, with similar birth dates, body weights, milk yields, and somatic cell scores.None of the selected cows had a history of revelation to the FMD virus and vaccine.Each selected cow was given 2 mL of the vaccine according to the vaccine instructions.During the experiment, all the cows were placed in the same room.The vaccine used in this study was traditional and commercial water in oil, bivalent inactivated vaccine (O 2 + AKT-III strain) (Tiankangbio, Xinjiang, China; http://www.tcsw.com.cn/html/html/pc/prodetail.html?id=66&t).Peripheral blood of cows was collected from vacuum blood collection vessels containing EDTA anticoagulant by jugular vein sampling at 0, 7, and 14 days post-immunization (dpi).Peripheral blood mononuclear cells (PBMC) and plasma samples were isolated from the collected blood.These plasma samples were used to detect virus-specific antibodies.

Analysis of bivalent inactivated FMD vaccine antigens
In the inactivated FMD vaccines, intact virion are important immune antigens, and their content, integrity, and stability determine the immune effect of the vaccine (14).The antigen of the bivalent inactivated FMD vaccine used in this study was analyzed by negative stain electron microscopy (Hitachi, Tokyo, Japan).Purified bivalent inactivated FMD vaccine samples was added to the glow-discharged, Formvar/carbon-coated copper grids to precipitated for 1 minute, and the float was absorbed by filter paper.Then uranium dioxyacetate drops were added to the copper grids to precipitate for 1 minute, and the filter paper absorbed the float.After drying at normal temperature, the images were detected by scanning projection electron microscopy at 80 kv.

Antibody detection and phenotypic classification
The plasma samples collected from vaccinated cows at 0, 7, and 14 dpi were assayed using commercial bovine FMDV-A-Ab and FMDV-O-Ab ELISA Kits (Qiaodu, Shanghai, China) to determine the specific antibody concentrations against the bivalent inactivated FMD vaccine.The optical density (OD) values of the samples were read at 450 nm using an ELISA plate reader (Tecan, Shanghai, China), and the OD values were corrected according to a previous method (15).
Previous studies have shown that antibody-mediated immune responses are characterized by quantitative characteristics that can be phenotypically classified (16).Therefore, we used previous methods to rank vaccine effectiveness that was assessed using antibody response levels.Briefly, the corrected OD values of all antibodies were logarithmically processed.The values on days 7 and 14 after vaccination were subtracted from the values before vaccination to obtain the antibody response level, reflecting the vaccine's efficacy.The first and third quartiles of the antibody response values were used as critical values to categorize cows into high response (HR) and low response (LR) groups for both serotype O and A specificities.Blood samples from cows in the HR (n = 15) and LR (n = 15) groups were used for the subsequent isolation of PBMCs.

Isolation of PBMC
Density gradient centrifugation was used to separate PBMC from dairy cows.In brief, the blood sample was diluted with equal-volume phosphate buffer solution (PBS).Next, the diluted blood sample is gently dripped along the wall of the tube onto an equal volume of separating liquid.After centrifugation at 650 g for 30 min, the second layer of cell fluid was absorbed into 10-mL PBS for washing.The cells were resuspended in 500 µL of RPMI-1640 nutritional medium (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Cytiva, Tauranga, New zealand) and counted using Automated Cell Counter (Invitrogen, Shanghai, China).The PBMCs were then diluted with nutritional medium containing 10% FBS to a concentration of 1 × 10 7 viable cells/mL.

Flow cytometry analysis
The PBMCs were plated in 96-well V-bottom plates, with 200 µL in each well.After centrifugation at 1,600 revolutions per minute for 5 min, the cells were suspended in a 50-μL mixture of antibodies with fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin [BSA]-PBS).The list of antibodies is shown in Table 1.Next, the cells were stained for 20 minutes at room temperature in the dark.The cells were then washed once with FACS buffer and rotated at 400 g for 5 min at 4°C.At least 100,000 cells were obtained for FACS analysis.FACS LSR Fortessa (BD Biosciences, Franklin Lakes, NJ, USA) was used for flow cytometry, and FlowJo software (Tree Star Inc., Ashland, OR, USA) was used for the data analysis.The gating policy is based on fluorescence minus one and is shown in Fig. 1.

Statistical analyses
All data were analyzed using SPSS software (IBM Corporation, Armonk, NY, USA).The normality and homogeneity of variance of the data were tested using the Kolmogorov-Smirnov test.Data are expressed as mean ± standard deviation (SD).For comparisons between two groups with normally distributed data, one-way Student t-tests were  performed.For comparisons among multiple groups, analysis of variance tests were used.A P value of less than 0.05 was considered significant.

Intact structure of FMDV capsid in bivalent inactivated FMD vaccine
Chemical inactivation of viral particles to produce commercial vaccines may make them more unstable and can rapidly transform them into immunogenically incompetent pentamer subunits (17,18).We observed the structure of the virus in the vaccine using negative stain electron microscopy.The results showed that many FMD virions were observed in the vaccine used in this study (Fig. 2A).The FMD virion appeared spherical; the capsid structure was complete; and the edges were smooth.No dissociated pentameric subunits were observed (Fig. 2B).

Significant increase in plasma antibody levels after vaccination
To assess the efficacy of the foot-and-mouth disease vaccine used in this study and to identify potential T-cell response markers, we measured plasma antibody concentrations of cows after vaccination using ELISA.As expected, higher levels of FMDV (serotypes O and A) antibodies were detected at 7 and 14 dpi (Fig. 3A and B).Additionally, plasma FMDV (serotype A) antibodies at 14 dpi were significantly lower than those at 7 dpi but remained significantly higher than before immunization.

Bivalent inactivated FMD vaccine induces decreased percentage of αβ and γδ T cells but not CD4 + CD8 + double-positive T cells
FMD is characterized by significant lymphocytopenia associated with immunosuppres sion (19).However, the dynamic changes in different T-cell subsets in peripheral blood of cows after inoculation with bivalent inactivated FMD vaccine have not been well characterized.To address this, we employed a seven-color flow cytometry panel to characterize T-cell immune responses in cows following FMD vaccination.As shown in Fig. 4, the percentages of αβ (WC1 − CD3 + ) and γδ (WC1 + CD3 + ) T cells in the peripheral blood of cows at 7 dpi with bivalent inactivated FMD vaccine were significantly reduced compared to those before vaccination.These reductions were recovered at 14 dpi, where the percentage of γδ T cells had fully returned to pre-vaccination levels, while αβ T cells may take longer.These changes did not significantly differ between the HR and LR groups, suggesting that these indicators may not replace existing methods of evaluating vaccines using antibody levels.Furthermore, it remained unclear whether the dynamics of different T-cell subsets differed between the two groups.Therefore, we distinguished three T-cell subsets (CD8, CD4, and CD4CD8 double-positive) in the WC1γδ − CD3 + T cells (Fig. 5A).As illustrated in Fig. 5B and C, FMD vaccination elicited significantly lower percentages of CD4 and CD8 T cells in the two groups at 7 and 14 dpi.Intriguingly, the percentage of CD4 + CD8 + T cells in the HR and LR groups increased significantly to varying degrees after inoculation with bivalent inactivated FMD vaccine (Fig. 5D).The percentage of CD4 + CD8 + double-positive (DP) T cells in the HR group was significantly higher than that in the LR group at 7 dpi.At 14 dpi, the percentage of CD4 + CD8 + DP T cells remained significantly higher than the pre-vaccination level.This suggests that the increase in the percentage of CD4 + CD8 + DP T cells in the peripheral blood of cows after vaccination may serve as a signal of the efficacy of the bivalent inactivated FMD vaccine.

Bivalent inactivated FMD vaccine induces differential activation of CD4, CD8, and γδ T cells in cows
To determine the activated phenotype of T cells in vaccinated cows, vaccine-experienced T cells were identified based on high or low expression of CD44.Phenotypic analysis of CD4 T cells revealed a reduced percentage of the CD44 +high phenotype in T cells from cows vaccinated with the bivalent inactivated FMD vaccine (Fig. 6A).The percentage of the CD44 +low phenotype and the mean fluorescence intensity (MFI) of CD44 in CD4 T cells remained unchanged (Fig. 6B and C).Following FMD vaccination, CD8 T cells exhibited a reduced percentage of the CD44 +high phenotype and an increased percentage of the CD44 +low phenotype (Fig. 6D and E).The MFI of CD44 in CD8 T cells showed no significant difference before and after vaccination (Fig. 6F).Similar phenotypic changes of CD44 +high or CD44 +low were observed in γδ T cells and CD8 T cells before and after vaccination (Fig. 6G through I).

Significant up-regulation of memory T cells at 7 dpi
The expression of cell surface memory markers CD45RO and CD27 is associated with functional segregation of T-cell memory subsets in Holstein cows.CD27, a costimulatory molecule, has been normally used to identify the stages of T-cell differentiation (20).Normally, bovine memory T cells are identified based on CD45RO expression (21).The naïve cells are defined as CD45RO − CD27 + , and central memory T cells are defined as CD45RO + CD27 + in this study.As proven in Fig. 7, the percentage of central memory T cells in CD4 and CD8 T cells increased significantly at 7 days after vaccination compared with pre-vaccination and returned to pre-immunization levels at 14 days.However, this difference was not significant in γδ T cells.The dynamic changes in naïve phenotype cells in CD4, CD8, and γδ T cells were different.In CD4 T cells, the percentage of naïve T cells was significantly up-regulated at 7 dpi and returned to the pre-immunization level at 14 dpi.In CD8 T cells, the percentage of naïve T cells was not significantly increased at 7 dpi but significantly decreased at 14 dpi.There existed no crucial distinction in the percentage of γδ T cells with naïve phenotype before and after vaccination.

DISCUSSION
Previous studies evaluating vaccine efficacy have commonly focused on antibody levels as a measure, overlooking the significance of T cell-mediated cellular immunity (22).However, robust T-cell responses are vital for pathogen clearance and the formation of strong memory responses (23).In this research, we utilized a seven-color flow cytometry method to evaluate cellular immunity in dairy cows, specifically the T-cell immune response to the bivalent inactivated FMD vaccine.By doing so, we aimed to establish a standardized method for evaluating the efficacy of bivalent inactivated FMD vaccine based on T-cell responses.While a previous study had established a cattle T-cell phenotyping method using an eight-color panel, the function of identified cell subsets in vaccinated or infected cattle had not been determined (24).In contrast, the cell subset functions identified in our study were well established, making them more reliable for the evaluation of T-cell responses in dairy cows.In this study, we did not set up virus-infected cows as controls.On the one hand, it is too costly to establish an adequately powered parallel group trial of FMDV infection.On the other hand, the level of virus-specific antibodies is sufficient to determine vaccine immunogenicity, which is widely accepted by researchers and farmers (22,25).In addition, a large number of viral particles with intact capsid structure were observed by negative staining scanning electron microscopy.This result further assured the immunogenicity of the vaccine.Before assessing the T-cell immune response to bivalent inactivated FMD vaccine in dairy cows, we detected virus-specific antibodies by ELISA tests; the result showed that a significant increase in antibody concentrations was detected in the plasma of Chinese Holstein cows at 7 dpi.Although antibody concentrations decreased at 14 dpi, they remained above pre-immunization levels.In our study, OD value and change regularity of antibody of vaccinated cows were compatible with earlier research (26,27).The FMD vaccine shows different efficacies in different individual cows (28).Therefore, we used the level of vaccine-specific antibodies produced by individuals to classify cows into high-and low-response groups, which represent high vaccine efficacy and low vaccine efficacy in cows, respectively.Since antibody-mediated immune response and cell-mediated immune response are mutually regulated (29), this grouping design may help us screen out T-cell response indicators that can take into account antibody-level factors to evaluate vaccine efficacy.This clustering strategy has been used in dairy cattle breeding efforts for disease resistance (30).Previous studies have assessed the possibility of phenotypic classification of immune responses in cows using mean ± SD and quartiles (31,32).In this study, ranking responses were divided into HR and LR using quartiles, which reduced the variability in the number of cows classified as HR or LR due to means and SD differences and allowed comparison of phenotypic with genetic rankings.
Based on the above grouping of cows, we characterized the dynamic changes in different T-cell subsets in the peripheral blood of cows after vaccination with inactivated FMDV.We noted that the percentage of CD4, CD8, and γδ T cells in the peripheral blood of cows after FMDV vaccination was significantly reduced and did not fully recover 14 days after vaccination.Peripheral blood lymphocytopenia, or lymphocytopenia, after FMDV infection is a common feature in pigs (33), cattle (34), and mice (35).Lymphopenia is considered to be one of the important mechanisms by which FMDV evades the host immune response and induces immune suppression (36).A previous research on cattle indicated that FMDV down-regulates CD4, CD8, and γδ T cells up to 48 h post-immuni zation (37).By contrast, Garcia-Valcarcel et al. recorded no significant difference in the numbers of circulating CD4 T cells in the first vaccinated/infected bovines (38).One possible reason is that the vaccine used in that study was not the inactivated virus but the capsid protein VP1.Our study is the first to report that an bivalent inactivated FMD vaccine causes T-cell depletion in dairy cows lasting up to 14 days.Lymphopenia may delay pathogen clearance in favor of macrophage stimulation (39) and the accompany ing "cytokine storm" that leads to host organ dysfunction (40) in previous cases of other viral infections (SARS-CoV-2, COVID-19, and HIV).These injuries may increase the risk of infections by other pathogens.This suggests to us that cows may have a higher risk of disease for at least 14 days after FMD vaccination and require additional special care.In our current results, we found no distinguished distinctions between the HR and LR groups, and changes in these T-cell subsets may not be directly related to B cell-mediated antibody production.Surprisingly, we observed a significant up-regulation of CD4 + CD8 + DP T cells in the peripheral blood of cows after vaccination with the bivalent inactivated FMD vaccine.CD4 + CD8 + DP T cells were previously considered a developmental stage of T cells in the thymus, and it was uncommon for mature CD4 + CD8 + DP T cells to be present in peripheral blood (41).Unexpectedly, previous studies in the past decades have shown the presence of mature CD4 + CD8 + DP T cells in peripheral blood of humans (42), dogs (43), and pigs (44).Earlier studies have indicated that it is primarily CD4 + CD8 + DP T cells that are involved in the interferon gamma responses among all subtypes of T cells in pigs (45).Besides, in porcine reproductive and respiratory syndrome virus disease, CD4 + CD8 + DP T cells appear to have a certain cytotoxic function (46).The CD4 + CD8 + DP T cells make up a enormous percentage of T lymphocytes in pigs (47), in stark contrast to cows, where the function of CD4 + CD8 + DP T cells in viral immunity has not been studied.Strikingly, The percentage of CD4 + CD8 + DP T cells in αβ T cells of cows in the HR group was distinguishably higher than that in the LR group at 7 dpi.This suggests that the mount in the proportion of CD4 + CD8 + DP T cells may be connected with antibody secretion in cows induced by FMDV vaccination.CD4 helper T cells regulate the activation and differentiation of B cells into antibody-producing plasma cells (48)(49)(50).In recent years, a new class of IL-21-producing memory CD4 helper T cells has been identified, suggesting that there may be many more helper T cells that have not been discovered (51).This new population, known as peripheral T helper cells (Tph), has essential similarities to Tfh cells.Therefore, we speculate that the increase in CD4 + CD8 + DP T cells in αβ T cells of dairy cows after inoculation with FMD vaccination might be a Tph.
The mechanisms of lymphopenia induced by FMDV are still ambiguous.One hypothesis is that the interaction of FMDV with integrin receptors alters leukocyte adhesion properties and thus lymphocyte trasport during the peak period of viremia, which might be responsible for the down-regulation of the T-cell subpopulation after FMDV infection, as it has been revealed that FMDV infects cells via integrins of αβ T cells (52).Like integrin expression, CD44 expression is up-regulated after lymphocyte action, thereby promoting their movement in the extracellular matrix through interactions with hyaluronic acid and fibulectin (53).In this research, the expression of CD44 in CD4, CD8, and γδ T cells was detected.The results showed that the percentage of CD4, CD8, and γδ T cells expressing CD44 decreased significantly after inoculation with FMDV vaccine, and their changes were consistent with those of CD4, CD8, and γδ T cells.Furthermore, we found no distinguished distinctions in the MFI of CD44 in CD4 and CD8 T cells before and after vaccination.The MFI of CD44 in γδ T cells of the HR group increased significantly at 7 dpi compared to before vaccination.Together, these results indicate that the percentage of CD4, CD8, and γδ T cells expressing CD44 was greater in the decreased T cells caused by FMD vaccination.Therefore, our experimental results seem to support the above hypothesis.
Memory T cells produce a rapid and powerful immune response that is a hallmark of efficient vaccination (21).Thus, the quality and quantity of induced immune memory are crucial to assessing the vaccine's effectiveness.In this study, the naïve cells are defined as CD45RO − CD27 + , and central memory T cells are defined as CD45RO + CD27 + .The consequences of the current research showed that the percentage of CD45RO + CD27 + T cells in CD4 and CD8 T cells increased significantly at 7 days after vaccination compared with pre-vaccination and returned to pre-immunization levels at 14 days.Over that past decades, numerous reports on humans and cattle have challenged the classical CD45RA/RO paradigm, which defined the memory phenotype as CD45RO + and CD45RA − cells (54,55).For instance, cell proliferation was not observed after stimulation of CD45RO + CD4 + and CD45RO + CD8 + T cells isolated from cattle using the homogenates of the parasites (56).In contrast, our results appear to support the original classical paradigm.
In conclusion, our study systematically evaluated the dynamic changes in T-cell subsets in Chinese Holstein cattle after FMD vaccination and compared the differences in T-cell responses among individuals with different vaccine antibody responses.We observed that the percentage of CD4, CD8, and γδ T cells in peripheral blood of dairy cows was significantly decreased after inoculation with bivalent inactivated FMD vaccine, and the expression of CD44 changed synchronously.Unexpectedly, CD4 + CD8 + DP T cells were significantly up-regulated after vaccination, suggesting their potential role as a T-cell response marker for the evaluation of bivalent inactivated FMD vaccine efficacy.However, the correlation between these results and challenge/protection has not been studied, and further research with different types of FMD vaccines and cattle species is recommended to generalize the results.Overall, our study highlights the importance of considering T-cell responses in evaluating vaccine effectiveness, and our findings contribute to a better understanding of the immune responses induced by bivalent inactivated FMD vaccine in dairy cows.

FIG 2 5 FIG 3 FIG 4
FIG 2 Demonstration of virus particles in vaccines.(A) Negative stain electron microscope (EM) images of FMD vaccine (serotypes O and A).The scale bar indicates 500 nm.(B) Negative stain EM images of FMD vaccine (serotypes O and A).The scale bar indicates 100 nm.

FIG 5
FIG 5 Dynamic changes in cow CD4, CD8, and DP T cells after FMD vaccination.(A) Representative dot plots depict CD4, CD8, and DP T cells in the peripheral bloods of indicated cows at 0, 7, and 14 dpi.Numbers indicate the percentages of CD4, CD8, and DP T cells.(B) Dynamic changes in CD4 T-cell percentage in αβ T cells after FMD vaccination.(C) Dynamic changes in CD8 T-cell percentage in αβ T cells after FMD vaccination.(D) Dynamic changes in CD4CD8 DP T-cell percentage in αβ T cells after FMD vaccination.Data shown are mean ± SD (n = 15).Different letters represent significant differences between groups (P < 0.05).HR, high antibody response against both serotypes O and A; LR, low antibody response against both serotypes O and A.

FIG 6
FIG 6 Differences in the expressions of CD44 in different T-cell subsets (CD4, CD8, and γδ) of HR and LR groups.The percentages of CD44 high (A) or low (B) expressing cells and MFI (C) were compared in CD4 T cells in blood between HR and LR cows.The percentages of CD44 high (D) or low (E) expressing cells and MFI (F) were compared in CD8 T cells in blood between HR and LR cows.The percentages of CD44 high (G) or low (H) expressing cells and MFI (I) were compared in γδ T cells in blood between HR and LR cows.Data shown are mean ± SD (n = 15).Different letters represent significant differences between groups (P < 0.05).HR, high antibody response against both serotypes O and A; LR, low antibody response against both serotypes O and A.

FIG 7
FIG 7 Dynamic changes in cow memory T cells after FMD vaccination.The percentages of CD27 + CD45RO + T cells (Tcm) were compared in CD4 (A), CD8 (C), and γδ (E) T cells in whole blood between HR and LR cows.The percentages of CD27 + CD45RO − T cells (naïve) were compared in CD4 (B), CD8 (D), and γδ (F) T cells in whole blood between HR and LR cows.HR, high antibody response against both serotypes O and A; LR, low antibody response against both serotypes O and A.

TABLE 1
Antibodies used for flow cytometry in this study