In Vivo Detection of Dendritic Cell Antigen Presentation to CD4+ T Cells

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4⁺ T cells specific for an OVA peptide–I-A^d complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell–rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.     

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103⁺ and CD11b^hi phenotype, respectively - are the primary DC presenting antigen to naïve CD4⁺ and CD8⁺ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103⁺ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8⁺ T cells responding to infection into effector cells, and only the CD103⁺ RDC subset can present to naïve CD8⁺ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103⁺ and CD11b^hi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.     

Turnover of CD4+ and CD8+ T Lymphocytes in HIV-1 Infection as Measured by Ki-67 Antigen

We investigated CD4⁺ and CD8⁺ T cell turnover in both healthy and HIV-1–infected adults by measuring the nuclear antigen Ki-67 specific for cell proliferation. The mean growth fraction, corresponding to the expression of Ki-67, was 1.1% for CD4⁺ T cells and 1.0% in CD8⁺ T cells in healthy adults, and 6.5 and 4.3% in HIV-1–infected individuals, respectively. Analysis of CD45RA⁺ and CD45RO⁺ T cell subsets revealed a selective expansion of the CD8⁺ CD45RO⁺ subset in HIV-1–positive individuals. On the basis of the growth fraction, we derived the potential doubling time and the daily turnover of CD4⁺ and CD8⁺ T cells. In HIV-1–infected individuals, the mean potential doubling time of T cells was five times shorter than that of healthy adults. The mean daily turnover of CD4⁺ and CD8⁺ T cells in HIV-1–infected individuals was increased 2- and 6-fold, respectively, with more than 40-fold interindividual variation. In patients with <200 CD4⁺ counts, CD4⁺ turnover dropped markedly, whereas CD8⁺ turnover remained elevated. The large variations in CD4⁺ T cell turnover might be relevant to individual differences in disease progression.     

Persistence of Peptide-induced CD4+ T cell Anergy In Vitro

Clonal T cell unresponsiveness, or anergy, has been proposed as a mechanism of peripheral tolerance in vivo, and as a potential means of curbing unwanted T cell responses. In this study, anergy was induced in a T helper cell (Th) clone reactive to hemoglobin (Hb) peptide 64–76 by coculture of the T cells with live antigen-presenting cells (APCs) and 74L, a peptide analog of Hb(64–76) that contains a single amino acid substitution of leucine for glycine at position 74, or with a low concentration of the agonist ligand. The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64–76), and was not the result of impaired TCR/CD3 downmodulation. The addition of exogenous IL-12 transiently restored proliferation of the anergic lines, but removal of IL-12 from culture returned the T cells to their nonproliferative state. Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2. Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.     

Resting Memory CD8+ T Cells are Hyperreactive to Antigenic Challenge In Vitro

The characteristics of CD8⁺ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8⁺ T cell populations. In most experimental systems, heterogeneous populations of primed CD8⁺ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8⁺ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8⁺ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8⁺ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8⁺ T cells were compared in vitro. We found that CD8⁺ T cells from primed mice are activated by peptide concentrations 10–50-fold lower than naive mice. In addition, the kinetics of interleukin 2Rα chain upregulation by primed CD8⁺ T cells differ from naive CD8⁺ T cells. These primed hyperresponsive CD8⁺ T cells might play an important role in the memory response.     

Endogenous Interleukin 4 Is Required for Development of Protective CD4+ T Helper Type 1 Cell Responses to Candida albicans

Interleukin (IL)-4–deficient mice were used to assess susceptibility to systemic or gastrointestinal Candida albicans infections, as well as parameters of innate and elicited T helper immunity. In the early stage of systemic infection with virulent C. albicans, an unopposed interferon (IFN)-γ response renders IL-4–deficient mice more resistant than wild-type mice to infection. Yet, IL-4–deficient mice failed to efficiently control infection in the late stage and succumbed to it. Defective IFN-γ and IL-12 production, but not IL-12 responsiveness, was observed in IL-4–deficient mice that failed to mount protective T helper type 1 cell (Th1)-mediated acquired immunity in response to a live vaccine strain of the yeast or upon mucosal immunization in vivo. In vitro, IL-4 primed neutrophils for cytokine release, including IL-12. However, late treatment with exogenous IL-4, while improving the outcome of infection, potentiated CD4⁺ Th1 responses even in the absence of neutrophils. These findings indicate that endogenous IL-4 is required for the induction of CD4⁺ Th1 protective antifungal responses, possibly through the combined activity on cells of the innate and adaptive immune systems.     

Commitment of Immature CD4+8+ Thymocytes to the CD4 Lineage Requires CD3 Signaling but Does Not Require Expression of Clonotypic T Cell Receptor (TCR) Chains

As a consequence of positive selection in the thymus, immature CD4⁺8⁺ double-positive, [DP] thymocytes selectively terminate synthesis of one coreceptor molecule and, as a result, differentiate into either CD4⁺ or CD8⁺ T cells. The decision by individual DP thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment. Previously, we reported that the intrathymic signals that induced commitment to the CD4 versus CD8 T cell lineages were markedly asymmetric. Notably, CD8 commitment appeared to require lineage-specific signals, whereas CD4 commitment appeared to occur in the absence of lineage-specific signals by default. Consequently, it was unclear whether CD4 commitment, as revealed by selective termination of CD8 coreceptor synthesis, occurred in all DP thymocytes, or whether CD4 commitment occurred only in T cell receptor (TCR)–CD3-signaled DP thymocytes. Here, we report that selective termination of CD8 coreceptor synthesis does not occur in DP thymocytes spontaneously. Rather, CD4 commitment in DP thymocytes requires signals transduced by either CD3 or ζ chains, which can signal CD4 commitment even in the absence of clonotypic TCR chains.     

Disturbed CD4+ T Cell Homeostasis and In Vitro HIV-1 Susceptibility in Transgenic Mice Expressing T Cell Line–tropic HIV-1 Receptors

T cell line–tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4⁺ T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4⁺ T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4⁺ T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4⁺ T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4⁺ T cell decline in patients.     

Direct Visualization of Antigen-specific CD8+T Cells during the Primary Immune Response to Epstein-Barr Virus In Vivo

Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex–peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8⁺ T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8⁺ T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr.     

Productive Infection of Neonatal CD8+ T Lymphocytes by HIV-1

CD8⁺ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8⁺ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell–tropic (T-tropic) HIV strains. Physiological activation of CD8-α/β⁺ CD4⁻ T cell receptor–α/β⁺ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1–activated cord blood CD8⁺ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8⁺ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.     

Transferable Anergy: Superantigen Treatment Induces CD4+ T Cell Tolerance That Is Reversible and Requires CD4−CD8− Cells and Interferon γ

Bacterial superantigens induce peripheral unresponsiveness in CD4⁺ T cell populations that express appropriate Vβ chains. We have used Vβ3/Vα11 T cell receptor transgenic (Tg) mice and the Vβ3-specific superantigen staphylococcal enterotoxin A (SEA) to further investigate the mechanisms that contribute to such unresponsiveness. As in other models, in vivo exposure to SEA rendered the Tg CD4⁺ cells unresponsive to subsequent restimulation in vitro with antigen or mitogens. However, when the SEA-treated CD4⁺ cells were completely purified away from all other contaminating cells, they regained the ability to proliferate and secrete cytokines. Moreover, enriched CD4⁻CD8⁻ cells from the SEA-treated mice suppressed the responses of fresh control CD4⁺ cells in mixed cultures indicating that the apparent “anergy” was both transferable and reversible. Further analysis demonstrated that interferon γ, but not the Fas receptor, played a critical role in the suppression.     

Single Cell Analysis Reveals Regulated Hierarchical T Cell Antigen Receptor Signaling Thresholds and Intraclonal Heterogeneity for Individual Cytokine Responses of CD4+ T Cells

T cell receptor (TCR) recognition of peptide–major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4⁺ cell population. Low concentrations of TCR ligand elicit only interferon-γ (IFN-γ) production. Increasing ligand recruits more cells into the IFN-γ⁺ pool, increases IFN-γ produced per cell, and also elicits IL-2, but only from cells already making IFN-γ. Most cells producing only IFN-γ show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-γ synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-γ/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-γ and not IL-2, and the amount of IFN-γ exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.     

HIV-1 Directly Kills CD4+ T Cells by a Fas-independent Mechanism

The mechanism by which HIV-1 induces CD4⁺ T cell death is not known. A fundamental issue is whether HIV-1 primarily induces direct killing of infected cells or indirectly causes death of uninfected bystander cells. This question was studied using a reporter virus system in which infected cells are marked with the cell surface protein placental alkaline phosphatase (PLAP). Infection by HIV-PLAP of peripheral blood mononuclear cells (PBMCs) and T cell lines leads to rapid depletion of CD4⁺ T cells and induction of apoptosis. The great majority of HIV-induced T cell death in vitro involves direct loss of infected cells rather than indirect effects on uninfected bystander cells. Because of its proposed role in HIV-induced cell death, we also examined the Fas (CD95/Apo1) pathway in killing of T cells by HIV-1. Infected PBMCs or CEM cells display no increase in surface Fas relative to uninfected cells. In addition, HIV-1 kills CEM and Jurkat T cells in the presence of a caspase inhibitor that completely blocks Fas-mediated apoptosis. HIV-1 also depletes CD4⁺ T cells in PBMCs from patients who have a genetically defective Fas pathway. These results suggest that HIV-1 induces direct apoptosis of infected cells and kills T cells by a Fas-independent mechanism.     

Thalidomide Costimulates Primary Human T Lymphocytes,Preferentially Inducing Proliferation,Cytokine Production,and Cytotoxic Responses in the CD8+ Subset

The efficacy of thalidomide (α-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor α. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2–mediated T cell proliferation and interferon γ production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset.