Transient T cell accumulation in lymph nodes and sustained lymphopenia in mice treated with FTY720

FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) is an orally available immunomodulatory agent that induces severe peripheral blood lymphopenia. Most studies of these lymphopenic effects have been limited to short-term exposure to FTY720. FTY720 alters the ability of lymphocytes to respond to sphingosine-1-phosphate (S1P) through S1P receptors, particularly S1P1. FTY720 affects different leukocyte populations and their trafficking through major lymphoid organs. We show the dynamics of CD4 T, CD8 T, and B lymphocyte recirculation in all major lymphoid compartments during 21-day FTY720 treatment of normal C57BL/6 mice. Following a transient increase in peripheral lymph nodes and Peyer's patches, lymphocyte recirculation reaches a new steady state. Other lymphoid organs show transient changes in lymphocyte composition with various patterns. At 21 days of FTY720 treatment, total body lymphocyte content is reduced by 20% and blood lymphocytes by 80%. Modeling suggests that the new steady state is due to a combination of reduced naive lymphocyte release from the thymus and a transient reduction of lymphocyte egress from lymph nodes. Our data indicate that the commonly held belief that FTY720 blocks lymphocyte egress from lymph nodes cannot fully explain the lymphocyte dynamics observed with prolonged treatment.     

Retention of Ag‐specific memory CD4+ T cells in the draining lymph node indicates lymphoid tissue resident memory populations

Several different memory T‐cell populations have now been described based upon surface receptor expression and migratory capabilities. Here we have assessed murine endogenous memory CD4⁺ T cells generated within a draining lymph node and their subsequent migration to other secondary lymphoid tissues. Having established a model response targeting a specific peripheral lymph node, we temporally labelled all the cells within draining lymph node using photoconversion. Tracking of photoconverted and non‐photoconverted Ag‐specific CD4⁺ T cells revealed the rapid establishment of a circulating memory population in all lymph nodes within days of immunisation. Strikingly, a resident memory CD4⁺ T cell population became established in the draining lymph node and persisted for several months in the absence of detectable migration to other lymphoid tissue. These cells most closely resembled effector memory T cells, usually associated with circulation through non‐lymphoid tissue, but here, these cells were retained in the draining lymph node. These data indicate that lymphoid tissue resident memory CD4⁺ T‐cell populations are generated in peripheral lymph nodes following immunisation.     

Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus

B1 cells have different origin and function from conventional B (B2) cells and are considered to be involved in autoantibody production in the development of autoimmune disease. We found that B1 cells preferentially accumulated in the target organs including thymus in aged BWF1 mice, a murine model for systemic lupus erythematosus, and that B lymphocyte chemoattractant (BLC/CXCL13) expression was increased in the thymus before the onset of lupus nephritis, while stromal cell-derived factor-1 (SDF-1/CXCL12) and secondary lymphoid tissue chemokine (SLC/CCL21) expression remained unchanged. Adhesion molecules such as peripheral node addressin (PNAd), ICAM-1, and VCAM-1 were also expressed on endothelial cells in the enlarged thymic perivascular space (PVS) in aged BWF1 mice. BLC protein and PNAd were co-localized on these high-endothelial-venules-like vessels in enlarged PVS. B1 cells expressed higher level of costimulatory molecules and showed a potent antigen-presenting activity in allogeneic mixed lymphocyte reaction comparable to splenic dendritic cells. Interestingly, B1 cells stimulated proliferation of autologous thymic CD4 T cells in the presence of IL-2. These results indicate that aberrant B1 cell trafficking into the thymus due to ectopic high expression of BLC may result in an activation of self-reactive T cells in the development of murine lupus.     

CD8 alpha is an activation marker for a subset of peripheral CD4 T cells

Rat CD4 T lymphocytes express CD8 alpha upon activation. Here, we show that double-positive cells express CD8 alpha alpha homodimers, and we study their phenotype and function. Most activated CD4(+) lymphocytes expressing CD8 alpha are recent thymic emigrants. Accordingly, most activated CD4 single-positive thymocytes express CD8 alpha, and thymectomy and aging decrease the frequency of CD4(+)CD8 alpha(+) lymphocytes. However, CD8 induction is not restricted to CD4(+) recent thymic emigrants. CD4(+)CD8 alpha(+) and CD4(+)CD8 alpha(-)cells were generated in vitro from naive or from primed donors and, to study their function, were transferred to normal rats. Both cell types helped primary humoral responses, but only CD4(+)CD8 alpha(-) cells promoted secondary responses. Thus, memory CD4 T cells mediating antibody responses and some naive CD4(+) lymphocytes do not express CD8 alpha. In addition, CD4(+)CD8 alpha(+) cells produce mainly Th1 cytokines while CD4(+)CD8 alpha(-) cells produce IL-10 and showed a sustained proliferative response. Hence, CD8 alpha expression after activation distinguishes two distinct CD4 T cell subsets.     

Intestinal double-positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines

Peripheral blood and intestinal CD4+CD8+ double-positive (DP) T cells have been described in several species including humans, but their function and immunophenotypic characteristics are still not clearly understood. Here we demonstrate that DP T cells are abundant in the intestinal lamina propria of normal rhesus macaques (Macaca mulatta). Moreover, DP T cells have a memory phenotype and are capable of producing different and/or higher levels of cytokines and chemokines in response to mitogen stimulation compared to CD4+ single-positive T cells. Intestinal DP T cells are also highly activated and have higher expression of CCR5, which makes them preferred targets for simian immunodeficiency virus/HIV infection. Increased levels of CD69, CD25 and HLA-DR, and lower CD62L expression were found on intestinal DP T cells populations compared to CD4+ single-positive T cells. Collectively, these findings demonstrate that intestinal and peripheral blood DP T cells are effector cells and may be important in regulating immune responses, which distinguishes them from the immature DP cells found in the thymus. Finally, these intestinal DP T cells may be important target cells for HIV infection and replication due to their activation, memory phenotype and high expression of CCR5.     

Demonstration of strong enterobacterial reactivity of CD4+CD25- T cells from conventional and germ-free mice which is counter-regulated by CD4+CD25+ T cells

Unfractionated CD4+ T cells from the gut-associated lymphoid tissue (GALT) and peripheral lymph nodes are unresponsive when exposed to enterobacterial antigens in vitro. Under similar conditions, CD4+ T cells depleted in vivo or in vitro of CD4+CD25+ T cells proliferate extensively. The CD4+CD25- T cell reactivity depends on MHC class II presentation, specific TCR stimulation, CD4 ligation, and antigen processing by antigen-presenting cells. The CD4+CD25- T cells respond to autologous and heterologous enterobacterial antigens, but not to antigens from the feces of germ-free mice. Surprisingly, CD4+CD25- T cells obtained from the GALT of germ-free mice also proliferate when exposed to enterobacterial antigens, and adding back the conventional or germ-free CD4+CD25+ T cells to the enteroantigen-stimulated CD4+CD25- T cells abolishes proliferation. As judged from carboxyfluorescein diacetate succinimidyl ester-labeling experiments, 4-5% of the CD4+CD25- T cells respond to enteroantigen. The data show for the first time that CD4+CD25- T cells with reactivity towards the enterobacterial flora and regulatory CD4+CD25- T cells are present in both conventional and germ-free mice. The data suggest that a significant proportion of the peripheral pool of CD4+CD25- T cells express anti-enterobacterial reactivity, which, due to the presence of regulatory CD4+CD25+ T cells, is kept in a quiescent state.     

Polymerase mu is up-regulated during the T cell-dependent immune response and its deficiency alters developmental dynamics of spleen centroblasts

Mammalian DNA polymerase mu (Polmu), preferentially expressed in secondary lymphoid organs, is shown here to be up-regulated in germinal centers after immunization. Alternative splicing appears to be part of Polmu regulation during an immune response. We generated Polmu-deficient mice that are viable and show no anatomical malformation or serious alteration in lymphoid populations, with the exception of an underrepresentation of the B cell compartment. Young and aged homozygous Polmu(-/-) mice generated similar immune responses after immunization with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gammaglobulin (CGG), compared with their wild-type littermates. Nonetheless, the kinetics of development of the centroblast population showed significant differences. Hypermutation analysis of the rearranged heavy chain intron region in centroblasts isolated from NP-CGG-immunized Polmu(-/-) mice showed a similar quantitative and qualitative somatic mutation spectrum, but a lower representation of heavily mutated clones. These results suggest that although it is not a critical partner, Polmu modulates the in vivo somatic hypermutation process.     

A very rapid and simple assay based on trogocytosis to detect and measure specific T and B cell reactivity by flow cytometry

Detection, quantification, separation and characterization of T and B cells reactive to specific antigens are important tasks in both basic and clinical immunology. Here, we describe an approach allowing the performance of all four tasks on a functional basis by flow cytometry. The assay is based on the property of lymphocytes to capture membrane components from the cells they interact with, in a process we call trogocytosis. Working with CD8+ CTL and target cells labeled with membrane markers, we describe the conditions allowing reactive lymphocytes to be detected rapidly and inexpensively within mixed populations. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this method to the detection of antigen-specific CD4+ T and B cells. While our method is, for the time being, not as sensitive as staining of CTL with MHC class I multimers, it allows the simultaneous quantitative identification of reactive CD8+, CD4+ and B cells. Altogether, our method offers a simple and general alternative to other methods previously described to detect and quantify lymphocyte reactivity, and it can also be used in combination with those.     

Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation

Multi-color flow cytometric analysis on human CD8(+) T cell subsets revealed that CXCR4 is predominantly expressed on CD8(+) T cells with the naive CD27(+)CD28(+)CD45RA(+) phenotype, and is down-regulated during differentiation into those with an effector phenotype. The down-regulation of CXCR4 expression during peripheral differentiation was supported by the fact that the expression of CXCR4 on CD8(+) T cells was negatively correlated with that of perforin. The analysis of CCR5, CCR7, and CXCR4 co-expression further showed that CD8(+) T cells expressing a high level of CXCR4 are CCR7(+)CCR5(-) naive or central memory subsets, and those expressing a low level of CXCR4 were included in the CCR7(-)CCR5(+/-) memory/effector and effector subsets. Epstein Barr virus-specific CD8(+) T cells, which mostly express the memory phenotype, expressed CXCR4, while human cytomegalovirus-specific CD8(+) T cells, which mostly express the effector phenotype, partially expressed this receptor, showing that the expression of CXCR4 is also down-regulated during differentiation of viral antigen-specific CD8(+) T cells. The classification of human CD8(+) T cells based on the expression of these chemokine receptors should prove useful for studies that clarify the differentiation of human CD8(+) T cells.     

Maintenance of memory CTL responses by T helper cells and CD40-CD40 ligand: antibodies provide the key

Cytotoxic T lymphocytes (CTL) are essential for control of primary infections by many pathogens and in particular by non-cytopathic viruses. It has been proposed that long-term maintenance of CTL memory and control of lymphocytic choriomeningitis virus (LCMV) is dependent upon the presence of T helper cells and interaction of antigen-presenting cells and CTL via CD40 and its ligand CD40L. However, we demonstrate here that CD40-CD40L interaction maintains CTL memory by induction of virus-specific antibodies. In fact, loss of CTL memory responses and spread of virus in mice lacking CD40 or its ligand is prevented by repetitive therapeutic injections of LCMV-specific antibodies. This indicates that antibodies are essential for long-term control of non-cytopathic virus and to maintain protective memory. Transfer of neutralizing antibodies or induction of antibodies by therapeutic vaccination within weeks after infection may therefore prove beneficial for the treatment of chronic virus infections such as HIV, hepatitis B, and hepatitis C. See accompanying article http://dx.doi.org/10.1002/eji.200324844     

CXCR3 is required for migration to dermal inflammation by normal and in vivo activated T cells: differential requirements by CD4 and CD8 memory subsets

Lymphocytes in inflamed tissues express numerous chemokine receptors. The relative importance of these receptors for migration in inflammation is unclear. The role of CXCR3 in T cell subset migration was examined using monoclonal antibodies developed to rat CXCR3. CXCR3 was expressed on sixfold more CD8(+) ( approximately 30%) than CD4(+) ( approximately 5%) T cells in spleen, lymph nodes and blood, and on approximately 10% of CD4(+)CD45RC(-) (memory) and approximately 50% of CD8(+)CD45RC(+) spleen T cells. After immunization, CXCR3 increased tenfold on CD4(+) lymph node lymphoblasts ( approximately 55%), and >90% of inflammatory exudate T cells were CXCR3(+). CXCR3(+) T cells migrated significantly better than CXCR3(-) T cells to all dermal inflammatory stimuli tested in vivo, even though these T cells are a minority of the memory T cells. Blocking CXCR3 inhibited recruitment of 60-85% of unstimulated T cells and up to 90% of CD8(+)CD45RC(+) effector T cells, but caused <50% inhibition of CD4(+) and CD8(+) memory (CD45RC(-)) T cells. About 90% of T lymphoblast migration to IFN-gamma, IFN-gamma plus TNF-alpha, polyinosinic polycytidylic acid, lipopolysaccharide, and delayed-type hypersensitivity (DTH)-induced inflammation was inhibited. Blockade also reduced DTH-induced induration. Thus, CXCR3 has a non-redundant role in T cell migration to dermal inflammation and is critical for activated T lymphoblast recruitment, but memory T cells are less dependent on CXCR3 for their infiltration.     

Identification of a CD4+CD25+ T cell subset committed in vivo to suppress antigen-specific T cell responses without additional stimulation

Naturally occurring CD4+ regulatory T cells can be identified on the basis of expression of CD25 and suppression of T cell responses in vitro after TCR triggering. Here, we demonstrate that a CD134+ subset of CD4+CD25+ T cells in naive rats suppresses antigen-specific T cell responses in vitro without additional TCR stimulation. In contrast, CD4+CD25+CD134- regulatory T cells and total CD4+CD25+ regulatory T cells have suppressive activity only during simultaneous activation of responder and regulatory T cells or after in vitro pre-activation. Furthermore CD4+CD25+CD134+ T cells have a more activated phenotype than CD4+CD25+CD134- T cells, as based on the expression of CD62L, CD45RC, and MHC class II. We propose that the CD134+ regulatory T cells contain an in vivo activated and highly suppressive regulatory T cell subset. CD4+CD25+CD134+ T cells can be found in several compartments of the immune system, including spleen, lymph nodes, and blood. Interestingly though, the relative amounts of these cells within the CD4+ population and their CD134 expression levels are highest in mucosa-draining lymph nodes and lowest in blood. This suggests that the presence of CD4+CD25+CD134+ T cells indicates sites of active immune suppression.     

CD4+ CD25+ regulatory T cells control the magnitude of T-dependent humoral immune responses to exogenous antigens

CD4+ CD25+ T reg cells are critical for peripheral tolerance and prevention of autoimmunity. Here we show that CD4+ CD25+ T reg also regulate the magnitude of humoral responses against a panel of T-dependent antigens of foreign origin during both primary and secondary immune responses. Depletion of CD4+ CD25+ T cells leads to increased antigen-specific antibody production and affinity maturation but does not affect T-independent B cell responses, suggesting that CD4+ CD25+ T reg exert a feedback mechanism on non-self antigen-specific antibody secretion by dampening the T cell help for B cell activation. Moreover, we show that CD4+ CD25+ T reg also suppress in vitro B cell immunoglobulin production by inhibiting CD4+ CD25- T cell help delivery, and that blockade of TGF-beta activity abolishes this suppression.     

ABCB1 transporter discriminates human resting naive B cells from cycling transitional and memory B cells

The exact identification of B cell subsets is instrumental to understand their dynamics under physiological and pathological conditions. Human memory B cells are currently identified according to the expression of CD27, which is absent on naive B cells. We report here that the ATP-binding cassette (ABC)B1 transporter is exclusively present on mature CD27- naive B cells, while it is absent in CD27+ memory B cells and in a heterogeneous subset of CD27- cells that comprise both switch memory and transitional B cells. Thus, ABCB1 activity precisely discriminates naive from transitional and all memory B cells. Using this improved method to discriminate human B cell subsets, and Ki67 staining to identify recently divided cells, we show that in both cord blood and adult peripheral blood, mature naive B cells are quiescent while transitional B cells and memory B cells have a high in vivo turnover.     

B cells can prime naive CD4+ T cells in vivo in the absence of other professional antigen-presenting cells in a CD154-CD40-dependent manner

The role of B cells as APC is well established. However, their ability to prime naive T cells in vivo has been difficult to examine because of the presence of dendritic cells. The current studies were undertaken to examine this issue in a model of adoptive transfer of antigen-specific B cells and T cells into histoincompatible Rag2(-/-) mice. By means of this system, we were able to demonstrate that antigen-specific B cells are competent APC for naive CD4(+) T cells specific for the same antigen. In vivo antigen presentation resulted in expansion of both CD4(+) T cells and B cells. The antigen-presenting function of the transferred B cells was dependent on the CD154-CD40 interaction, as transfer of CD154-deficient antigen-specific CD4(+) T cells or CD40-deficient B cells failed to induce T and B cell expansion in response to immunization. These results indicate that antigen-specific B cells have the capacity to induce primary T cell responses in the absence of other competent APC.     

The fate of heterologous CD4+ T cells during Leishmania donovani infection

Little is currently understood about the consequences of chronic parasitic infection for the fate of memory CD4+ T cells that recognize heterologous antigens, e.g. resulting from prior infections or vaccination. Here, we address how Leishmania donovani infection affected the fate of non-cross-reactive (OVA)-specific memory CD4+ T cells. DO11 cells were adoptively transferred into naive recipient mice, which were then immunized to generate memory DO11 cells. After 6 weeks, mice were infected with L. donovani and the fate of DO11 cells was determined. L. donovani infection stimulated an approximately threefold expansion in the total number of CD4+ T cells and DO11 cells, compared to that observed in uninfected mice. DO11 T cells were more actively dividing in infected mice, as judged by 5-bromo-2' deoxyuridine labeling, whereas their rate of apoptosis in control and infected mice was identical. Both CD45RBhiCD44lo naive T cells and to a greater extent CD45RBloCD44hi memory DO11 cells increased in number in the spleens of infected mice, whereas no changes occurred to DO11 cell number or phenotype in the draining lymph nodes. These data indicate that heterologous CD4+ T cells may actively divide during chronic infectious diseases, with important implications for how chronic infection may impact on heterologous immunity.     

Phenotypic heterogeneity of antigen-specific CD4 T cells under different conditions of antigen persistence and antigen load

The factors responsible for the phenotypic heterogeneity of memory CD4 T cells are unclear. In the present study, we have identified a third population of memory CD4 T cells characterized as CD45RA(+)CCR7(-) that, based on its replication history and the homeostatic proliferative capacity, was at an advanced stage of differentiation. Three different phenotypic patterns of memory CD4 T cell responses were delineated under different conditions of antigen (Ag) persistence and load using CD45RA and CCR7 as markers of memory T cells. Mono-phenotypic CD45RA(-)CCR7(+) or CD45RA(-)CCR7(-) CD4 T cell responses were associated with conditions of Ag clearance (tetanus toxoid-specific CD4 T cell response) or Ag persistence and high load (chronic HIV-1 and primary CMV infections), respectively. Multi-phenotypic CD45RA(-)CCR7(+), CD45RA(-)CCR7(-) and CD45RA(+)CCR7(-) CD4 T cell responses were associated with protracted Ag exposure and low load (chronic CMV, EBV and HSV infections and HIV-1 infection in long-term nonprogressors). The mono-phenotypic CD45RA(-)CCR7(+) response was typical of central memory (T(CM)) IL-2-secreting CD4 T cells, the mono-phenotypic CD45RA(-)CCR7(-) response of effector memory (T(EM)) IFN-gamma-secreting CD4 T cells and the multi-phenotypic response of both IL-2- and IFN-gamma-secreting cells. The present results indicate that the heterogeneity of different Ag-specific CD4 T cell responses is regulated by Ag exposure and Ag load.     

Naive and memory B cells respond differentially to T-dependent signaling but display an equal potential for differentiation toward the centroblast-restricted CD77/globotriaosylceramide phenotype

Resting (CD38(low)) tonsillar B cells differentiate to express the centroblast-restricted CD77/globotriaosylceramide antigen on high-level engagement of CD154. As the CD38(low) population comprises both naive and memory subsets, we wished to compare the propensity of each to develop this germinal center phenotype; particularly as the capacity of memory B cells to re-enter afollicular reaction remains unclear. Resting B lymphocytes were therefore separated into CD27(-)IgA(-)IgG(-) and IgD(-) fractions to generate subsets enriched for naive and memory cells, respectively. Following stimulation via BCR and/or CD40 - surrogate signals for B cells engaged in T-dependent signaling - differences between the two subsets were seen in the kinetics and/or magnitude of responses such as entry into DNA synthesis, induction of the costimulatory molecules CD80 and CD86; up-regulation of CD23, and changes in BCL-6 mRNA expression. Nevertheless, naive and memory cells revealed a nigh identical capacity for acquiring CD77: both appeared equally sensitive in this regard, with high-level CD40 engagement via cell-bound CD154 being required for both subsets to achieve the hallmark centroblast phenotype. These findings suggest that, provided with the opportunity to encounter cell membrane CD154 in abundance, both naive and memory B cells display the potential to be diverted towards a germinal center pathway of differentiation.     

Bone marrow-derived cells expand memory CD8+ T cells in response to viral infections of the lung and skin

While naive CD8(+) T cells have been shown to require bone marrow-derived dendritic cells (DC) to initiate immunity, such a requirement for memory CD8(+) T cells has had limited assessment. By generating bone marrow chimeras that express the appropriate antigen-presenting molecules on either radiation-sensitive bone marrow-derived or radiation-resistant non-bone marrow-derived compartments, we showed that both primary and secondary immune responses to influenza virus infection of the lung were initiated in the draining LN. This required cells of bone marrow origin, most likely DC, for optimal expansion within the secondary lymphoid compartment. This was similarly the case with HSV-1 infection of the skin. As Langerhans cells are radioresistant, unlike other DC populations, these studies also demonstrate that the radiosensitive DC responsible for secondary expansion of HSV-specific memory are not Langerhans cells.     

Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll-like receptor 9 ligand

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 T cells in culture. The CD8+ DC showed a reduced capacity to stimulate T cell proliferation compared to either of the CD8- DC subsets, regardless of antigen and DC dose. In contrast to CD8- DC, the quiescent CD8+ DC did not induce IFN-gamma production from CD4 T cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC class II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll-like receptor 9 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 T cell proliferation and IFN-gamma production. In contrast, similar activation of CD8- DC produced a more modest increase in capacity to stimulate CD4 T cell proliferation and no increase in capacity to stimulate IFN-gamma production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8- DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.