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Vβ profile in toxic shock syndrome: A possible diagnostic test
International Congress Series 1289 (2006) 129 – 132
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Vh profile in toxic shock syndrome: A possible diagnostic test Christopher M. MacIsaac a,*, Nigel Curtis b,c, John Cade a, Kumar Visvanathan b,d a
Intensive Care Unit, The Royal Melbourne Hospital, Grattan St, Parkville, Victoria, 3050, Australia b Murdoch Childrens Research Institute, Australia c Department of General Medicine, Royal Childrens Hospital, University of Melbourne, Australia d Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Australia
Abstract. There is currently no diagnostic test for Toxic shock syndrome (TSS) which is caused by superantigens (SAg). SAg bind to the lateral aspect of a T cell receptor at its Vh region. We studied the expression of Vh segments on circulating T cells from 7 patients with TSS and 11 controls. The Vh profile was abnormal in all patients. This could in the future be a possible diagnostic test for TSS. D 2005 Elsevier B.V. All rights reserved. Keywords: T-cell receptor; Superantigen; Flow cytometry; Lymphocyte; Toxic shock; T-cell receptor beta
1. Introduction Toxic Shock Syndrome (TSS), which is caused by superantigens (SAg) from Staphylococcus aureus and Streptococcus pyogenes, was first described in 1978 [1]. SAg bypass the normal antigen presentation via MHC class II molecules and directly bind the lateral aspect of the T cell receptor (TCR) at the TCR Vh region. Due to this method of binding, the signature feature of SAg activity is the expansion of T lymphocyte populations bearing the particular Vh chain(s) that the SAg binds to [2]. The diagnosis of TSS is difficult in its early phases and there is currently no diagnostic test. An accurate diagnostic test may facilitate better treatments for toxic shock such as
* Corresponding author. Tel.: +61 3 9342 7463; fax: +61 3 9342 8812. E-mail address: [email protected] (C.M. MacIsaac). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.09.142
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Fig. 1. Typical flow cytometry dotplots from 1 of 8 tubes stained with antibodies to 3 different Vh families. Each quadrant represents a population positive for a different Vh segment save the lower left quadrant which is negative for all 3. In this figure CD4+ cells are shown on the left and CD8+ on the right. The numbers in the corner of each quadrant represent the percentage of cells present in each quadrant.
intravenous immunoglobulin [3]. We sought to determine the Vh profile of circulating T cells from a cohort of patients with TSS. 2. Methods Following institutional Ethics committee approval, 1.1 ml of whole blood was sampled serially from 7 patients with TSS and 11 non-septic ICU adult controls (negative controls). The samples were stained with a panel of monoclonal antibodies directed against 24 Vh segments (IO Test Beta Mark, Immunotech, Mareille, France) in addition to CD4 (Becton Dickinson, San Jose, CA, USA) and CD8 (Immunotech, Mareille, France) [4]. Following staining, the samples were analysed by flow cytometry to determine the expression of each of the 24 Vh segments assayed for both CD4+ and CD8+ lymphocytes (Fig. 1). A normal range for each of the 24 Vh segments assayed for CD4+ and CD8+ cells was defined as the mean (F 2 S.D.) of the negative control data. The results obtained from an individual patient sample were compared to this normal range. Results were expressed as the number of CD4+ and CD8+ T cell Vh segments that were outside the normal range. In addition, to quantify the degree of abnormality, the maximum Z score (Number of S.D.
Fig. 2. CD4 Vh profile on day 5 post onset of TSS from a 12-year-old girl with non-menstruation associated TSS. Note the expansion (*) of Vh3, Vh12, Vh14 and Vh17. In addition Vh2 is under-represented (#). For negative controls mean of sample (N = 11) is shown, error bars indicated (F) 2 S.D. from mean.
C.M. MacIsaac et al. / International Congress Series 1289 (2006) 129–132
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Table 1 Summary statistics of Vh data for CD4 and CD8 positive cells from negative controls and patients with toxinmediated disease Control (N = 11) Toxin mediated disease (N = 7) Maximal number of CD4 segments skewed, 1 median (range) Maximal Z score for CD4 segments, median (range) 2.4 Maximal number of CD8 segments skewed, 1 median (range) Maximal Z score for CD8 segments, median (range) 2.3
(0–3) (1.6–2.8) (0–3) (1.3–3.1)
4 (1–7)
P (Mann–Whitney) 0.01
10.94 (2.4–13.0) 0.004 3 (2–5) 0.003 4.1 (2.5–8.6)
0.02
from mean) of each Vh segment was recorded for each sample. Thus for each sample from the raw data of percentage of Vh segments expressed, 4 variables were obtained, (1) number of abnormal CD4 segments, (2) number of abnormal CD8 segments, (3) maximum Z score for CD4+ segments and (4) maximum Z score for CD8+ segments. To account for serial samples, for each patient a summary statistic of the maximum of each of the preceding 4 variables was analysed [5]. 3. Results Seven patients with TSS aged from 3 to 35 years of age were sampled. Three patients had a single sample, two were bled twice, one had four samples while another had nine samples over a 4-month period. Eleven single samples were taken from 11 negative controls. All patients with TSS showed a skewed Vh profile. As an example in Fig. 2, data from a 12-year-old girl on day 5 post onset of TSS shows an expansion of Vh segments 3, 12, 14 and 17 which have known affinity for Staphylococcal Enterotoxin B (SEB). The group of 7 patients with toxin mediated disease had a different Vh profile compared to the non-septic ICU controls (Table 1) and this difference was also present when Vh profile from the first bleed only was compared between groups (Table 1). Of the 3 patients with TSS who were sampled serially from day 1 post onset of TSS there was an under-representation followed by an expansion of several Vh segments. Fig. 3 gives an example of this finding.
Fig. 3. Vh results from a patient with TSS samples on days 1, 5 and 7 post onset of TSS highlighting 4 Vh segments that were initially under-represented and then underwent expansion. For negative controls mean of sample (N = 11) is shown, error bars indicated (F) 2 S.D. from mean.
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4. Conclusions In patients with TSS skewing of the T cell Vh repertoire can be detected rapidly and reliably. This could form the basis of a diagnostic test that may allow application of more targeted therapies, such as immunoglobulin in patients with TSS. References [1] J. Todd, et al., Toxic-shock syndrome associated with phage-group-I Staphylococci, Lancet 2 (8100) (1978) 1116 – 1118. [2] J.K. McCormick, J.M. Yarwood, P.M. Schlievert, Toxic shock syndrome and bacterial superantigens: an update, Annu. Rev. Microbiol. 55 (2001) 77 – 104. [3] R. Kaul, et al., Intravenous immunoglobulin therapy for streptococcal toxic shock syndrome—a comparative observational study. The Canadian Streptococcal Study Group, Clin. Infect. Dis. 28 (4) (1999) 800 – 807. [4] C. MacIsaac, et al., Rapid analysis of the Vbeta repertoire of CD4 and CD8 T lymphocytes in whole blood, J. Immunol. Methods 283 (1–2) (2003) 9 – 15. [5] J.N. Matthews, et al., Analysis of serial measurements in medical research, BMJ 300 (6719) (1990) 230 – 235.
www.ics-elsevier.com
Vh profile in toxic shock syndrome: A possible diagnostic test Christopher M. MacIsaac a,*, Nigel Curtis b,c, John Cade a, Kumar Visvanathan b,d a
Intensive Care Unit, The Royal Melbourne Hospital, Grattan St, Parkville, Victoria, 3050, Australia b Murdoch Childrens Research Institute, Australia c Department of General Medicine, Royal Childrens Hospital, University of Melbourne, Australia d Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Australia
Abstract. There is currently no diagnostic test for Toxic shock syndrome (TSS) which is caused by superantigens (SAg). SAg bind to the lateral aspect of a T cell receptor at its Vh region. We studied the expression of Vh segments on circulating T cells from 7 patients with TSS and 11 controls. The Vh profile was abnormal in all patients. This could in the future be a possible diagnostic test for TSS. D 2005 Elsevier B.V. All rights reserved. Keywords: T-cell receptor; Superantigen; Flow cytometry; Lymphocyte; Toxic shock; T-cell receptor beta
1. Introduction Toxic Shock Syndrome (TSS), which is caused by superantigens (SAg) from Staphylococcus aureus and Streptococcus pyogenes, was first described in 1978 [1]. SAg bypass the normal antigen presentation via MHC class II molecules and directly bind the lateral aspect of the T cell receptor (TCR) at the TCR Vh region. Due to this method of binding, the signature feature of SAg activity is the expansion of T lymphocyte populations bearing the particular Vh chain(s) that the SAg binds to [2]. The diagnosis of TSS is difficult in its early phases and there is currently no diagnostic test. An accurate diagnostic test may facilitate better treatments for toxic shock such as
* Corresponding author. Tel.: +61 3 9342 7463; fax: +61 3 9342 8812. E-mail address: [email protected] (C.M. MacIsaac). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.09.142
130
C.M. MacIsaac et al. / International Congress Series 1289 (2006) 129–132
Fig. 1. Typical flow cytometry dotplots from 1 of 8 tubes stained with antibodies to 3 different Vh families. Each quadrant represents a population positive for a different Vh segment save the lower left quadrant which is negative for all 3. In this figure CD4+ cells are shown on the left and CD8+ on the right. The numbers in the corner of each quadrant represent the percentage of cells present in each quadrant.
intravenous immunoglobulin [3]. We sought to determine the Vh profile of circulating T cells from a cohort of patients with TSS. 2. Methods Following institutional Ethics committee approval, 1.1 ml of whole blood was sampled serially from 7 patients with TSS and 11 non-septic ICU adult controls (negative controls). The samples were stained with a panel of monoclonal antibodies directed against 24 Vh segments (IO Test Beta Mark, Immunotech, Mareille, France) in addition to CD4 (Becton Dickinson, San Jose, CA, USA) and CD8 (Immunotech, Mareille, France) [4]. Following staining, the samples were analysed by flow cytometry to determine the expression of each of the 24 Vh segments assayed for both CD4+ and CD8+ lymphocytes (Fig. 1). A normal range for each of the 24 Vh segments assayed for CD4+ and CD8+ cells was defined as the mean (F 2 S.D.) of the negative control data. The results obtained from an individual patient sample were compared to this normal range. Results were expressed as the number of CD4+ and CD8+ T cell Vh segments that were outside the normal range. In addition, to quantify the degree of abnormality, the maximum Z score (Number of S.D.
Fig. 2. CD4 Vh profile on day 5 post onset of TSS from a 12-year-old girl with non-menstruation associated TSS. Note the expansion (*) of Vh3, Vh12, Vh14 and Vh17. In addition Vh2 is under-represented (#). For negative controls mean of sample (N = 11) is shown, error bars indicated (F) 2 S.D. from mean.
C.M. MacIsaac et al. / International Congress Series 1289 (2006) 129–132
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Table 1 Summary statistics of Vh data for CD4 and CD8 positive cells from negative controls and patients with toxinmediated disease Control (N = 11) Toxin mediated disease (N = 7) Maximal number of CD4 segments skewed, 1 median (range) Maximal Z score for CD4 segments, median (range) 2.4 Maximal number of CD8 segments skewed, 1 median (range) Maximal Z score for CD8 segments, median (range) 2.3
(0–3) (1.6–2.8) (0–3) (1.3–3.1)
4 (1–7)
P (Mann–Whitney) 0.01
10.94 (2.4–13.0) 0.004 3 (2–5) 0.003 4.1 (2.5–8.6)
0.02
from mean) of each Vh segment was recorded for each sample. Thus for each sample from the raw data of percentage of Vh segments expressed, 4 variables were obtained, (1) number of abnormal CD4 segments, (2) number of abnormal CD8 segments, (3) maximum Z score for CD4+ segments and (4) maximum Z score for CD8+ segments. To account for serial samples, for each patient a summary statistic of the maximum of each of the preceding 4 variables was analysed [5]. 3. Results Seven patients with TSS aged from 3 to 35 years of age were sampled. Three patients had a single sample, two were bled twice, one had four samples while another had nine samples over a 4-month period. Eleven single samples were taken from 11 negative controls. All patients with TSS showed a skewed Vh profile. As an example in Fig. 2, data from a 12-year-old girl on day 5 post onset of TSS shows an expansion of Vh segments 3, 12, 14 and 17 which have known affinity for Staphylococcal Enterotoxin B (SEB). The group of 7 patients with toxin mediated disease had a different Vh profile compared to the non-septic ICU controls (Table 1) and this difference was also present when Vh profile from the first bleed only was compared between groups (Table 1). Of the 3 patients with TSS who were sampled serially from day 1 post onset of TSS there was an under-representation followed by an expansion of several Vh segments. Fig. 3 gives an example of this finding.
Fig. 3. Vh results from a patient with TSS samples on days 1, 5 and 7 post onset of TSS highlighting 4 Vh segments that were initially under-represented and then underwent expansion. For negative controls mean of sample (N = 11) is shown, error bars indicated (F) 2 S.D. from mean.
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C.M. MacIsaac et al. / International Congress Series 1289 (2006) 129–132
4. Conclusions In patients with TSS skewing of the T cell Vh repertoire can be detected rapidly and reliably. This could form the basis of a diagnostic test that may allow application of more targeted therapies, such as immunoglobulin in patients with TSS. References [1] J. Todd, et al., Toxic-shock syndrome associated with phage-group-I Staphylococci, Lancet 2 (8100) (1978) 1116 – 1118. [2] J.K. McCormick, J.M. Yarwood, P.M. Schlievert, Toxic shock syndrome and bacterial superantigens: an update, Annu. Rev. Microbiol. 55 (2001) 77 – 104. [3] R. Kaul, et al., Intravenous immunoglobulin therapy for streptococcal toxic shock syndrome—a comparative observational study. The Canadian Streptococcal Study Group, Clin. Infect. Dis. 28 (4) (1999) 800 – 807. [4] C. MacIsaac, et al., Rapid analysis of the Vbeta repertoire of CD4 and CD8 T lymphocytes in whole blood, J. Immunol. Methods 283 (1–2) (2003) 9 – 15. [5] J.N. Matthews, et al., Analysis of serial measurements in medical research, BMJ 300 (6719) (1990) 230 – 235.