Similar to hematopoietic stem cells, memory lymphocytes self renew, while their clonally expanded effector progeny differentiate to fight infection and tumors. Recently, Muranski et al. report in Immunity (2011) that a subset of Th17 effector cells functions as memory cells and retain stem cell properties.

Regulatory B cells that are functionally defined by their capacity to express IL-10 (B10 cells) downregulate inflammation and autoimmunity. In studies using well-defined IL-10-reporter mice, this rare B10 cell subset was also found to maintain a capacity for plasma cell differentiation. During a transient period of il10 transcription, the blimp1 and irf4 transcription factors were induced in B10 cells while pax5 and bcl6 were downregulated as a significant fraction of B10 cells completed the genetic and phenotypic program leading to antibody-secreting cell differentiation in vitro and in vivo. B10 cell-derived IgM reacted with both self and foreign Ags, whereas B10 cells generated Ag-specific IgG in response to immunizations. Moreover, B10 cells represented a significant source of serum IgM and IgG during adoptive transfer experiments, and produced Ag-specific, polyreactive and autoreactive antibody specificities that were consistent with their expression of a diverse Ag receptor repertoire. Thereby, B10 cells not only limit inflammation and immune responses by the transient production of IL-10, but may also facilitate clearance of their eliciting Ags through an inherent capacity to quickly generate polyreactive and/or Ag-specific antibodies during humoral immune responses.

Summary

Human neonates are at significantly greater risk of serious infection than immunocompetent adults. In particular, very low birth weight infants in the neonatal intensive care nursery are at high risk of developing life-threatening bacterial and fungal infections. Recent studies have identified Th17 cells as critical mediators of immunity to bacterial and fungal infections at epithelial barriers. Little is known, however, about the ontogeny of Th17 responses in humans. The frequency of serious bacterial infections in preterm infants and the importance of Th17 cells in providing protection against such infections in animal studies prompted us to study Th17 development in human neonates. NaÔve CD4 T cells from extremely preterm infants, term infants, and adults were assayed for their capacity to develop into Th17 effector cells. Surprisingly, Th17 capacity was inversely related to developmental age. Neonates expressed higher levels of IL-23R, RORγt, and STAT3 prior to activation and showed a significant Th17 bias after activation. In contrast, adult cells expressed more TBX21 with a corresponding Th1 bias. CD161 expression on Th17 precursors was also developmentally regulated. Our results suggest there is significant developmental regulation of CD4 effector lineages with a strong bias toward Th17 development early in life.

B cells contribute to the pathogenesis of chronic autoimmune disorders like systemic lupus erythematosus (SLE) via multiple effector functions. However, B cells are also implicated in regulating SLE and other autoimmune syndromes via release of IL-10. B cells secreting IL-10 have been termed “Breg” and have been proposed as a separate subset of cells, a concept that remains controversial. The balance between pro- and anti-inflammatory effects could determine the success of B cell targeted therapies for autoimmune disorders and it is therefore pivotal to understand the significance of B cell-secreted IL-10 in spontaneous autoimmunity. By lineage specific deletion of Il10 from B cells we demonstrate that B cell-derived IL-10 is ineffective in suppressing the spontaneous activation of self-reactive B and T cells during lupus. Correspondingly, severity of organ disease and survival rates in mice harboring Il10 deficient B cells are unaltered. Genetic marking of cells that transcribe Il10 illustrates that the pool of IL-10 competent cells is dominated by CD4 T cells and macrophages. IL-10 competent cells of the B lineage are rare in vivo and among them short-lived plasmablasts have the highest frequency, suggesting an activation rather than lineage-driven phenotype. Putative Breg phenotypic subsets such as CD1dhiCD5+ and CD21hiCD23hi B cells are not enriched in Il10 transcription. These genetic studies demonstrate that in a spontaneous model of murine lupus, IL-10 dependent B cell regulation does not restrain disease and thus the pathogenic effects of B cells are not detectably counterbalanced by their IL-10 dependent regulatory functions.

SUMMARY

The interleukin-17 (IL-17) family of cytokines phylogenetically pre-dates the evolution of T cells in jawed vertebrates, suggesting that the ontogeny of the Th17 cell lineage must have arisen to confer an evolutionary advantage to the host over innate sources of IL-17. Using a model of mucosal immunization with the encapsulated bacteria Klebsiella pneumoniae, B cells largely recognized polysaccharide capsular antigens, which limited protection to only the vaccine strain. In contrast, memory Th17 cells proliferated in response to conserved outer membrane proteins and conferred protection against several serotypes of K. pneumoniae, including the recently described multi-drug resistant New Dehli metallolactamase strain. Notably, this heterologous, clade specific protection was antibody-independent, demonstrating the Th17 cell lineage confers a host advantage by providing heterologous mucosal immunity independent of serotype specific antibody.

We have previously reported that mice deficient in the beta-glucan receptor Dectin-1 displayed increased susceptibility to Aspergillus fumigatus lung infection in the presence of lower interleukin 23 (IL-23) and IL-17A production in the lungs and have reported a role for IL-17A in lung defense. As IL-23 is also thought to control the production of IL-22, we examined the role of Dectin-1 in IL-22 production, as well as the role of IL-22 in innate host defense against A. fumigatus. Here, we show that Dectin-1-deficient mice demonstrated significantly reduced levels of IL-22 in the lungs early after A. fumigatus challenge. Culturing cells from enzymatic lung digests ex vivo further demonstrated Dectin-1-dependent IL-22 production. IL-22 production was additionally found to be independent of IL-1β, IL-6, or IL-18 but required IL-23. The addition of recombinant IL-23 augmented IL-22 production in wild-type (WT) lung cells and rescued IL-22 production by lung cells from Dectin-1-deficient mice. In vivo neutralization of IL-22 in the lungs of WT mice resulted in impaired A. fumigatus lung clearance. Moreover, mice deficient in IL-22 also demonstrated a higher lung fungal burden after A. fumigatus challenge in the presence of impaired IL-1α, tumor necrosis factor alpha (TNF-α), CCL3/MIP-1α, and CCL4/MIP-1β production and lower neutrophil recruitment, yet intact IL-17A production. We further show that lung lavage fluid collected from both A. fumigatus-challenged Dectin-1-deficient and IL-22-deficient mice had compromised anti-fungal activity against A. fumigatus in vitro. Although lipocalin 2 production was observed to be Dectin-1 and IL-22 dependent, lipocalin 2-deficient mice did not demonstrate impaired A. fumigatus clearance. Moreover, lung S100a8, S100a9, and Reg3g mRNA expression was not lower in either Dectin-1-deficient or IL-22-deficient mice. Collectively, our results indicate that early innate lung defense against A. fumigatus is mediated by Dectin-1-dependent IL-22 production.

Regulatory T (Treg) cells are plastic, but the in vivo mechanisms by which they are converted into Foxp3+interferon (IFN)-γ+ T cells, and whether these converted cells retain the ability to inhibit colitis, are not clear.

Methods

Foxp3+ Treg cells were generated by culture of naïve CD4+ T cells from Foxp3GFP CBir1 T-cell receptor (TCR) transgenic (CBir1-Tg) mice, which are specific for CBir1 flagellin (an immunodominant microbiota antigen), with transforming growth factor (TGF)-β. Foxp3GFP+ CBir1-Tg Treg cells were isolated by fluorescence-activated cell sorting and transferred into TCRβxδ−/− mice. Colitis was induced by transfer of naïve CBir1-Tg CD4+ T cells into immunodeficient mice.

Results

Microbiota antigen-specific Foxp3+ Treg cells were converted, in the intestine, to IFN-γ+ T-helper (Th)1 cells, interleukin (IL)-17+ Th17 cells, and Foxp3+ T cells that coexpress IFN-γ and/or IL-17. Conversion of Treg cells into IFN-γ-producing Th1 cells and Foxp3+IFN-γ+ T cells required innate cell production of IL-12 in the intestine; blocking IL-12 with an antibody inhibited their conversion to Th1 and Foxp3+IFN-γ+ T cells in the intestines of mice that were recipients of Treg cells. Addition of IL-12, but not IL-23, promoted conversion of Treg cells into Th1 and Foxp3+IFN-γ+ T cells, in vitro. Foxp3+IFN-γ+ T cells had regulatory activity, because they suppressed proliferation of naïve T cells, in vitro, and inhibited induction of colitis by microbiota antigen-specific T cells. IFN-γ+ Th1 cells were not converted into Treg cells; Foxp3+IFN-γ+ T cells differentiated into IFN-γ+ but not Foxp3+ T cells.

Conclusions

IL-12 promotes conversion of Treg cells into IFN-γ-expressing cells; Foxp3+IFN-γ+ T cells retain their regulatory functions and develop during the transition of Foxp3+ Treg cells into IFN-γ+ Th1 cells.

We have previously reported that compromised interleukin 17A (IL-17A) production in the lungs increased susceptibility to infection with the invasive fungal pathogen Aspergillus fumigatus. Here we have shown that culturing lung cells from A. fumigatus-challenged mice ex vivo demonstrated Dectin-1-dependent IL-17A production. In this system, neutralization of IL-23 but not IL-6, IL-1β, or IL-18 resulted in attenuated IL-17A production. Il23 mRNA expression was found to be lower in lung cells from A. fumigatus-challenged Dectin-1-deficient mice, whereas bone marrow-derived dendritic cells from Dectin-1-deficient mice failed to produce IL-23 in response to A. fumigatus in vitro. Addition of recombinant IL-23 augmented IL-17A production by wild-type (WT) and Dectin-1-deficient lung cells, although the addition of IL-6 or IL-1β did not augment the effect of IL-23. Intracellular cytokine staining of lung cells revealed lower levels of CD11b+ IL-17A+ and Ly-6G+ IL-17A+ cells in A. fumigatus-challenged Dectin-1-deficient mice. Ly-6G+ neutrophils purified from the lungs of A. fumigatus-challenged Dectin-1-deficient mice displayed lower Il17a mRNA expression but surprisingly had intact Rorc and Rora mRNA expression. We further demonstrated that Ly-6G+ neutrophils required the presence of myeloid cells for IL-17A production. Finally, upon in vitro stimulation with A. fumigatus, thioglycolate-elicited peritoneal neutrophils were positive for intracellular IL-17A expression and produced IL-17A in a Dectin-1- and IL-23-dependent manner. In summary, Dectin-1-dependent IL-17A production in the lungs during invasive fungal infection is mediated in part by CD11b+ Ly-6G+ neutrophils in an IL-23-dependent manner.

Summary

Study of the development of distinct CD4+ T-cell subsets from naive precursors continues to provide excellent opportunities for dissection of mechanisms that control lineage-specific gene expression or repression. Whereas it had been thought that the induction of transcription networks that control T-lineage commitment were highly stable, reinforced by epigenetic processes that confer heritability of functional phenotypes by the progeny of mature T cells, recent findings support a more dynamic view of T-lineage commitment. Here, we highlight advances in the mapping and functional characterization of cis elements in the Ifng locus that have provided new insights into the control of the chromatin structure and transcriptional activity of this signature T-helper 1 cell gene. We also examine epigenetic features of the Ifng locus that have evolved to enable its re-programming for expression by other T-cell subsets, particularly T-helper 17 cells, and contrast features of the Ifng locus with those of the Il17a–Il17f locus, which appears less promiscuous.

During many chronic infections the responding CD8 T cells become exhausted as they progressively lose their ability to elaborate key effector functions. Unlike prototypic memory CD8 cells, which rapidly synthesize IFN-γ following activation, severely exhausted T cells fail to produce this effector molecule. Nevertheless, the ontogeny of exhausted CD8 T cells as well as the underlying mechanisms that account for their functional inactivation remains ill-defined. We have utilized cytokine reporter mice, which mark the transcription of IFN-γ mRNA by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that virus-specific CD8 T cells clearly respond during the early stages of chronic lymphocytic choriomeningitis virus (LCMV) infection, and that this early T cell response is more pronounced than that initially observed in acutely infected hosts. Thus, exhausted CD8 T cells appear to emerge from populations of potently activated precursors. Unlike acute infections, which result in massive expansion of the responding T cells, there is a rapid attenuation of further expansion during chronic infections. The exhausted T cells that subsequently emerge in chronically infected hosts are incapable of producing the IFN-γ protein. Surprisingly, high levels of the IFN-γ transcript are still present in exhausted cells, demonstrating that ablation of IFN-γ production by exhausted cells is not due to transcriptional silencing. Thus, post-transcription regulatory mechanisms likely disable this effector module.

SUMMARY

Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three transacting factors – NF-κB, STAT4 and T-bet – with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and co-recruitment of STAT4. STAT4 and NF-κB therefore cooperate at multiple cis elements to enable NF-κB–dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in Th1 and Tc1 effector cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals.

Phenotypic plasticity of T helper 17 (Th17) cells suggests instability of chromatin structure of key genes of this lineage. Here we identify epigenetic modifications across the clustered Il17a and Il17f, and Ifng loci before and after differential IL-12 or TGFβ signaling, which induce divergent fates of Th17 cell precursors. We find that Th17 precursors have substantial remodeling of the Ifng locus but undergo critical additional modifications to enable high-level expression when stimulated by IL-12. Permissive modifications across the Il17a-Il17f locus are amplified by TGFβ signaling in Th17 cells, but are rapidly reversed downstream of IL-12–induced, STAT4– and T-bet–mediated silencing of the Rorc gene. These findings reveal substantial chromatin instability of key transcription factor and cytokine genes of Th17 cells and support a model of Th17 lineage plasticity in which cell-extrinsic factors modulate Th17 cell fates through differential effects on the epigenetic status of Th17 lineage factors.

Summary

Crohn’s disease and ulcerative colitis are the two major forms of chronic relapsing inflammatory disorders of the human intestines collectively referred to as inflammatory bowel disease (IBD). Though a complex set of autoinflammatory disorders that can be precipitated by diverse genetic and environmental factors, a feature that appears common to IBD pathogenesis is a dysregulated effector T cell response to the commensal microbiota. Due to the heightened effector T cell activity in IBD, developmental and functional pathways that give rise to these cells are potential targets for therapeutic intervention. In this review, we highlight recent advances in our understanding of effector T cell biology in the context of intestinal immune regulation and speculate on their potential clinical significance.

Th2 lymphocytes deliver essential signals for induction of asthmatic airway inflammation. We previously found that airway antigen challenge induces recruitment of Gr-1+ neutrophils prior to the recruitment of Th2 cells. We examined, therefore, whether Gr-1+ cells contribute to the development of Th2-dependent airway inflammation. Systemic depletion of Gr-1+ cells using the RB6-8C5 monoclonal antibody reduced Th2 cell recruitment following intranasal antigen challenge. The levels of both matrix metalloproteinase (MMP)-9 and the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA were up-regulated in the lungs of mice 12 h after intranasal antigen challenge. Up-regulation of TIMP-1 was independent of Gr-1+ cells, whereas up-regulation of MMP-9 RNA and total gelatinolytic activity were dramatically reduced in mice depleted of Gr-1+ cells. At 24 h after challenge, total lung collagenolytic activity was also up-regulated, in a Gr-1+ cell-dependent fashion. Systemic inhibition of MMP-8 and MMP-9 reduced the airway recruitment of Th cells, resulting in significantly reduced eosinophilic inflammation. These data suggest that antigen challenge via the airway activates Gr-1+ cells and consequently MMPs to facilitate the recruitment of Th cells in the airway inflammatory response.

TGF-β, together with IL-6 and IL-21, promotes Th17 cell development. IL-6 and IL-21 induce activation of STAT3, which is crucial for Th17 cell differentiation, as well as the expression of SOCS3, a major negative feedback regulator of STAT3-activating cytokines that negatively regulates Th17 cells. However, it is still largely unclear how TGF-β regulates Th17 cell development, and which TGF-β signaling pathway is involved in Th17 cell development. In this report, we demonstrate that TGF-β inhibits IL-6- and IL-21-induced SOCS3 expression, thus enhancing as well as prolonging STAT3 activation in naïve CD4+CD25− T cells. TGF-β inhibits IL-6-induced SOCS3 promoter activity in T cells. Also, SOCS3 siRNA knockdown partially compensates for the action of TGF-β on Th17 cell development. In mice with a dominant-negative form of TGF-β receptor II (TGF-β RII DN) and impaired TGF-β signaling, IL-6-induced CD4+ T cell expression of SOCS3 is higher, whereas STAT3 activation is lower compared to wild type B6 CD4+ T cells. Addition of a TGF-β RI kinase inhibitor that blocks Smad-dependent TGF-β signaling greatly, but not completely, abrogates the effect of TGF-β on Th17 cell differentiation. Our data indicate that inhibition of SOCS3 and thus enhancement of STAT3 activation is at least one of the mechanisms of TGF-β promotion of Th17 cell development.

We report that like other T cells cultured in the presence of transforming growth factor (TGF) β, Th17 cells also produce interleukin (IL) 9. Th17 cells generated in vitro with IL-6 and TGF-β as well as purified ex vivo Th17 cells both produced IL-9. To determine if IL-9 has functional consequences in Th17-mediated inflammatory disease, we evaluated the role of IL-9 in the development and progression of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. The data show that IL-9 neutralization and IL-9 receptor deficiency attenuates disease, and this correlates with decreases in Th17 cells and IL-6–producing macrophages in the central nervous system, as well as mast cell numbers in the regional lymph nodes. Collectively, these data implicate IL-9 as a Th17-derived cytokine that can contribute to inflammatory disease.

Activator protein 1 (AP-1) transcription factors are dimers of Jun, Fos, MAF and activating transcription factor (ATF) family proteins characterized by basic region and leucine zipper domains1. Many AP-1 proteins contain defined transcriptional activation domains (TADs), but Batf and the closely related Batf3 (refs 2, 3) contain only a basic region and leucine zipper and have been considered inhibitors of AP-1 activity3–8. Here we show that Batf is required for the differentiation of IL-17-producing T helper (TH17) cells9. TH17 cells comprise a CD4+ T cell subset that coordinates inflammatory responses in host defense but is pathogenic in autoimmunity10–13.Batf −/−mice have normal TH1 and TH2 differentiation, but show a defect in TH17 differentiation, and are resistant to experimental autoimmune encephalomyelitis (EAE).Batf −/−T cells fail to induce known factors required for TH17 differentiation, such as RORγt11 and the cytokine IL-21 (refs 14–17). Neither addition of IL-21 nor overexpression of RORγt fully restores IL-17 production in Batf−/− T cells. The IL-17 promoter is Batf-responsive, and upon TH17 differentiation, Batf binds conserved intergenic elements in the IL-17A/F locus and to the IL-17, IL-21 and IL-22 (ref 18) promoters. These results demonstrate that the AP-1 protein Batf plays a critical role in TH17 differentiation.

LFA-1 (CD11a/CD18) is a member of the β2-integrin family of adhesion molecules important in leukocyte trafficking and activation. Although LFA-1 is thought to contribute to the development of experimental autoimmune encephalomyelitis (EAE) primarily through its functions on effector T cells, its importance on other leukocyte populations remains unexplored. To address this question, we performed both adoptive transfer EAE experiments involving CD11a-/- mice and trafficking studies using bioluminescent T cells expressing luciferase under the control of a CD2 promoter (T-lux cells). Transfer of encephalitogenic CD11a-/- T cells to wild type mice resulted in a significant reduction in overall EAE severity compared to control transfers. We also observed, using in vivo imaging techniques, that CD11a-/- T-lux cells readily infiltrated lymph nodes and the CNS of wild type recipients with kinetics comparable to CD11a+/+ transfers, although their overall numbers in these organs were reduced. Surprisingly, transfer of encephalitogenic wild type T cells to CD11a-/- mice induced a severe and sometimes fatal EAE disease course, associated with massive T cell infiltration and proliferation in the CNS. These data indicate that LFA-1 expression on leukocytes in recipient mice plays an important immunomodulatory role in EAE. Thus, LFA-1 acts as a key regulatory adhesion molecule during the development of EAE, serving both pro- and anti-inflammatory roles in disease pathogenesis.

Summary

Recent progress in our understanding of mechanisms by which the immunosuppressive cytokine interleukin-10 (IL-10) participates in an ever-increasing diversity of T-cell lineages to maintain immune homeostasis has broadened the framework for defining regulatory and effector T cells and has blurred the lines between them. In this review, we highlight established and emerging roles for IL-10 produced by distinct CD4+ T-cell lineages that underlie its non-redundant role in curbing immune responses to the intestinal microbiota at steady state and its role to limit T-cell-driven inflammation in responses to pathogens.

Extrathymic induction of regulatory T (T reg) cells is essential to the regulation of effector T cell responses in the periphery. In addition to Foxp3, T reg cell expression of suppressive cytokines, such as IL-10, is essential for peripheral tolerance, particularly in the intestines. TGF-β has been shown to induce expression of Foxp3 as well as IL10 and the vitamin A metabolite; all-trans retinoic acid (RA [at-RA]) has been found to enhance the former. We report that in contrast to its enhancement of TGF-β–mediated Foxp3 induction, at-RA potently inhibits the TGF-β–mediated induction of Il10 in naive CD4 T cells. Thus, mucosal DC subsets that are active producers of at-RA inhibit induction of Il10 in naive CD4 T cells while promoting induction of Foxp3. Accordingly, mice with vitamin A deficiency have increased numbers of IL-10–competent T reg cells. Activation of DCs by certain Toll-like receptors (TLRs), particularly TLR9, suppresses T cell induction of Foxp3 and enables induction of Il10. Collectively, our data indicate that at-RA has reciprocal effects on the induction of Foxp3 and Il10 in developing CD4+ T reg cells and suggest that TLR9-dependent inhibition of at-RA production by antigen-presenting cells might represent one mechanism to promote the development of IL-10–expressing T cells.

Background

Rapid clonal expansion of T cells occurs in response to antigenic challenges. The kinetics of the T cell response has previously been described using tissue-based studies performed at defined time points. Luciferase bioluminescence has recently been utilized for non-invasive analysis of in vivo biologic processes in real-time.

Results

We have created a novel transgenic mouse model (T-Lux) using a human CD2 mini-gene to direct luciferase expression specifically to the T cell compartment. T-Lux T cells demonstrated normal homing patterns within the intact mouse and following adoptive transfer. Bioluminescent signal correlated with T cell numbers in the whole body images as well as within specific organ regions of interest. Following transfer into lymphopenic (RAG2-/-) recipients, homeostatic proliferation of T-Lux T cells was visualized using bioluminescent imaging. Real-time bioluminescent analysis of CD4+ T cell antigen-specific responses enabled real-time comparison of the kinetics and magnitude of clonal expansion and contraction in the inductive lymph node and tissue site of antigen injection. T cell expansion was dose-dependent despite the presence of supraphysiologic numbers of OVA-specific OT-II transgenic TCR T-Lux T cells. CD4+ T cells subsequently underwent a rapid (3–4 day) contraction phase in the draining lymph node, with a delayed contraction in the antigen delivery site, with bioluminescent signal diminished below initial levels, representing TCR clonal frequency control.

Conclusion

The T-Lux mouse provides a novel, efficient model for tracking in vivo aspects of the CD4+ T cell response to antigen, providing an attractive approach for studies directed at immunotherapy or vaccine design.

Each of the effector CD4 T-cell lineages - Th1, Th2, and the more recently identified Th17 - arises from pluripotent naïve precursors whose developmental fate is largely controlled by cytokines that act in concert with antigenic signals. Remarkably, development of the Th17 lineage has been linked to that of regulatory T cells, which obviate or downregulate Th17 responses to preserve immune homeostasis, through a shared requirement for the cytokine transforming growth factor-beta. Several new studies offer insights into the mechanism whereby the precursors of these subsets are directed into distinct lineages.

There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.

Limited frequencies of T cells express IL-2 in primary antigenic responses, despite activation marker expression and proliferation by most clonal members. To define the basis for restricted IL-2 expression, a videomicroscopic system and IL-2 reporter transgenic model were used to characterize dendritic cell (DC)–T cell interactions. T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2. Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression. Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

The migration of antigen-specific T cells to nonlymphoid tissues is thought to be important for the elimination of foreign antigens from the body. However, recent results showing the migration of activated T cells into many nonlymphoid tissues raised the possibility that antigen-specific T cells do not migrate preferentially to nonlymphoid tissues containing antigen. We addressed this question by tracking antigen-specific CD4 T cells in the whole body after a localized subcutaneous antigen injection. Antigen-specific CD4 T cells proliferated in the skin-draining lymph nodes and the cells that underwent the most cell divisions acquired the ability to bind to CD62P. As time passed, CD62P-binding antigen-specific CD4 T cells with interferon γ production potential accumulated preferentially at the site of antigen injection but only in recipients that expressed CD62E. Surprisingly, these T cells did not proliferate in the injection site despite showing evidence of more cell divisions than the T cells in the draining lymph nodes. The results suggest that the most divided effector CD4 T cells from the lymph nodes enter the site of antigen deposition via recognition of CD62E on blood vessels and are retained there in a nonproliferative state via recognition of peptide–major histocompatibility complex II molecules.