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Differential B7 expression by individual astrocyte lines correlates with T cell activation and cytokine production
84
P o s t e r Abstracts' / J o u r n a l o f N e u r o i m m u n o l o g y 9 0 ( 1 9 9 8 ) 1 3 - 1 0 5
473
476
Lymphocytes in Cerebral Spinal Fluid are Part of the Normal Rccireulating Lymphocyte Pool
I n t e r f e r o n Tau Induction o f T H 2 Cytokines and Synergy w i t h O r a l
11.1. Seabrook. M. Johnston, Universityof Toronto,Dept. ofLab. Medicineand Pathobiology,Canada, J.B. Hay, Universityof Toronto,Dept. ofImmunology,Canada
J,M. Sons, Brigham and Women'sHospital, Boston, USA,H.M. Johnson, Universityof Florzda, USA,H.L. Weiner, S.S. Zamvil, Brigharnand Women'sHospital,Boston, USA
We investigated the recirculation properties of lymphocytes found in cerebral spinal fluid (CSF) in sheep under normal physiological conditions. Subcutaneous efferent lymphatics w e r e cannulated, lymphocytes collected, labeled with fluorescein isothiocyanate and reinfased intravenously. CSF, blood and lymph were collected at varioas times and the percentage of labeled cells determined using flow cytometry. If lymphocytes in CSF are part of the recirculating pool we would expect the percentage of labeled cells to be similar in lymph and CSF. In these experiments there was no significant differerrce between these compartments, demonstrating that lymphocytes can enter the CSF and lymph with similar kinetics. To determine the egress of lymphocytes, labeled cells were infused into the lateral ventricles of the brain and they could be found in higher concentrations in cervical lymph nodes than in other lymph nodes. These data suggest that although the numbers of lymphocytes are relatively small they enter and leave normal CSF much as they do in other tissues and are part of the normal recireulating pool.
MBP for lYeatment of Experimental Allergic Encephalomyelitis
IFN-x is a novel type I IFN which has been shown to prevent development of disease as well as reverse ongoing disease in the animal model for multiple sclerosis, experimental allergic encephalomyelitis. Amelioration of clinical paralysis in the EAE model by IFN-'~ can occur by multiple routes of administration including both oral administration and intraperitoneal (i.p.) injection. We have examined both oral IFN-x and oral MBP in order to study antigen non-specific therapy (IFN-x) in combination with oral tolerance. Oral administration of IFN-'c was as effective as i.p. injection for induction of Th2 cytokines. Increased~levels oflL-4 and IL-10 were observed when IFN-'c and a low dose of oral MBP were administered in combination as compared to IFN-'t: alone. The combination of oral IFN-'~ and oral MBP were also shown to prevent development of EAE using doses at which each individual therapy was ineffective but in combination were efficacious. Thus, combination therapy using oral IF'N-t in accordance with oral tolerance may prove to be of considerable potential for treatment of autoimmune disorders such as MS.
474
477
The V~6
Differential 117 Expression by Individual Astroeyte Lines Correlates
Gene Usage of the 7 ~ T C R in EAE B. Szym anska, E. Tronczynska, T. Berkowiez, M. Marcia, MedicalAcademyof Lodz, Poland, C.F. Brosnan, C.S. Raine, AlbertEinstein College ofMedicine, New York, USA,K. Selmaj. MedicalAcademyofLodz, Poland
w i t h T C e i l A c t i v a t i o n and Cytokine P r o d u c t i o n J.M. Soos~ "11A.Ashley, Brighamand Women'sHospital,Boston, USA, J. Morrow, Bridgham and Women'sHospital, Boston, USA,B.E. Szente, S.S. Zam vii, Brigham and
Women'sHospital, Boston, USA
I has been suggestedtliaf'~"T'tzeil~;"are activated by stress-induced proteilrs in the inflamed CNS. In the mouse major subset of gd T cells reactive to hsp-60 has been shown to reside within the V86 subset, particularly the V~,6.4 subset. To determine whether these cells are present in EAE lesions we have cloned with pCR-Script Amp SK(+) vector ,and sequenced automatically by dideoxy chain termination method V~6+ TCR in the CNS and spleen of mice sensitized to develop EAE. In both the spleen and the CNS, the V~,6 sequences could be divided into 2 major subfamilies V86 and V~6.4. Most of these clones used elements of both D81 and D82 with frequent use of the entire coding region for D~2. In spleen extensive diversity was evident within the CD3 region. In contrast, in the CNS homological sequences were frequently fotmd within each sumplc, with some identical sequences in different animals. These data support the conclusion that there are specific accumulation of gd T cells in the CNS in EAE. and further suggest that some of these cells may be responding to hsp60.
A panel of individual immortalized marine astrocyte lines has been examined lbr their capability to activate mature and naive MBP-reactive T cells. After exposure to IFN-y, these astrocyte lines all expressed MHC class II and B7-2 molecules, although some lines also expressed B7-1. All lines stimulated proliferation of encephalitogenic Thl cells utilizing B7-2 for B7:CD28 costimulation. However, those lines that expressed B7-1 and B7-2 were more effective in stimulating proliferation of naive MBP Acl-ll-specific CD4 + T cells. Astrocyte lines that expressed B7-1 and B7-2 stimulated Thp cells to secrete proinflammatory Thl cytokines whereas lines that expressed B7-2 only, stimulated Thp to produce an anti-inflammatory Th2 cytokine pattern. Oar results demonstrate that astrocytes can differentially express B7-1 molecules, wldch may correlate with their capability to stimulate proinflammatory and regulatory patterns of cytokine production. Astrocytes may have potential for both promoting and downregulating T cell responses in vivo.
475
478
OralAdministration of IL-4 and IL-10 Enhanee the Induction of Low Dose Oral Tolerance
R e g u l a t i o n of MOG p35-55 induced EAE by Gamma Delta T-cells T.W. Spahn, S. Issazadeh, A. Slavia, H.L. Weiner, Brigham and Women'sHospital,
A. Slavia. R~ Maron, Y. Komagata, H.L. Weiner, Brigham & Women'sHospital, USA
USA
Low dose feeding induces regulatory cells secreting IL-4, IL-10 (Th2 type) and TGF-[], (Th3 type) that regulate pro-inflammatory responses such as proliferation and IFN-y production. IL-4 and IL-10 have been shown to preferentially direct T cells toward a Th2/Th3 phenotype, thus we investigated their possible use to enhance oral tolerance. IL-4 and IL-10 were administered orally alone or in conjunction with sub-tolerogenic doses of MBP to (SJL x PL/J) F1 mice that were then immunized for EAE with MBP/CFA. EAE was significantly suppressed in animals receiving oral IL-4 or IL-10 plus MBP as compared to those receiving MBP or oral IL-4 or IL-10 alone. Disease suppression was associated with decreased IFN-y/IL-2 cytokine production plus increased TGF-~3 (IL-4 fed) and IL-10 (IL-10 fed) production and was reversed by IP administration of neutralizing anti~TGF-[3 antibody. Deacreased expression of IFN-y was observed in the Peyer's patch of animals fed MBP which was enhanced when IL4 was given with MBP. Similar enhancing effects on oral tolerance weie observed feeding IL-10 plus MOG to NOD mice immunized for EAE with MOG/CFA. These results show that orally administered IL-4 and IL-10 retain their biologic activity and serve as mucosal adjuvants to enhance oral tolerance.
Immunization of C57/BL6 mice with MOG p35-55 induces chronic EAE. The role of y~5 T cells in the regulation of EAE is unclear. We investigated y~ T cells in C57/BL6 wild type and TCR k-chain disrupted mice using MOG p3555. We found significantly less disease in ~-k.o. mice immunized with MOG/CFA (44% mortality in wild type vs. 11% mortality in 5-k.o. mice). In addition, secretion ofy-IFN, IL-2, IL-5 and 1L-10 was decreased in ~-k.o. mice. Decreased immune responses were also seen in 8-k.o. animals immunized with OVA peptide or protein. Furthermore, when EAE was induced by adoptive transfer of an anti-MOG 35-55 ~[3 T-cell line, there was a striking reduction of disease incidence (83%) and severity in 5-k.o. as compared to wild type mice (8% incidence). Clinically asymptomatic fy-k.o, mice showed no cellular infiltration in the spinal cord whereas wild type animals had infiltration of macrophages, Bcells, ccl3- and y~i T-cells independent of clinical disease. In adoptive transfer EAE, there was reduced y-IFN secretion in fi-k.o, mice accompanied by an increase in IL-10. These findings suggest an important role for y~ T-cells in the regulation of both actively induced and adoptively transferred EAE.
P o s t e r Abstracts' / J o u r n a l o f N e u r o i m m u n o l o g y 9 0 ( 1 9 9 8 ) 1 3 - 1 0 5
473
476
Lymphocytes in Cerebral Spinal Fluid are Part of the Normal Rccireulating Lymphocyte Pool
I n t e r f e r o n Tau Induction o f T H 2 Cytokines and Synergy w i t h O r a l
11.1. Seabrook. M. Johnston, Universityof Toronto,Dept. ofLab. Medicineand Pathobiology,Canada, J.B. Hay, Universityof Toronto,Dept. ofImmunology,Canada
J,M. Sons, Brigham and Women'sHospital, Boston, USA,H.M. Johnson, Universityof Florzda, USA,H.L. Weiner, S.S. Zamvil, Brigharnand Women'sHospital,Boston, USA
We investigated the recirculation properties of lymphocytes found in cerebral spinal fluid (CSF) in sheep under normal physiological conditions. Subcutaneous efferent lymphatics w e r e cannulated, lymphocytes collected, labeled with fluorescein isothiocyanate and reinfased intravenously. CSF, blood and lymph were collected at varioas times and the percentage of labeled cells determined using flow cytometry. If lymphocytes in CSF are part of the recirculating pool we would expect the percentage of labeled cells to be similar in lymph and CSF. In these experiments there was no significant differerrce between these compartments, demonstrating that lymphocytes can enter the CSF and lymph with similar kinetics. To determine the egress of lymphocytes, labeled cells were infused into the lateral ventricles of the brain and they could be found in higher concentrations in cervical lymph nodes than in other lymph nodes. These data suggest that although the numbers of lymphocytes are relatively small they enter and leave normal CSF much as they do in other tissues and are part of the normal recireulating pool.
MBP for lYeatment of Experimental Allergic Encephalomyelitis
IFN-x is a novel type I IFN which has been shown to prevent development of disease as well as reverse ongoing disease in the animal model for multiple sclerosis, experimental allergic encephalomyelitis. Amelioration of clinical paralysis in the EAE model by IFN-'~ can occur by multiple routes of administration including both oral administration and intraperitoneal (i.p.) injection. We have examined both oral IFN-x and oral MBP in order to study antigen non-specific therapy (IFN-x) in combination with oral tolerance. Oral administration of IFN-'c was as effective as i.p. injection for induction of Th2 cytokines. Increased~levels oflL-4 and IL-10 were observed when IFN-'c and a low dose of oral MBP were administered in combination as compared to IFN-'t: alone. The combination of oral IFN-'~ and oral MBP were also shown to prevent development of EAE using doses at which each individual therapy was ineffective but in combination were efficacious. Thus, combination therapy using oral IF'N-t in accordance with oral tolerance may prove to be of considerable potential for treatment of autoimmune disorders such as MS.
474
477
The V~6
Differential 117 Expression by Individual Astroeyte Lines Correlates
Gene Usage of the 7 ~ T C R in EAE B. Szym anska, E. Tronczynska, T. Berkowiez, M. Marcia, MedicalAcademyof Lodz, Poland, C.F. Brosnan, C.S. Raine, AlbertEinstein College ofMedicine, New York, USA,K. Selmaj. MedicalAcademyofLodz, Poland
w i t h T C e i l A c t i v a t i o n and Cytokine P r o d u c t i o n J.M. Soos~ "11A.Ashley, Brighamand Women'sHospital,Boston, USA, J. Morrow, Bridgham and Women'sHospital, Boston, USA,B.E. Szente, S.S. Zam vii, Brigham and
Women'sHospital, Boston, USA
I has been suggestedtliaf'~"T'tzeil~;"are activated by stress-induced proteilrs in the inflamed CNS. In the mouse major subset of gd T cells reactive to hsp-60 has been shown to reside within the V86 subset, particularly the V~,6.4 subset. To determine whether these cells are present in EAE lesions we have cloned with pCR-Script Amp SK(+) vector ,and sequenced automatically by dideoxy chain termination method V~6+ TCR in the CNS and spleen of mice sensitized to develop EAE. In both the spleen and the CNS, the V~,6 sequences could be divided into 2 major subfamilies V86 and V~6.4. Most of these clones used elements of both D81 and D82 with frequent use of the entire coding region for D~2. In spleen extensive diversity was evident within the CD3 region. In contrast, in the CNS homological sequences were frequently fotmd within each sumplc, with some identical sequences in different animals. These data support the conclusion that there are specific accumulation of gd T cells in the CNS in EAE. and further suggest that some of these cells may be responding to hsp60.
A panel of individual immortalized marine astrocyte lines has been examined lbr their capability to activate mature and naive MBP-reactive T cells. After exposure to IFN-y, these astrocyte lines all expressed MHC class II and B7-2 molecules, although some lines also expressed B7-1. All lines stimulated proliferation of encephalitogenic Thl cells utilizing B7-2 for B7:CD28 costimulation. However, those lines that expressed B7-1 and B7-2 were more effective in stimulating proliferation of naive MBP Acl-ll-specific CD4 + T cells. Astrocyte lines that expressed B7-1 and B7-2 stimulated Thp cells to secrete proinflammatory Thl cytokines whereas lines that expressed B7-2 only, stimulated Thp to produce an anti-inflammatory Th2 cytokine pattern. Oar results demonstrate that astrocytes can differentially express B7-1 molecules, wldch may correlate with their capability to stimulate proinflammatory and regulatory patterns of cytokine production. Astrocytes may have potential for both promoting and downregulating T cell responses in vivo.
475
478
OralAdministration of IL-4 and IL-10 Enhanee the Induction of Low Dose Oral Tolerance
R e g u l a t i o n of MOG p35-55 induced EAE by Gamma Delta T-cells T.W. Spahn, S. Issazadeh, A. Slavia, H.L. Weiner, Brigham and Women'sHospital,
A. Slavia. R~ Maron, Y. Komagata, H.L. Weiner, Brigham & Women'sHospital, USA
USA
Low dose feeding induces regulatory cells secreting IL-4, IL-10 (Th2 type) and TGF-[], (Th3 type) that regulate pro-inflammatory responses such as proliferation and IFN-y production. IL-4 and IL-10 have been shown to preferentially direct T cells toward a Th2/Th3 phenotype, thus we investigated their possible use to enhance oral tolerance. IL-4 and IL-10 were administered orally alone or in conjunction with sub-tolerogenic doses of MBP to (SJL x PL/J) F1 mice that were then immunized for EAE with MBP/CFA. EAE was significantly suppressed in animals receiving oral IL-4 or IL-10 plus MBP as compared to those receiving MBP or oral IL-4 or IL-10 alone. Disease suppression was associated with decreased IFN-y/IL-2 cytokine production plus increased TGF-~3 (IL-4 fed) and IL-10 (IL-10 fed) production and was reversed by IP administration of neutralizing anti~TGF-[3 antibody. Deacreased expression of IFN-y was observed in the Peyer's patch of animals fed MBP which was enhanced when IL4 was given with MBP. Similar enhancing effects on oral tolerance weie observed feeding IL-10 plus MOG to NOD mice immunized for EAE with MOG/CFA. These results show that orally administered IL-4 and IL-10 retain their biologic activity and serve as mucosal adjuvants to enhance oral tolerance.
Immunization of C57/BL6 mice with MOG p35-55 induces chronic EAE. The role of y~5 T cells in the regulation of EAE is unclear. We investigated y~ T cells in C57/BL6 wild type and TCR k-chain disrupted mice using MOG p3555. We found significantly less disease in ~-k.o. mice immunized with MOG/CFA (44% mortality in wild type vs. 11% mortality in 5-k.o. mice). In addition, secretion ofy-IFN, IL-2, IL-5 and 1L-10 was decreased in ~-k.o. mice. Decreased immune responses were also seen in 8-k.o. animals immunized with OVA peptide or protein. Furthermore, when EAE was induced by adoptive transfer of an anti-MOG 35-55 ~[3 T-cell line, there was a striking reduction of disease incidence (83%) and severity in 5-k.o. as compared to wild type mice (8% incidence). Clinically asymptomatic fy-k.o, mice showed no cellular infiltration in the spinal cord whereas wild type animals had infiltration of macrophages, Bcells, ccl3- and y~i T-cells independent of clinical disease. In adoptive transfer EAE, there was reduced y-IFN secretion in fi-k.o, mice accompanied by an increase in IL-10. These findings suggest an important role for y~ T-cells in the regulation of both actively induced and adoptively transferred EAE.