Linda M. Bradley, Ph.D.
Professor

Dr. Bradley's Active Grants
Dr. Bradley's Publications
lbradley@skcc.org

Immunology Program

Laboratory Staff: Bas J. G. Baaten, Ph.D., Daphne Summers-Torres, Melissa Lin, Michael Hanna, Larissa Low, and Thomas Pham

Regulation of CD4 T Cells: From the Development of Immunity to Control of Autoimmunity

Immunologic memory is a fundamental attribute of the immune system that accounts for protection against repeat infections. Despite a wealth of information pertaining to functional and phenotypic characteristics of naive, effector, and memory CD4 cells, mechanisms that underlie their development, homeostasis, and migration are not well understood. My research program is focused on identifying parameters that control memory CD4 cells and on determining how to best elicit or downregulate responses of memory subsets. CD4 cells promote immunity through effector cells that expand after encounter with antigen and regulate B cell responses in the lymphoid tissue sites where they are activated, and also relocate to nonlymphoid tissues where they produce mediators that orchestrate inflammatory processes. The expansion phase of the effector response proceeds for as long as antigen and costimulation can support it. Afterwards, effector populations contract dramatically due to regulated cell death that serves to reestablish homeostasis and leaves only a small cohort of cells to maintain memory. Although memory can be long-lived, often for the life of an individual, for many antigens memory wanes with time. In addition, in some circumstances memory cells can promote autoimmunity rather than immunity after challenge with the immunizing antigen. Understanding how CD4 memory is regulated is essential for exploiting the immune system to protect against disease and to dampen immunopathology.

Regulation of Effector and Memory CD4 Cell Development

When T cell memory can arise during a response is not yet understood. Our studies indicate that memory T cells develop from effectors (1). We find that antigen is necessary only for the initial activation of naive CD4 cells, and that effector expansion and further differentiation is then mediated by costimulation via B cells as well as cytokines (2). Of the co-receptors that are expressed by B cells, we identified the TNFR family member, OX40L (CD154) as critical not only for optimal CD4 cell expansion but also for development of Th2 cells that produce IL-4 and IL-13 (3). Temporally, OX40 expression on activated CD4 cells coincides with OX40L induction on B cells, which become necessary to support T cell expansion as dendritic cells become limiting. This extended proliferative response is needed for epigenetic remodeling that enables transcription the linked IL-4 and IL-13 genes. In studies with Dr. Phyllis Linton at the center, we determined that fewer effector CD4 cells are generated in the absence of B cells, and this translates to limited development of memory (4). Since IL-4 regulates B cells growth and differentiation, our work supports the concept of a coordinated interplay of CD4 and B cells which enables survival and expansion of both populations as the response progresses to the germinal centers where OX40L is necessary for CD4 cell accumulation.

As the expansion phase of the CD4 effector response wanes, cells with characteristics of memory cells remain, suggesting that the decline of costimulation and growth factors might be a key signal for generation of memory cells. Indeed, we find that withdrawal of stimulation is sufficient to initiate linear progression of effector cell to memory cells, in the absence of further differentiation (1). The data indicate that memory develops in CD4 cells as a default pathway when no additional signals to remain engaged in a response are received. From these observations, we propose that availability of antigen and associated inflammatory stimuli determine the onset of memory development at the level of individual CD4 cells, which may occur in both lymphoid and nonlymphoid sites. Our findings suggest that the generation of memory is a stochastic rather than an instructional process. One prediction of this hypothesis is that the memory population would be heterogeneous and reflects the stages of differentiation achieved by individual cells at the time of their disengagement from the response (5).

Activation produces profound alterations in CD4 cells. By Affymetrix microarray analysis, we find that ~7000 genes are differentially expressed after activation of naive CD4 cells. Receptors that regulate adhesion are variably modulated, with the most notable upregulation occurring in CD44, integrins that regulate retention of cells in tissues, and catenins, which control anchoring of cadherins that mediate cell-cell adhesion. Several adhesion molecules that are upregulated on effectors remain elevated on memory cells, in part accounting for decreased costimulation requirements and increased migratory capacity (6). However, loss of the lymph node homing receptor, CD62L, which we showed accompanies CD4 cell effector generation (7), is a key determinant of migration and an attribute of effectors that enter nonlymphoid sites. Our work indicates that memory can be carried by CD62L- cells that reside in the spleen and blood (8). The majority of naive CD4 cells can acquire this phenotype after immunization and those memory cells that initially remain CD62L+ convert to CD62L- with time. CD62L- effectors also distribute throughout the body and give rise to memory cells that persist in nonlymphoid tissues where they may provide a first-line of defense in sites of reexposure to antigen and be instrumental in marshalling new recruits from the memory population that resides in the lymphoid compartment. Our current studies are focused on this aspect of memory using and influenza model to study a response to an infection in that is localized in the lungs. In addition, we are investigating the adhesion mechanisms which regulate recruitment of effector and memory CD4 cells to the lungs during infection, and which control their egress once a response subsides.

Regulation of Memory and Effector CD4 Cell Survival and Homeostasis

Once a state of memory is achieved, the size and persistence of the memory CD4 cell pool is subject to regulation by homeostatic mechanisms. By generating CD4 memory cells with a defined history of exposure to antigen, we discovered that the elusive critical regulator of memory CD4 cell development and survival is the common-gamma chain receptor family cytokine, IL-7 (9). Our studies show that naive CD4 cells can lose IL-7R after activation, and that IL-7 then fails to protect effectors from AICD. However, IL-7R is regained during the transition to memory. Thus, IL-7R does not distinguish progenitors of CD4 memory cells as reported for CD8 cells (10), revealing further differences in the cytokine regulation of CD4 and CD8 memory cells. Our current work is aimed at defining the stages at which IL-7 can regulate the survival of responding CD4 cells and enable them to make a transition to memory.

Although IL-7 rescues CD4 effectors during their transition to memory, the survival of effectors, themselves, is regulated by other factors. We recently discovered that the adhesion receptor, CD44, which interacts with the extracellular matrix component, hyaluronic acid, controls survival in CD4 effectors. Dendritic cells express hyaluronic acid when induced to mature. However, ligation of CD44 on CD4 cells is not necessary for the development of effectors. Instead, in the absence of signaling via CD44, responding CD4 effectors fail to survive the contraction phase after a primary response in vivo, leading to impaired generation of memory due to a profound attrition of effectors. By gene array analysis, we find that multiple pro-apoptotic genes become upregulated in CD44 knockout CD4 cells in response to activation. The results demonstrate a previously unidentified role for this adhesion receptor in regulating CD4 cell survival during the effector phase of an immune response. We also find that CD44 contributes to survival of effectors during the transition to memory in the absence of an overt response, and that TCR signals are required. These findings indicate that CD4 cell survival during the transition to memory is a regulated process that requires adhesive interactions via CD44 with the surrounding environment, a previously unknown mechanism governing the return of responding CD4 cells to homeostasis. Of note, CD8 cells do not show obligatory dependence on CD44 for survival. Once established, the memory CD4 cell pool is then impacted by changes that occur as a consequence of subsequent immune responses. We find that repeated boosting with antigen does not produce significant alterations in the frequencies of memory cells that are maintained, but rather promotes their further differentiation to populations that are more effective in terms of capacity to produce effector cytokines. In new studies, we are evaluating if this functional change translates to greater levels protective memory in the response to influenza viruses in either the lymphoid and nonlymphoid compartments (11).

As part of our research on immunity to influenza viruses, we have begun to analyze the role of Toll-like receptors in modulating the innate response to the virus, and the consequences for the development of T cell memory. The recent discovery of TLR-7 as an important endosomal ligand for single stranded RNA viruses that include influenza, has prompted our collaboration with Dr. Dennis Carson from UCSD to analyze novel agonists and antagonists of this receptor in the T cell response to these viruses, and the generation of immunity that is protective against newly arising or divergent strains (heterosubtypic immunity). We are using the agonist compounds conjugated to internal influenza proteins as a vaccine strategy to augment T cell memory and will have the opportunity to study efficacy in the murine model with the avian influenza virus, H5N1, which is highly pathogenic, raising concerns that a pandemic is on the horizon. In addition, together with Dr. Phyllis Linton at SKCC, we recently initiated a collaboration with Medimmune, Inc. to study T cell immune responses to the live, attenuated, cold adapted vaccine (FluMist) with aging to expand her ongoing work on the challenge of improving immunity in the aged (12). In addition to their clinical relevance, we expect these studies to reveal new insights into mechanisms that link the innate and adaptive immune responses and the potential to modulate protective immunity.

CD4 Effector and Memory Cells in the Control of Autoimmunity

To understand potential immunopathogenic consequences of effector and memory CD4 cell responses, we are studying the NOD mouse model of type I diabetes in which inflammatory destruction of insulin producing ? islet cells triggers disease onset. We use islet-specific TCR transgenic CD4 cells to analyze factors that regulate autoaggressive vs protective responses. Our work in collaboration with Dr.Marc Horwitz (University of British Columbia) determined that islet antigens that are released as a consequence of Cocksackie virus infection of the pancreas induce autoreactive CD4 cells that orchestrate the autoimmune response (13). Our studies revealed that cytokines and chemokines produced by Th1 cells are crucial for progression to disease, while those of nonpathogenic Th2 cells fail to elicit pathogenic inflammation (14). However, while islet expression of Th2 cytokines, and IL-4 in particular, can confer protection against disease (15), Th2 cells, themselves cannot. To determine if immunoregulatory CD4 cells might modulate disease, we have generated islet-specific effector CD4 cells in the presence of TGF-?1 that completely prevent spontaneous and induced diabetes development (16). These T cells acquire and maintain expression of the FoxP3 transcription factor that is associated regulatory function in CD4 cells (TR cells), but are otherwise phenotypically distinct from endogenous TR CD4 cells that express CD25. They produce the immunosuppressive cytokines,TGF-?1 and IL-10, but mediate the disappearance of Th1 cells in the draining pancreatic lymph nodes, in part via by the Fas/FasL pathway. We are currently studying the roles of antigen and cytokines produced by adaptive TR cells in their function. Our results reveal that these adaptive regulatory CD4 cells may have the potential to be used as a cell-based therapy and suggest that one mechanism by which they may contribute to control of diabetes is by preventing relocation of autoaggressive Th1 cells from the lymphoid compartment to the pancreatic islets. Our recent studies are focused on the clinical potential of adaptive regulatory CD4 cells. We have found that regulatory cells with the capacity to prevent diabetes can be generated from polyclonal CD4 cells. We are now examining their functional and phenotypic stability in vivo after Cocksackie virus infection and whether such cells can be induced from responding autoreactive CD4 cells. We find that adaptive regulatory CD4 cells share characteristics of memory cells that persist indefinitely in protected animals. In ongoing studies, we are examining the regulation of memory in this unique regulatory population, which is maintained in the context of chronic exposure to self-antigens. By in vitro generation of regulatory CD4 cells, and in vivo manipulation of inflammatory mediators to intervene in mechanisms that control cellular recruitment during effector and memory CD4 responses, we hope to gain insights into strategies to ameliorate this disease.

Selected References

1. Harbertson, J., Biederman, E., Bennett, K.E., Kondrack, R.M., and Bradley, L.M. 2002. Withdrawal of stimulation may initiate the transition of effector to memory CD4 cells. J. Immunol. 168:1095-1102.
2. Bradley, L.M., Harbertson, J., Biederman, E., Zhang, Y., Bradley, S. M., and Linton, P.-J. 2002. Availability of APC can determine the extent of CD4 effector expansion and priming for secretion of Th2 cytokines. Eur. J. Immunol. 32:2338-2346.
3. Linton, P.-J., Bautista, B., Biederman, E., Bradley, E.S., Harbertson, J., Kondrack, R.M., Padrick, R.C., and Bradley, L.M. 2003. Costimulation via OX40L expressed by B cells is sufficient to determine the extent of primary CD4 cell expansion and Th2 cytokine secretion in vivo. J. Exp. Med. 197:875-883.
4. Linton, P.-J., Harbertson, J., and Bradley, L.M. 2000. A critical role for B cells in the development of memory CD4 cells, J. Immunol.165:5558-5565.
5. Bradley, L.M. 2003. Migration and T-lymphocyte effector function. Curr. Opin. Immunol. 15:343-348
6. Watson, S. R. and Bradley, L. M. 1998. The recirculation of naive and memory lymphocytes. Cell Adhes. Comm. 6:105-110.
7. Bradley, L.M., Duncan, D.D., Tonkonogy, S., and Swain, S.L. 1991. Characterization of antigen-specific CD4+ effector T cells in vivo: Immunization results in a transient population of MEL-14-, CD45RB- helper cells that secretes interleukin-2 (IL-2), IL-3, IL-4, and interferon-g. J. Exp. Med. 174:547-559.
8. Bradley, L.M., Atkins, G.G., and Swain, S.L. 1992. Long-term CD4+ memory T cells from the spleen lack MEL-14, the lymph node homing receptor. J. Immunol. 148:324-331.
9. Kondrack, R.M., Harbertson, J., Tan, J.T., McBreen, M.E., Surh, C.D., and Bradley, L.M. 2003. Interleukin 7 regulates the survival and generation of memory CD4 cells. J. Exp. Med. 198:1797-1806.
10. Bradley, L.M., Haynes, L., and Swain, S.L. 2005. Interleukin-7: Maintaining T cell memory and achieving homeostasis. Trends Immunol. 26:172-176.
11. Chapman, T.J., Castrucci, M.R., Padrick, R.C., Bradley, L.M., and Topham, D. J. 2005. Antigen-specific and non-specific CD4+ T cell recruitment and proliferation during influenza infection. J. Virol. 340:296-306.
12. Zhang, Y., Diago, O., Li, S., Bradley, L.M., Sherman, L.A., Smith, B., and Linton, P.-J. 2006. Defective dendritic cell migration with aging, submitted.
13. Horwitz, M.S., Bradley, L.M., Harbertson, J., Krahl, T., Lee, J., and Sarvetnick, N. 1998. Coxsackie virus-induced diabetes: Initiation by bystander damage and not molecular mimicry, Nature Med. 4:781-785.
14. Bradley, L.M., Asensio, V.C., Schioetz, L.-K, Harberston, J., Krahl, T., Patstone, G., Woolf, N., Campbell, I., and Sarvetnick, N. 1999. Islet-specific Th1 cells but not Th2 cells secrete multiple chemokines and promote rapid induction of autoimmune diabetes, J. Immunol.162:2511-2520.
15. Mueller, R., Bradley, L. M., Krahl, T., and Sarvetnick, N. 1997. Mechanism underlying counter-regulation of autoimmune diabetes by IL-4. Immunity 7:411-418.
16. Weber. S.E., Harbertson, J., Godebu, E., Mros, G,A., Padrick, R.C., Carson, B.D., Ziegler, S.F., and Bradley, L.M. 2006. Adaptive islet specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Th1 cells in vivo. J. Immunol. 176:4730-4739.