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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 5, 2002 as doi:10.1096/fj.02-0248fje. |
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2
* Department of Biological Sciences, Rutgers University, Newark, New Jersey, USA; and
Departamento Biologia Celular, Facultad de Biologia, Universidad Complutense, Madrid 28040, Spain
2Correspondence: Department Cell Biology, School of Biology, Complutense University, Madrid 28040, Spain. E-mail: mariodm{at}bio.ucm.es
SPECIFIC AIMS
Clonal expansion of antigen-specific lymphocytes leads to the generation of effector T cells and is followed by removal of excess cells, although a small number survive and differentiate into memory T cells. The mechanisms by which some effector T cells escape from apoptosis and become memory T cells are not elucidated. Here we hypothesize that neuropeptides such as the vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), which inhibit antigen-induced apoptosis of CD4 T cells, play a role in the generation of memory T cells; we asked the question whether the neuropeptides will differentially promote differentiation into Th1 and Th2 memory cells.
PRINCIPAL FINDINGS
VIP and PACAP promote clonal expansion and survival of CD4 T cells in vivo
By using an in vivo model involving the administration of a superantigen that induces an initial clonal expansion, followed by a massive deletion of specific effector T cells, we have demonstrated that VIP and PACAP prevent superantigen-induced CD4 but not CD8 T cell clonal deletion in vivo (not shown).
In a second in vivo model involving specific peptide antigens, CD4 T cells from TRC transgenic mice were transferred into syngeneic nontransgenic hosts, which were immunized with the antigen (Ag) specific for the transgenic T cells in the presence or absence of VIP or PACAP. Exposure to the Ag for 7 days resulted in complete clonal deletion of Ag-specific T cells; administration of VIP or PACAP concomitant with Ag prevented the Ag-induced deletion of TCR transgenic T cells in both spleen and lymph nodes (not shown). These data indicate that VIP and PACAP enhance clonal expansion and survival of Ag-specific T cells in vivo.
VIP and PACAP promote long-term memory Th2 cell survival
To study whether the increased survival of CD4 Ag-specific T cells could lead to higher numbers of Ag-specific memory cells, we used a long-term Ag-specific in vivo model where TCR transgenic CD4 T cells were adoptively transferred into syngeneic, nontransgenic thymectomized recipients that were immunized with the antigen specific for the transgenic T cells in the presence or absence of VIP or PACAP, and analyzed for transgenic-bearing T cells 62 days later. VIP and PACAP promote long-term T cell survival of Ag-activated CD4 T cells in vivo (Fig. 1
a).
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Flow cytometry analysis of TCR transgenic cells harvested from hosts treated with VIP or PACAP indicated that they exhibited a memory-type phenotype: the cells were small (as assessed by low forward scatter) and expressed high levels of CD44 and low levels of L-selectin and CD45RB (Fig. 1b
, filled histograms). In contrast, nontransgenic resident T cells recovered from the same recipients expressed a naive-type phenotype (Fig. 1b
, open histograms),—i.e., CD44lo, L-selectinhi, and CD45RBhi—similar to freshly isolated CD4 T cells. These findings suggest that surviving VIP/PACAP-induced, Ag-stimulated T cells express a memory phenotype.
VIP/PACAP-induced surviving memory T cells express a Th2 phenotype, because the cells recovered from recipients treated with VIP or PACAP produced markedly higher levels of interleukin-4 (IL-4) and IL-5, little interferon-
and IL-2 (determined by ELISA), and expressed significantly higher frequencies of IL-4-producing T cells (determined by ELISPOT) after restimulation with Ag (Fig. 1c, d
). Therefore, VIP/PACAP contribute to the generation of Th2 Ag-specific memory T cells. This conclusion is supported by fact that in an in vitro helper assay, CD4 T cells from the VIP/PACAP-treated recipients, but not naive T cells (fresh), provided effective help for IgG1 Ab production (characteristic of a Th2 response) (Fig. 1e
).
VIP and PACAP induce long-term memory T cell survival of Ag-activated Th2 but not Th1 cells
The fact that VIP/PACAP promote generation of Th2 memory T cells was confirmed by transfer of TCR transgenic Th1 or Th2 effectors into syngeneic thymectomized hosts, which were then challenged with the specific antigen in the presence or absence of the neuropeptides. VIP and PACAP promote the long-term survival in vivo of transgenic CD4 T cells in Th2 but not in Th1 adoptively transferred mice (not shown).
VIP and PACAP promote expansion of Ag-induced CD4 Th2 effectors in vitro by inhibiting antigen-induced apoptosis in Th2, but not in Th1, effectors
In vitro experiments indicated that proliferation of Th2 (but not Th1) TCR transgenic cell lines was supported by VIP/PACAP (Fig. 2
a).The effect of VIP/PACAP appears to be mediated through an inhibition of antigen-induced apoptosis in the Th2 cell lines, but not in Th1 cells (Fig. 2b
).
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VIP and PACAP inhibit Ag-induced expression of Fas ligand in Th2 effectors
Next we investigated the mechanisms by which VIP/PACAP specifically inhibit Th2 apoptosis. Th1 and Th2 effectors both express the same VIP/PACAP receptors and VIP/PACAP do not affect bcl-2 and FAP-1 expression in Th2 cells, arguing against an effect on the Fas-induced intracellular pathways leading to cell death (not shown). In contrast, VIP/PACAP reduce Fas ligand expression in Th2 but not Th1 effectors (not shown), suggesting that the specific protective effect on Th2 cells is mediated through the down-regulation of Fas ligand expression.
CONCLUSIONS AND SIGNIFICANCE
During a primary immune response, naive Ag-specific CD4 T cells proliferate and differentiate into effector cells able to rapidly produce large amounts of cytokines on restimulation. At this stage, the effector T cells are susceptible to activation-induced cell death mediated primarily through FasL/Fas interactions. A low percentage of the activated effector T cells are protected from apoptosis and differentiate into long-lived memory T cells. During an immune response, mechanisms operate to destroy no longer needed or even potentially damaging T cells, but also allow the survival of a small number of activated T cells. Although all CD4 T cells express Fas, naive T cells are apoptosis resistant and gradually switch to an apoptosis-sensitive phenotype during the proliferative stage after activation. The mechanisms controlling this switch are not completely understood. The present study demonstrates that VIP and PACAP, two multifunctional neuropeptides present in the lymphoid organs, protect certain CD4 Th2 cells from apoptosis and allow survival of Th2 effectors and the generation of long-lived memory cells. This is the first report of neuropeptides participating in the generation of memory T cells, particularly in the development of a specific memory T cell subset, and contributes to understanding how the nervous and immune systems interact in vivo and the role played by the central nervous system (CNS) in overseeing immune functions. This is especially relevant in view of the immune deviation existing in certain immune privileged sites such as the brain and eye, where Th2, but not Th1, responses are allowed to occur in nonpathological conditions. This work might also lead to new developments in vaccination procedures and a new understanding of immune dysregulation occurring in pathological conditions in the CNS.
The conclusions reached in this manuscript agree with our previous reports that neuropeptides protect CD4 T cells from antigen-induced apoptosis (J. Immunol. 2000, vol. 164: p. 1200), inhibit Fas ligand expression (J. Immunol. 2001, vol. 166: p.1028), and inhibit Th1 responses while promoting Th2-type responses in vivo (J. Immunol. 1999, vol. 163: p. 3629).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0248fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0248fje 
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