Regional and mucosal memory T cells.

After infection, most antigen-specific memory T cells reside in nonlymphoid tissues. Tissue-specific programming during priming leads to directed migration of T cells to the appropriate tissue, which promotes the development of tissue-resident memory in organs such as intestinal mucosa and skin. Mechanisms that regulate the retention of tissue-resident memory T cells include transforming growth factor-ß (TGF-ß)-mediated induction of the E-cadherin receptor CD103 and downregulation of the chemokine receptor CCR7. These pathways enhance protection in internal organs, such as the nervous system, and in the barrier tissues--the mucosa and skin. Memory T cells that reside at these surfaces provide a first line of defense against subsequent infection, and defining the factors that regulate their development is critical to understanding organ-based immunity.

Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function.

The intestinal mucosa promotes T cell responses that might be beneficial for effective mucosal vaccines. However, intestinal resident memory T (Trm) cell formation and function are poorly understood. We found that oral infection with Listeria monocytogenes induced a robust intestinal CD8 T cell response and blocking effector T cell migration showed that intestinal Trm cells were critical for secondary protection. Intestinal effector CD8 T cells were predominately composed of memory precursor effector cells (MPECs) that rapidly upregulated CD103, which was needed for T cell accumulation in the intestinal epithelium. CD103 expression, rapid MPEC formation, and maintenance in intestinal tissues were dependent on T cell intrinsic transforming growth factor ß signals. Moreover, intestinal Trm cells generated after intranasal or intravenous infection were less robust and phenotypically distinct from Trm cells generated after oral infection, demonstrating the critical contribution of infection route for directing the generation of protective intestinal Trm cells.

Environmental cues dictate the fate of individual CD8+ T cells responding to infection.

Many studies have examined pathways controlling effector T cell differentiation, but less is known about the fate of individual CD8+ T cells during infection. Here, we examine the antiviral and antibacterial responses of single CD8+ T cells from the polyclonal repertoire. The progeny of naive clonal CD8+ T cells displayed unique profiles of differentiation based on extrinsic pathogen-induced environmental cues, with some clones demonstrating extreme bias toward a single developmental pathway. Moreover, even within the same animal, a single naive CD8+ T cell exhibited distinct fates that were controlled by tissue-specific events. However, memory CD8+ T cells relied on intrinsic factors to control differentiation upon challenge. Our results demonstrate that stochastic and instructive events differentially contribute to shaping the primary and secondary CD8+ T cell response and provide insight into the underlying forces that drive effector differentiation and protective memory formation.

γδ T cells exhibit multifunctional and protective memory in intestinal tissues.

The study of T cell memory and the target of vaccine design have focused on memory subsumed by T cells bearing the αß T cell receptor. Alternatively, γδ T cells are thought to provide rapid immunity, particularly at mucosal borders. Here, we have shown that a distinct subset of mucosal γδ T cells mounts an immune response to oral Listeria monocytogenes (Lm) infection and leads to the development of multifunctional memory T cells capable of simultaneously producing interferon-γ and interleukin-17A in the murine intestinal mucosa. Challenge infection with oral Lm, but not oral Salmonella or intravenous Lm, induced rapid expansion of memory γδ T cells, suggesting contextual specificity to the priming pathogen. Importantly, memory γδ T cells were able to provide enhanced protection against infection. These findings illustrate that γδ T cells play a role with hallmarks of adaptive immunity in the intestinal mucosa.

B cells, not just for antibody anymore.

B cell antibody production is thought to be crucial for protection against virus infection. In this issue of Immunity, Moseman et al. (2012) illustrate an antibody-independent role for B cells in macrophage activation that prevents virus dissemination after subcutaneous infection.

IL-17A-producing resident memory γδ T cells orchestrate the innate immune response to secondary oral Listeria monocytogenes infection.

Memory γδ T cells are important for the clearance of Listeria monocytogenes infection in the intestinal mucosa. However, the mechanisms by which memory γδ T cells provide protection against secondary oral infection are poorly understood. Here we used a recombinant strain of L. monocytogenes that efficiently invades the intestinal epithelium to show that Vγ4(+) memory γδ T cells represent a resident memory (Trm) population in the mesenteric lymph nodes (MLNs). The γδ Trm exhibited a remarkably static pattern of migration that radically changed following secondary oral L. monocytogenes infection. The γδ Trms produced IL-17A early after rechallenge and formed organized clusters with myeloid cells surrounding L. monocytogenes replication foci only after a secondary oral infection. Antibody blocking studies showed that in addition to IL-17A, the chemokine receptor C-X-C chemokine receptor 3 (CXCR3) is also important to enable the local redistribution of γδ Trm cells and myeloid cells specifically near the sites of L. monocytogenes replication within the MLN to restrict bacterial growth and spread. Our findings support a role for γδ Trms in orchestrating protective immune responses against intestinal pathogens.

IL-15 receptor α signaling constrains the development of IL-17-producing γδ T cells.

The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of IL-15 and its unique receptor, IL-15Rα, in the development of IL-17-producing γδ (γδ-17) T cells. Phenotypic analysis has shown that CD44(high) γδ-17 cells express IL-15Rα and the common gamma chain (CD132), yet lack the IL-2/15Rß chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice, but not of IL-15 KO mice. The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. γδ-17 cells also were increased in the peripheral lymph nodes of transgenic knock-in mice, where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). Finally, an analysis of neonatal thymi revealed that the CD44(lo/int) precursors of γδ-17 cells, which also expressed IL-15Rα, were increased in newborn mice deficient in IL-15Rα signaling, but not in IL-15 itself. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny, with long-term effects on their peripheral homeostasis in the adult.

Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection.

Through genetic recombination, the adaptive immune system generates a diverse T cell repertoire allowing recognition of a vast spectrum of foreign antigens. Any given CD8+ T cell specificity is thought to be rare, but none have been directly quantified. Here, major histocompatibility complex tetramer and magnetic-bead technology were coupled to quantitate naive antigen-specific CD8+ T cells and the early response to infection. Among six specificities measured, the number of naive antigen-specific precursors ranged from approximately 80 to 1200 cells/mouse. After vesicular stomatitis virus infection, the antigen-specific CD8+ T cell response occurred in discrete phases: prolonged activation of a subset of cells over the first 72 hr followed by a rapid proliferative burst. Naive precursor frequency altered response kinetics and regulated immunodominance, as well as the time required for the responding population to shift toward CD62L(hi) memory cells. Thus, initial endogenous precursor frequencies were surprisingly diverse and not only regulated initial immune response characteristics but also controlled memory CD8+ T cell lineage decisions.

CD8 T Cells Enter the Splenic T Cell Zones Independently of CCR7, but the Subsequent Expansion and Trafficking Patterns of Effector T Cells after Infection Are Dysregulated in the Absence of CCR7 Migratory Cues.

CCR7 is an important chemokine receptor that regulates T cell trafficking and compartmentalization within secondary lymphoid organs. However, the T cell-intrinsic role of CCR7 during infection in the spleen is not well understood. This study was designed to understand how CCR7-dependent localization and migration of CD8(+) T cells in different compartments of the spleen affected the primary and recall responses after infection. To this end, we used adoptive transfer of naive Ag-specific CD8 T cells (OT-I) that either lacked CCR7 or constitutively expressed CCR7 (CD2-CCR7) in mice that were subsequently infected i.v. with Listeria monocytogenes. We show that naive CCR7(-/-)CD8(+) T cells failed to enter the T cell zone, whereas CD2-CCR7 OT-I cells were exclusively confined to the T cell zones of the spleen. Surprisingly, however, CCR7(-/-) OT-I cells entered the T cell zones after infection, but the entry and egress migratory pattern of these cells was dysregulated and very distinct compared with wild-type OT-I cells. Moreover, CCR7-deficient OT-I cells failed to expand robustly when compared with wild-type OT-I cells and were preferentially skewed toward a short-lived effector cell differentiation pattern. Interestingly, CCR7(-/-), CD2-CCR7, and wild-type OT-I memory cells responded equally well to rechallenge infection. These results highlight a novel role of CCR7 in regulating effector CD8 T cell migration in the spleen and demonstrate differential requirement of CCR7 for primary and secondary CD8 T cell responses to infection.

The descent of memory T-cell subsets.

The immune system has evolved by continuously increasing its complexity to provide the host with an advantage over infectious agents. The development of immunological memory engenders long-lasting protection and lengthens the lifespan of the host. The generation of subsets of memory T cells with distinct homing and functional properties increases our defensive capabilities. However, the developmental relationship of memory T-cell subsets is a matter of debate. In this Opinion article, in light of recent developments, we suggest that it is probable that two distinct lineages comprise the memory CD8+ T-cell population generated in response to infection.

CD11a is essential for normal development of hematopoietic intermediates.

The process of lymphopoiesis begins in the bone marrow (BM) and requires multiple cellular intermediates. For T cell production, lymphoid progenitors exit the BM and home to the thymus where maturation and selection ensue. These processes are dependent on a number of factors, including chemokines and adhesion molecules. Although the ß2 integrin CD11a plays an important role in the migration of lymphocytes to lymph nodes, the role of CD11a in T cell development is largely undefined. Our studies now show that, in CD11a(-/-) mice, thymic cellularity was decreased and early T cell development was partially impaired. Remarkably, CD11a was critical for generation of common lymphoid progenitors (CLPs) and lymphoid-primed multipotent progenitors. However, in intact CD11a(-/-) mice, peripheral B and T cell subsets were only modestly altered, suggesting that compensatory mechanisms were operating. In contrast, competitive BM-reconstitution assays revealed an essential role for CD11a in the generation of thymocytes and mature T and B cells. This defect was linked to the requirement for CD11a in the development of CLPs. Furthermore, our results identified CLPs, and not lymphoid-primed multipotent progenitors, as the requisite CD11a-dependent precursor for lymphocyte development. Thus, these findings established a key role for CD11a in lymphopoiesis.

Transcriptional regulation of IL-15 expression during hematopoiesis.

Dendritic cells (DCs) are the most commonly studied source of the cytokine IL-15. Using an IL-15 reporter transgenic mouse, we have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8⁺ and CD8⁻). In this study, we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (lineage⁻Sca-1⁺c-Kit⁺) expressed high levels of IL-15, suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was Notch-dependent and occurred in a stepwise pattern coincident with increasing maturation and commitment to a T cell fate. Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory activity of IL-15 production occurring at the pre-DC branch point, leading to the generation of both IL-15⁺CD8⁺ and IL-15(⁻/low)CD8⁻ DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells.

Distinct mechanisms mediate naive and memory CD8 T-cell tolerance.

Peripheral tolerance to developmentally regulated antigens is necessary to sustain tissue homeostasis. We have now devised an inducible and reversible system that allows interrogation of T-cell tolerance induction in endogenous naïve and memory CD8 T cells. Our data show that peripheral CD8 T-cell tolerance can be preserved through two distinct mechanisms, antigen addiction leading to anergy for naïve T cells and ignorance for memory T cells. Induction of antigen in dendritic cells resulted in substantial expansion and maintenance of endogenous antigen-specific CD8 T cells. The self-reactive cells initially exhibited effector activity but eventually became unresponsive. Upon antigen removal, the antigen-specific population waned, resulting in development of a self-specific memory subset that recalled to subsequent challenge. In striking contrast to naïve CD8 T cells, preexisting antigen-specific memory CD8 T cells failed to expand after antigen induction and essentially ignored the antigen despite widespread expression by dendritic cells. The inclusion of inflammatory signals partially overcame memory CD8 T-cell ignorance of self-antigen. Thus, peripheral CD8 T-cell tolerance for naïve CD8 T cells depended on the continuous presence of antigen, whereas memory CD8 T cells were prohibited from autoreactivity in the absence of inflammation.

Cytokine control of memory T-cell development and survival.

Evidence has accumulated that cytokines have a fundamental role in the differentiation of memory T cells. Here, we follow the CD8+ T cell from initial activation to memory-cell generation, indicating the checkpoints at which cytokines determine the fate of the T cell. Members of the common cytokine-receptor gamma-chain (gammac)-cytokine family--in particular, interleukin-7 (IL-7) and IL-15--act at each stage of the immune response to promote proliferation and survival. In this manner, a stable and protective, long-lived memory CD8+ T-cell pool can be propagated and maintained.

Splenic priming of virus-specific CD8 T cells following influenza virus infection.

In healthy individuals, influenza virus (IAV) infection generally remains localized to the epithelial cells of the respiratory tract. Previously, IAV-specific effector CD8 T cells found systemically during the course of IAV infection were thought to have been primed in lung-draining lymph nodes with subsequent migration to other tissues. However, little is known about whether other lymphoid sites participate in the generation of virus-specific CD8 T cells during localized IAV infection. Here, we present evidence of early CD8 T cell priming in the spleen following respiratory IAV infection independent of lung-draining lymph node priming of T cells. Although we found early indications of CD8 T cell activation in the lymph nodes draining the respiratory tract, we also saw evidence of virus-specific CD8 T cell activation in the spleen. Furthermore, CD8 T cells primed in the spleen differentiated into memory cells of equivalent longevity and with similar recall capacity as CD8 T cells primed in the draining lymph nodes. These data showed that the spleen contributes to the virus-specific effector and memory CD8 T cell populations that are generated in response to respiratory infection.

Cutting edge: the role of IFN-α receptor and MyD88 signaling in induction of IL-15 expression in vivo.

IL-15 plays a multifaceted role in immune homeostasis, but the unreliability of IL-15 detection has stymied exploration of IL-15 regulation in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in innate cells including dendritic cells (DCs), macrophages, and monocytes. However, DC subsets expressed varying levels of EmGFP/IL-15 with CD8(+) DCs constitutively expressing EmGFP/IL-15 and CD8(-) DCs expressing low EmGFP/IL-15 levels. Virus infection resulted in IL-15 upregulation in both subsets. By crossing the transgenic mice to mice deficient in specific elements of innate signaling, we found a cell-intrinsic dependency of DCs and Ly6C(+) monocytes on IFN-α receptor expression for EmGFP/IL-15 upregulation after vesicular stomatitis virus infection. In contrast, myeloid cells did not require the expression of MyD88 to upregulate EmGFP/IL-15 expression. These findings provide evidence of previously unappreciated regulation of IL-15 expression in myeloid lineages during homeostasis and following infection.

Cutting edge: intravascular staining redefines lung CD8 T cell responses.

Nonlymphoid T cell populations control local infections and contribute to inflammatory diseases, thus driving efforts to understand the regulation of their migration, differentiation, and maintenance. Numerous observations indicate that T cell trafficking and differentiation within the lung are starkly different from what has been described in most nonlymphoid tissues, including intestine and skin. After systemic infection, we found that >95% of memory CD8 T cells isolated from mouse lung via standard methods were actually confined to the pulmonary vasculature, despite perfusion. A respiratory route of challenge increased virus-specific T cell localization within lung tissue, although only transiently. Removing blood-borne cells from analysis by the simple technique of intravascular staining revealed distinct phenotypic signatures and chemokine-dependent trafficking restricted to Ag-experienced T cells. These results precipitate a revised model for pulmonary T cell trafficking and differentiation and a re-evaluation of studies examining the contributions of pulmonary T cells to protection and disease.

Once a killer, always a killer: from cytotoxic T cell to memory cell.

The control of the differentiation pathways followed by responding CD8(+) T cells to produce protective memory cells has been intensely studied. Recent developments have identified heterogeneity at the effector cytotoxic T-lymphocyte level within which a bona fide memory cell precursor has emerged. The challenge now is to identify the cellular and molecular factors that control this developmental pathway. This review considers aspects of the regulation of the induction of effectors, the transition of effectors to memory cells, and the dynamics of the memory population.

CD11a regulates effector CD8 T cell differentiation and central memory development in response to infection with Listeria monocytogenes.

ß2 (CD18) integrins with α-chains CD11a, -b, -c, and -d are important adhesion molecules necessary for leukocyte migration and cellular interactions. CD18 deficiency leads to recurrent bacterial infections and poor wound healing due to reduced migration of leukocytes to inflammatory sites. CD8 T cells also upregulate CD11a, CD11b, and CD11c upon activation. However, the role these molecules play for CD8 T cells in vivo is not known. To determine the function of individual ß2 integrins, we examined CD8 T cell responses to Listeria monocytogenes infection in CD11a-, CD11b-, and CD11c-deficient mice. The absence of CD11b or CD11c had no effect on the generation of antigen-specific CD8 T cells. In contrast, the magnitude of the primary CD8 T cell response in CD11a-deficient mice was significantly reduced. Moreover, the response in CD11a(-/-) mice exhibited reduced differentiation of short-lived effector cells (KLRG1(hi) CD127(lo)), although cytokine and granzyme B production levels were unaffected. Notably, CD11a deficiency resulted in greatly enhanced generation of CD62L(+) central memory cells. Surprisingly, CD8 T cells lacking CD11a mounted a robust secondary response to infection. Taken together, these findings demonstrated that CD11a expression contributes to expansion and differentiation of primary CD8 T cells but may be dispensable for secondary responses to infection.