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Probing for the main immunogenic region of the human acetylcholine receptor
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A SUPPRESSOR CELL LINE WHICH PREVENTS THE ADOPTIVE TRANSFER OF EAE IN LEWIS RATS Karen Eilerman and Steven Brostoff, Department of Neurology, Medical University of South Carolina, Charleston, SC 29425 The observations that Lewis rats, which spontaneously recover from active EAE, are resistant to relnduction of active disease even though effector cell lines can be "rescued" from these recovered rats, suggests the presence of a suppressor mechanism. Using recovered rats we succeeded in generating a guinea pig myelin basic protein (GP-MBP) specific T cell line (TCL) (manuscript in preparation) with characteristics of a suppressor cell. These cells can be maintained in culture by alternately activating with GP-MBP and expanding with Con A supernatant. The TCL cells generated can prevent the adoptive transfer of paralytic EAE. The phenotype of this T cell line is CD4 +, 0X-22-, IL2-R + and becomes weakly CD8 + after antigenic stimulation. Adoptive transfer of EAE can be achieved with spleen cells (SpC) taken from GP-MBP sensitized Lewis rats on day ii or 12 post immunization and incubated in vitro in the presence of antigen for 72 hours. Recipients of 3xlO 7 of these S T develop severe paralytic EAE 4-6 days after transfer. In contrast, recipients of SpC mixed with TCL cells in an 8:1 (SpC:TCL) ratio fail to exhibit clinical and histologic evidence of disease after transfer of up to 5xlO 7 cells. This observation is not the non-specific result of T blasts since normal splenocytes incubated for 3 days in culture in Con A do not prevent the transfer of paralytic disease when added to the SpC in place of TCL in the protocol described above. The mechanism of action of these TCL cells is unknown although experimental evidence suggests that the cells are directed against antigen-specific effector cells. During the generation of the TCL we noted that direct injection of TCL ceils into recipients resulted in mild clinical disease which decreased in severity with the increased number of the cycles of antigenic stimulation until all evidence of clinical disease finally disappeared after 7-8 cycles. The TCL cells, nevertheless, continued to exhibit a proliferative response from a resting state in vitro to irradiated MBP-activated blasts derived from EAE rats in the absence of antigen or antigen-presenting cells. The above results suggest that a T suppressor cell directed at a specific determinant on the T effector cell may play a role in regulating EAE in the Lewis rat. Supported in part by NIH Grant No. NSI1867.
PROBING FOR THE MAIN IMMUNOGENIC REGION OF THE HUMAN ACETYLCHOLINE RECEPTOR. H. Eng 1, H. JSrnvall 2, M. Carlquist 3 and A.K. Lefvert I (1Dept. of Medicine, Karolinska Hospital, and Dept.'s of 2Medical Chemistry and 3Biochemistry, Karolinska Instit., Stockholm, Sweden). The main immunogenic region (MIR) of the mouse nicotinic acetylcholine receptor (AChR) alpha subunit has been shown to reside within amino acids 5 to 85, though its exact location is still unknown. Antigenic determinants within this region of the human AChR alpha-subunit were probed using a monoclonal Ab against the MIR and myasthenia gravis (MG) patient sera. We have examined two synthetic peptides comprising amino acids 66 79 (peptide 1) and 44 - 59 (peptide 2) and found both to react with the monoclonal Ab and MG sera using a direct binding ELISA assay with the
52
peptides as coating antigen. The monoclonal Ab reacted better to peptide 2 than 1, whereas the patient sera demonstrated variation as to which peptide was more immunogenic. Free peptide was able to inhibit binding of sera to Torpedo AChR in a concentration dependent manner. As the monoclonal Ab reacts with both peptides it appears that the MIR is a conformational determinant composed of at least two regions that are in close spatial proximity.
ANTI-IDIOTYPIC ANTIBODIES THAT BIND CHOLINERGIC LIGANDS ISOLATED FROM A MYASTHENIC PATIENT. H. Eng and A.K. Lefvert (Dept. of Medicine, Karolinska Hospital, Stockholm, Sweden) In myasthenia gravis, a subpopulation of the anti-acetylcholine receptor antibody repertoire is directed against the ligand binding site. The network theory predicts that the anti-ld antibody will be the internal image of the initial stimulus, that is, these anti-ld should resemble the acetylcholine receptor binding site and hence may also bind cholinergic ligands. We have utilized this theoretical specificity to isolate and characterize these anti-ld antibodies from the serum of one myasthenia gravis patient using a choline hemiglutarate affinity column. Antibodies eluted from the column, designated as anti-Ch Ab, exhibited binding properties similar to the receptor for various cholinergic ligands and were found to be predominantly IgG subclass 3. An inverse relationship between the presence of the anti-Ch Ab and anti-AChR Ab has been observed for several MG patients. Whether this anti-ld Ab functions to suppress or enhance the anti-AChR response is uncertain but of importance when considering future alternatives for immunotherapy in myasthenia gravis.
A different cell distribution in CSF compared to blood: Immunoenzymatic cell enumeration in patients with lymphoproliferative and inflammatory neurological diseases. Jan Ernerudh, M.D, Department of Neurology, University Hospital, LinkSping and Tomas Olsson, M.D., Department of Neurology, Huddlnge Hospital, Karolinska Institute, Sweden. The origin of CSF leukocytes under normal and pathological conditions remain unknown. Since morphologically distinguishable cells in CSF differ in number and distribution from those in blood, there may exist selection mechanisms for cell passage into the CSF, which is also supported by experimental studies.
A SUPPRESSOR CELL LINE WHICH PREVENTS THE ADOPTIVE TRANSFER OF EAE IN LEWIS RATS Karen Eilerman and Steven Brostoff, Department of Neurology, Medical University of South Carolina, Charleston, SC 29425 The observations that Lewis rats, which spontaneously recover from active EAE, are resistant to relnduction of active disease even though effector cell lines can be "rescued" from these recovered rats, suggests the presence of a suppressor mechanism. Using recovered rats we succeeded in generating a guinea pig myelin basic protein (GP-MBP) specific T cell line (TCL) (manuscript in preparation) with characteristics of a suppressor cell. These cells can be maintained in culture by alternately activating with GP-MBP and expanding with Con A supernatant. The TCL cells generated can prevent the adoptive transfer of paralytic EAE. The phenotype of this T cell line is CD4 +, 0X-22-, IL2-R + and becomes weakly CD8 + after antigenic stimulation. Adoptive transfer of EAE can be achieved with spleen cells (SpC) taken from GP-MBP sensitized Lewis rats on day ii or 12 post immunization and incubated in vitro in the presence of antigen for 72 hours. Recipients of 3xlO 7 of these S T develop severe paralytic EAE 4-6 days after transfer. In contrast, recipients of SpC mixed with TCL cells in an 8:1 (SpC:TCL) ratio fail to exhibit clinical and histologic evidence of disease after transfer of up to 5xlO 7 cells. This observation is not the non-specific result of T blasts since normal splenocytes incubated for 3 days in culture in Con A do not prevent the transfer of paralytic disease when added to the SpC in place of TCL in the protocol described above. The mechanism of action of these TCL cells is unknown although experimental evidence suggests that the cells are directed against antigen-specific effector cells. During the generation of the TCL we noted that direct injection of TCL ceils into recipients resulted in mild clinical disease which decreased in severity with the increased number of the cycles of antigenic stimulation until all evidence of clinical disease finally disappeared after 7-8 cycles. The TCL cells, nevertheless, continued to exhibit a proliferative response from a resting state in vitro to irradiated MBP-activated blasts derived from EAE rats in the absence of antigen or antigen-presenting cells. The above results suggest that a T suppressor cell directed at a specific determinant on the T effector cell may play a role in regulating EAE in the Lewis rat. Supported in part by NIH Grant No. NSI1867.
PROBING FOR THE MAIN IMMUNOGENIC REGION OF THE HUMAN ACETYLCHOLINE RECEPTOR. H. Eng 1, H. JSrnvall 2, M. Carlquist 3 and A.K. Lefvert I (1Dept. of Medicine, Karolinska Hospital, and Dept.'s of 2Medical Chemistry and 3Biochemistry, Karolinska Instit., Stockholm, Sweden). The main immunogenic region (MIR) of the mouse nicotinic acetylcholine receptor (AChR) alpha subunit has been shown to reside within amino acids 5 to 85, though its exact location is still unknown. Antigenic determinants within this region of the human AChR alpha-subunit were probed using a monoclonal Ab against the MIR and myasthenia gravis (MG) patient sera. We have examined two synthetic peptides comprising amino acids 66 79 (peptide 1) and 44 - 59 (peptide 2) and found both to react with the monoclonal Ab and MG sera using a direct binding ELISA assay with the
52
peptides as coating antigen. The monoclonal Ab reacted better to peptide 2 than 1, whereas the patient sera demonstrated variation as to which peptide was more immunogenic. Free peptide was able to inhibit binding of sera to Torpedo AChR in a concentration dependent manner. As the monoclonal Ab reacts with both peptides it appears that the MIR is a conformational determinant composed of at least two regions that are in close spatial proximity.
ANTI-IDIOTYPIC ANTIBODIES THAT BIND CHOLINERGIC LIGANDS ISOLATED FROM A MYASTHENIC PATIENT. H. Eng and A.K. Lefvert (Dept. of Medicine, Karolinska Hospital, Stockholm, Sweden) In myasthenia gravis, a subpopulation of the anti-acetylcholine receptor antibody repertoire is directed against the ligand binding site. The network theory predicts that the anti-ld antibody will be the internal image of the initial stimulus, that is, these anti-ld should resemble the acetylcholine receptor binding site and hence may also bind cholinergic ligands. We have utilized this theoretical specificity to isolate and characterize these anti-ld antibodies from the serum of one myasthenia gravis patient using a choline hemiglutarate affinity column. Antibodies eluted from the column, designated as anti-Ch Ab, exhibited binding properties similar to the receptor for various cholinergic ligands and were found to be predominantly IgG subclass 3. An inverse relationship between the presence of the anti-Ch Ab and anti-AChR Ab has been observed for several MG patients. Whether this anti-ld Ab functions to suppress or enhance the anti-AChR response is uncertain but of importance when considering future alternatives for immunotherapy in myasthenia gravis.
A different cell distribution in CSF compared to blood: Immunoenzymatic cell enumeration in patients with lymphoproliferative and inflammatory neurological diseases. Jan Ernerudh, M.D, Department of Neurology, University Hospital, LinkSping and Tomas Olsson, M.D., Department of Neurology, Huddlnge Hospital, Karolinska Institute, Sweden. The origin of CSF leukocytes under normal and pathological conditions remain unknown. Since morphologically distinguishable cells in CSF differ in number and distribution from those in blood, there may exist selection mechanisms for cell passage into the CSF, which is also supported by experimental studies.