Reactivation of dormant infections causes an immense burden of morbidity and mortality in the world at large. Reactivation can occur as a result of immunosuppression, environmental insult, or aging; however, the cause of reactivation of such infections is often not clear. We have previously shown that persistence of the parasite Leishmania major is controlled by endogenous CD4+ CD25+ regulatory T (T reg) cells. In this report, we show that despite efficient parasite clearance at secondary sites of infection, Leishmania superinfection can cause disease reactivation at the primary site. Our results strongly suggest that T reg cells, whose numbers increase in sites of reactivation, are directly responsible for such reactivation. Depletion of CD25+ cells at the time of secondary challenge prevented disease reactivation at the site of persistent infection while strengthening the expression of immunity at the site of secondary challenge. Finally, transfer of T reg cells purified from infected mice into chronically infected mice was sufficient to trigger disease reactivation and prevent the expression of an effector memory response. Our results demonstrate that after persistence is achieved, an equilibrium between T reg cells and effector lymphocytes, which can be disturbed by superinfection, controls the efficiency of recall immune responses and disease reactivation.

Endogenous regulatory T cells (Treg) play a central role in the control of excessive or misdirected immune responses against self or foreign Ags. To date, virtually no data are available on the nature of the molecules and signals involved in the trafficking and retention of Treg in tissues where regulation is required. Here, we show that expression of αEβ7 integrin is necessary for the homing of Treg at site of Leishmania major infection. The vast majority of Treg present in the dermis at steady-state conditions or during L. major infection express the αE chain (CD103) of αEβ7. Genetically susceptible BALB/c mice that lack CD103 become resistant to infection, a phenotype that is associated with a poor capacity of Treg to be retained in the infected site. Such susceptible phenotype can be restored when Treg from wild-type mice were transferred in CD103−/− mice. The central role of CD103 in Treg retention was further demonstrated by usage of blocking Abs against CD103 and the transfer of Treg purified from CD103−/− mice. Our results strongly suggest that this molecule is induced and maintained on Treg following or just prior to their arrival in tissues. Furthermore, the expression of CD103 and the subsequent retention of Treg in tissues is highly regulated by their exposure to Leishmania Ag and the level of activation of the APCs they encounter. Thus, CD103, by controlling Treg retention, can contribute to the outcome of chronic infection by Leishmania.

Natural regulatory T (T reg) cells are involved in control of the immune response, including response to pathogens. Previous work has demonstrated that the repertoire of natural T reg cells may be biased toward self-antigen recognition. Whether they also recognize foreign antigens and how this recognition contributes to their function remain unknown. Our studies addressed the antigenic specificity of natural T reg cells that accumulate at sites of chronic infection with Leishmania major in mice. Our results support the idea that natural T reg cells are able to respond specifically to foreign antigens in that they strongly proliferate in response to Leishmania-infected dendritic cells, they maintain Foxp3 expression, and Leishmania-specific T reg cell lines can be generated from infected mice. Surprisingly, the majority of natural T reg cells at the infected site are Leishmania specific. Further, we showed that parasite-specific natural T reg cells are restricted to sites of infection and that their survival is strictly dependent on parasite persistence.

Following the peak of the T-cell response, most of the activated effector T cells die by apoptosis driven by the proapoptotic Bcl-2 family member Bim (Bcl-2-interacting mediator of death). Whether the absence of Bimmediated T-cell apoptosis can affect protective immunity remains unclear. Here, we used a mouse model of Leishmania major infection, in which parasite persistence and protective immunity are controlled by an equilibrium reached between parasite-specific gamma interferon (IFN-␥)-producing effector T cells and interleukin-10 (IL-10)-producing CD4 ؉ CD25 ؉ T regulatory cells. To further understand the role of Bimmediated apoptosis in persistent infection and protective immunity, we infected Bim ؊/؊ mice with L. major. We found that the initial parasite growth and lesion development were similar in Bim ؊/؊ and wild-type mice after primary L. major infection. However, at later times after infection, Bim ؊/؊ mice had significantly increased L. major-specific CD4 ؉ T-cell responses and were resistant to persistent infection. Interestingly, despite their resistance to primary L. major infection, Bim ؊/؊ mice displayed significantly enhanced protection against challenge with L. major. Increased resistance to challenge in Bim ؊/؊ mice was associated with a significant increase in the number of L. major-specific IFN-␥-producing CD4 ؉ T cells and a lack of IL-10 production at the challenge site. Taken together, these data suggest that Bim limits protective immunity and that the absence of Bim allows the host to bypass antigen persistence for maintenance of immunity against reinfection.

Regulatory T cells (Treg) are increased and directly infected by feline immunodeficiency virus (FIV) and likely play a role in other feline autoimmune, neoplastic, and infectious diseases. Phenotypically, Treg are best characterized by surface expression of CD4 and CD25 and intranuclear expression of the forkhead transcription factor Foxp3. Our objective was to clone and sequence feline FOXP3 for the purpose of developing assays to enhance studies of feline Treg. We determined the feline FOXP3 is 1293 nucleotides in length and codes for a protein that shares high homology to other species. A splice variant devoid of exon 2 was also identified. A real-time PCR assay was developed and used to show Foxp3 mRNA expression occurs primarily in CD4+CD25+ T cells. Two crossreacting antibodies were identified by immunocytochemical staining of HEK293 cells transfected with feline FOXP3. The antibody labeling confirmed the nuclear localization of the protein. A flow cytometric assay was also validated and used to correlate the phenotypic and functional characteristics of feline Treg induced by treatment of lymph node lymphocytes with flagellin or LPS in combination with mitogen or IL2. Together, these studies provide useful tools to further investigate Foxp3 and Tregs in cats. KeywordsFoxp3; feline; Treg; regulatory T cells; Toll-like receptors ReportRegulatory T cells (Treg) can functionally suppress CD4 and CD8 T cells, B cells, NK cells, NKT cells, monocytes/macrophages, dendritic cells and neutrophils, thereby playing a key role in limiting both innate and adaptive immune responses (Azuma et al., 2003;Fallarino et al., 2003;Lewkowicz et al., 2006;Lim et al., 2005;Piccirillo and Shevach, 2004;Ralainirina et al., 2007;Taams et al., 2005;Wing et al., 2005 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The forkhead transcription factor Foxp3 has been identified as essential and sufficient to impart Treg function in mice (Ziegler, 2006). This understanding has come from two lines of study. The first involved determination of the genetic basis of autoimmune diseases observed in the scurfy mouse ) and human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy (IPEX) (Bennett et al., 2001). In both cases, mutations in the FOXP3 gene were shown to be the sole cause of these diseases that result from a lack of Treg. Second, transfection of FOXP3 into CD4+CD25− cells imparts Treg suppressive function (Fontenot et al., 2003;Hori et al., 2003;Khattri et al., 2003). NIH Public AccessRegulatory T cells have been phenotypically and functionally characterized in the cat ...