Selective resistance of CD44hi T cells to p53-dependent cell death results in persistence of immunologic memory after total body irradiation

Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation markedly changes the balance of residual T cell subsets to favor CD4(+)CD44(hi) NKT cells because of the differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results showed that CD4(+) T cells were markedly more resistant than CD8(+) T cells, and CD44(hi) T cells, including NKT cells and memory T cells, were markedly more resistant than CD44(lo) (naive) T cells. The memory T cells immunized to alloantigens persisted even after myeloablative (1000 cGy) TBI and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1000 cGy was prevented in p53(-/-) mice, there was progressive T cell death in p53(-/-) mice at higher doses. Although p53-dependent T cell death changed the balance of subsets, p53-independent T cell death did not. In conclusion, resistance of CD44(hi) T cells to p53-dependent cell death results in the persistence of immunological memory after TBI and can explain the immune-mediated rejection of marrow transplants in sensitized recipients.     

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.     

CD4+ T cells mediate abscess formation in intra-abdominal sepsis by an IL-17-dependent mechanism

Abscess formation associated with intra-abdominal sepsis causes severe morbidity and can be fatal. Previous studies have implicated T cells in the pathogenesis of abscess formation, and we have recently shown that CD4(+) T cells activated in vitro by zwitterionic capsular polysaccharides from abscess-inducing bacteria such as Staphylococcus aureus and Bacteroides fragilis initiate this host response when transferred to naive rats. In this study, we show that mice deficient in alphabetaTCR-bearing T cells or CD4(+) T cells fail to develop abscesses following challenge with B. fragilis or abscess-inducing zwitterionic polysaccharides, compared with CD8(-/-) or wild-type animals. Transfer of CD4(+) T cells from wild-type mice to alphabetaTCR(-/-) animals reconstituted this ability. The induction of abscesses required T cell costimulation via the CD28-B7 pathway, and T cell transfer experiments with STAT4(-/-) and STAT6(-/-) mice demonstrated that this host response is dependent on STAT4 signaling. Significantly higher levels of IL-17, a proinflammatory cytokine produced almost exclusively by activated CD4(+) T cells, were associated with abscess formation in Th2-impaired (STAT6(-/-)) mice, while STAT4(-/-) mice had significantly lower levels of this cytokine than control animals. The formation of abscesses was preceded by an increase in the number of activated CD4(+) T cells in the peritoneal cavity 24 h following bacterial challenge. Confocal laser-scanning microscopy analysis revealed that CD4(+) T cells comprise the abscess wall in these animals and produce IL-17 at this site. Administration of a neutralizing Ab specific for IL-17 prevented abscess formation following bacterial challenge in mice. These data delineate the specific T cell response necessary for the development of intra-abdominal abscesses and underscore the role of IL-17 in this disease process.     

CD4+CD25+ regulatory T cells control innate immune reactivity after injury

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1(-/-)) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-alpha, IL-1beta, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1(-/-), CD4(-/-), or CD8(-/-) mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8(-/-) mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4(-/-) mice produced cytokines at significantly higher levels, equivalent to those in Rag1(-/-) mice. Moreover, reconstitution of Rag1(-/-) or CD4(-/-) mice with WT CD4(+) T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4(+) T cells into CD4(+)CD25(+) and CD4(+)CD25(-) subpopulations before their adoptive transfer into Rag1(-/-) mice showed that CD4(+)CD25(+) T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4(+)CD25(-) T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses after injury.     

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.     

Vgamma4(+) T cells promote autoimmune CD8(+) cytolytic T-lymphocyte activation in coxsackievirus B3-induced myocarditis in mice: role for CD4(+) Th1 cells

T cells expressing the Vgamma4 T-cell receptor (TCR) promote myocarditis in coxsackievirus B3 (CVB3)-infected BALB/c mice. CD1, a major histocompatibility complex (MHC) class I-like molecule, is required for activation of Vgamma4(+) cells. Once activated, Vgamma4(+) cells initiate myocarditis through gamma interferon (IFN-gamma)-mediated induction of CD4(+) T helper type 1 (Th1) cells in the infected animal. These CD4(+) Th1 cells are required for activation of an autoimmune CD8(+) alphabeta TCR(+) effector, which is the predominant pathogenic agent in this model of CVB3-induced myocarditis. Activated Vgamma4(+) cells can adoptively transfer myocarditis into BALB/c mice infected with a nonmyocarditic variant of CVB3 (H310A1) but cannot transfer myocarditis into either uninfected or CD1(-/-) recipients, demonstrating the need for both infection and CD1 expression for Vgamma4(+) cell function. In contrast, CD8(+) alphabeta TCR(+) cells transfer myocarditis into either infected CD1(-/-) or uninfected recipients, showing that once activated, the CD8(+) alphabeta TCR(+) effectors function independently of both virus and CD1. Vgamma4(+) cells given to mice lacking CD4(+) T cells minimally activate the CD8(+) alphabeta TCR(+) cells. These studies show that Vgamma4(+) cells determine CVB3 pathogenicity by their ability to influence both the CD4(+) and CD8(+) adaptive immune response. Vgamma4(+) cells enhance CD4(+) Th1 (IFN-gamma(+)) cell activation through IFN-gamma- and CD1-dependent mechanisms. CD4(+) Th1 cells promote activation of the autoimmune CD8(+) alphabeta TCR(+) effectors.     

Differential expression of a 70 kDa O-glycoprotein on T cells: a possible marker for naive and early activated murine T cells

We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.     

Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria

Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4(+) T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44(hi) memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure.     

PD-1 protects against inflammation and myocyte damage in T cell-mediated myocarditis

PD-1, a member of the CD28 family of immune regulatory molecules, is expressed on activated T cells, interacts with its ligands, PD-L1/B7-H1 and PD-L2/B7-DC, on other cells, and delivers inhibitory signals to the T cell. We studied the role of this pathway in modulating autoreactive T cell responses in two models of myocarditis. In a CD8(+) T cell-mediated adoptive transfer model, we found that compared with Pd1(+/+) CD8(+) T cells, Pd1(-/-) CD8(+) T cells cause enhanced disease, with increased inflammatory infiltrate, particularly rich in neutrophils. Additionally, we show enhanced proliferation in vivo and enhanced cytotoxic activity of PD-1-deficient T lymphocytes against myocardial endothelial cells in vitro. In experimental autoimmune myocarditis, a disease model dependent on CD4(+) T cells, we show that mice lacking PD-1 develop enhanced disease compared with wild-type mice. PD-1-deficient mice displayed increased inflammation, enhanced serum markers of myocardial damage, and an increased infiltration of inflammatory cells, including CD8(+) T cells. Together, these studies show that PD-1 plays an important role in limiting T cell responses in the heart.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

Contribution of T cell subsets to the pathophysiology of Pneumocystis-related immunorestitution disease

Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4(+) and CD8(+) T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4(+) cells were more abundant than CD8(+) cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8(+) cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4(+) T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8(+) T cell response was observed. In contrast, mice depleted of CD8(+) T cells efficiently cleared the infection but developed more severe disease, an increased frequency of IFN-gamma-producing CD4(+) cells, and a prolonged CD4(+) T cell response than mice with both CD4(+) and CD8(+) cells. These data suggest that CD4(+) T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8(+) T cells contributed to neither lung injury nor organism clearance when CD4(+) cells were present, but instead served to modulate CD4 function. In the absence of CD4(+) cells, CD8(+) T cells produced a nonprotective, pathological immune response. These data suggest that the interplay of CD4(+) and CD8(+) T cells affects the ultimate outcome of PcP-related IRD.     

Alloreactive memory T cells are responsible for the persistence of graft-versus-host disease

Graft-vs-host disease (GVHD) is caused by a donor T cell anti-host reaction that evolves over several weeks to months, suggesting a requirement for persistent alloreactive T cells. Using the C3H.SW anti-C57BL/6 (B6) mouse model of human GVHD directed against minor histocompatibility Ags, we found that donor CD8(+) T cells secreting high levels of IFN-gamma in GVHD B6 mice receiving C3H.SW naive CD8(+) T cells peaked by day 14, declined by day 28 after transplantation, and persisted thereafter, corresponding to the kinetics of a memory T cell response. Donor CD8(+) T cells recovered on day 42 after allogeneic bone marrow transplantation expressed the phenotype of CD44(high)CD122(high)CD25(low), were able to homeostatically survive in response to IL-2, IL-7, and IL-15 and rapidly proliferated upon restimulation with host dendritic cells. Both allogeneic effector memory (CD44(high)CD62L(low)) and central memory (CD44(high)CD62L(high)) CD8(+) T cells were identified in B6 mice with ongoing GVHD, with effector memory CD8(+) T cells as the dominant (>80%) population. Administration of these allogeneic memory CD8(+) T cells into secondary B6 recipients caused virulent GVHD. A similar allogeneic memory CD4(+) T cell population with the ability to mediate persistent GVHD was also identified in BALB/b mice receiving minor histocompatibility Ag-mismatched B6 T cell-replete bone marrow transplantation. These results indicate that allogeneic memory T cells are generated in vivo during GVH reactions and are able to cause GVHD, resulting in persistent host tissue injury. Thus, in vivo blockade of both alloreactive effector and memory T cell-mediated host tissue injury may prove to be valuable for GVHD prevention and treatment.     

Increased protection from vaccinia virus infection in mice genetically prone to lymphoproliferative disorders

Mutations in the genes that encode Fas or Fas ligand (FasL) can result in poor restraints on lymphocyte activation and in increased susceptibility to autoimmune disorders. Because these mutations portend a continuously activated immune state, we hypothesized that they might in some cases confer resistance to infection. To examine this possibility, the immune response to, morbidity caused by, and clearance of vaccinia virus (VACV) Western Reserve was examined in 5- to 7-week-old Fas mutant (lpr) mice, before an overt lymphoproliferative disorder was observable. On day 6 after VACV infection, C57BL/6-lpr (B6-lpr) mice had decreased morbidity, decreased viral titers, and an increased percentage and number of CD4(+) and CD8(+) T cells. As early as day 2 after infection, B6-lpr mice had decreased liver and spleen viral titers and increased numbers of and increased gamma interferon (IFN-γ) production by several different effector cell populations. Depletion of individual effector cell subsets did not inhibit the resistance of B6-lpr mice. Uninfected B6-lpr mice also had increased numbers of NK cells, γδ(+) T cells, and CD44(+) CD4(+) and CD44(+) CD8(+) T cells compared to uninfected B6 mice. Antibody to IFN-γ resulted in increased virus load in both B6 and B6-lpr mice and eliminated the differences in viral titers between them. These results suggest that IFN-γ produced by multiple activated leukocyte populations in Fas-deficient hosts enhances resistance to some viral infections.     

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

IL-15 expands unconventional CD8alphaalphaNK1.1+ T cells but not Valpha14Jalpha18+ NKT cells

Despite recent gains in knowledge regarding CD1d-restricted NKT cells, very little is understood of non-CD1d-restricted NKT cells such as CD8(+)NK1.1(+) T cells, in part because of the very small proportion of these cells in the periphery. In this study we took advantage of the high number of CD8(+)NK1.1(+) T cells in IL-15-transgenic mice to characterize this T cell population. In the IL-15-transgenic mice, the absolute number of CD1d-tetramer(+) NKT cells did not increase, although IL-15 has been shown to play a critical role in the development and expansion of these cells. The CD8(+)NK1.1(+) T cells in the IL-15-transgenic mice did not react with CD1d-tetramer. Approximately 50% of CD8(+)NK1.1(+) T cells were CD8alphaalpha. In contrast to CD4(+)NK1.1(+) T cells, which were mostly CD1d-restricted NKT cells and of which approximately 70% were CD69(+)CD44(+), approximately 70% of CD8(+)NK1.1(+) T cells were CD69(-)CD44(+). We could also expand similar CD8alphaalphaNK1.1(+) T cells but not CD4(+) NKT cells from CD8alpha(+)beta(-) bone marrow cells cultured ex vivo with IL-15. These results indicate that the increased CD8alphaalphaNK1.1(+) T cells are not activated conventional CD8(+) T cells and do not arise from conventional CD8alphabeta precursors. CD8alphaalphaNK1.1(+) T cells produced very large amounts of IFN-gamma and degranulated upon TCR activation. These results suggest that high levels of IL-15 induce expansion or differentiation of a novel NK1.1(+) T cell subset, CD8alphaalphaNK1.1(+) T cells, and that IL-15-transgenic mice may be a useful resource for studying the functional relevance of CD8(+)NK1.1(+) T cells.     

Phenotypic characteristics associated with the acquisition of HSV-specific CD8 T-lymphocyte-mediated cytolytic function in vitro.

Class I MHC-restricted, HSV-1-specific CD8(+) cytolytic T lymphocyte (CTL) function is rarely detected in lymphocytes isolated directly from the lymph node draining the site of infection. However, culture in vitro for 24 to 72 h in the absence of exogenous antigen results in the development of easily detectable levels of HSV-1-specific CTL effectors. The inability to detect virus-specific CTL in HSV-1-infected mice is not well understood. However, since the in vitro culture of HSV-1-immune lymphocytes results in the transition to CTL function, studies of the changes occurring to the CD8(+) T cell subpopulation may provide important insights into the development of virus-specific CTL. Therefore, the phenotypic changes taking place in the CD8(+) population of T cells from draining popliteal lymph nodes of HSV-1-infected C57BL/6 (B6) mice were investigated, focusing on changes in the expression of cell surface markers associated with T lymphocyte activation. The results demonstrate an increase in the percentage of CD8(+) T cells expressing the activation markers CD44 and CD25 in parallel with the acquisition of HSV-specific CTL effector function. Cytolytic function was found exclusively within the CD8(+) CD44(hi) CD25(hi) fraction of cells in culture, but, surprisingly, was not detectable in CD8(+) CD44(hi) CD25(lo) T cells. This suggested that the acquisition of high levels of the high-affinity IL-2 receptor was closely linked to cytolytic function and may define an important developmental stage in the transition from noncytolytic to cytolytic effector cell. In support of this, CD8(+) CD25(hi) T cells isolated from the regional lymph node exhibited direct ex vivo cytolytic function, indicating that cytolytic effector cells were present in the lymph node, but must emigrate rapidly after attaining this level of differentiation.     

Down-regulation of p27Kip1 expression is required for development and function of T cells

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27(Kip1) is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4(-)CD8(-) thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27(Kip1) expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27(Kip1) expression. The development of thymocytes in the transgenic strain in which p27(Kip1) is most abundant (p27-Tg(high) mice) appeared to be blocked at the CD4(-)CD8(-)CD25(+)CD44(low) stage. Peripheral T cells from p27-Tg(high) mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg(high) mice. These results suggest that down-regulation of p27(Kip1) expression is required for the development, proliferation, and immunoresponsiveness of T cells.     

Cell-based immunotherapy with suppressor CD8+ T cells in rheumatoid arthritis

The chronic persistence of rheumatoid synovitis, an inflammation driven by activated T cells, macrophages, and fibroblasts causing irreversible joint damage, suggests a failure in physiologic mechanisms that down-regulate and terminate chronic immune responses. In vitro CD8(+)CD28(-)CD56(+) T cells tolerize APCs, prevent the priming of naive CD4(+) T cells, and suppress memory CD4(+) T cell responses. Therefore, we generated CD8(+)CD28(-)CD56(+) T cell clones from synovial tissues, expanded them in vitro, and adoptively transferred them into NOD-SCID mice engrafted with synovial tissues from patients with rheumatoid arthritis. Adoptively transferred CD8(+)CD28(-)CD56(+) T cells displayed strong anti-inflammatory activity. They inhibited production of IFN-gamma, TNF-alpha, and chemokines in autologous and HLA class I-matched heterologous synovitis. Down-regulation of costimulatory ligands CD80 and CD86 on synovial fibroblasts was identified as one mechanism of immunosuppression. We propose that rheumatoid synovitis can be suppressed by cell-based immunotherapy with immunoregulatory CD8(+) T cells.     

Effector function-deficient memory CD8+ T cells clonally expand in the liver and give rise to peripheral memory CD8+ T cells

Upon adoptive transfer into histocompatible mice, naive CD8(+) T cells stimulated ex vivo by TCR+IL-4 turn into long-lived functional memory cells. The liver contains a large number of so formed memory CD8(+) T cells, referred to as liver memory T cells (T(lm)) in the form of cell clusters. The CD62L(low) expression and nonlymphoid tissue distribution of T(lm) cells are similar to effector memory (T(em)) cells, yet their deficient cytotoxicity and IFN-γ inducibility are unlike T(em) cells. Adoptive transfer of admixtures of TCR+IL-4-activated Vβ8(+) and Vβ5(+) CD8(+) T cells into congenic hosts reveals T(lm) clusters that are composed of all Vβ5(+) or Vβ8(+), not mixed Vβ5(+)/Vβ8(+) cells, indicating that T(lm) clusters are formed by clonal expansion. Clonally expanded CD8(+) T cell clusters are also seen in the liver of Listeria monocytogenes-immune mice. T(lm) clusters closely associate with hepatic stellate cells and their formation is IL-15/IL-15R-dependent. CD62L(low) T(LM) cells can home to the liver and secondary lymphoid tissues, remain CD62L(low), or acquire central memory (T(cm))-characteristic CD62L(hi) expression. Our findings show the liver as a major site of CD8(+) memory T cell growth and that T(lm) cells contribute to the pool of peripheral memory cells. These previously unappreciated T(lm) characteristics indicate the inadequacy of the current T(em)/T(cm) classification scheme and help ongoing efforts aimed at establishing a unifying memory T cell development pathway. Lastly, our finding of T(lm) clusters suggests caution against interpreting focal lymphocyte infiltration in clinical settings as pathology and not normal physiology.     

Characterization of a new mouse model for peripheral T cell lymphoma in humans

Peripheral T cell lymphomas (PTCLs) are associated with a poor prognosis due to often advanced disease at the time of diagnosis and due to a lack of efficient therapeutic options. Therefore, appropriate animal models of PTCL are vital to improve clinical management of this disease. Here, we describe a monoclonal CD8(+) CD4(-) αβ T cell receptor Vβ2(+) CD28(+) T cell lymphoma line, termed T8-28. T8-28 cells were isolated from an un-manipulated adult BALB/c mouse housed under standard pathogen-free conditions. T8-28 cells induced terminal malignancy upon adoptive transfer into syngeneic BALB/c mice. Despite intracellular expression of the cytotoxic T cell differentiation marker granzyme B, T8-28 cells appeared to be defective with respect to cytotoxic activity as read-out in vitro. Among the protocols tested, only addition of interleukin 2 in vitro could partially compensate for the in vivo micro-milieu in promoting growth of the T8-28 lymphoma cells.