Immune Activation Profiles Elicited by Distinct, Repeated TLR Agonist Infusions in Rhesus Macaques.

TLR agonists are a promising class of immune system stimulants investigated for immunomodulatory applications in cancer immunotherapy and viral diseases. In this study, we sought to characterize the safety and immune activation achieved by different TLR agonists in rhesus macaques (Macaca mulatta), a useful preclinical model of complex immune interactions. Macaques received one of three TLR agonists, followed by plasma cytokine, immune cell subset representation, and blood cell activation measurements. The TLR4 agonist LPS administered i.v. induced very transient immune activation, including TNF-α expression and monocyte activation. The TLR7/8 agonist 2BXy elicited more persistent cytokine expression, including type I IFN, IL-1RA, and the proinflammatory IL-6, along with T cell and monocyte activation. Delivery of 2BXy i.v. and i.m. achieved comparable immune activation, which increased with escalating dose. Finally, i.v. bacillus Calmette-Guérin (BCG) vaccination (which activates multiple TLRs, especially TLR2/4) elicited the most pronounced and persistent innate and adaptive immune response, including strong induction of IFN-γ, IL-6, and IL-1RA. Strikingly, monocyte, T cell, and NK cell expression of the proliferation marker Ki67 increased dramatically following BCG vaccination. This aligned with a large increase in total and BCG-specific cells measured in the lung. Principal component analysis of the combined cytokine expression and cellular activation responses separated animals by treatment group, indicating distinct immune activation profiles induced by each agent. In sum, we report safe, effective doses and routes of administration for three TLR agonists that exhibit discrete immunomodulatory properties in primates and may be leveraged in future immunotherapeutic strategies.

Broad coverage of neutralization-resistant SIV strains by second-generation SIV-specific antibodies targeting the region involved in binding CD4.

Both SIV and SHIV are powerful tools for evaluating antibody-mediated prevention and treatment of HIV-1. However, owing to a lack of rhesus-derived SIV broadly neutralizing antibodies (bnAbs), testing of bnAbs for HIV-1 prevention or treatment has thus far been performed exclusively in the SHIV NHP model using bnAbs from HIV-1-infected individuals. Here we describe the isolation and characterization of multiple rhesus-derived SIV bnAbs capable of neutralizing most isolates of SIV. Eight antibodies belonging to two clonal families, ITS102 and ITS103, which target unique epitopes in the CD4 binding site (CD4bs) region, were found to be broadly neutralizing and together neutralized all SIV strains tested. A rare feature of these bnAbs and two additional antibody families, ITS92 and ITS101, which mediate strain-specific neutralizing activity against SIV from sooty mangabeys (SIVsm), was their ability to achieve near complete (i.e. 100%) neutralization of moderately and highly neutralization-resistant SIV. Overall, these newly identified SIV bnAbs highlight the potential for evaluating HIV-1 prophylactic and therapeutic interventions using fully simian, rhesus-derived bnAbs in the SIV NHP model, thereby circumventing issues related to rapid antibody clearance of human-derived antibodies, Fc mismatch and limited genetic diversity of SHIV compared to SIV.

Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV.

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

Blocking α4ß7 integrin binding to SIV does not improve virologic control.

A study in nonhuman primates reported that infusions of an antibody against α4ß7 integrin, in combination with antiretroviral therapy, showed consistent, durable control of simian immunodeficiency virus (SIV) in rhesus macaques. The antibody used has pleiotropic effects, so we set out to gain insight into the underlying mechanism by comparing this treatment to treatment with non-neutralizing monoclonal antibodies against the SIV envelope glycoprotein that only block α4ß7 binding to SIV Env but have no other host-directed effects. Similar to the initial study, we used an attenuated strain of SIV containing a stop codon in nef. The study used 30 macaques that all began antiretroviral therapy and then were divided into five groups to receive different antibody treatments. Unlike the published report, we found no sustained virologic control by these treatments in vivo.

The peripheral differentiation of human natural killer T cells.

The peripheral maturation of human CD1d-restricted natural killer T (NKT) cells has not been well described. In this study, we identified four major subsets of NKT cells in adults, distinguished by the expression of CD4, CD8 and CCR5. Phenotypic analysis suggested a hierarchical pattern of differentiation, whereby immature CD4+ CD8- CCR5- cells progressed to an intermediate CD4+ CD8- CCR5+ stage, which remained less differentiated than the CD4- CD8- and CD4- CD8+ subsets, both of which expressed CCR5. This interpretation was supported by functional data, including clonogenic potential and cytokine secretion profiles, as well as T-cell receptor (TCR) excision circle analysis. Moreover, conventional and high-throughput sequencing of the corresponding TCR repertoires demonstrated significant clonotypic overlap within individuals, especially between the more differentiated CD4- CD8- and CD4- CD8+ subsets. Collectively, these results mapped a linear differentiation pathway across the post-thymic landscape of human CD1d-restricted NKT cells.

A high throughput lentivirus sieving assay identifies neutralization resistant Envelope sequences and predicts in vivo sieving.

An effective prophylactic vaccine against human immunodeficiency virus (HIV) will likely require a potent antibody response that can neutralize the virus at the mucosal portal of entry. The elicitation of potent broadly-neutralizing anti-sera will be an iterative process, optimizing candidates that only block a fraction of potential viral strains. This effect, termed "sieving", is evidence of a partially efficacious vaccine. Understanding the mechanisms of resistance of the breakthrough viruses is important for improving vaccines. We developed a high-throughput assay that can be used on vaccine-elicited antisera or monoclonal antibodies. Using the SIVsmE660 swarm stock and sera from a large NHP vaccine/challenge study, our in vitro sieving assay identified the same viral subspecies as in the animal study-those with a canonical C1 amino acid variants conferring global neutralization resistance to antibodies. Using a genetically divergent swarm stock, we identified five other amino acid variants that confer global resistance; the C1 mutations in this stock were not selected, also in agreement with in vivo challenge studies. Thus, the in vitro sieving assay can be used with genetically diverse challenge stocks to predict the coverage of a vaccine-elicited sera and possibly inform candidate vaccine development efforts.

Unique cellular and humoral immunogenicity profiles generated by aerosol, intranasal, or parenteral vaccination in rhesus macaques.

Respiratory mucosa immunization is capable of eliciting both local and distal mucosal immune responses; it is a potentially powerful yet largely unused modality for vaccination against respiratory diseases. Targeting the lower versus upper airways by aerosol delivery alters the immunogenicity profile of a vaccine, although the full extent of this impact is not well characterized. We set out to define the cellular and humoral response profiles elicited by immunization via intranasal, small aerosol droplets, and large aerosol droplets. We compared responses following adenovirus-vectored vaccination by these routes in macaques, either for the generation of primary immune responses or for the boosting of previously primed systemic responses. Aerosol delivery (4 or 10µm diameter droplets, addressing lower or upper airways, respectively) generated the highest magnitude lung CD4 and CD8 T-cell responses, reaching 10-30% vaccine-specific levels in bronchoalveolar lavage cells. In contrast, intranasal delivery was less immunogenic with >10-fold lower peak lung T-cell responses. Systemic (blood) T-cell responses were only observed following 4µm aerosol (and parenteral) immunization, while all delivery routes elicited similar humoral responses. These data demonstrate distinct immune response profiles with each respiratory tract vaccination modality and suggest that small droplet aerosol offers several immunological advantages over other respiratory routes.

Natural Functions of PLIN2 Mediating Wnt/LiCl Signaling and Glycogen Synthase Kinase 3 (GSK3)/GSK3 Substrate-Related Effects Are Modulated by Lipid.

Belonging to the PLIN family, PLIN2 associates with lipid storage droplets (LSDs), but other functions of PLIN2 remain unclear. Here, we suggest that PLIN2 mediates Wnt signaling because PLIN2 small interfering RNA (siRNA) suppresses activation of Wnt/coreceptor pathways. The mediation in the Wnt/Frizzled pathway seems to occur from Dishevelleds to axin/glycogen synthase kinase 3(GSK3)/ß-catenin complexes (AGßC) as Wnt decreases Dishevelled/PLIN2 but increases AGßC/PLIN2 associations. Augmenting cellular LSDs that affect PLIN2 associations with these proteins, oleic acid (OA) treatment inhibits Wnt-increased AGßC/PLIN2 associations and ß-catenin T-cell factor signaling (ß-CTS). Revealing that PLIN2 is a GSK3-associated protein, the study explored PLIN2-mediated effects on GSK3/GSK3 substrates. PLIN2 siRNA reduces inhibitory GSK3 levels and lithium chloride (LiCl)-upregulated ß-catenin or CCAAT/enhancer binding protein α (c/EBPα) expression. OA treatment decreases LiCl-increased c/EBPα via PLIN2-c/EBPα dissociation. In addition to PLIN2 overexpression increasing ß-CTS, PLIN2 depletion or overexpression drops or adds expression of GSK3 substrates, such as ß-catenin, c/EBPα,c-Myc, cyclin D1, and insulin receptor substrate 1, and cell growth/survival. PLIN2 N or C terminus overexpression that is associated with higher levels of the substrates suggests that those substrates bind to specific regions of PLIN2. Mimicking the possible high lipid concentrations in cells in the human body under conditions of hyperlipidemia/obesity, OA-treated cells gain or reduce GSK3 substrate expression in parallel with a decrease (a Wnt-like effect) or increase in GSK3 activity, likely regulated by GSK3/PLIN2/GSK3 substrate associations.

Superior T memory stem cell persistence supports long-lived T cell memory.

Long-lived memory T cells are able to persist in the host in the absence of antigen; however, the mechanism by which they are maintained is not well understood. Recently, a subset of human T cells, stem cell memory T cells (TSCM cells), was shown to be self-renewing and multipotent, thereby providing a potential reservoir for T cell memory throughout life. However, their in vivo dynamics and homeostasis still remain to be defined due to the lack of suitable animal models. We identified T cells with a TSCM phenotype and stem cell-like properties in nonhuman primates. These cells were the least-differentiated memory subset, were functionally distinct from conventional memory cells, and served as precursors of central memory. Antigen-specific TSCM cells preferentially localized to LNs and were virtually absent from mucosal surfaces. They were generated in the acute phase of viral infection, preferentially survived in comparison with all other memory cells following elimination of antigen, and stably persisted for the long term. Thus, one mechanism for maintenance of long-term T cell memory derives from the unique homeostatic properties of TSCM cells. Vaccination strategies designed to elicit durable cellular immunity should target the generation of TSCM cells.

Early immunologic and virologic predictors of clinical HIV-1 disease progression.

OBJECTIVE: To identify early determinants of HIV-1 disease progression, which could potentially enable individualized patient treatment, and provide correlates of progression applicable as reference phenotypes to evaluate breakthrough infections in vaccine development. DESIGN: High-throughput technologies were employed to interrogate multiple parameters on cryopreserved, retrospective peripheral blood mononuclear cell (PBMC) samples from 51 individuals from São Paulo, Brazil, obtained within 1 year of diagnosing early Clade B HIV-1 infection. Fast Progressors, Slow Progressors, and Controllers were identified based on a 2-year clinical follow-up. METHODS: Phenotypic and functional T-cell parameters were tested by flow cytometry and qPCR to identify potential early determinants of subsequent HIV-1 disease progression. RESULTS: Major differences were observed between Controllers and Progressors, especially in cell-associated viral load (CAVL), the differentiation pattern and CD38 expression of CD8 T cells, and the cytokine pattern and activation phenotype of HIV-1-specific CD8 T cells. Despite remarkably few other differences between the two Progressor groups, the CAVL had predictive power independent of plasma viral load. CONCLUSION: Analysis of three parameters (% CD38 CD8 T cells, total CAVL, % CCR5 CD8 T cells) was sufficient to predict subsequent disease progression (P < 0.001). Use of such prognostic correlates may be crucial when early CD4 T-cell counts and plasma viral load levels fail to discriminate among groups with differing subsequent clinical progression.

Priming T-cell responses with recombinant measles vaccine vector in a heterologous prime-boost setting in non-human primates.

Licensed live attenuated virus vaccines capable of expressing transgenes from other pathogens have the potential to reduce the number of childhood immunizations by eliciting robust immunity to multiple pathogens simultaneously. Recombinant attenuated measles virus (rMV) derived from the Edmonston Zagreb vaccine strain was engineered to express simian immunodeficiency virus (SIV) Gag protein for the purpose of evaluating the immunogenicity of rMV as a vaccine vector in rhesus macaques. rMV-Gag immunization alone elicited robust measles-specific humoral and cellular responses, but failed to elicit transgene (Gag)-specific immune responses, following aerosol or intratracheal/intramuscular delivery. However, when administered as a priming vaccine to a heterologous boost with recombinant adenovirus serotype 5 expressing the same transgene, rMV-Gag significantly enhanced Gag-specific T lymphocyte responses following rAd5 immunization. Gag-specific humoral responses were not enhanced, however, which may be due to either the transgene or the vector. Cellular response priming by rMV against the transgene was highly effective even when using a suboptimal dose of rAd5 for the boost. These data demonstrate feasibility of using rMV as a priming component of heterologous prime-boost vaccine regimens for pathogens requiring strong cellular responses.

The simian immunodeficiency virus targets central cell cycle functions through transcriptional repression in vivo.

A massive and selective loss of CD4+ memory T cells occurs during the acute phase of immunodeficiency virus infections. The mechanism of this depletion is poorly understood but constitutes a key event with implications for progression. We assessed gene expression of purified T cells in Rhesus Macaques during acute SIVmac239 infection in order to define mechanisms of pathogenesis. We observe a general transcriptional program of over 1,600 interferon-stimulated genes induced in all T cells by the infection. Furthermore, we identify 113 transcriptional changes that are specific to virally infected cells. A striking downregulation of several key cell cycle regulator genes was observed and shared promotor-region E2F binding sites in downregulated genes suggested a targeted transcriptional control of an E2F regulated cell cycle program. In addition, the upregulation of the gene for the fundamental regulator of RNA polymerase II, TAF7, demonstrates that viral interference with the cell cycle and transcriptional regulation programs may be critical components during the establishment of a pathogenic infection in vivo.

Genetic immunization in the lung induces potent local and systemic immune responses.

Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.

CCR2 identifies a stable population of human effector memory CD4+ T cells equipped for rapid recall response.

Because T cells act primarily through short-distance interactions, homing receptors can identify colocalizing cells that serve common functions. Expression patterns for multiple chemokine receptors on CD4(+) T cells from human blood suggested a hierarchy of receptors that are induced and accumulate during effector/memory cell differentiation. We characterized CD4(+)CD45RO(+) T cells based on expression of two of these receptors, CCR5 and CCR2, the principal subsets being CCR5(-)CCR2(-) (∼70%), CCR5(+)CCR2(-) (∼25%), and CCR5(+)CCR2(+) (∼5%). Relationships among expression of CCR5 and CCR2 and CD62L, and the subsets' proliferation histories, suggested a pathway of progressive effector/memory differentiation from the CCR5(-)CCR2(-) to CCR5(+)CCR2(-) to CCR5(+)CCR2(+) cells. Sensitivity and rapidity of TCR-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The subsets also showed increasing responsiveness to IL-7, and the CCR5(+)CCR2(+) cells were CD127(bright) and invariably showed the greatest response to tetanus toxoid. CCR5(+)CCR2(+) cells also expressed the largest repertoire of chemokine receptors and migrated to the greatest number of chemokines. By contrast, the CCR5(+)CCR2(-) cells had the greatest percentages of regulatory T cells, activated/cycling cells, and CMV-reactive cells, and were most susceptible to apoptosis. Our results indicate that increasing memory cell differentiation can be uncoupled from susceptibility to death, and is associated with an increase in chemokine responsiveness, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells that are highly enriched in the CCR5(+)CCR2(+) subset and ideally equipped for rapid recall responses in tissue.

Trafficking, persistence, and activation state of adoptively transferred allogeneic and autologous Simian Immunodeficiency Virus-specific CD8(+) T cell clones during acute and chronic infection of rhesus macaques.

Despite multiple lines of evidence suggesting their involvement, the precise role of CD8(+) T cells in controlling HIV replication remains unclear. To determine whether CD8(+) T cells can limit retroviral replication in the absence of other immune responses, we transferred 1-13 x 10(9) allogeneic in vitro expanded SIV-specific CD8(+) T cell clones matched for the relevant restricting MHC-I allele into rhesus macaques near the time of i.v. SIV challenge. Additionally, in vitro expanded autologous SIV-specific CD8(+) T cell clones were infused 4-9 mo postinfection. Infused cells did not appreciably impact acute or chronic viral replication. The partially MHC-matched allogeneic cells were not detected in the blood or most tissues after 3 d but persisted longer in the lungs as assessed by bronchoalveolar lavage (BAL). Autologous cells transferred i.v. or i.p. were found in BAL and blood samples for up to 8 wk postinfusion. Interestingly, despite having a nominally activated phenotype (CD69(+)HLA-DR(+)), many of these cells persisted in the BAL without dividing. This suggests that expression of such markers by T cells at mucosal sites may not reflect recent activation, but may instead identify stable resident memory T cells. The lack of impact following transfer of such a large number of functional Ag-specific CD8(+) T cells on SIV replication may reflect the magnitude of the immune response required to contain the virus.

Reduced protection from simian immunodeficiency virus SIVmac251 infection afforded by memory CD8+ T cells induced by vaccination during CD4+ T-cell deficiency.

Adaptive CD4(+) and CD8(+) T-cell responses have been associated with control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication. Here, we have designed a study with Indian rhesus macaques to more directly assess the role of CD8 SIV-specific responses in control of viral replication. Macaques were immunized with a DNA prime-modified vaccinia virus Ankara (MVA)-SIV boost regimen under normal conditions or under conditions of antibody-induced CD4(+) T-cell deficiency. Depletion of CD4(+) cells was performed in the immunized macaques at the peak of SIV-specific CD4(+) T-cell responses following the DNA prime dose. A group of naïve macaques was also treated with the anti-CD4 depleting antibody as a control, and an additional group of macaques immunized under normal conditions was depleted of CD8(+) T cells prior to challenge exposure to SIV(mac251). Analysis of the quality and quantity of vaccine-induced CD8(+) T cells demonstrated that SIV-specific CD8(+) T cells generated under conditions of CD4(+) T-cell deficiency expressed low levels of Bcl-2 and interleukin-2 (IL-2), and plasma virus levels increased over time. Depletion of CD8(+) T cells prior to challenge exposure abrogated vaccine-induced protection as previously shown. These data support the notion that adaptive CD4(+) T cells are critical for the generation of effective CD8(+) T-cell responses to SIV that, in turn, contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will be afforded only by T-cell vaccines for HIV that provide a balanced induction of CD4(+) and CD8(+) T-cell responses and protect against early depletion of CD4(+) T cells postinfection.

The homophilic binding of junctional adhesion molecule-C mediates tumor cell-endothelial cell interactions.

The junctional adhesion molecule C (JAM-C) was recently shown to undergo a heterophilic interaction with the leukocyte beta2 integrin Mac-1, thereby mediating interactions between vascular cells in inflammatory cell recruitment. Here, the homophilic interaction of JAM-C is presented and functionally characterized to mediate tumor cell-endothelial cell interactions. Recombinant soluble JAM-C in fluid phase bound to immobilized JAM-C as assessed in a purified system; moreover, JAM-C-transfected Chinese hamster ovary (CHO) cells adhered to immobilized JAM-C. The homophilic interaction of JAM-C was mediated by the isolated amino-terminal Ig domain (D1), but not the carboxyl-terminal Ig domain (D2), of the molecule. Dimerization of JAM-A is dependent on the sequence RVE in the amino-terminal Ig domain. This motif is conserved in JAM-C (Arg64-Ile65-Glu66), and a single amino acid mutation in this motif (E66R) abolished the homophilic interaction of JAM-C. The lung carcinoma cell line NCI-H522 was found to express JAM-C. NCI-H522 cells adhered to immobilized JAM-C, as well as to JAM-C-transfected CHO cells, but not to mock-transfected CHO cells or to CHO cells transfected with the JAM-C mutant (E66R). Adhesion of NCI-H522 cells to JAM-C protein or JAM-C-transfected CHO cells was abolished in the presence of soluble JAM-C or the isolated D1. Furthermore, the adhesion of NCI-H522 cells to endothelial cells was significantly blocked by soluble JAM-C or the isolated D1. Thus, JAM-C undergoes a homophilic interaction via the Arg64-Ile65-Glu66 motif on the membrane-distal Ig domain of the molecule. The homophilic interaction of JAM-C can mediate tumor cell-endothelial cell interactions and may thereby be involved in the process of tumor cell metastasis.

Characterization of subsets of CD4+ memory T cells reveals early branched pathways of T cell differentiation in humans.

The pathways for differentiation of human CD4(+) T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naive CD4(+) T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-receptor(+) cells suggest that they are very early memory CD4(+) T cells that have "rested down" before acquiring the phenotypes described for "central" or "effector" memory T cells. In addition, the chemokine-receptor(+) "naive" populations contain Th1 and Th2 cells, respectively, demonstrating that Th1/Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-receptor(+) subsets may be recruited and contribute to segregated, polarized microenvironments within lymphoid organs. Importantly, our data suggest that CD4(+) T cells do not differentiate according to a simple schema from naive --> CD45RO(+) noneffector/central memory --> effector/effector memory cells. Rather, developmental pathways branch early on to yield effector/memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells.

Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3(high)CD69+ thymocytes and gammadeltaTCR+ thymocytes preferentially respond to CCL25.

CCR9 mediates chemotaxis of thymocytes in response to CCL25/thymus-expressed chemokine, and its mRNA is selectively expressed in thymus and small intestine, the two known sites of T lymphopoiesis. To examine the expression of CCR9 during lymphocyte development, we generated polyclonal Ab that recognizes murine CCR9. CCR9 was expressed on the majority of immature CD4+CD8+ (double-positive) thymocytes, but not on immature CD4(-)CD8(-) (double-negative) thymocytes. CCR9 was down-regulated during the transition of double-positive thymocytes to the CD4+ or CD8+ (single-positive) stage, and only a minor subset of CD8+ lymph node T cells expressed CCR9. All CCR9+ thymocyte subsets migrated in response to CCL25; however, CD69+ thymocytes demonstrated enhanced CCL25-induced migration compared with CD69(-) thymocytes. Ab-mediated TCR stimulation also enhanced CCL25 responsiveness, indicating that CCL25-induced thymocyte migration is augmented by TCR signaling. Approximately one-half of all gammadeltaTCR+ thymocytes and peripheral gammadeltaTCR+ T cells expressed CCR9 on their surface, and these cells migrated in response to CCL25. These findings suggest that CCR9 may play an important role in the development and trafficking of both alphabetaTCR+ and gammadeltaTCR+ T cells.