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Investigating the Antigen Specificity of Multiple Sclerosis Central Nervous System-Derived Immunoglobulins.

Willis SN, Stathopoulos P, Chastre A, Compton SD, Hafler DA, O'Connor KC - Front Immunol (2015)

Bottom Line: This infiltrate often includes B cells that are found in multiple locations throughout the CNS, including the cerebrospinal fluid (CSF), parenchyma, and the meninges, frequently forming tertiary lymphoid structures in the latter.However, the antigen(s) driving this response have yet to be conclusively defined.We conclude that while MS CNS resident B cells display the characteristics of an antigen-driven B cell response, the antigen(s) driving this response remain at large.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Yale School of Medicine , New Haven, CT , USA ; Walter and Eliza Hall Institute of Medical Research , Parkville, VIC , Australia ; Department of Medical Biology, University of Melbourne , Parkville, VIC , Australia.

ABSTRACT
The central nervous system (CNS) of patients with multiple sclerosis (MS) is the site where disease pathology is evident. Damaged CNS tissue is commonly associated with immune cell infiltration. This infiltrate often includes B cells that are found in multiple locations throughout the CNS, including the cerebrospinal fluid (CSF), parenchyma, and the meninges, frequently forming tertiary lymphoid structures in the latter. Several groups, including our own, have shown that B cells from distinct locations within the MS CNS are clonally related and display the characteristics of an antigen-driven response. However, the antigen(s) driving this response have yet to be conclusively defined. To explore the antigen specificity of the MS B cell response, we produced recombinant human immunoglobulin (rIgG) from a series of expanded B cell clones that we isolated from the CNS tissue of six MS brains. The specificity of these MS-derived rIgG and control rIgG derived from non-MS tissues was then examined using multiple methodologies that included testing individual candidate antigens, screening with high-throughput antigen arrays and evaluating binding to CNS-derived cell lines. We report that while several MS-derived rIgG recognized particular antigens, including neurofilament light and a protocadherin isoform, none were unique to MS, as non-MS-derived rIgG used as controls invariably displayed similar binding specificities. We conclude that while MS CNS resident B cells display the characteristics of an antigen-driven B cell response, the antigen(s) driving this response remain at large.

No MeSH data available.


Related in: MedlinePlus

MS and control-derived rIgG binding to protocadherin gamma (PCDHGC3) by solid phase ELISA. MS-derived rIgG (n = 11) and control rIgG (n = 10) derived from a germinoma and muscle tissue were tested by solid phase ELISA for binding to protocadherin. The specific samples included in the assay are shown in the Supplementary Material. Each dot or square represents the binding of a single rIgG. The dashed line indicates the mean +2 SD of the control-derived cohort (0.87). Values above this line were determined to be positive (95% CI). The mean and SD are shown for each data set. Statistical differences are indicated when significant. Data associated with each rIgG for the MS and control groups are shown in the Supplementary Material.
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Figure 3: MS and control-derived rIgG binding to protocadherin gamma (PCDHGC3) by solid phase ELISA. MS-derived rIgG (n = 11) and control rIgG (n = 10) derived from a germinoma and muscle tissue were tested by solid phase ELISA for binding to protocadherin. The specific samples included in the assay are shown in the Supplementary Material. Each dot or square represents the binding of a single rIgG. The dashed line indicates the mean +2 SD of the control-derived cohort (0.87). Values above this line were determined to be positive (95% CI). The mean and SD are shown for each data set. Statistical differences are indicated when significant. Data associated with each rIgG for the MS and control groups are shown in the Supplementary Material.

Mentions: Having shown no specificity for the MS-derived rIgG to several candidate antigens, we sought to expand the search by using an unbiased library of antigens that could be screened in a high-throughput manner. To this end, we examined the rIgG specificity from the MS and control cohorts with a commercially available protein array composed of approximately 9,400 unique full-length human proteins that were expressed in a system such that the products included some physiologic post-translational modifications and processing. The rIgGs from both the MS and controls groups were pooled so that three rIgG were included on each array during the initial scouting to maximize efficient use of the arrays. A total of three MS and three control arrays were run. Target antigens that were identified by at least one MS antibody pool that did not react with any of the control groups are shown in Figure S2 in Supplementary Material. In most instances, antigen targets were found on a single MS array; however, several were found on two of the three MS arrays. Of these, protocadherin gamma subfamily C, three (PCDHGC3), transcript variant three was of particular interest as a candidate autoantigen as protocadherin isoforms, include extracellular domains, are predominantly expressed in the nervous system and have been implicated in human neurological disorders (40, 41). Given their attractive role as candidate MS antigens we investigated this specificity further. To do this we tested binding to PCDHGC3 protein by ELISA with individual rIgGs rather than pooled mixtures (Figure 3). The MS-derived rIgG MS-C2 that was present in the pool that bound PCDHGC3 on the ProtoArray (MS array 2) bound to the protein. However, binding was not observed for the individual rIgGs present on the second array that also identified this target (MS array 3). Furthermore, the difference between the MS and the control group was not significant (p = 0.3432, Mann–Whitney test) and binding was not restricted to MS-derived antibodies as a germinoma-derived antibody (GCT-A10) also was positive in the ELISA, indicating a lack of specificity for MS in the rIgG cohorts.


Investigating the Antigen Specificity of Multiple Sclerosis Central Nervous System-Derived Immunoglobulins.

Willis SN, Stathopoulos P, Chastre A, Compton SD, Hafler DA, O'Connor KC - Front Immunol (2015)

MS and control-derived rIgG binding to protocadherin gamma (PCDHGC3) by solid phase ELISA. MS-derived rIgG (n = 11) and control rIgG (n = 10) derived from a germinoma and muscle tissue were tested by solid phase ELISA for binding to protocadherin. The specific samples included in the assay are shown in the Supplementary Material. Each dot or square represents the binding of a single rIgG. The dashed line indicates the mean +2 SD of the control-derived cohort (0.87). Values above this line were determined to be positive (95% CI). The mean and SD are shown for each data set. Statistical differences are indicated when significant. Data associated with each rIgG for the MS and control groups are shown in the Supplementary Material.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: MS and control-derived rIgG binding to protocadherin gamma (PCDHGC3) by solid phase ELISA. MS-derived rIgG (n = 11) and control rIgG (n = 10) derived from a germinoma and muscle tissue were tested by solid phase ELISA for binding to protocadherin. The specific samples included in the assay are shown in the Supplementary Material. Each dot or square represents the binding of a single rIgG. The dashed line indicates the mean +2 SD of the control-derived cohort (0.87). Values above this line were determined to be positive (95% CI). The mean and SD are shown for each data set. Statistical differences are indicated when significant. Data associated with each rIgG for the MS and control groups are shown in the Supplementary Material.
Mentions: Having shown no specificity for the MS-derived rIgG to several candidate antigens, we sought to expand the search by using an unbiased library of antigens that could be screened in a high-throughput manner. To this end, we examined the rIgG specificity from the MS and control cohorts with a commercially available protein array composed of approximately 9,400 unique full-length human proteins that were expressed in a system such that the products included some physiologic post-translational modifications and processing. The rIgGs from both the MS and controls groups were pooled so that three rIgG were included on each array during the initial scouting to maximize efficient use of the arrays. A total of three MS and three control arrays were run. Target antigens that were identified by at least one MS antibody pool that did not react with any of the control groups are shown in Figure S2 in Supplementary Material. In most instances, antigen targets were found on a single MS array; however, several were found on two of the three MS arrays. Of these, protocadherin gamma subfamily C, three (PCDHGC3), transcript variant three was of particular interest as a candidate autoantigen as protocadherin isoforms, include extracellular domains, are predominantly expressed in the nervous system and have been implicated in human neurological disorders (40, 41). Given their attractive role as candidate MS antigens we investigated this specificity further. To do this we tested binding to PCDHGC3 protein by ELISA with individual rIgGs rather than pooled mixtures (Figure 3). The MS-derived rIgG MS-C2 that was present in the pool that bound PCDHGC3 on the ProtoArray (MS array 2) bound to the protein. However, binding was not observed for the individual rIgGs present on the second array that also identified this target (MS array 3). Furthermore, the difference between the MS and the control group was not significant (p = 0.3432, Mann–Whitney test) and binding was not restricted to MS-derived antibodies as a germinoma-derived antibody (GCT-A10) also was positive in the ELISA, indicating a lack of specificity for MS in the rIgG cohorts.

Bottom Line: This infiltrate often includes B cells that are found in multiple locations throughout the CNS, including the cerebrospinal fluid (CSF), parenchyma, and the meninges, frequently forming tertiary lymphoid structures in the latter.However, the antigen(s) driving this response have yet to be conclusively defined.We conclude that while MS CNS resident B cells display the characteristics of an antigen-driven B cell response, the antigen(s) driving this response remain at large.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Yale School of Medicine , New Haven, CT , USA ; Walter and Eliza Hall Institute of Medical Research , Parkville, VIC , Australia ; Department of Medical Biology, University of Melbourne , Parkville, VIC , Australia.

ABSTRACT
The central nervous system (CNS) of patients with multiple sclerosis (MS) is the site where disease pathology is evident. Damaged CNS tissue is commonly associated with immune cell infiltration. This infiltrate often includes B cells that are found in multiple locations throughout the CNS, including the cerebrospinal fluid (CSF), parenchyma, and the meninges, frequently forming tertiary lymphoid structures in the latter. Several groups, including our own, have shown that B cells from distinct locations within the MS CNS are clonally related and display the characteristics of an antigen-driven response. However, the antigen(s) driving this response have yet to be conclusively defined. To explore the antigen specificity of the MS B cell response, we produced recombinant human immunoglobulin (rIgG) from a series of expanded B cell clones that we isolated from the CNS tissue of six MS brains. The specificity of these MS-derived rIgG and control rIgG derived from non-MS tissues was then examined using multiple methodologies that included testing individual candidate antigens, screening with high-throughput antigen arrays and evaluating binding to CNS-derived cell lines. We report that while several MS-derived rIgG recognized particular antigens, including neurofilament light and a protocadherin isoform, none were unique to MS, as non-MS-derived rIgG used as controls invariably displayed similar binding specificities. We conclude that while MS CNS resident B cells display the characteristics of an antigen-driven B cell response, the antigen(s) driving this response remain at large.

No MeSH data available.


Related in: MedlinePlus