- Email: [email protected]
The antibody response to Hib polysaccharide: Regulation by T cells
254
B-cell and T-cell memory
P.2.05.05
The antibody response to Hib pofysaccharide: Regulatlon by T cells
Mijke A. Breukels, Elly A. To&es, Cobi Heijnen, Ben J.M. Zegers, Ger T. Rijkers. Dept.of Immunology, Univeffiity Hospital for Children and Youth “Het wilhelmina Kindatziakanhuis” PO. Bax 18009,3!701 CA Utmcht, The Netherlands Conjugated Haemophilus inftuenzae type b (Hib) polysacchartde (PRP) to Tetanus toxoid (TT) induces an anti-PRP response with the characteristics of a T cell dependent antigen. In order to study the T cell regulation of this response, we immunized adult volunteen with PRP-TT conjugate vaccine, and after 21 days restimulated B cells in vitro with PRP. The in vitro anti-PRP B cell response required, next to PRP, TT (either conjugated to or mixed with PRP) as well as T cells. Two lT peptides (encompassing T helper epitopes) were generated: 156 (aa 830-844) and 158 (aa 947-967) which induced in approx. 50% of donors a proliferative T cell response after PRP-lT vaccination. While these peptides were unable to induce an anti-l-f B cell response, we did find a strict correlation between the ability to induce T cell proliferation and supporting the in vitro anti-PRP antibody response. Preliminary data from transwell studies indicate that physical contact between T and B cells is necessary to induce the anti-PRP antibody response. Future experiments are aimed to identii the interacting receptors and cytokines in this response.
1P.2.05.06]
T cell response to HAV after hepatltis A vaccination
X.Q.Chen ‘, G.J. Boland ‘, J. van Hattum ‘, G.C. de Gast2. ‘Department of Gastmenterolog~ University Hospital of Utrscht, The Netherlands, 2Department of Immunohaematol~ University Hospital of Utmcht, The Netherlands
Intrcductlon:The T cell response to hepatitis A virus (HAV) was investigated several years after vaccination wlth formalin-inactivated HAV vaccine. Twenty six subjects were included. Seventeen vaccinees had previously received a complete course of immunization with Vaqta (Merck & Co., Inc., USA). Among them, 11 had received the first injection in 1992 (group 1) and 6 in 1994 (group 2). Nine non-vaccinated and uninfected healthy volunteers sewed as negative controls (group 3). Methods: The in vitmT cell response was measured by lymphocyte proliferation assay. Freshly isolated peripheral blood mononuclear cells (PBMC) were incubated with different concentrations of HAV antigen for 5, 7 and 9 days, in order to determine the optimal concentrations and time course of HAV stimulation. Tetanus toxoid was used as positive controls. Results were expressed as Stimulation Index (SI), values of SI ? 3.0 were considered as significant increases of proliferation. Surface antigens on lymphocytes were detected by immunofluorescence staining of incubated PBMC on day 0, 3, 5, 7 and 9 of culture respectively and these two-colour immunofluorescence stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson). The in vivo anti-HAV response was determined by HAVAB-IMx (Abbott) and was evaluated in term of geometrtc mean titer (GMT). Reeufte: A significant proliferative T cell response to HAV was found in all vaccinees, while no proliferation to HAV was observed in the non-vaccinated controls. This response to inactivated HAV in vitro was mainly due to CD4+ (helper) T cells, as shown by concomitant expression of HLA-DR and CD25 on 23-27% of CD4+ T cells after 9 days of culture versus only 3-7% of CD8+ T cells. All groups exhibited a similar strono resoonse to the recall anttoen tetanus toxoid. T-cell.HAV responses of recent6 vaccinated persons (medien SI 36.0 on day 7 and 68.6 on day 9) were as high as to tetanus toxoid (median SI 39.2). All vaccinees still had protective anti-HAV titers (110 mlU/ ml). No correlation between the height of the titer and that of the SI value was detected. Conclusion: CD4+ T cell response after HA vaccination can be effectively monitored by using an HAV-specific lymphocyte proliferation assay.
[ P.2.05.07 1 Model system to study the mechanisms of D memory cell generation T.K. Borisova, E.V. Sidorova. Institute for Vim/ Preparations of RAMS, Moscow, Russia
Introduction:Cellular mechanisms of generation of B memory cells (Bm) to T-independent antigens of type 2 (TI-2) are still unclear. The present study was done to develop an experimental system for investigation of this process. MaterIsIsand Mathods:Experiments were performed on CBA mice. DNPFicoll was used as TI-2 antigen. Total splenocytes or different cell subpopulations were cultured with or without the antigen for 4 days at 3PC, washed and transferred into unimmunized syngeneic mice. One month later mice were immunized by the antigen in vivo. Antibody-forming cells (AFC) were detenined by the local hemolysis method. Anti-DNP antibodies were determined by ELISA. Results: The transfer of immune splenocytes resulted in two-fold increase of immune response to DNP as compared with that of recipients of normal splenocytes. Anti-DNP antibodies in recipient sera were not only of IgM, but
24 June 1997 - Poster presentations also of IgG isotypes (of all subclasses). This effect was due mainly to adherent cells, but not to T or B cells; the transfer of primed adherent cells induced the same increase in AFC number and the appearance of the same isotopes of antibodies. These data can be explained by more efficient presentation of DNP-Ficoll by primed adherent cells. Conclusion: The experimental system described can be used for the investigation of Bm cell generation and for study the role of different cell subpopulations in this process.
1P2.05.08 ] Reduction of memory cytotoxic T cell numbers by heterologous viral infections: A mathematical model Katsuhisa Takumi, Rob de Boer. 77reoreficalBiology, Utrscht Univsrsirv; Padualaan 8,3584 CH Utrecht, The Netherlands
Intmcluctlon:Dynamic changes of cytotoxic T lymphocyte (CTL) clones in response to infection by heterologous viruses were experimentally observed in mice. If a mouse immune to lymphocyti~ chortomeningitis virus (LCMV) is subsequently infected by PMinds virus (PV), the number of LCMV specific memory CTLs decreases. Addltional infection by another virus further reduces the number of LCMV specific CTL. A plausible explanation Is that competition between two or more memory CTL clones took place and consequently changed the numbers of each CTL clone. Methods:Basedon the experimental data we develop a mathematical model which describes the changes of CTL numbers after an infection (Selin et al., 1998). The model illustrates a general process of competition and its consequences. Rwub After a viral infection, the total number of CTLs increases transiently and returns to a homeostattc level. As a new memory CTL clone is accommodated for every new infection within a limited pool of lymphocyte population, CD8+ T cell responses are continuously changed by each infectious agents. Conclusion: The change in the numbers of virus-specific CTLs by heterologous viral infections can be explained by competition among memory CTLs. [l] Selin, L. K., Vergilis, K., Welsh, R. M. & Nahill, S. R. (1996). Reduction of Otherwise Remarkably Stable Virus-specific Cytotoxic T Lymphocyte Memory by Heterologous Viral Infections J. Exp. Med. 189,2489-2499.
1P.2.05.09 1 T cell receptor usage by CD45RA+ and CD4!SRO+T lymphocytes J.M. Faint’, G.D. Kitas’, D. Pilling I, A.N. Akbar2, P.A. Bacon ‘, M. Salmon l. ’ Department of Rheumatdogy, Univemity of Binningham, Binningham. UK, 2Department of Clinical Immunol~ Royal Free Hospital, London, UK Introduction: The T cell receptor (TCR) repertoire in peripheral T lymphocytes consists of the apparently naive or resting repertoire (CD45RA+ cells) and the primed repertoire (CD45RO+ cells). Changes in the repertoire use between these populations may reflect underlying immune responses to antigen. The recent availability of a wide range of human TCR specific antibodies has made the rapid assessment of T cell receptor usage by flow cytometry possible. Interpretation of changes in the repertoire in disease requires a thorough understanding of the TCR repertoire in healthy subjects. Our experiments have defined the limits of sensitivity of flow cytometry for this purpose. We then assessed the TCR repertoire expressed by the CD45RA+ and CD45RO+ subsets of CD4+ and CD8+ T cells in healthy individuals. Yaterlals and Methods: Flow Cytometry Sensttlvlty. Peripheral blood mononuclear cells (PBMC) were stained with anti- CD3. CD4, CD8 or one of 4 Vfi specific antibodies and analywd using a Coulter EPICS XL flow cytometer. The number of cells positively stained with each antibody was determined by counting a predetermined number of lymphocytes. These values were used to calculate the coefficient of variation (CV = SD/Mean) for the number of positively stained cells for each given number of lymphocytes ranging from 100 to 105 cells. TCR RepertoireAnalysis.Freshly isolated PBMC were stained with antibodies to either CD4 or CD& plus CD45RA and a panel of 19 VP family specificities. Information for 10.000 cells was collected using the Coulter EPICS XL cytometer. Results:Flow Cytometry!Sensltlvtty.Increasing the number of cells counted reduced the variability between samples, but no improvement in the precision of analysis was obtained by collecting more than 10,000 cells, even for a VP family specificity present at a frequency less than 0.5%. TCR RepertclreAnalysis.(1) Comparison of the TCR repertoire of CD4 and CD8 cells of 25 healthy individuals (aged 20-35) revealed a skewed expression of some VP specificities toward CD4 cells ([email protected], 6.7, 13.1, 18) and some toward CD8 cells (V,314, 23). (2) The T cell receptors expressed by the CD4CD45RA and CD4CD45RO subsets showed an extremely close relationship suggesting tight homeostatic control.
B-cell and T-cell memory
P.2.05.05
The antibody response to Hib pofysaccharide: Regulatlon by T cells
Mijke A. Breukels, Elly A. To&es, Cobi Heijnen, Ben J.M. Zegers, Ger T. Rijkers. Dept.of Immunology, Univeffiity Hospital for Children and Youth “Het wilhelmina Kindatziakanhuis” PO. Bax 18009,3!701 CA Utmcht, The Netherlands Conjugated Haemophilus inftuenzae type b (Hib) polysacchartde (PRP) to Tetanus toxoid (TT) induces an anti-PRP response with the characteristics of a T cell dependent antigen. In order to study the T cell regulation of this response, we immunized adult volunteen with PRP-TT conjugate vaccine, and after 21 days restimulated B cells in vitro with PRP. The in vitro anti-PRP B cell response required, next to PRP, TT (either conjugated to or mixed with PRP) as well as T cells. Two lT peptides (encompassing T helper epitopes) were generated: 156 (aa 830-844) and 158 (aa 947-967) which induced in approx. 50% of donors a proliferative T cell response after PRP-lT vaccination. While these peptides were unable to induce an anti-l-f B cell response, we did find a strict correlation between the ability to induce T cell proliferation and supporting the in vitro anti-PRP antibody response. Preliminary data from transwell studies indicate that physical contact between T and B cells is necessary to induce the anti-PRP antibody response. Future experiments are aimed to identii the interacting receptors and cytokines in this response.
1P.2.05.06]
T cell response to HAV after hepatltis A vaccination
X.Q.Chen ‘, G.J. Boland ‘, J. van Hattum ‘, G.C. de Gast2. ‘Department of Gastmenterolog~ University Hospital of Utrscht, The Netherlands, 2Department of Immunohaematol~ University Hospital of Utmcht, The Netherlands
Intrcductlon:The T cell response to hepatitis A virus (HAV) was investigated several years after vaccination wlth formalin-inactivated HAV vaccine. Twenty six subjects were included. Seventeen vaccinees had previously received a complete course of immunization with Vaqta (Merck & Co., Inc., USA). Among them, 11 had received the first injection in 1992 (group 1) and 6 in 1994 (group 2). Nine non-vaccinated and uninfected healthy volunteers sewed as negative controls (group 3). Methods: The in vitmT cell response was measured by lymphocyte proliferation assay. Freshly isolated peripheral blood mononuclear cells (PBMC) were incubated with different concentrations of HAV antigen for 5, 7 and 9 days, in order to determine the optimal concentrations and time course of HAV stimulation. Tetanus toxoid was used as positive controls. Results were expressed as Stimulation Index (SI), values of SI ? 3.0 were considered as significant increases of proliferation. Surface antigens on lymphocytes were detected by immunofluorescence staining of incubated PBMC on day 0, 3, 5, 7 and 9 of culture respectively and these two-colour immunofluorescence stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson). The in vivo anti-HAV response was determined by HAVAB-IMx (Abbott) and was evaluated in term of geometrtc mean titer (GMT). Reeufte: A significant proliferative T cell response to HAV was found in all vaccinees, while no proliferation to HAV was observed in the non-vaccinated controls. This response to inactivated HAV in vitro was mainly due to CD4+ (helper) T cells, as shown by concomitant expression of HLA-DR and CD25 on 23-27% of CD4+ T cells after 9 days of culture versus only 3-7% of CD8+ T cells. All groups exhibited a similar strono resoonse to the recall anttoen tetanus toxoid. T-cell.HAV responses of recent6 vaccinated persons (medien SI 36.0 on day 7 and 68.6 on day 9) were as high as to tetanus toxoid (median SI 39.2). All vaccinees still had protective anti-HAV titers (110 mlU/ ml). No correlation between the height of the titer and that of the SI value was detected. Conclusion: CD4+ T cell response after HA vaccination can be effectively monitored by using an HAV-specific lymphocyte proliferation assay.
[ P.2.05.07 1 Model system to study the mechanisms of D memory cell generation T.K. Borisova, E.V. Sidorova. Institute for Vim/ Preparations of RAMS, Moscow, Russia
Introduction:Cellular mechanisms of generation of B memory cells (Bm) to T-independent antigens of type 2 (TI-2) are still unclear. The present study was done to develop an experimental system for investigation of this process. MaterIsIsand Mathods:Experiments were performed on CBA mice. DNPFicoll was used as TI-2 antigen. Total splenocytes or different cell subpopulations were cultured with or without the antigen for 4 days at 3PC, washed and transferred into unimmunized syngeneic mice. One month later mice were immunized by the antigen in vivo. Antibody-forming cells (AFC) were detenined by the local hemolysis method. Anti-DNP antibodies were determined by ELISA. Results: The transfer of immune splenocytes resulted in two-fold increase of immune response to DNP as compared with that of recipients of normal splenocytes. Anti-DNP antibodies in recipient sera were not only of IgM, but
24 June 1997 - Poster presentations also of IgG isotypes (of all subclasses). This effect was due mainly to adherent cells, but not to T or B cells; the transfer of primed adherent cells induced the same increase in AFC number and the appearance of the same isotopes of antibodies. These data can be explained by more efficient presentation of DNP-Ficoll by primed adherent cells. Conclusion: The experimental system described can be used for the investigation of Bm cell generation and for study the role of different cell subpopulations in this process.
1P2.05.08 ] Reduction of memory cytotoxic T cell numbers by heterologous viral infections: A mathematical model Katsuhisa Takumi, Rob de Boer. 77reoreficalBiology, Utrscht Univsrsirv; Padualaan 8,3584 CH Utrecht, The Netherlands
Intmcluctlon:Dynamic changes of cytotoxic T lymphocyte (CTL) clones in response to infection by heterologous viruses were experimentally observed in mice. If a mouse immune to lymphocyti~ chortomeningitis virus (LCMV) is subsequently infected by PMinds virus (PV), the number of LCMV specific memory CTLs decreases. Addltional infection by another virus further reduces the number of LCMV specific CTL. A plausible explanation Is that competition between two or more memory CTL clones took place and consequently changed the numbers of each CTL clone. Methods:Basedon the experimental data we develop a mathematical model which describes the changes of CTL numbers after an infection (Selin et al., 1998). The model illustrates a general process of competition and its consequences. Rwub After a viral infection, the total number of CTLs increases transiently and returns to a homeostattc level. As a new memory CTL clone is accommodated for every new infection within a limited pool of lymphocyte population, CD8+ T cell responses are continuously changed by each infectious agents. Conclusion: The change in the numbers of virus-specific CTLs by heterologous viral infections can be explained by competition among memory CTLs. [l] Selin, L. K., Vergilis, K., Welsh, R. M. & Nahill, S. R. (1996). Reduction of Otherwise Remarkably Stable Virus-specific Cytotoxic T Lymphocyte Memory by Heterologous Viral Infections J. Exp. Med. 189,2489-2499.
1P.2.05.09 1 T cell receptor usage by CD45RA+ and CD4!SRO+T lymphocytes J.M. Faint’, G.D. Kitas’, D. Pilling I, A.N. Akbar2, P.A. Bacon ‘, M. Salmon l. ’ Department of Rheumatdogy, Univemity of Binningham, Binningham. UK, 2Department of Clinical Immunol~ Royal Free Hospital, London, UK Introduction: The T cell receptor (TCR) repertoire in peripheral T lymphocytes consists of the apparently naive or resting repertoire (CD45RA+ cells) and the primed repertoire (CD45RO+ cells). Changes in the repertoire use between these populations may reflect underlying immune responses to antigen. The recent availability of a wide range of human TCR specific antibodies has made the rapid assessment of T cell receptor usage by flow cytometry possible. Interpretation of changes in the repertoire in disease requires a thorough understanding of the TCR repertoire in healthy subjects. Our experiments have defined the limits of sensitivity of flow cytometry for this purpose. We then assessed the TCR repertoire expressed by the CD45RA+ and CD45RO+ subsets of CD4+ and CD8+ T cells in healthy individuals. Yaterlals and Methods: Flow Cytometry Sensttlvlty. Peripheral blood mononuclear cells (PBMC) were stained with anti- CD3. CD4, CD8 or one of 4 Vfi specific antibodies and analywd using a Coulter EPICS XL flow cytometer. The number of cells positively stained with each antibody was determined by counting a predetermined number of lymphocytes. These values were used to calculate the coefficient of variation (CV = SD/Mean) for the number of positively stained cells for each given number of lymphocytes ranging from 100 to 105 cells. TCR RepertoireAnalysis.Freshly isolated PBMC were stained with antibodies to either CD4 or CD& plus CD45RA and a panel of 19 VP family specificities. Information for 10.000 cells was collected using the Coulter EPICS XL cytometer. Results:Flow Cytometry!Sensltlvtty.Increasing the number of cells counted reduced the variability between samples, but no improvement in the precision of analysis was obtained by collecting more than 10,000 cells, even for a VP family specificity present at a frequency less than 0.5%. TCR RepertclreAnalysis.(1) Comparison of the TCR repertoire of CD4 and CD8 cells of 25 healthy individuals (aged 20-35) revealed a skewed expression of some VP specificities toward CD4 cells ([email protected], 6.7, 13.1, 18) and some toward CD8 cells (V,314, 23). (2) The T cell receptors expressed by the CD4CD45RA and CD4CD45RO subsets showed an extremely close relationship suggesting tight homeostatic control.