ORIGINAL RESEARCH ARTICLE
Journal of
CD40 Signaling Drives B Lymphocytes Into an Intermediate Memory-Like State, Poised Between Naïve and Plasma Cells
Cellular Physiology
MALA UPADHYAY,1 G. KRISHNA PRIYA,1 P. RAMESH,1 M.B. MADHAVI,1 SATYAJIT RATH,2 VINEETA BAL,2 ANNA GEORGE,2 AND TUSHAR VAIDYA1* 1
Centre for Cellular and Molecular Biology, Hyderabad 500007, India
2
National Institute of Immunology, New Delhi 110067, India
Immunological memory comprising of antigen-specific B and T cells contributes to the acquisition of long-term resistance to pathogens. Interactions between CD40 on B cells and CD40L on T cells are responsible for several aspects of acquired immune responses including generation of memory B cells. In order to gain insights into events leading to memory B cell formation, we analyzed the genome-wide expression profile of murine naive B cells stimulated in the presence of anti-CD40. We have identified over 8,000 genes whose expression is altered minimally 1.5-fold at least at one time point over a 3-day time course. The array analysis indicates that changes in expression level of maximum number of these genes occur within 24 h of anti-CD40 treatment. In parallel, we have studied the events following CD40 ligation by examining the expression of known regulators of naive B cell to plasma cell transition, including Pax5 and BLIMP1. The expression profile of these regulatory genes indicates firstly, that CD40 signaling activates naïve B cells to a phenotype that is intermediate between the naive and plasma cell stages of the B cell differentiation. Secondly, the major known regulator of plasma cell differentiation, BLIMP1, gets irreversibly downregulated upon anti-CD40 treatment. Additionally, our data reveal that CD40 signaling mediated BLIMP1 downregulation occurs by non-Pax5/non-Bcl6 dependent mechanisms, indicating novel mechanisms at work that add to the complexity of understanding of B cell master regulatory molecules like BLIMP1 and Pax5. J. Cell. Physiol. 229: 1387–1396, 2014. © 2014 Wiley Periodicals, Inc.
B-Lymphocytes constitute an important aspect of our immune system by virtue of their ability to produce highly specific antibodies in response to foreign antigens and pathogens. When naïve B lymphocytes encounter the particular antigen that they are responsive to, they differentiate into two types of cells: antibody-secreting plasma cells and long-lived memory cells. While plasma cells are responsible for the rapid resolution of a current infection or immune challenge, memory B cells are responsible for mounting a rapid response to subsequent challenges. Although activation of individual naïve B cells is initiated by the interaction between a specific B cell receptor and its cognate antigen, the intensity and duration of interaction is modulated by various co-receptors such as CD19, CD21, CD22, CD45, and FcgRIIB (O’Rourke et al., 1997). Members of the TNFR family that include CD27 and CD40 also control B cell activation (Dadgostar et al., 2002; Raman et al., 2000, 2003). The concerted binding of the antigen: BCR and the various co-receptors in turn lead to the recruitment and participation of various protein tyrosine kinases (such as Syk, Lyn, Blk, Lck, Fyn) and phosphatases (such as SHP1 and SHIP) (Tedder, 1998). The orchestrated reciprocal phosphorylation/ de-phosphorylation activities of these signaling molecules leads to the generation of second messengers like diacyl glycerol (DAG) and inositol-(1,4,5)-triphosphate (IP3) which ultimately results in the activation and nuclear localization of transcription factors, which now control the manifestation of the final phenotype (Baeuerle and Henkel, 1994). However, exactly how a proliferating B cell makes the choice to become a memory cell is poorly understood. Commitment to memory cells seems to occur in germinal centers (Vinuesa et al., 2010) but the mechanisms by which decisions are made to follow memory or plasma cell differentiation remain unclear. © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
In humans, proxy markers such as CD27 can identify memory B lymphocytes; a facility that is lacking in the murine system (Klein et al., 1998; Xiao et al., 2004). Hence, it is expected that there will be some differences in the generation of immunological memory between murine and human system. However, it continues to be important to understand murine immune memory generation because the murine system allows us to use various genetic means as well as functional assays to validate findings. The TNFR family receptor CD40, expressed on a number of antigen presenting cells, including B lymphocytes, has a cognate ligand on T lymphocytes, CD154 (CD40 L). CD40 mediated signaling is involved in a broad variety of immune activities including antibody class switching, memory B cell development, and germinal center formation (Grewal and Flavell, 1998; Randall et al., 1998; van Kooten and Banchereau, 2000). Additionally, previous work had
The authors declare that they have no conflict of interest. Contract grant sponsor: Department of Biotechnology, Ministry of Science and Technology, Government of India; Contract grant number: BT/PR6351/BRB/10/429/2005. *Correspondence to: Tushar Vaidya, Centre for Cellular and Molecular Biology, Uppal Road, Habshiguda, Hyderabad 500007, India. E-mail: [email protected] Manuscript Received: 6 September 2013 Manuscript Accepted: 16 January 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 31 January 2014. DOI: 10.1002/jcp.24572
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UPADHYAY ET AL.
demonstrated that treatment of mice with a single dose of antiCD40 at the time of immunization lead to improved secondary responses (Randall et al., 1998; Raman et al., 2003). With this in mind, we have chosen to study the molecular mechanisms underlying the generation of memory B cells, using CD40 ligation as an induction signal. LPS is a known mitogen and stimulator of plasma cell differentiation (Schliephake and Schimpl, 1996). We have carried out a comparative genome wide gene expression study of naïve murine B lymphocytes stimulated by LPS as compared to LPS þ anti-CD40. Microarray analyses, carried out over a time course of 24, 48, and 72 h indicated that maximal events occurring within the initial 24 h. A significant number of genes modulated in the early time points are in common with gene expression profiles of long-term memory cells. Additionally, we have carried out quantitative real-time PCR analyses of candidate regulators of plasma cell differentiation. Our results indicate that CD40-mediated signaling places activated B lymphocytes in a memory-like state: not naive any longer and yet not differentiated into plasma cells. We also find that CD40 signaling invokes novel mechanisms for the irreversible downregulation of the B lymphocyte induced maturation protein, BLIMP1. Results Microarray analyses
To elucidate molecular contribution of CD40 signaling mediated events in the differentiation of naive B cells into memory or plasma cells, we used DNA microarrays to analyze genes regulated by the engagement of the CD40 receptor on primary lymphocytes. We stimulated IgDþ primary murine B cells with LPS or LPS þ anti-CD40 over a time course extending up to 72 h. At 24 h intervals, total RNA was isolated from each group. The RNA was used to generate Cy3 or Cy5 labeled cDNA and hybridized to Operon’s Array ready mouse chips. The array set contains 35,000 spots, each containing 70mers, which represent 29,000 mouse genes. Changes in gene expression were profiled from 24, 48, and 72 h post-treatment. In addition, we have also compared untreated cells (Day 0) to cells treated with LPS for 24 h. For each time point: four independent hybridizations were carried out with three slides being replicates þ 1 slide, a dye reversal between the samples. After data acquisition, the signal data were subjected to two different normalizations: Lowess normalization, which normalized the signal by individual block comparisons and Flip dye normalization, which addressed the Dye bias in signal levels. Gene spots were included in analysis if they met the following criteria: The signal was non-zero; present on each slide; directionality same in both normalizations. Comparison of the normalizations showed approximately 10% differences between the two normalization. Hence, for all subsequent analyses, only Lowess Normalization was used. Combining the data from all the time points, we initially generated a composite list of 10,462 genes that are consistently detectable at least one time point (unpublished results). We then analyzed this list of valid genes in each experiment for their pattern of expression in response to the treatment. In all experiments, the change in expression changes was always determined in terms of antiCD40 treatment versus LPS alone. In the case of the Day 0 experiment, the expression change was interpreted in terms of no treatment versus LPS. Thus, in the Day 0 experiment, induced gene ¼ NOT repressed by LPS. We culled our array results further to focus on identification of relevant genes. Criteria applied for induced or repressed genes were: Induced ¼ fold change >1.5; Repressed ¼ Fold change
Journal of
CD40 Signaling Drives B Lymphocytes Into an Intermediate Memory-Like State, Poised Between Naïve and Plasma Cells
Cellular Physiology
MALA UPADHYAY,1 G. KRISHNA PRIYA,1 P. RAMESH,1 M.B. MADHAVI,1 SATYAJIT RATH,2 VINEETA BAL,2 ANNA GEORGE,2 AND TUSHAR VAIDYA1* 1
Centre for Cellular and Molecular Biology, Hyderabad 500007, India
2
National Institute of Immunology, New Delhi 110067, India
Immunological memory comprising of antigen-specific B and T cells contributes to the acquisition of long-term resistance to pathogens. Interactions between CD40 on B cells and CD40L on T cells are responsible for several aspects of acquired immune responses including generation of memory B cells. In order to gain insights into events leading to memory B cell formation, we analyzed the genome-wide expression profile of murine naive B cells stimulated in the presence of anti-CD40. We have identified over 8,000 genes whose expression is altered minimally 1.5-fold at least at one time point over a 3-day time course. The array analysis indicates that changes in expression level of maximum number of these genes occur within 24 h of anti-CD40 treatment. In parallel, we have studied the events following CD40 ligation by examining the expression of known regulators of naive B cell to plasma cell transition, including Pax5 and BLIMP1. The expression profile of these regulatory genes indicates firstly, that CD40 signaling activates naïve B cells to a phenotype that is intermediate between the naive and plasma cell stages of the B cell differentiation. Secondly, the major known regulator of plasma cell differentiation, BLIMP1, gets irreversibly downregulated upon anti-CD40 treatment. Additionally, our data reveal that CD40 signaling mediated BLIMP1 downregulation occurs by non-Pax5/non-Bcl6 dependent mechanisms, indicating novel mechanisms at work that add to the complexity of understanding of B cell master regulatory molecules like BLIMP1 and Pax5. J. Cell. Physiol. 229: 1387–1396, 2014. © 2014 Wiley Periodicals, Inc.
B-Lymphocytes constitute an important aspect of our immune system by virtue of their ability to produce highly specific antibodies in response to foreign antigens and pathogens. When naïve B lymphocytes encounter the particular antigen that they are responsive to, they differentiate into two types of cells: antibody-secreting plasma cells and long-lived memory cells. While plasma cells are responsible for the rapid resolution of a current infection or immune challenge, memory B cells are responsible for mounting a rapid response to subsequent challenges. Although activation of individual naïve B cells is initiated by the interaction between a specific B cell receptor and its cognate antigen, the intensity and duration of interaction is modulated by various co-receptors such as CD19, CD21, CD22, CD45, and FcgRIIB (O’Rourke et al., 1997). Members of the TNFR family that include CD27 and CD40 also control B cell activation (Dadgostar et al., 2002; Raman et al., 2000, 2003). The concerted binding of the antigen: BCR and the various co-receptors in turn lead to the recruitment and participation of various protein tyrosine kinases (such as Syk, Lyn, Blk, Lck, Fyn) and phosphatases (such as SHP1 and SHIP) (Tedder, 1998). The orchestrated reciprocal phosphorylation/ de-phosphorylation activities of these signaling molecules leads to the generation of second messengers like diacyl glycerol (DAG) and inositol-(1,4,5)-triphosphate (IP3) which ultimately results in the activation and nuclear localization of transcription factors, which now control the manifestation of the final phenotype (Baeuerle and Henkel, 1994). However, exactly how a proliferating B cell makes the choice to become a memory cell is poorly understood. Commitment to memory cells seems to occur in germinal centers (Vinuesa et al., 2010) but the mechanisms by which decisions are made to follow memory or plasma cell differentiation remain unclear. © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
In humans, proxy markers such as CD27 can identify memory B lymphocytes; a facility that is lacking in the murine system (Klein et al., 1998; Xiao et al., 2004). Hence, it is expected that there will be some differences in the generation of immunological memory between murine and human system. However, it continues to be important to understand murine immune memory generation because the murine system allows us to use various genetic means as well as functional assays to validate findings. The TNFR family receptor CD40, expressed on a number of antigen presenting cells, including B lymphocytes, has a cognate ligand on T lymphocytes, CD154 (CD40 L). CD40 mediated signaling is involved in a broad variety of immune activities including antibody class switching, memory B cell development, and germinal center formation (Grewal and Flavell, 1998; Randall et al., 1998; van Kooten and Banchereau, 2000). Additionally, previous work had
The authors declare that they have no conflict of interest. Contract grant sponsor: Department of Biotechnology, Ministry of Science and Technology, Government of India; Contract grant number: BT/PR6351/BRB/10/429/2005. *Correspondence to: Tushar Vaidya, Centre for Cellular and Molecular Biology, Uppal Road, Habshiguda, Hyderabad 500007, India. E-mail: [email protected] Manuscript Received: 6 September 2013 Manuscript Accepted: 16 January 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 31 January 2014. DOI: 10.1002/jcp.24572
1387
1388
UPADHYAY ET AL.
demonstrated that treatment of mice with a single dose of antiCD40 at the time of immunization lead to improved secondary responses (Randall et al., 1998; Raman et al., 2003). With this in mind, we have chosen to study the molecular mechanisms underlying the generation of memory B cells, using CD40 ligation as an induction signal. LPS is a known mitogen and stimulator of plasma cell differentiation (Schliephake and Schimpl, 1996). We have carried out a comparative genome wide gene expression study of naïve murine B lymphocytes stimulated by LPS as compared to LPS þ anti-CD40. Microarray analyses, carried out over a time course of 24, 48, and 72 h indicated that maximal events occurring within the initial 24 h. A significant number of genes modulated in the early time points are in common with gene expression profiles of long-term memory cells. Additionally, we have carried out quantitative real-time PCR analyses of candidate regulators of plasma cell differentiation. Our results indicate that CD40-mediated signaling places activated B lymphocytes in a memory-like state: not naive any longer and yet not differentiated into plasma cells. We also find that CD40 signaling invokes novel mechanisms for the irreversible downregulation of the B lymphocyte induced maturation protein, BLIMP1. Results Microarray analyses
To elucidate molecular contribution of CD40 signaling mediated events in the differentiation of naive B cells into memory or plasma cells, we used DNA microarrays to analyze genes regulated by the engagement of the CD40 receptor on primary lymphocytes. We stimulated IgDþ primary murine B cells with LPS or LPS þ anti-CD40 over a time course extending up to 72 h. At 24 h intervals, total RNA was isolated from each group. The RNA was used to generate Cy3 or Cy5 labeled cDNA and hybridized to Operon’s Array ready mouse chips. The array set contains 35,000 spots, each containing 70mers, which represent 29,000 mouse genes. Changes in gene expression were profiled from 24, 48, and 72 h post-treatment. In addition, we have also compared untreated cells (Day 0) to cells treated with LPS for 24 h. For each time point: four independent hybridizations were carried out with three slides being replicates þ 1 slide, a dye reversal between the samples. After data acquisition, the signal data were subjected to two different normalizations: Lowess normalization, which normalized the signal by individual block comparisons and Flip dye normalization, which addressed the Dye bias in signal levels. Gene spots were included in analysis if they met the following criteria: The signal was non-zero; present on each slide; directionality same in both normalizations. Comparison of the normalizations showed approximately 10% differences between the two normalization. Hence, for all subsequent analyses, only Lowess Normalization was used. Combining the data from all the time points, we initially generated a composite list of 10,462 genes that are consistently detectable at least one time point (unpublished results). We then analyzed this list of valid genes in each experiment for their pattern of expression in response to the treatment. In all experiments, the change in expression changes was always determined in terms of antiCD40 treatment versus LPS alone. In the case of the Day 0 experiment, the expression change was interpreted in terms of no treatment versus LPS. Thus, in the Day 0 experiment, induced gene ¼ NOT repressed by LPS. We culled our array results further to focus on identification of relevant genes. Criteria applied for induced or repressed genes were: Induced ¼ fold change >1.5; Repressed ¼ Fold change
