Chemokine Signatures of Pathogen-Specific T Cells II: Memory T Cells in Acute and Chronic Infection.

Pathogen-specific memory T cells (TM) contribute to enhanced immune protection under conditions of reinfection, and their effective recruitment into a recall response relies, in part, on cues imparted by chemokines that coordinate their spatiotemporal positioning. An integrated perspective, however, needs to consider TM as a potentially relevant chemokine source themselves. In this study, we employed a comprehensive transcriptional/translational profiling strategy to delineate the identities, expression patterns, and dynamic regulation of chemokines produced by murine pathogen-specific TM CD8+TM, and to a lesser extent CD4+TM, are a prodigious source for six select chemokines (CCL1/3/4/5, CCL9/10, and XCL1) that collectively constitute a prominent and largely invariant signature across acute and chronic infections. Notably, constitutive CCL5 expression by CD8+TM serves as a unique functional imprint of prior antigenic experience; induced CCL1 production identifies highly polyfunctional CD8+ and CD4+TM subsets; long-term CD8+TM maintenance is associated with a pronounced increase of XCL1 production capacity; chemokines dominate the earliest stages of the CD8+TM recall response because of expeditious synthesis/secretion kinetics (CCL3/4/5) and low activation thresholds (CCL1/3/4/5/XCL1); and TM chemokine profiles modulated by persisting viral Ags exhibit both discrete functional deficits and a notable surplus. Nevertheless, recall responses and partial virus control in chronic infection appear little affected by the absence of major TM chemokines. Although specific contributions of TM-derived chemokines to enhanced immune protection therefore remain to be elucidated in other experimental scenarios, the ready visualization of TM chemokine-expression patterns permits a detailed stratification of TM functionalities that may be correlated with differentiation status, protective capacities, and potential fates.

Chemokine Signatures of Pathogen-Specific T Cells I: Effector T Cells.

The choreography of complex immune responses, including the priming, differentiation, and modulation of specific effector T cell populations generated in the immediate wake of an acute pathogen challenge, is in part controlled by chemokines, a large family of mostly secreted molecules involved in chemotaxis and other patho/physiological processes. T cells are both responsive to various chemokine cues and a relevant source for certain chemokines themselves; yet, the actual range, regulation, and role of effector T cell-derived chemokines remains incompletely understood. In this study, using different in vivo mouse models of viral and bacterial infection as well as protective vaccination, we have defined the entire spectrum of chemokines produced by pathogen-specific CD8+ and CD4+T effector cells and delineated several unique properties pertaining to the temporospatial organization of chemokine expression patterns, synthesis and secretion kinetics, and cooperative regulation. Collectively, our results position the "T cell chemokine response" as a notably prominent, largely invariant, yet distinctive force at the forefront of pathogen-specific effector T cell activities and establish novel practical and conceptual approaches that may serve as a foundation for future investigations into the role of T cell-produced chemokines in infectious and other diseases.

Aging boosts antiviral CD8+T cell memory through improved engagement of diversified recall response determinants.

The determinants of protective CD8+ memory T cell (CD8+TM) immunity remain incompletely defined and may in fact constitute an evolving agency as aging CD8+TM progressively acquire enhanced rather than impaired recall capacities. Here, we show that old as compared to young antiviral CD8+TM more effectively harness disparate molecular processes (cytokine signaling, trafficking, effector functions, and co-stimulation/inhibition) that in concert confer greater secondary reactivity. The relative reliance on these pathways is contingent on the nature of the secondary challenge (greater for chronic than acute viral infections) and over time, aging CD8+TM re-establish a dependence on the same accessory signals required for effective priming of naïve CD8+T cells in the first place. Thus, our findings reveal a temporal regulation of complementary recall response determinants that is consistent with the recently proposed "rebound model" according to which aging CD8+TM properties are gradually aligned with those of naïve CD8+T cells; our identification of a broadly diversified collection of immunomodulatory targets may further provide a foundation for the potential therapeutic "tuning" of CD8+TM immunity.

Aging of Antiviral CD8+ Memory T Cells Fosters Increased Survival, Metabolic Adaptations, and Lymphoid Tissue Homing.

Aging of established antiviral T cell memory can foster a series of progressive adaptations that paradoxically improve rather than compromise protective CD8+ T cell immunity. We now provide evidence that this gradual evolution, the pace of which is contingent on the precise context of the primary response, also impinges on the molecular mechanisms that regulate CD8+ memory T cell (TM) homeostasis. Over time, CD8+ TM generated in the wake of an acute infection with the natural murine pathogen lymphocytic choriomeningitis virus become more resistant to apoptosis and acquire enhanced cytokine responsiveness without adjusting their homeostatic proliferation rates; concurrent metabolic adaptations promote increased CD8+ TM quiescence and fitness but also impart the reacquisition of a partial effector-like metabolic profile; and a gradual redistribution of aging CD8+ TM from blood and nonlymphoid tissues to lymphatic organs results in CD8+ TM accumulations in bone marrow, splenic white pulp, and, particularly, lymph nodes. Altogether, these data demonstrate how temporal alterations of fundamental homeostatic determinants converge to render aged CD8+ TM poised for greater recall responses.

Multiple layers of CD80/86-dependent costimulatory activity regulate primary, memory, and secondary lymphocytic choriomeningitis virus-specific T cell immunity.

The lymphocytic choriomeningitis virus (LCMV) system constitutes one of the most widely used models for the study of infectious disease and the regulation of virus-specific T cell immunity. However, with respect to the activity of costimulatory and associated regulatory pathways, LCMV-specific T cell responses have long been regarded as relatively independent and thus distinct from the regulation of T cell immunity directed against many other viral pathogens. Here, we have reevaluated the contribution of CD28-CD80/86 costimulation in the LCMV system by use of CD80/86-deficient mice, and our results demonstrate that a disruption of CD28-CD80/86 signaling compromises the magnitude, phenotype, and/or functionality of LCMV-specific CD8(+) and/or CD4(+) T cell populations in all stages of the T cell response. Notably, a profound inhibition of secondary T cell immunity in LCMV-immune CD80/86-deficient mice emerged as a composite of both defective memory T cell development and a specific requirement for CD80 but not CD86 in the recall response, while a related experimental scenario of CD28-dependent yet CD80/86-independent secondary CD8(+) T cell immunity suggests the existence of a CD28 ligand other than CD80/86. Furthermore, we provide evidence that regulatory T cells (T(REG)s), the homeostasis of which is altered in CD80/86(-/-) mice, contribute to restrained LCMV-specific CD8(+) T cell responses in the presence of CD80/86. Our observations can therefore provide a more coherent perspective on CD28-CD80/86 costimulation in antiviral T cell immunity that positions the LCMV system within a shared context of multiple defects that virus-specific T cells acquire in the absence of CD28-CD80/86 costimulation.

Aging promotes acquisition of naive-like CD8+ memory T cell traits and enhanced functionalities.

Protective T cell memory is an acquired trait that is contingent upon the preservation of its constituents and therefore vulnerable to the potentially deleterious effects of organismal aging. Here, however, we have found that long-term T cell memory in a natural murine host-pathogen system can substantially improve over time. Comprehensive molecular, phenotypic, and functional profiling of aging antiviral CD8+ memory T cells (CD8+ TM) revealed a pervasive remodeling process that promotes the gradual acquisition of distinct molecular signatures, of increasingly homogeneous phenotypes, and of diversified functionalities that combine to confer a CD8+ TM-autonomous capacity for enhanced recall responses and immune protection. Notably, the process of CD8+ TM aging is characterized by a progressive harmonization of memory and naive T cell traits, is broadly amenable to experimental acceleration or retardation, and serves as a constitutional component for the "rebound model" of memory T cell maturation. By casting CD8+ TM populations within the temporal framework of their slowly evolving properties, this model establishes a simple ontogenetic perspective on the principal organization of CD8+ T cell memory that may directly inform the development of improved diagnostic, prophylactic, and therapeutic modalities.

High efficiency of antiviral CD4(+) killer T cells.

The destruction of infected cells by cytotxic T lymphocytes (CTL) is integral to the effective control of viral and bacterial diseases, and CTL function at large has long been regarded as a distinctive property of the CD8(+)T cell subset. In contrast, and despite their first description more than three decades ago, the precise contribution of cytotoxic CD4(+)T cells to the resolution of infectious diseases has remained a matter of debate. In particular, the CTL activity of pathogen-specific CD4(+) "helper" T cells constitutes a single trait among a diverse array of other T cell functionalities, and overall appears considerably weaker than the cytolytic capacity of CD8(+) effector T cells. Here, using an in vivo CTL assay, we report that cytotoxic CD4(+)T cells are readily generated against both viral and bacterial pathogens, and that the efficiency of MHC-II-restricted CD4(+)T cell killing adjusted for effector:target cell ratios, precise specificities and functional avidities is comparable in magnitude to that of CD8(+)T cells. In fact, the only difference between specific CD4(+) and CD8(+)T cells pertains to the slightly delayed killing kinetics of the former demonstrating that potent CTL function is a cardinal property of both antiviral CD8(+) and CD4(+)T cells.

The Ikaros transcription factor regulates responsiveness to IL-12 and expression of IL-2 receptor alpha in mature, activated CD8 T cells.

The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

Expression and regulation of chemokines in murine and human type 1 diabetes.

More than one-half of the ~50 human chemokines have been associated with or implicated in the pathogenesis of type 1 diabetes, yet their actual expression patterns in the islet environment of type 1 diabetic patients remain, at present, poorly defined. Here, we have integrated a human islet culture system, murine models of virus-induced and spontaneous type 1 diabetes, and the histopathological examination of pancreata from diabetic organ donors with the goal of providing a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family. Chemokine (C-C motif) ligand (CCL) 5 (CCL5), CCL8, CCL22, chemokine (C-X-C motif) ligand (CXCL) 9 (CXCL9), CXCL10, and chemokine (C-X3-C motif) ligand (CX3CL) 1 (CX3CL1) were the major chemokines transcribed (in an inducible nitric oxide synthase-dependent but not nuclear factor-κB-dependent fashion) and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients, whereas CCL5, CCL8, CXCL9, and CX3CL1 proteins were present at lower levels in the islets of both species. Of importance, additional expression of the same chemokines in human acinar tissues emphasizes an underappreciated involvement of the exocrine pancreas in the natural course of type 1 diabetes that will require consideration for additional type 1 diabetes pathogenesis and immune intervention studies.

Comprehensive assessment of chemokine expression profiles by flow cytometry.

The chemokines are a large family of mainly secreted molecules involved in the regulation of numerous physiological and pathophysiological processes. Despite many years of investigation, the precise cellular sources of most chemokines have remained incompletely defined as a consequence of the limited availability of suitable reagents to visualize the expression of chemokine proteins at the single-cell level. Here, we developed a simple flow cytometry-based assay using commercially available chemokine-specific antibodies for efficient cell-associated detection of 37 of 39 murine chemokines. To demonstrate the utility of this methodology, we used it to reevaluate the nature of homeostatic chemokines in the hematopoietic compartment, to delineate the complete chemokine profiles of NK cells and B cells in response to major polyclonal stimuli, and to assess the chemokine response of DCs to bacterial infection. The versatility of this analytical methodology was further demonstrated by its application to selected human chemokines and should greatly facilitate any future investigation into chemokine biology at large.

Mapping and restriction of a dominant viral CD4+ T cell core epitope by both MHC class I and MHC class II.

Virus-specific CD4(+) T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as "help" for B cell and CD8(+) T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4(+) T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4(+) T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8(+) T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.