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P D Hodgkin

Publications and source records attributed to P D Hodgkin.

At least 19 recordsLinked to original sources
PubMed2005

Quantitative rules for lymphocyte regulation: the cellular calculus and decisions between tolerance and activation.

The innovation of fluorescent division-tracking techniques has elevated our understanding of lymphocytes to a new level. These techniques applied in vitro have identified quantitative rules for lymphocyte differentiation, proliferation and survival that had previously been hidden. The many patterns of quantitative response revealed by these analyses provide a sharp contrast to the traditional idea that T cells must make a binary choice between tolerance and activation. Here, evidence for the classic dogma of two-signal theory and T-T help is re-examined in the light of the new quantitative view to show how logical difficulties can be resolved.

Animals↗
PubMed2001

B cells activated via CD40 and IL-4 undergo a division burst but require continued stimulation to maintain division, survival and differentiation.

T cell stimulation of B cell proliferation during T-B collaboration requires membrane-bound stimulatory ligands, such as CD40 ligand and the secretion of soluble cytokines, such as IL-4. Nevertheless, it remains unclear whether T cell contact is required to provoke each consecutive B cell division, or whether B cells divide in a T cell-free burst following the initial stimulation. To test this, naive B cells were cultured with anti-CD40 monoclonal antibody (mAb) and IL-4 and, after various times, these stimuli were removed and the cells re-cultured with or without further stimulation. Following stimulus removal, B cells were able to continue proliferating, with the size of the B cell burst dependent on the strength of the initial anti-CD40 mAb stimulus. Furthermore, in the absence of activating signals from anti-CD40 and/or IL-4, re-cultured B cells died rapidly. In addition, B cells undergoing a stimulus-free division burst could switch to IgG1. Thus, maximal B cell proliferation, differentiation and survival may require continued, although not necessarily consecutive, cognate interactions with T cells. These results suggest that antigen persistence and T cell help are necessary to sustain B cell proliferation and differentiation in vivo.

Animals↗
PubMed2000

Flow cytometric cell division tracking using nuclei.

BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle.

Animals↗
PubMed2000

A cellular calculus for signal integration by T cells.

During an immune response numerous receptor-mediated signals delivered to T cells direct their proliferation, survival and differentiation. Here, we describe a quantitative model and in vitro methods for assessing the "calculus" used by T cells to process these multiple signals. The model reveals how T cells convert independently received signals into linear additive effects on division times which, in turn, amplify T cell number exponentially. These results explain why so many ligands can each appear obligatory for T cell activation and argue for a re-examination of the two-signal theory as the basis for decisions between tolerance and activation.

Animals↗
PubMed2000

Absence of lipopolysaccharide high-dose paralysis in B-cell responses: implications for the one-signal theory.

Over 20 years ago, Coutinho and Möller reported that high concentrations of LPS were paralytic for the development of antibody secreting cells (ASC). This data was used to explain bell-shaped dose-response curves observed for antihapten antibody formation in response to haptenated LPS. In turn, this bell curve was used to formulate the one-signal model of B cell activation, which argued that antigen signalling was generally unimportant to B cell responses. The present paper re-examines LPS dose-response curves and finds results that do not support the view that high doses of LPS inhibit B cell differentiation to ASC. If high-dose paralysis is not an attribute of LPS stimulation, then the bell-shaped dose curve for hapten-specific ASC originally observed by Coutinho and Möller required an alternative explanation. Through the use of haptenated Ficoll, it was possible to show that the generation of LPS-induced antitrinitrophenol ASC could be inhibited by antigen presented on an inert substrate. Thus, the transmission of surface Ig-mediated (antigen) signals at higher concentrations can explain the antihapten bell-shaped dose curves, in contradiction to the conclusions of the one-signal model.

Animals↗
PubMed2000

Regulation of lipopolysaccharide-induced B-cell activation: evidence that surface immunoglobulin mediates two independently regulated signals.

An antigen-specific B cell response can be induced by low concentrations of haptenated lipopolysaccharide (LPS), whereas high concentrations are inhibitory. Two explanations have been proposed for the latter phenomenon. In the first, specific surface Ig focuses LPS to the B cell membrane, where high local concentrations of the mitogen become paralytic for B cell responses. In the alternative, transmission of an antigen signal at higher concentrations of hapten LPS actively inhibits the development of antibody secreting cells (ASC). In the present paper, the immunosuppressant cyclosporine A (CsA) was used to attempt to distinguish between these two models. Cyclosporine A did not block the inhibitory effects of goat anti-IgM (galphaIgM) on development of ASC induced by LPS and therefore was unsuitable for testing between the two models. However, surprisingly, in the presence but not the absence of CsA, even low concentrations of galphaIgM became inhibitory for LPS-induced B cell proliferation. Thus, a CsA-insensitive signal could inhibit both B cell proliferation and the development of ASC. In contrast, the CsA-sensitive signal induced by sIg required high concentrations of galphaIgM for triggering and enhanced the LPS proliferative response without affecting development of ASC. Evidence is presented that these two signals are regulated independently, suggesting that together they may transmit information about the physical form of an antigen to the B cell.

Animals↗
PubMed1999

Switching to IgG3, IgG2b, and IgA is division linked and independent, revealing a stochastic framework for describing differentiation.

LPS was used to induce switching of B cells to IgG3 and, in the presence of TGF-beta, to IgG2b and IgA. Switching to all three isotypes increased with division number according to a consistent relationship that was independent of time in culture. The mode of activation altered the relationship with division, as CD40 ligand increased switching to IgA and decreased switching to IgG2b and IgG3 when measured per division. This division-linked switching behavior could be described by Gaussian probability distributions centered around a mean division number. The divisions at which switching to IgG3 and IgG2b occurred overlapped, raising the possibility that the two switching mechanisms were linked. However, when IgG3+ and IgG3- B cells were sorted and placed back in culture, they switched to IgG2b at an equivalent rate, indicating that alternative switching decisions were made independently within a single cell. As a consequence, isotype switching could be predicted at the population level by standard probability laws. Therefore, division number provides a framework for a stochastic description of differentiation that may be widely applicable.

Animals↗
PubMed1999

Integrating signals from IFN-gamma and IL-4 by B cells: positive and negative effects on CD40 ligand-induced proliferation, survival, and division-linked isotype switching to IgG1, IgE, and IgG2a.

IL-4 and IFN-gamma each have potent effects on B cell responses as well as strong mutual antagonism. Here we have examined the quantitative effects of these cytokines on CD40 ligand-induced B cell proliferation, cell survival, and division-linked isotype switching. Both IL-4 (strongly) and IFN-gamma (weakly) enhanced the number of B cells found in culture by reducing the average time cells take to enter the first division cycle and by promoting B cell survival. When added in combination, the net effect of IL-4 and IFN-gamma on time to division and survival was a response intermediate between that of the two cytokines alone, indicating a partial antagonism of IL-4 by IFN-gamma. By modulating both time to division and cell survival, these small effects of IFN-gamma are amplified and give rise to large reductions in cell number in the presence of IL-4. At higher concentrations, IFN-gamma had minor inhibitory effects on IL-4-induced isotype switching to IgG1 and greater effects on IgE. A reciprocal relation was observed between the ability to inhibit IgE at late cell divisions vs induction of IgG2a. In contrast, IL-4 did not prevent switching to IgG2a induced by IFN-gamma alone. Therefore, antagonism between IFN-gamma and IL-4 is observed at multiple levels and over different concentration ranges, resulting in complex net outcomes. The evolutionary significance of this complexity is discussed.

Adjuvants, Immunologic↗
PubMed1999

Switch recombination and germ-line transcription are division-regulated events in B lymphocytes.

Treatment of resting murine B lymphocytes with CD40 ligand (CD40L) and IL-4 induces proliferation and a switch in immunoglobulin (Ig) isotype surface expression from IgM and IgD to IgG1 and IgE. Using a fluorescent dye to enable cell sorting according to cell division cycle number, we have examined molecular events associated with B cell differentiation, namely, germ-line transcription and DNA recombination. Digestion-circularisation polymerase chain reaction experiments showed that DNA recombination leading to isotype switching from IgM to IgG1 surface expression is division-dependent and was first detected after B cells had divided three times. Similarly, DNA rearrangement involving the IgE switch region was detectable only after five division cycles. These division cycle numbers correlate with the numbers of divisions required before surface expression of the switched isotype [P.D. Hodgkin, J.-H. Lee, A.B. Lyons, J. Exp. Med. 184 (1996) 277-281]. RT-PCR analyses also revealed that germ-line transcripts for both IgG1 and IgE increased with division number suggesting a threshold expression level may be required for recombination to occur.

Animals↗
PubMed1999

Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester.

Mature T and B lymphocytes respond to receptor-delivered signals received during and following activation. These signals regulate the rates of cell death, growth, differentiation and migration that ultimately establish the behaviour patterns collectively referred to as immune regulation. We have been pursuing the philosophy that in vitro systems of lymphocyte stimulation, when analysed quantitatively, help reveal the logical attributes of lymphocyte behaviour. The development of carboxyfluorescein diacetate succinimidyl ester (CFSE) to track division has enabled the variable of division number to be incorporated into these quantitative analyses. Our studies with CFSE have established a fundamental link between differentiation and division number. Isotype switching, expression of T cell cytokines, surface receptor alterations and changes to intracellular signalling components all display independent patterns of change with division number. The stochastic aspects of these changes and the ability of external signals to independently regulate them argue for a probabilistic modelling framework for describing and understanding immune regulation.

Antigens, Differentiation↗
PubMed1998

Generation of splenic follicular structure and B cell movement in tumor necrosis factor-deficient mice.

Secondary lymphoid tissue organogenesis requires tumor necrosis factor (TNF) and lymphotoxin alpha (LTalpha). The role of TNF in B cell positioning and formation of follicular structure was studied by comparing the location of newly produced naive recirculating and antigen-stimulated B cells in TNF-/- and TNF/LTalpha-/- mice. By creating radiation bone marrow chimeras from wild-type and TNF-/- mice, formation of normal splenic B cell follicles was shown to depend on TNF production by radiation-sensitive cells of hemopoietic origin. Reciprocal adoptive transfers of mature B cells between wild-type and knockout mice indicated that normal follicular tropism of recirculating naive B cells occurs independently of TNF derived from the recipient spleen. Moreover, soluble TNF receptor-IgG fusion protein administered in vivo failed to prevent B cell localization to the follicle or the germinal center reaction. Normal T zone tropism was observed when antigen-stimulated B cells were transferred into TNF-/- recipients, but not into TNF/LTalpha-/- recipients. This result appeared to account for the defect in isotype switching observed in intact TNF/LTalpha-/- mice because TNF/LTalpha-/- B cells, when stimulated in vitro, switched isotypes normally. Thus, TNF is necessary for creating the permissive environment for B cell movement and function, but is not itself responsible for these processes.

Animals↗
PubMed1998

Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation.

Naive T lymphocytes have the potential to differentiate and produce a range of cytokines crucial for appropriate immune responses. How T lymphocytes vary their cytokine output during differentiation is unknown, although they are clearly influenced by the cytokines already present in the environment. Here we show that the number of divisions taken by the cells after activation is a critical element in T cell differentiation. Our experiments used the dye 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester to track cells in different divisions after activation by anti-CD3 in the presence of the differentiating cytokine interleukin (IL)-4. The patterns of acquisition or loss of secretion of IL-2, IL-3, IL-4, IL-5, IL-10, and interferon gamma all varied markedly with division number. These relationships were consistent regardless of the time-dependent variation in distribution of T cells among divisions. Thus, the observed combination of complex asynchronous T cell growth, overlaying a fixed probability of acquisition or loss of a cytokine at each division can explain why T cell differentiation displays the contradictory features of being both highly stochastic and highly controlled. Furthermore, these data reveal that T cells share a common regulatory strategy with B cells, whereby changes in the class of immune response are linked to the process of clonal expansion.

Animals↗
PubMed1998

Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4.

CD40 ligand (CD40L) and IL-4 are sufficient to induce resting murine B cells to divide and switch isotypes from IgM and IgD to IgG1 and IgE. Tracking of cell division following (5- and 6) carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling revealed that B cells expressed IgG1 after three cell divisions, and IgE after five. The probability of isotype switching at each division was independent of both time after stimulation and of the dose of CD40L. IL-4 concentration regulated the number of divisions that preceded isotype switching. Loss of surface IgM and IgD was also related to cell division and appeared to be differentially regulated. B cell proliferation was typically asynchronous with the proportion of cells in consecutive divisions being markedly affected by the concentration of CD40L and IL-4. Simultaneous (5-bromo)-2'-deoxyuridine labeling and CFSE staining revealed that B cells in each division cycle were dividing at the same rate. Therefore, division cycle asynchrony resulted from dose-dependent variation in the time taken to enter the first division cycle. These results suggest that T-dependent B cell expansion is linked to predictable functional changes that may, in part, explain why IgE is produced in response to prolonged antigenic stimulation.

Animals↗
PubMed1998

The logic of intercellular communication in the immune system.

The collaboration between T and B lymphocytes is used as an example to illustrate how the key features of immune regulation (cell interaction, reciprocal exchange of signals by cell contact, and dependence on soluble cytokines) serve as amplifying reactions. By linking cell-based amplifiers in sequence, the resulting immune response is made highly sensitive to small changes in the environment. Thus, intercellular communication in the immune system can be viewed as a higher level analogue to the kinase cascades that amplify intracellular signalling mechanisms.

Animals↗
PubMed1998

Role of anti-donor antibody in the rejection of pig proislet xenografts in mice.

CBA/H mice produced serum anti-pig IgG1, IgG2a, and IgG2b following xenotransplantation of pig proislets beneath the kidney capsule; anti-pig IgM was present as pre-existing antibody in the serum of normal CBA/H mice and was also produced in response to pig proislet xenografts. Serum anti-pig IgG3 was not detected post-xenotransplantation. Rejection of pig proislet xenografts and the production of anti-pig IgG1, IgG2a, and IgG2b isotypes were CD4 T cell-dependent. The capacity for adoptively transferred hyperimmune CBA/H mouse anti-pig PBL serum to induce the rejection of intact pig proislet xenografts in CD4 T cell-depleted mice was dose dependent and correlated with markedly elevated levels of serum anti-pig IgG3. Levels of anti-pig IgG1, IgG2a, IgG2b, and IgM comparable to control mice acutely rejecting pig proislet xenografts and achieved following adoptive transfer of hyperimmune serum did not correlate with induced xenograft rejection. These findings suggest that anti-pig Ig isotypes produced during the conventional process of acute proislet xenograft rejection do not play a major role in mediating graft damage. The acute rejection of pig proislet xenografts in the absence of serum anti-pig Ig in microMT-/- hosts confirmed that anti-pig antibody is not essential for proislet xenograft destruction.

Adoptive Transfer↗
PubMed1997

An antigen valence theory to explain the evolution and organization of the humoral immune response.

The three modes of antibody production, natural, T independent (TI) and T dependent (TD) are conserved among vertebrate species suggesting an important role for each in protection against pathogens. Here, I use an artificial 'universe' to argue that the three modes of antibody production represent layers that evolved to deal optimally with antigens of different valence. Thus, the apparently more sophisticated TD response has not superseded the natural and TI components of the humoral immune response. Furthermore, the characteristic differences in isotype, somatic mutation and memory displayed by each antibody layer are appropriate for their targeted range of surface structures. It is also suggested that the TD and TI activation arms are at the extremes of a continuum, with signal integration of antigen and T cell-derived signals contributing to B cell decisions about isotype selection, proliferation and secretion that minimize the time to protection.

Animals↗
PubMed1997

The importance of efficacy and partial agonism in evaluating models of B lymphocyte activation.

Immunologists have developed a range of in vitro techniques for probing the receptor mediated response of cells comprising the immune system. An important and ubiquitous method is the use of antibodies in either soluble or aggregated form to engage cell surface receptors and transmit a signal. Models of cell and molecular interactions, derived from the use of these antibodies, form the basis of our efforts to understand and explain the corresponding in vivo systems. However, interpreting in vitro experiments and distinguishing between alternative models is difficult. This complexity is illustrated here using B cell stimulation by surface immunoglobulin and CD40. The fluorescent cell labelling dye carboxyfluorescein, diacetate, succinimidyl ester (CFSE) is used to show that many anti-Ig and CD40 stimulatory agents, used to assess the role of B cells and lymphokines, are partial agonists. By modelling each step in B cell signalling, activation and division it is possible to show that small changes in signal contributed by a second receptor can generate numerous distinct dose response curves that are highly dependent on the "efficacy" of signal transmission by the primary ligand and the number of cell divisions taken in culture. Differences in dose response curves become particularly striking if the primary activating stimulus is a partial agonist. Although exemplified here with B cell stimulation the conclusions are applicable to other in vitro activation systems and suggest ways to improve both the design and interpretation of in vitro experiments.

Animals↗