Marc K. Jenkins

Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo

An adoptive transfer system was used to monitor physically the behavior of a trace population of TCR transgenic T cells in vivo. After subcutaneous injection of antigen in adjuvant, the antigen-specific cells accumulated first in the paracortical region of the draining lymph nodes, proliferated there for several days, and then moved into lymph node follicles, where they accounted for most of the T cells. They then disappeared slowly from the draining nodes, and the remaining cells were hypersensitive to antigenic stimulation in vitro. In contrast, when the antigen was introduced into the blood, the antigen-specific cells rapidly accumulated in the paracortical regions of all lymph nodes, proliferated there for a short time, but never entered follicles. Most of the cells then rapidly disappeared, leaving behind a population that was hyporesponsive to antigenic stimulation. These results provide a physical basis for the classical finding that antigen-specific memory and tolerance can be influenced by the form of antigen administration.

Distinct Dendritic Cell Populations Sequentially Present Antigen to CD4 T Cells and Stimulate Different Aspects of Cell-Mediated Immunity

Peptide:MHC II complexes derived from a fluorescent antigen were detected in vivo to identify the cells that present subcutaneously injected antigen to CD4 T cells. Skin-derived dendritic cells (DCs) that acquired the antigen while in the draining lymph nodes were the first cells to display peptide:MHC II complexes. Presentation by these cells induced CD69, IL-2 production, and maximal proliferation by the T cells. Later, DCs displaying peptide:MHC II complexes migrated from the injection site via a G protein-dependent mechanism. Presentation by these migrants sustained expression of the IL-2 receptor and promoted delayed type hypersensitivity. Therefore, presentation of peptide:MHC II complexes derived from a subcutaneous antigen occurs in two temporally distinct waves with different functional consequences.

Antigen presentation to naive CD4 T cells in the lymph node.

Although the presentation of peptide–major histocompatibility complex class II (pMHC class II) complexes to CD4 T cells has been studied extensively in vitro, knowledge of this process in vivo is limited. Unlike the in vitro situation, antigen presentation in vivo takes place within a complex microenvironment in which the movements of antigens, antigen-presenting cells (APCs) and T cells are governed by anatomic constraints. Here we review developments in the areas of lymph node architecture, APC subsets and T cell activation that have shed light on how antigen presentation occurs in the lymph nodes.

Linked T Cell Receptor and Cytokine Signaling Govern the Development of the Regulatory T cell Repertoire

Appropriate development of regulatory T (Treg) cells is necessary to prevent autoimmunity. Neonatal mice, unlike adults, lack factors required for Treg cell development. It is unclear what these missing factors are. However, signals emanating from the T cell receptor (TCR), the costimulatory receptor CD28, and the family of gammac-dependent cytokine receptors are required for Treg cell development. Herein we demonstrate that expression of a constitutively active Stat5b transgene (Stat5b-CA) allowed for Treg cell development in neonatal mice and restored Treg cell numbers in Cd28(-/-) mice. Sequence analysis of TCR genes in Stat5b-CA Treg cells indicated that ectopic STAT5 activation resulted in a TCR repertoire that more closely resembled that of naive T cells. Using MHCII tetramers to identify antigen-specific T cells, we showed that STAT5 signals diverted thymocytes normally destined to become naive T cells into the Treg cell lineage. Our data support a two-step model of Treg cell differentiation in which TCR and CD28 signals induce cytokine responsiveness and STAT5-inducing cytokines then complete the program of Treg cell differentiation.

Different B cell populations mediate early and late memory during an endogenous immune response.

A cell enrichment technique reveals the dynamics of the endogenous memory B cell response. Memory B cells formed in response to microbial antigens provide immunity to later infections; however, the inability to detect rare endogenous antigen-specific cells limits current understanding of this process. Using an antigen-based technique to enrich these cells, we found that immunization with a model protein generated B memory cells that expressed isotype-switched immunoglobulins (swIg) or retained IgM. The more numerous IgM+ cells were longer lived than the swIg+ cells. However, swIg+ memory cells dominated the secondary response because of the capacity to become activated in the presence of neutralizing serum immunoglobulin. Thus, we propose that memory relies on swIg+ cells until they disappear and serum immunoglobulin falls to a low level, in which case memory resides with durable IgM+ reserves.

Normalizing the environment recapitulates adult human immune traits in laboratory mice.

Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside. Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice—like newborn, but not adult, humans—lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.Our current understanding of immunology was largely defined in laboratory mice because of experimental advantages including inbred homogeneity, tools for genetic manipulation, the ability to perform kinetic tissue analyses starting with the onset of disease, and tractable models. Comparably reductionist experiments are neither technically nor ethically possible in humans. Despite revealing many fundamental principals of immunology, there is growing concern that mice fail to capture relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside1–8. Laboratory mice live in abnormally hygienic “specific pathogen free” (SPF) barrier facilities. Here we show that the standard practice of laboratory mouse husbandry has profound effects on the immune system and that environmental changes result in better recapitulation of features of adult humans. Laboratory mice lack effector-differentiated and mucosally distributed memory T cells, which more closely resembles neonatal than adult humans. These cell populations were present in free-living barn populations of feral mice, pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting a role for environment. Consequences of altering mouse housing profoundly impacted the cellular composition of the innate and adaptive immune system and resulted in global changes in blood cell gene expression patterns that more closely aligned with immune signatures of adult humans rather than neonates, altered the mouse’s resistance to infection, and impacted T cell differentiation to a de novo viral infection. These data highlight the impact of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modeling immunological events in free-living organisms, including humans.

Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cells for the study of T-cell activation in vivo

Summary: Adoptive transfer of TCR‐transgenic T cells uniformly expressing an identifiable TCR of known peptide/MHC specificity can be used to monitor the in vivo behavior of antigen‐specific T cells. We have used this system to show that naive T cells are initially activated within the T‐cell zones of secondary lymphoid tissue to prohferate in a B7‐dependent manner. If adjuvants or inflammatory cytokines are present during this period, enhanced numbers of T cells accumulate, migrate into B‐cell‐rich follicles, and acquire the capacity to produce IFN‐7 and help B cells produce IgG2a. If inflammation is absent, most of the initially activated antigen‐specific T cells disappear without entering the follicles and the survivors are poor producers of IL‐2 and IFN‐γ Our results indicate that inflammatory mediators play a key role in regulating the anatomic location, clonal expansion, survival and lymphokine production potential of antigen‐stimulated T cells in vivo.

Characterization of CD4+ T Cell Responses During Natural Infection with Salmonella typhimurium

CD4+ T cells are important for resistance to infection with Salmonella typhimurium. However, the Ag specificity of this T cell response is unknown. Here, we demonstrate that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect naive mice from lethal Salmonella infection. To characterize this Ag-specific response further, we generated FliC-specific CD4+ T cell clones from mice that had resolved infection with an attenuated strain of Salmonella. These clones were found to respond to an epitope from a constant region of FliC, enabling them to cross-react with flagellar proteins expressed by a number of distinct Salmonella serovars.

Different routes of bacterial infection induce long-lived T H 1 memory cells and short-lived T H 17 cells

We used a sensitive method based on tetramers of peptide and major histocompatibility complex II (pMHCII) to determine whether CD4+ memory T cells resemble the T helper type 1 (TH1) and interleukin 17 (IL-17)-producing T helper (TH17) subsets described in vitro. Intravenous or intranasal infection with Listeria monocytogenes induced pMHCII-specific CD4+ naive T cells to proliferate and produce effector cells, about 10% of which resembled TH1 or TH17 cells, respectively. TH1 cells were also present among the memory cells that survived 3 months after infection, whereas TH17 cells disappeared. The short lifespan of TH17 cells was associated with small amounts of the antiapoptotic protein Bcl-2, the IL-15 receptor and the receptor CD27, and little homeostatic proliferation. These results suggest that TH1 cells induced by intravenous infection are more efficient at entering the memory pool than are TH17 cells induced by intranasal infection.

Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.

A naive CD4(+) T cell population specific for a microbial peptide:major histocompatibility complex II ligand (p:MHCII) typically consists of about 100 cells, each with a different T cell receptor (TCR). Following infection, this population produces a consistent ratio of effector cells that activate microbicidal functions of macrophages or help B cells make antibodies. We studied the mechanism that underlies this division of labor by tracking the progeny of single naive T cells. Different naive cells produced distinct ratios of macrophage and B cell helpers but yielded the characteristic ratio when averaged together. The effector cell pattern produced by a given naive cell correlated with the TCR-p:MHCII dwell time or the amount of p:MHCII. Thus, the consistent production of effector cell subsets by a polyclonal population of naive cells results from averaging the diverse behaviors of individual clones, which are instructed in part by the strength of TCR signaling.