T cell activation is known to be critically regulated by the extent and duration of TCR-induced signaling pathways. The NFAT family of transcription factors is believed to play an important role in coupling these quantitative differences in TCR-induced signaling events into changes in gene expression. In this study we have specifically investigated the effects of sustained NFAT signaling on T cell activation by introducing a constitutively active mutant version of NFATc1 (caNFATc1) into primary murine CD4(+) T cells and examining its effects on gene expression. We now report that ectopic expression of caNFATc1 partially mimics TCR signaling, resulting in enhanced expression of CD25 and CD40 ligand and down-regulation of CD62L. More importantly, we find that expression of caNFATc1 in T cells maintained under either nonpolarizing or Th1-skewing conditions leads to a marked selective increase in the number of cells expressing the prototypical Th1 cytokine, IFN-gamma. Furthermore, when expressed in Th2-skewed cells, caNFATc1 appears to attenuate Th2 differentiation by decreasing production of IL-4 and promoting the expression of IFN-gamma. Finally, we find that caNFATc1 enhances expression of functional P-selectin glycoprotein ligand-1, up-regulates Fas ligand expression, and increases susceptibility to activation-induced cell death, cellular traits that are preferentially associated with Th1 effector cells. Taken together, these results suggest that sustained NFAT signaling, mediated by ectopic expression of caNFATc1, acts to promote a Th1-like pattern of gene expression and thereby serves to highlight the important relationship between the degree of NFAT signaling and the qualitative pattern of gene expression induced during T cell activation.

To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56CD3 donor NK cells during stimulation with 1000 U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16 and CD16 subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR) NK cells were overgrown by KIR proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-gamma, tumor necrosis factor-beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCR and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2-stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell immunotherapy.

Forkhead transcription factors play critical roles in leukocyte homeostasis. To study further the immunological functions of Foxo1, we generated mice that selectively lack Foxo1 in T cells (Foxo1(flox/flox) Lck.cre(+)conditional knockout mice (cKO)). Although thymocyte development appeared relatively normal, Foxo1 cKO mice harbored significantly increased percentages of mature single positive T cells in the thymus as compared with WT mice, yet possessed smaller lymph nodes and spleens that contained fewer T cells. Foxo1 cKO T cells were not more prone to apoptosis, but instead were characterized by a CD62L(lo) CCR7(lo) CD44(hi) surface phenotype, a poorly populated lymphoid compartment in the periphery, and were relatively refractory to TCR stimulation, all of which were associated with reduced expression of Sell, Klf2, Ccr7, and S1pr1. Thus, Foxo1 is critical for naïve T cells to populate the peripheral lymphoid organs by coordinating a molecular program that maintains homeostasis and regulates trafficking.

Recently, traces of double-positive FoxP3(+)RORgammat(+) T cells were identified and viewed as dual programming differentiation intermediates geared toward development into T regulatory or Th17 cells. In this study, we report that FoxP3(+)RORgammat(+) intermediates arise in the NOD mouse T cell repertoire prior to inflammation and can be expanded with tolerogen without further differentiation. Furthermore, FoxP3(+)RORgammat(+) cells express both CD62L and membrane-bound TGFbeta and use the former to traffic to the pancreas and the latter to suppress effector T cells both in vitro and in vivo. The cells perform these functions as FoxP3(+)RORgammat(+) intermediates, despite being able to terminally differentiate into either FoxP3(+)RORgammat(-) T regulatory or FoxP3(-)RORgammat(+) Th17 cells on polarization. These previously unrecognized observations extend plasticity to both differentiation and function and indicate that the intermediates are poised to traffic to sites of inflammation and target diverse pathogenic T cells, likely without prior conditioning by effector T cells, thus broadening efficacy against autoimmunity.

To test whether homeostasis-driven T cell proliferation in reconstituted lymphodepleted hosts would improve the therapeutic efficacy of tumor vaccines, normal mice and reconstituted lymphopenic mice (RLM; C57BL/6 mice rendered lymphopenic with sublethal total-body irradiation and reconstituted with naive splenocytes) were used in the vaccination and challenge experiments with weakly immunogenic F10 melanoma cells. Only limited protection was observed in vaccinated normal mice (16.7%), whereas significantly greater protection was induced in vaccinated RLM (63.2%). Protective immunity in RLM depended on CD8 T cells. Following vaccination, a significant increase in the percentage of CD44(hi)CD62L(lo) T cells was detected in the tumor vaccine-draining lymph node (TVDLN) of vaccinated RLM compared to that of vaccinated normal mice. After in vitro stimulation, effector T cells generated from TVDLN of vaccinated RLM produced more IFN-gamma than T cells from vaccinated normal mice, and contained more melanoma-specific T cells, as assessed by ELISA and intracellular cytokine staining. This study suggests that vaccination of reconstituted lymphopenic hosts could elicit superior anti-tumor immunity compared to normal hosts, highlighting the potential clinical benefit of performing tumor vaccination during immune reconstitution.

Bone marrow of breast cancer patients was found to contain CD8(+) T cells specific for peptides derived from breast cancer-associated proteins MUC1 and Her-2/neu. Most of these cells had a central or effector memory phenotype (CD45RA(-)CD62L(+) or CD45RA(-)CD62L(-), respectively). To test their in vivo function, we separated bone marrow-derived CD45RA(+) naive or CD45RA(-)CD45RO(+) memory T cells, stimulated them with autologous dendritic cells pulsed with tumor lysate, and transferred them into NOD/SCID mice bearing autologous breast tumors and normal skin transplants. CD45RA(-) memory but not CD45RA(+) naive T cells infiltrated autologous tumor but not skin tissues after the transfer. These tumor-infiltrating cells had a central or effector memory phenotype and produced perforin. Many of them expressed the P-selectin glycoprotein ligand 1 and were found around P-selectin(+) tumor endothelium. Tumor infiltration included cluster formation in tumor tissue by memory T cells with cotransferred dendritic cells. It was associated with the induction of tumor cell apoptosis and significant tumor reduction. We thus demonstrate selective homing of memory T cells to human tumors and suggest that tumor rejection is based on the recognition of tumor-associated antigens on tumor cells and dendritic cells by autologous specifically activated central and effector memory T cells.

Thymus transplantation is a promising investigational therapy for infants born with no thymus. Because of the athymia, these infants lack T cell development and have a severe primary immunodeficiency. Although thymic hypoplasia or aplasia is characteristic of DiGeorge anomaly, in "complete" DiGeorge anomaly, there is no detectable thymus as determined by the absence of naive (CD45RA(+), CD62L(+)) T cells. Transplantation of postnatal allogeneic cultured thymus tissue was performed in sixty subjects with complete DiGeorge anomaly who were under the age of 2 years. Recipient survival was over 70%. Naive T cells developed 3-5 months after transplantation. The graft recipients were able to discontinue antibiotic prophylaxis, and immunoglobulin replacement. Immunosuppression was used in a subset of subjects but was discontinued when naive T cells developed. The adverse events have been acceptable with thyroid disease being the most common. Research continues on mechanisms underlying immune reconstitution after thymus transplantation.

Memory T cells are heterogeneous in expression of lymph node homing receptors, delineating "central-memory" (TCM, CD62Lhi/CCR7+) and "effector-memory" (TEM, CD62Llo/CCR7-) subsets that migrate to lymphoid and non-lymphoid tissues, respectively. It is not known how these subsets arise or how homing receptor expression and tissue origin determine their functional and migratory properties. Here, we investigated the role of CD62L expression in the generation, function, distribution and migration of heterogeneous memory CD4 T cells specific for influenza hemagglutinin (HA). We found that CD62Lhi and CD62Llo memory subsets are generated independent of CD62L expression by the activated precursor, and both subsets distribute into spleen and lung. Functionally, spleen- and lung-derived CD62L memory subsets produce effector cytokines at similar kinetics but differ strikingly in cell surface phenotype and migration: the CD62Llo memory subset expresses a classic memory phenotype (CD45RBlo/CD44hi/CD11a(hi)), while the CD62Lhi subset expresses an unconventional phenotype (CD45RBhi/CD44int/CD11a(int)), defining a new polyclonal memory subset. The CD62Lhi subset also trafficked more efficiently than CD62Llo cells into lymph nodes; however, only lung but not spleen CD62Llo memory T cells homed to lung. Our results reveal novel phenotypic heterogeneity of memory CD4 T cells co-segregating with CD62L expression and tissue-specific tropism of non-lymphoid memory CD4 T cells.

Naive CD4+ T cells use L-selectin (CD62L) expression to facilitate immune surveillance. However, the reasons for its expression on a subset of memory CD4+ T cells are unknown. We show that memory CD4+ T cells expressing CD62L were smaller, proliferated well in response to tetanus toxoid, had longer telomeres, and expressed genes and proteins consistent with immune surveillance function. Conversely, memory CD4+ T cells lacking CD62L expression were larger, proliferated poorly in response to tetanus toxoid, had shorter telomeres, and expressed genes and proteins consistent with effector function. These findings suggest that CD62L expression facilitates immune surveillance by programming CD4+ T cell blood and lymph node recirculation, irrespective of naive or memory CD4+ T cell phenotype.

HLA-G is a tolerogenic molecule whose detection in sera and within allografted tissues is associated with better graft acceptance. HLA-G mediates T-cell differentiation into suppressor cells, which are thought to promote tolerance. Here, we investigated such T cells phenotypically and functionally and assessed their clinical relevance in the peripheral blood of patients who have undergone transplantation. Our results demonstrate that HLA-G expressed by antigen-presenting cells or present as soluble protein down-regulates the expression of CD4 and CD8 on allostimulated T cells at both transcriptional and posttranslational levels. These CD3(+)CD4(low) and CD3(+)CD8(low) T-cell subsets are characterized by an increased proportion of cells expressing CD45RA and HLA-DR, and a decreased number of cells expressing CD62L. In addition, these HLA-G-induced CD3(+)CD4(low) and CD3(+)CD8(low) subpopulations are Foxp3-negative suppressor T cells whose function involves IL-10. Biologic relevance came from analysis of patients who underwent transplantation, with high HLA-G plasma concentrations associated with better graft survival. Peripheral blood from these patients contains increased levels of IL-10 concomitantly to an enhanced representation of CD3(+)CD4(low) and CD3(+)CD8(low) T cells compared with HLA-G-negative patients who underwent transplantation and healthy individuals. These data define novel immunosuppressive subpopulations of peripheral blood T cells induced by HLA-G with potent implications in peripheral tolerance.

BACKGROUND

Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27(negative) B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated.

METHODS

Epratuzumab binding specificity and the surface expression of adhesion molecules (CD62L, β7 integrin and β1 integrin) after culture with epratuzumab was studied on B-cell subsets of SLE patients by flow cytometry. In addition, in vitro transwell migration assays were performed to analyze the effects of epratuzumab on migration towards different chemokines such as CXCL12, CXCL13 or to CXCR3 ligands, and to assess the functional consequences of altered adhesion molecule expression.

RESULTS

Epratuzumab binding was considerably higher on B-cells relative to other cell types assessed. No binding of epratuzumab was observed on T-cells, while weak non-specific binding of epratuzumab on monocytes was noted. On B-cells, binding of epratuzumab was particularly enhanced on CD27(negative) B-cells compared to CD27(positive) B-cells, primarily related to a higher expression of CD22 on CD27(negative) B-cells. Moreover, epratuzumab binding led to a decrease in the cell surface expression of CD62L and β7 integrin, while the expression of β1 integrin was enhanced. The effects on the pattern of adhesion molecule expression observed with epratuzumab were principally confined to a fraction of the CD27(negative) B-cell subpopulation and were associated with enhanced spontaneous migration of B-cells. Furthermore, epratuzumab also enhanced the migration of CD27(negative) B-cells towards the chemokine CXCL12.

CONCLUSIONS

The current data suggest that epratuzumab has effects on the expression of the adhesion molecules CD62L, β7 integrin and β1 integrin as well as on migration towards CXCL12, primarily of CD27(negative) B-cells. Therefore, induced changes in migration appear to be part of the mechanism of action of epratuzumab and are consistent with the observation that CD27(negative) B-cells were found to be preferentially reduced in the peripheral blood under treatment.

We investigated long-term memory and recall cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (VSVs) expressing Env and Gag. More than 7 months after a single vaccination with VSV-Env, approximately 6% of CD8(+) splenocytes stained with major histocompatibility complex class I tetramers containing the Env p18-I10 immunodominant peptide and showed a memory phenotype (CD44(Hi)). The level of tetramer-positive cells in memory was about 14% of the peak primary response. Recall responses elicited in these mice 5 days after boosting with a heterologous recombinant vaccinia virus expressing HIV-1 Env showed that 40 to 45% of CD8(+) splenocytes were tetramer positive and activated (CD62L(Lo)), and these cells produced gamma interferon after stimulation with Env peptide, indicating that they were functional. Five months after the boost, the long-term memory cell population (tetramer positive, CD44(Hi)) constituted 30% of the CD8(+) splenocytes. Recall responses to HIV-1 Gag were examined in mice primed with VSV recombinants expressing HIV-1 Gag protein and boosted with a vaccinia virus recombinant expressing Gag. Using this protocol, we found that approximately 40% of CD8(+) splenocytes were activated (CD62L(Lo)) and specific for a Gag immunodominant peptide (tetramer positive). The high-level Gag recall response elicited by the vaccinia virus-Gag was greater than that obtained by boosting with a VSV-Gag vector with a different VSV glycoprotein. The corresponding levels of CD44(Hi) memory cells were also higher long after boosting with vaccinia virus-Gag than after boosting with a glycoprotein exchange VSV-Gag. Our results show that VSV vectors elicit high-level memory CTL responses and that these can be amplified as much as six- to sevenfold using a heterologous boosting vector.

Recent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonpersistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct "central memory" CD62L(+) phenotype.

Pathogenic autoreactive T lymphocytes are mediators of spontaneous insulin-dependent diabetes in nonobese diabetic (NOD) mice. This is demonstrated by their capacity to transfer diabetes into syngeneic immunoincompetent recipients. In addition, especially in prediabetic NOD mice, peripheral CD4+ T lymphocytes were identified that are highly effective, in conventional mixing cotransfer experiments, at preventing disease transfer. The present data demonstrate that mature heat-stable Ag-TCR alpha beta+CD8-thymocytes from prediabetic NOD mice also express this inhibitory capacity. Selection using an L-selectin (CD62L)-specific Ab showed that TCR alpha/beta+CD4+CD62L+ thymocytes, emerging from the mainstream differentiation pathway, concentrate this ability to regulate autoreactive effectors. Compared with mature TCR alpha beta+CD8- thymocytes, significantly lower numbers of TCR alpha beta+CD4+CD62L+ were sufficient to achieve an efficient inhibition of disease transfer into NOD-scid recipients. This protective ability was potentiated following in vitro culture in the presence of IL-7. In contrast, TCR alpha beta+CD62L- thymocytes, highly enriched in class I-restricted NK T cells, were unable to influence diabetes transfer. Identical results were obtained using thymocytes that have been cultured in vitro for 4 days in the presence of IL-7. These results support the active role in NOD mice of a thymus-derived CD4+ subset that controls peripheral pathogenic autoimmune effectors.

Leishmania braziliensis is a parasite that can induce at least two clinical forms of leishmaniasis in humans: cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). In humans, the specific mechanisms that determine which form will develop following infection are not well established. In this study, peripheral blood mononuclear cells from 17 CL and 9 ml patients were compared both ex vivo and after culture with soluble leishmania antigen (SLA). Patients with ML presented a higher frequency of activated T cells as measured by ex vivo frequencies of (CD4+)(CD69+), (CD4+)(CD28-), (CD4+)(CD62L-) and (CD8+)(CD69+) than those with CL. Moreover, after stimulation with SLA, patients with ML presented a higher frequency of TNF-alpha-producing CD4+ and CD14+ cells than CL individuals. While CL patients displayed a positive correlation between the frequency of IL-10 and TNF-alpha-producing monocytes, the ML patients did not. This lack of a positive correlation between IL-10-producing and TNF-alpha-producing monocytes in ML patients could lead to a less controlled inflammatory response in vivo. These results corroborate with a model of an exacerbated, unregulated, immune response in ML patients and point to key immunomodulatory leucocyte populations and cytokine networks that may be involved in the development of immunopathology in ML patients.

Tyrosine kinase inhibitors (TKIs) are the current standard treatment in chronic myeloid leukemia (CML). In addition to the BCR-ABL target oncoprotein, they also inhibit off-target kinases (e.g. c-KIT, TEC, SRC), some of which have physiological functions in immune responses. In vitro studies have implied immunosuppressive effects of TKI treatment. As comprehensive in vivo data are missing, we aimed at analyzing the detailed immunoprofile of patients with CML at diagnosis and during therapy. We collected 88 peripheral blood (PB) and 73 bone marrow (BM) samples from 54 patients with CML at diagnosis, during imatinib and dasatinib therapies. Leukocytes and lymphocyte subclasses were analyzed with an extensive flow cytometry panel including markers for activation, differentiation and memory status. At diagnosis, a lower proportion of B cells and dendritic cells and an increased amount of NKT-like cells were detected in the BM. During imatinib therapy, all these changes normalized and the immunoprofile resembled healthy controls. However, dasatinib patients were clearly divided into two distinct groups: one similar to healthy controls and the other showing immunoactivation characterized by significant elevations of CD8+, NK- and NKT-like cells in PB. T cells of the latter group strongly expressed CD57+, HLA-DR and CD45RO and had low CD62L antigen levels characteristic of late memory cytotoxic lymphocytes. Our results indicate that while both TKIs show immunosuppressive effects in vitro, they have a significant and differential effect on the numbers and proportions of immune effector cells in vivo. In particular, in a distinct subgroup of dasatinib-treated patients, immune reactivity is markedly enhanced warranting careful follow-up.

BACKGROUND

CD4CD25 regulatory T (Treg) cells are essential for the induction and maintenance of immunologic self-tolerance as well as transplant tolerance. The effects of cyclosporin A (CsA), a widely used immunosuppressive agent, on CD4CD25Treg cells in mice were investigated.

METHODS

Balb/c mice were injected with CsA or control solution for one month. The levels, phenotype, and function of CD4CD25Treg cells in these mice were then assayed.

RESULTS

The percentages and total cell numbers of CD4CD25Treg cells in the peripheral blood and spleen were significantly reduced after the treatment with CsA. The total numbers of CD4CD25Treg cells in the thymus of CsA-treated mice were markedly reduced as compared to the control mice. However, the percentage of CD4CD25Treg cells in the thymus of CsA-treated mice was markedly enhanced. More CD4CD25Treg cells expressing high levels of CD44 and CD45RB, and less CD4CD25Treg cells expressing CD62L were observed in CsA-treated mice, compared with the control mice. CD4CD25Treg cells expressed slightly lower levels of Foxp3 in CsA-treated mice. Furthermore, CsA markedly impaired the immunosuppressive function of CD4CD25Treg cells.

CONCLUSIONS

CsA significantly impaired the development and function of CD4CD25Treg cells. The present studies suggest that CsA may block the potential induction of immune tolerance and increase the susceptibility to develop autoimmune diseases while preventing graft rejection.

We systematically investigated the comparative efficacy of three different cytokine regimens, administered after a reperfused myocardial infarction, in regenerating cardiac tissue and improving left ventricular (LV) function. Wild-type (WT) mice underwent a 30-minute coronary occlusion followed by reperfusion and received vehicle, granulocyte colony-stimulating factor (G-CSF)+Flt-3 ligand (FL), G-CSF+stem cell factor (SCF), or G-CSF alone starting 4 hours after reperfusion. In separate experiments, chimeric mice generated by reconstitution of radioablated WT mice with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice underwent identical protocols. Mice were euthanized 5 weeks later. Echocardiographically, LV function was improved in G-CSF+FL- and G-CSF+SCF-treated but not in G-CSF-treated mice, whereas LV end-diastolic dimensions were smaller in all three groups. Morphometrically, cytokine-treated hearts had smaller LV diameter and volume. Numerous EGFP-positive cardiomyocytes, capillaries, and arterioles were noted in the infarcted region in cytokine-treated chimeric mice treated with G-CSF+FL or G-CSF+SCF, but the numbers were much smaller in G-CSF-treated mice. G-CSF+FL therapy mobilized bone marrow-derived cells exhibiting increased expression of surface antigens (CD62L and CD11a) that facilitate homing. We conclude that postinfarct cytokine therapy with G-CSF+FL or G-CSF+SCF limits adverse LV remodeling and improves LV performance by promoting cardiac regeneration and probably also by exerting other beneficial actions unrelated to regeneration, and that G-CSF alone is less effective.

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea-induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4(+) T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-kappaB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4(+) T cells adopt a CD44(high)CD62L(low)CD69(low) phenotype and show reduced IL-7ralpha expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor-induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

The recent description of an early T-lineage progenitor (ETP) population in adult mouse thymus implies the presence of a bone marrow predecessor that has not yet been identified. Here we describe a Lin(Neg) Sca-1(Pos) c-kit(Hi) Thy-1.1(Neg) L-selectin(Pos) adult mouse bone marrow population that resembles the thymic ETP in both antigen expression phenotype and posttransplantation lineage potential. These cells produce wavelike kinetics of thymic seeding and reconstitute the irradiated thymus with kinetics comparable to a thymocyte graft after intravenous transplantation. Transient B-lineage reconstitution is also observed, but little myeloid potential can be detected in transplant experiments. A second subset of progenitors is L-selectin(Neg) and is highly enriched for rapid and persistent T- and B-lineage potential, as well as some myeloid potential. L-selectin (CD62L) is therefore an effective marker for separating lymphoid progenitors from myeloid progenitors and hematopoietic stem cells in mouse bone marrow.

The determinants of the prevalence of CD8(+) T cells in the inflamed myocardium of Trypanosoma cruzi-infected patients and experimental animals are undefined. Using C3H/He mice infected with the Colombiana strain of T. cruzi, we found that the distribution of CD4(+)/CD8(-) and CD4(-)/CD8(+) T cells in the myocardium mirrors the frequency of cells expressing the CD62L(Low)LFA-1(High)VLA-4(High) activation phenotype among CD4(+)/CD8(-) and CD4(-)/CD8(+ )peripheral blood T cells. Consistently, vascular cell adhesion molecule-1-positive endothelial cells and a fine fibronectin network surrounding VLA-4(+) mononuclear cells were found in the inflamed myocardium. Further, interferon gamma (IFN-gamma) and IFN-gamma-induced chemokines (RANTES, MIG and CRG-2/IP-10), as well as JE/MCP-1 and MIP1-alpha, were found to be the dominant cytokines expressed in situ during acute and chronic myocarditis elicited by T. cruzi. In contrast, interleukin 4 mRNA was only detected during the chronic phase. Altogether, the results indicate that the distribution of T-cell subsets in the myocardium of T. cruzi-infected mice reflects the particular profile of adhesion molecules acquired by most peripheral CD8(+) T lymphocytes and point to the possibility that multiple IFN-gamma-inducible molecules present in the inflamed tissue contribute to the establishment and maintenance of T. cruzi-induced myocarditis.

The function and phenotype of monocytes and granulocytes in the elderly is consistently remodelled. Because leucocyte adhesion molecules play important roles in mediating a wide variety of leucocyte functions, age-related changes in their expression on granulocyte and monocyte surfaces could be partially responsible for immune dysfunctions during senescence. Considering the central role of innate immunity in the process of immunosenescence and the involvement of cell adhesion molecules (CAM) in the great majority of leucocyte functions, we studied the expression of CD50 and CD62L adhesion molecules in peripheral blood granulocytes and monocytes from healthy elderly and young subjects. We show here that the percentage of granulocytes and monocytes expressing CD62L is decreased in the elderly, whereas its density expression is unchanged on both cell types. A downregulation of the density expression of CD50 at a per cell level characterizes granulocytes in the elderly, whereas CD50 expression on monocytes from old subjects shows a peculiar attitude: its density expression decreases whereas the number of positive cells is expanded. The downregulation of this receptor on granulocytes from aged people could determine a state of hyperactivation contributing to the proinflammatory status of the elderly, while the lower expression on monocytes could therefore contribute to the impaired antigen presentation in the elderly. On the other hand, the increased number of CD50 positive monocytes in the elderly, despite its decreased density expression at a per cell level, could be interpreted as an attempt to counteract the inability to mount strong immune responses. Both CD50 and CD62L changes in ageing polymorphonuclear (PMN) cells allow recognition as non-self or senescent self to permit macrophages in the liver and spleen to remove them from the circulation. The increased proportion of granulocytes and monocytes lacking CD62L and the downregulation of CD50 intensity expression on both cell types may suggest a state of in vivo activation. Therefore, CD50 and CD62L shedding from the cell surface of activated granulocytes and monocytes could be interpreted as a tentative to counteract the dangerous effects of an excessive chronic inflammation in the elderly. However, the increased proportion of CD62L negative granulocytes in the elderly leads to an impairment in cell adhesion which is the first line of response to acute inflammatory stimuli. This phenomenon likely contributes to the increased susceptibility to acute infections of elderly people.

To evaluate the therapeutic effects of the new synthetic sphingosine-1-phosphate (S1P) receptor modulator, FTY720, we investigated how FTY720 affects the development of dextran sulfate sodium (DSS)-induced colitis and CD4+CD62L+ T cell transfer colitis. BALB/c mice were fed a chow containing 3.5% (wt/wt) DSS to induce colitis. The CD4+CD62L+ T cell transfer colitis was induced by an intraperitoneal injection of CD4+CD62L+ spleen T cells into recipient CB17 SCID mice. The FTY720 was administered by lavage at a dose of 0.3 mg/kg/day. FTY720 was effective in preventing the body weight loss in the DSS-colitis model and the CD4+CD62L+ T cell transfer model. The disease activity index, histological colitis score, and MPO activity were all significantly lower in FTY720-treated mice than in the non-treated mice. Microscopically, mucosal edema, cellular infiltration and epithelial disruption were much more moderate in the FTY720-treated mice than in the non-treated mice. In both colitis models, FTY720 prevented the infiltration of CD4+ T cells into the inflamed colonic lamina propria. In conclusion, the development of DSS-colitis and CD4+CD62L+ T cell transfer colitis were significantly attenuated by FTY720. Since FTY720 is an immunosuppressive product that does not modulate T cell functions, it could be useful in the treatment of IBD patients.

It is well-known that acute stress, presumably as a first defense against pathogens, enhances PBMC counts by mobilizing these beta2-adrenoceptor positive cells from the marginal pool. Yet, only select leukocyte subsets participate in this phenomenon of adrenergic leukocytosis and underlying mechanisms are obscure. In this study, we analyzed in human blood adhesion molecule and chemokine receptor profiles in 14 leukocyte subsets, and responsiveness of subsets to epinephrine in vivo and in vitro. Five subsets, namely, CCR7(-)CD45RA(+)CD8(+) effector T cells, CD4(-)CD8(-) gamma/delta T cells, CD3(+)CD56(+) NKT-like cells, CD16(+)CD56(dim) cytotoxic NK cells, and CD14(dim)CD16(+) proinflammatory monocytes showed a rapid and transient increase after infusion of epinephrine at physiological concentrations. These cells were characterized by a CD62L(-)CD11a(bright)CX3CR(bright) phenotype, whereby expression of both CD11a and CX3CR1 was strongly correlated with adrenergic leukocytosis in vivo (r = 0.86 and 0.78, p < 0.005). The same subsets showed highest adherence to activated endothelium in vitro, which (except for proinflammatory monocytes) was reversed by epinephrine. We conclude that these five cytotoxic effector leukocyte subsets comprise the marginal pool by a CD11a/CX3CR1-mediated attachment to the endothelium. Epinephrine rapidly attenuates this attachment to allow demargination and release of the cells into the circulation that, because of their cytotoxic effector function, provide immediate protection from invading pathogens.