Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.     

IL-7 activates the phosphatidylinositol 3-kinase/AKT pathway in normal human thymocytes but not normal human B cell precursors

IL-7 signaling culminates in different biological outcomes in distinct lymphoid populations, but knowledge of the biochemical signaling pathways in normal lymphoid populations is incomplete. We analyzed CD127/IL-7Ralpha expression and function in normal (nontransformed) human thymocytes, and human CD19(+) B-lineage cells purified from xenogeneic cord blood stem cell/MS-5 murine stromal cell cultures, to further clarify the role of IL-7 in human B cell development. IL-7 stimulation of CD34(+) immature thymocytes led to phosphorylation (p-) of STAT5, ERK1/2, AKT, and glycogen synthase kinase-3 beta, and increased AKT enzymatic activity. In contrast, IL-7 stimulation of CD34(-) thymocytes (that included CD4(+)/CD8(+) double-positive, and CD4(+) and CD8(+) single-positive cells) only induced p-STAT5. IL-7 stimulation of CD19(+) cells led to robust induction of p-STAT5, but minimal induction of p-ERK1/2 and p-glycogen synthase kinase-3 beta. However, CD19(+) cells expressed endogenous p-ERK1/2, and when rested for several hours following removal from MS-5 underwent de-phosphorylation of ERK1/2. IL-7 stimulation of rested CD19(+) cells resulted in robust induction of p-ERK1/2, but no induction of AKT enzymatic activity. The use of a specific JAK3 antagonist demonstrated that all IL-7 signaling pathways in CD34(+) thymocytes and CD19(+) B-lineage cells were JAK3-dependent. We conclude that human CD34(+) thymocytes and CD19(+) B-lineage cells exhibit similarities in activation of STAT5 and ERK1/2, but differences in activation of the PI3K/AKT pathway. The different induction of PI3K/AKT may at least partially explain the different requirements for IL-7 during human T and B cell development.     

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.     

Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers

We show that the lymphoid hyperplasia observed in IL-2Ralpha- and IL-2-deficient mice is due to the lack of a population of regulatory cells essential for CD4 T cell homeostasis. In chimeras reconstituted with bone marrow cells from IL-2Ralpha-deficient donors, restitution of a population of CD25(+)CD4(+) T cells prevents the chaotic accumulation of lymphoid cells, and rescues the mice from autoimmune disease and death. The reintroduction of IL-2-producing cells in IL-2-deficient chimeras establishes a population of CD25(+)CD4(+) T cells, and restores the peripheral lymphoid compartments to normal. The CD25(+)CD4(+) T cells regulated selectively the number of naive CD4(+) T cells transferred into T cell-deficient hosts. The CD25(+)CD4(+)/naive CD4 T cell ratio and the sequence of cell transfer determines the homeostatic plateau of CD4(+) T cells. Overall, our findings demonstrate that IL-2Ralpha is an absolute requirement for the development of the regulatory CD25(+)CD4(+) T cells that control peripheral CD4 T cell homeostasis, while IL-2 is required for establishing a sizeable population of these cells in the peripheral pools.     

Long-term control of simian immunodeficiency virus mac251 viremia to undetectable levels in half of infected female rhesus macaques nasally vaccinated with simian immunodeficiency virus DNA/recombinant modified vaccinia virus Ankara

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIV(mac251) low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4β7(high+)/CD4(+) T cells and of circulating and mucosal CD4(+)/C(M) T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4(+)/IFN-γ(+) and CD8(+)/IFN-γ(+) T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4(+)/IL-2(+), CD4(+)/IFN-γ(+), and CD8(+)/TNF-α(+) T cells and vaginal anti-SIV Gag + Env CD8(+) T cell total monofunctional 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.     

Human bone marrow hosts polyfunctional memory CD4+ and CD8+ T cells with close contact to IL-15-producing cells

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4(+) and CD8(+) T cells have not yet been elucidated. In this study, we demonstrate that CD4(+) and CD8(+) effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4(+) and CD8(+) T cells are in the immediate vicinity of IL-15-producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4(+) and CD8(+) T cell subsets. Moreover, the IL-15-inducible molecules Bcl-x(L), MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15-producing cells, is important for the maintenance of a polyfunctional memory CD4(+) and CD8(+) T cell pool.     

CD8+ T lymphocytes regulating Th2 pathology escape neonatal tolerization

Transplantation tolerance induced by neonatal injection of semiallogeneic spleen cells is associated in several strain combinations with a pathological syndrome caused by Th2 differentiation of donor-specific CD4(+) T lymphocytes. We investigated the role of host CD8(+) T cells in the regulation of this Th2 pathology. IgE serum levels and eosinophilia significantly increased in BALB/c mice neonatally injected with (A/J x BALB/c)F(1) spleen cells when CD8(+) T cells were depleted by administration of anti-CD8 mAb or when beta(2)-microglobulin-deficient mice were used as recipients. In parallel, increased serum levels of IL-5 and IL-13 were measured in blood of tolerant CD8(+) T cell-deficient mice. Whereas neonatally injected mice were unable to generate anti-donor cytotoxic effectors, their CD8(+) T cells were as efficient as control CD8(+) T cells in reducing the severity of Th2 pathology and in restoring donor-specific cytotoxicity in vitro after in vivo transfer in beta(2)-microglobulin-deficient mice. Likewise, CD8(+) T cells from control and tolerant mice equally down-regulated the production of Th2 cytokines by donor-specific CD4(+) T cells in vitro. The regulatory activity of CD8(+) T cells depended on their secretion of IFN-gamma for the control of IL-5 production but not for IL-4 or IL-13. Finally, we found that CD8(+) T cells from 3-day-old mice were already able to down-regulate IL-4, IL-5, and IL-13 production by CD4(+) T cells. We conclude that regulatory CD8(+) T cells controlling Th2 responses are functional in early life and escape neonatal tolerization.     

Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common gamma-chain deficient NOD/LtSz-scid (NOD-scid IL2Rgamma(null)) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rgamma(null) mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rgamma(null) mice with human HSC generate 6-fold higher percentages of human CD45(+) cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rgamma(null) mice contain human Ig(+) B cells and lower numbers of human CD3(+) T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4(+)CD8(+) thymocytes as well human CD4(+)CD8(-) and CD4(-)CD8(+) peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rgamma(null) mice was validated by 1) high levels of TCR excision circles, 2) complex TCRbeta repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rgamma(null) mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.     

The schistosome oligosaccharide lacto-N-neotetraose expands Gr1(+) cells that secrete anti-inflammatory cytokines and inhibit proliferation of naive CD4(+) cells: a potential mechanism for immune polarization in helminth infections.

Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1(+)/CD11b(+)/F4/80(+), as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1(+)/F4/80(+) population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-beta ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1(+) cells adoptively suppressed naive CD4(+) T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4(+) cells involved cell contact and was dependent on IFN-gamma and NO, with a discrete role played by IL-10. Coculture of naive CD4(+)T cells with Gr1(+) suppressor cells did not lead to CD4(+) T cell apoptosis, although it did imprint on naive CD4(+) T cells a response characterized by lower levels of IFN-gamma, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.     

Ultraviolet irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway

Direct UV irradiation of dendritic cells and Langerhans cells reduces their Ag presenting ability. However, the effects of UV on CD11c(+) cells located distally to the point of irradiation are poorly understood. Three days after UV irradiation (8 kJ/m(2)) of BALB/c mice, bone marrow cells were isolated and cultured for 7 d with IL-4 and GM-CSF for the propagation of CD11c(+) cells. Bone marrow-derived CD11c(+) cells from UV-irradiated or nonirradiated mice were loaded with dinitrobenzene sulfonic acid and injected into the ear pinnas of naive BALB/c mice. After 7 d, the ears were painted with 2,4-dinitro-1-fluorobenzene and the ear swelling determined 24 h later. A reduced contact hypersensitivity response was found in mice injected with CD11c(+) cells from the UV-irradiated animals compared with those injected with cells from the nonirradiated animals. Further, a long-lasting suppression of the memory response to 2,4-dinitro-1-fluorobenzene was created. This suppressed response corresponded to increased IL-10 and PGE(2) secretion by freshly isolated bone marrow cells from UV-irradiated mice, and to increased myelopoiesis. The reduction in competence of bone marrow-derived CD11c(+) cells from UV-irradiated mice was not due to delayed maturation, as it was maintained upon LPS exposure prior to CD11c(+) cell purification. The UV-induced effect was reversed by the administration of indomethacin to mice prior to UV irradiation and could be reproduced by s.c. PGE(2). These results show that UV irradiation of mice can affect the function of bone marrow-derived CD11c(+) cells via a mechanism inhibitable by indomethacin; this pathway is likely to contribute to systemic UV-induced immunosuppression.     

Role of the indigenous microbiota in maintaining the virus-specific CD8 memory T cells in the lung of mice infected with murine cytomegalovirus

The potent role of indigenous microbiota in maintaining murine CMV (MCMV)-specific memory T cells, which were measured by multimer staining, was investigated using germfree (GF) mice. When the BALB/c mice bred under specific pathogen-free (SPF) conditions were i.p. infected with 0.2 LD(50) of MCMV, high frequencies of CD69(+)/CD44(+) MCMV-specific CD8 T cells were noted in the lungs even at 6-12 mo after infection (11.1 +/- 3.2 and 9.8 +/- 0.9%, respectively). In contrast, even though the viral load and expression levels of mRNA of such cytokines as IL-2, IL-7, IL-15, and IFN-gamma in the lungs of MCMV-infected GF mice were comparable to those of infected SPF mice, the frequencies of MCMV-specific CD8 T cells in the lungs of infected GF mice were kept lower than 1% at 6-12 mo after infection. In addition, the reconstitution of microbiota of MCMV-infected GF mice by orally administering a fecal suspension prepared from SPF mice restored the frequencies of both CD8(+)/multimer(+) and CD8(+)/multimer(-) T cells to levels similar to those found in SPF mice. These results suggested the indigenous microbiota to play a crucial role in the expansion and maintenance of viral-specific CD8 memory T cells, probably by cross-reactivity between the antigenic epitope of the MCMV-specific memory T cells and the variety of peptides derived from the members of the microbiota. Such cross-reactivity may thus be a major feature of those cells.     

Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)β(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.     

Selective induction of dendritic cells using granulocyte macrophage-colony stimulating factor,but not fms-like tyrosine kinase receptor 3-ligand,activates thyroglobulin-specific CD4+/CD25+ T cells and suppresses experimental autoimmune thyroiditis

Fms-like tyrosine kinase receptor 3-ligand (Flt3-L) and GM-CSF cause expansion of different subsets of dendritic cells and skew the immune response toward predominantly Th1 and Th2 type, respectively. In the present study, we investigated their effects on experimental autoimmune thyroiditis in CBA/J mice. Relative to mouse thyroglobulin (mTg) immunized controls, mTg-immunized mice treated with Flt3-L showed more severe thyroiditis characterized by enhanced lymphocytic infiltration of the thyroid, and IFN-gamma and IL-2 production. In contrast, mice treated with GM-CSF, either before or after immunization with mTg, showed suppressed T cell response to mTg and failed to develop thyroiditis. Lymphocytes from these mice, upon activation with mTg in vitro, produced higher levels of IL-4 and IL-10. Additionally, GM-CSF-treated mice showed an increase in the frequency of CD4(+)/CD25(+) T cells, which suppressed the mTg-specific T cell response. Neutralization of IL-10, but not IL-4, or depletion of CD4(+)/CD25(+) cells resulted in increased mTg-specific in vitro T cell proliferation suggesting that IL-10 produced by the Ag-specific CD4(+)/CD25(+) regulatory T cells might be critical for disease suppression. These results indicate that skewing immune response toward Th2, through selective activation of dendritic cells using GM-CSF, may have therapeutic potential in Th1 dominant autoimmune diseases including Hashimoto's thyroiditis.     

Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation

Deficient thymopoiesis and retarded recovery of newly developed CD4(+) T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage(-)sca-1(+)c-kit(+)flt3(+) lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.     

A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.     

Resistance to metastatic disease in STAT6-deficient mice requires hemopoietic and nonhemopoietic cells and is IFN-gamma dependent

Mice deficient for the STAT6 gene (STAT6(-/-) mice) have enhanced immunosurveillance against primary and metastatic tumors. Because STAT6 is a downstream effector of the IL-4R, and IL-13 binds to the type 2 IL-4R, IL-13 has been proposed as an inhibitor that blocks differentiation of tumor-specific CD8(+) T cells. Immunity in STAT6(-/-) mice is unusually effective in that 45-80% of STAT6(-/-) mice with established, spontaneous metastatic 4T1 mammary carcinoma, whose primary tumors are surgically excised, survive indefinitely, as compared with <10% of STAT(+/+) (BALB/c) mice. Surprisingly, STAT6(-/-) and BALB/c reciprocal bone marrow chimeras do not have increased immunosurveillance, demonstrating that immunity requires STAT6(-/-) hemopoietic and nonhemopoietic components. Likewise, CD1(-/-) mice that are NKT deficient and therefore IL-13 deficient also have heightened tumor immunity. However, STAT6(-/-) and CD1(-/-) reciprocal bone marrow chimeras do not have increased survival, suggesting that immunity in STAT6(-/-) and CD1(-/-) mice is via noncomplementing mechanisms. Metastatic disease is not reduced in BALB/c mice treated with an IL-13 inhibitor, indicating that IL-13 alone is insufficient for negative regulation of 4T1 immunity. Likewise, in vivo depletion of CD4(+)CD25(+) T cells in BALB/c mice does not increase survival, demonstrating that CD4(+)CD25(+) cells do not regulate immunity. Cytokine production and tumor challenges into STAT6(-/-)IFN-gamma(-/-) mice indicate that IFN-gamma is essential for immunity. Therefore, immunosurveillance in STAT6(-/-) mice facilitates survival against metastatic cancer via an IFN-gamma-dependent mechanism involving hemopoietic and nonhemopoietic derived cells, and is not exclusively dependent on counteracting IL-13 or CD4(+)CD25(+) T cells.     

Systemic dissemination and persistence of Th2 and type 2 cells in response to infection with a strictly enteric nematode parasite

Oral infection with the nematode parasite Heligmosomoides polygyrus H. polygyrus is entirely restricted to the small intestine. Although the evoked Th2 response has been extensively studied in secondary lymphoid organs, little is known about the systemic dissemination of Th2 cells or type 2 associated eosinophils and basophils. In this study we use bicistronic 4get IL-4 reporter mice to directly visualize the type 2 response to H. polygyrus infection. We observed that CD4(+)/GFP(+) Th2 cells spread systemically and found that these cells accumulated in nonlymphoid "hot spots" in the liver, the lung airways, and the peritoneal cavity. Interestingly, the total number of Th2 cells in the peritoneal cavity was comparable to those found in the draining mesenteric lymph node or the spleen. Peritoneal Th2 cells were distinguished by an exceptionally low apoptotic potential and high expression of the intestinal homing receptor alpha(4)beta(7) integrin. CD4(+)/GFP(+) Th2 cells from these peripheral sites were fully functional as indicated by rapid IL-4 production upon polyclonal or Ag-specific restimulation. Th2 cells persisted in the intestinal tissue and the peritoneal cavity of drug-cured mice for weeks. The presence of peripheral memory Th2 cells in the intestine might be crucial for immunity to recall infections. These findings have important implications for the design of vaccination strategies because it may be necessary to establish and maintain memory CD4(+) T cells at the potential future site of infection.