Topical Adjuvant Application during Subcutaneous Vaccination Promotes Resident Memory T Cell Generation.

Skin tissue resident memory T cells (TRM) provide superior protection to a second infection. In this study, we evaluated the use of topical CpG oligodeoxynucleotide (ODN) as adjuvant to generate skin TRM in mice. Topical or s.c. CpG ODN adjuvant administration at the time of a s.c. Ag injection led to an accumulation of CD103- CD8 T cells in the epidermis. However, only mice with CpG ODN administered topically had significant numbers of CD103+ Ag-specific CD8 T cells persisting in the local epidermal skin, enhanced circulating memory cells in the blood, and showed protection from intradermal challenge with melanoma cells. Generation of Ag-specific CD8 T cells was dependent on TLR9 expression on hematopoietic cells and partially dependent on receptor expression on stromal cells. Topical challenge of immunized mice at a distal site led to significant expansion of Ag-specific T cells in the blood and accumulation in the challenged skin. We demonstrate that local and systemic T cell memory can be generated with topical CpG ODN at the time of s.c. immunization, suggesting a new method of enhancing current vaccine formulations to generate tissue TRM.

Interleukin-7 in the transition of bone marrow progenitors to the thymus.

Interleukin-7 (IL-7) is essential for the development of T cells in humans and mice where deficiencies in IL-7 signaling result in severe immunodeficiency. T cells require IL-7 at multiple points during development; however, it is unclear when IL-7 is first necessary. We observed that mice with impaired IL-7 signaling had a large reduction in the number of early thymic progenitors (ETPs) while mice that overexpress IL-7 had greatly increased numbers of ETPs. These results indicated that the development of ETPs is sensitive to IL-7. Bone marrow progenitors of ETP are present in normal numbers in mice with impaired IL-7 signaling (IL-7Rα449F) and were efficiently recruited to the thymus. Furthermore, ETPs and their progenitors from IL-7Rα449F mice did not undergo increased apoptosis and proliferate normally compared to WT cells. Mixed bone marrow chimeras demonstrated that IL-7 signaling has a cell-intrinsic role in ETP development but was not required for development of bone marrow progenitors. We have shown a novel role for IL-7 signaling in the development of ETPs that is distinct from classic mechanisms of IL-7 regulating survival and proliferation.

PSGL-1 regulates the migration and proliferation of CD8(+) T cells under homeostatic conditions.

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.

PSGL-1 function in immunity and steady state homeostasis.

The substantial importance of P-selectin glycoprotein ligand 1 (PSGL-1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL-1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin-PSGL-1 interaction requires branched O-glycan extensions on specific PSGL-1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O-glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL-1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O-glycans on PSGL-1 and provide the molecular interactions responsible for the velcro-like catch bonds that support leukocyte rolling. Such binding of PSGL-1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL-1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T-cell homing to secondary lymphoid organs. PSGL-1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O-glycan modifications of PSGL-1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL-1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T-cell traffic. The multi-level influence of PSGL-1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O-glycans. However, central as specific O-glycosylation is to PSGL-1 function, in vivo regulation of PSGL-1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL-1 glycosylation and to update understanding of PSGL-1 functional scope.

Cutaneous CpG adjuvant conditioning to enhance vaccine responses.

Adjuvant activity of the Toll receptor 9 agonist CpG 1826 was compared when given subcutaneously (s.c.) together with ovalbumin (s.c.[CpG + Ova]), or when given by either s.c. or intradermally (i.d.) routes two days prior to s.c. ovalbumin. Frequencies of CD8 + effector (TEFF) and central memory (TCM) T cells along with total IgG, IgG2c, and IgG1 titres were measured to ascertain how timing and location of CpG conditioning influenced vaccination outcome. Prior treatment with CpG enhanced TEFF, TCM, as well as total IgG responses. TEFF and TCM responses were greatest when CpG was given intradermally and prior to s.c. ovalbumin, conditions that eliminated the fraction of TCM 'non-responders' observed after s.c.[CpG + Ova] vaccination. IgG responses were polarized toward IgG2c after early s.c. CpG but toward IgG1 after early i.d. CpG. Separating CpG adjuvant and antigen application in time and space can improve vaccination outcome.

Lymphocytes in the peritoneum home to the omentum and are activated by resident dendritic cells.

The omentum is of interest in the context of obesity-related metabolic disease where adipose tissue exhibits inflammatory changes; however, the immunology of the omentum is underexplored. The greater omentum is draped from the stomach and consists predominantly of adipose tissue studded with lymphoreticular aggregations (milky spots) that distinguish it from other visceral adipose tissues. Milky spots are thought to contain and conduct leukocytes in transit from the blood to the peritoneal cavity, particularly during peritonitis. We show here that both B and T lymphocytes counterflow from the peritoneal cavity to the omentum in mice. Residence in the omentum was brief with a t(1/2) residence time of 6 h. Omentum access was pertussis toxin-sensitive, dependent on activation of the Rap1 GTPase, and on the integrin LFA-1. B cells and CD44(high) T cells accessed the omentum most efficiently, but homing of resting CD44(low) T cells was also observed. Omental tissue from normal healthy mice was found to contain CD8(-)CD11b(high)MHC class II(high)CD11c(high) dendritic cells that promoted the rapid activation of T cells entering the omentum and cross-presented soluble OVA or OVA acquired from either OVA-expressing Escherichia coli or OVA-pulsed spleen cells. We conclude that the omentum incorporates two key features of immunological sentinel function, actively supported lymphocyte traffic and dendritic cells, that reinforce a conceptual framework for function in stimulating adaptive immunity. These results extend basic understanding of omental and peritoneal cavity immunology and of how proinflammatory events occurring within the peritoneal cavity might affect adipocyte and hepatocyte metabolism.

A cell-extrinsic ligand acquired by activated T cells in lymph node can bridge L-selectin and P-selectin.

P-selectin expressed on activated endothelia and platelets supports recruitment of leukocytes expressing P-selectin ligand to sites of inflammation. While monitoring P-selectin ligand expression on activated CD8+ T cells in murine adoptive transfer models, we observed two distinct ligands on responding donor cells, the canonical cell-intrinsic P-selectin ligand PSGL-1 and a second undocumented P-selectin ligand we provisionally named PSL2. PSL2 is unusual among selectin ligands in that it is cell-extrinsic, loaded onto L-selectin expressed by activated T cells but not L-selectin on resting naïve CD8+ T cells. PSL2 display is highest on activated T cells responding in peripheral lymph nodes and low on T cells responding in spleen suggesting that the original source of PSL2 is high endothelial venules, cells known to produce L-selectin ligands. PSL2 is a ligand for both P-selectin and L-selectin and can physically bridge the two selectins. The L-selectin/PSL2 complex can mediate P-selectin-dependent adherence of activated T cells to immobilized P-selectin or to activated platelets, either independently or cooperatively with PSGL-1. PSL2's capacity to bridge between L-selectin on activated T cells and P-selectin reveals an undocumented and unanticipated activity of cell-extrinsic selectin ligands in mediating selectin-selectin connectivity. The timing and circumstances of PSL2 detection on T cells, together with its capacity to support adherence to P-selectin-bearing substrates, are consistent with P-selectin engagement of both PSGL1 and the L-selectin/PSL2 complex during T cell recruitment. Engagement of PSGL-1 and L-selectin/PSL2 would likely deliver distinct signals known to be relevant in this process.

Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs.

P-selectin glycoprotein ligand 1 (PSGL-1) is central to the trafficking of immune effector cells to areas of inflammation through direct interactions with P-selectin, E-selectin and L-selectin. Here we show that PSGL-1 was also required for efficient homing of resting T cells to secondary lymphoid organs but functioned independently of selectin binding. PSGL-1 mediated an enhanced chemotactic T cell response to the secondary lymphoid organ chemokines CCL21 and CCL19 but not to CXCL12 or to inflammatory chemokines. Our data show involvement of PSGL-1 in facilitating the entry of T cells into secondary lymphoid organs, thereby demonstrating the bifunctional nature of this molecule.

CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency.

Using noncompetitive methodologies comparing CD43(+/+) and CD43(-/-) mice, it has been reported that CD43(-/-) leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43(-/-) T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43(+/+) vs CD43(-/-) activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43(+/+) and congenic CD43(-/-) mice in either B6 or P-selectin(-/-) recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

Inducing P-selectin ligand formation in CD8 T cells: IL-2 and IL-12 are active in vitro but not required in vivo.

In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2(null) CD8 T cells responding in doubly deficient IL-2(null)IL-12(null) or IL-2(null)IL-15(null) male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.

Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1.

The molecular mechanisms that direct the migration of early T lymphocyte progenitors to the thymus are unknown. We show here that P-selectin is expressed by thymic endothelium and that lymphoid progenitors in bone marrow and thymus bind P-selectin. Parabiosis, competitive thymus reconstitution and short-term homing assays indicated that P-selectin and its ligand PSGL-1 are functionally important components of the thymic homing process. Accordingly, thymi of mice lacking PSGL-1 contained fewer early thymic progenitors and had increased empty niches for prothymocytes compared with wild-type mice. Furthermore, the number of resident thymic progenitors controls thymic expression of P-selectin, suggesting that regulation of P-selectin expression by a thymic 'niche occupancy sensor' may be used to direct progenitor access.

Modulation of O-glycans and N-glycans on murine CD8 T cells fails to alter annexin V ligand induction by galectin 1.

Thymic negative selection and contraction of responding T cell oligoclones after infection represent important cell ablation processes required for maintaining T cell homeostasis. It has been proposed that galectin 1 contributes to these processes through interaction with lactosyl sequences principally on cell surface glycoproteins bearing core 2 (C2GnT1)-branched O-glycans. According to this model, specific T cell surface proteins cross-linked by galectin 1 induce signaling, ligand redistribution, and apoptosis in both immature thymocytes and activated T cells. The influence of lactosyl residues contained in branched O-glycans or complex N-glycans on galectin 1 binding and induction of annexin V ligand in murine CD8 T cells was assessed. Neither galectin binding nor galectin-induced expression of annexin V ligand was perturbed under conditions in which: 1) C2GnT1 activity was differentially induced by CD8 T cell activation/culture with IL-2 vs IL-4; 2) activated CD8(+) T cells lacked C2GnT1 expression; or 3) complex N-glycan formation was blocked by swainsonine. The maintenance of galectin 1 binding and induced annexin V expression under conditions that alter lactosamine abundance on O- or complex N-glycans suggest that galectin 1-mediated apoptosis is neither a simple function of fluctuating C2GnT1 activity nor a general C2GnT1-dependent mechanism underlying contraction of CD8 T cells subsequent to activation.

Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model.

OBJECTIVE: To establish the generality of cerebrovascular pathology frequently observed with Alzheimer disease, we have assessed blood-brain barrier (BBB) integrity using the Alzheimer disease model Tg2576 mice in which cognitive deficits and neuritic plaque formation develop around 10-12 months of age. METHODS: We assessed BBB integrity using well-established methods involving albumin and Evans blue uptake and introduce the use of a novel perfusion protocol using succinimidyl ester of carboxyfluorescein diacetate. RESULTS: BBB permeability is increased in the cerebral cortex of 10-month-old Tg2576 mice preceding Alzheimer disease pathology presentation. Furthermore, when compared with their nontransgenic littermates, 4-month-old Tg2576 mice exhibit compromised BBB integrity in some areas of the cerebral cortex. An age-related increase in albumin uptake by the brains of Tg2576 mice, compared with nontransgenic mice, was also observed. These findings were supported by quantitative Evans blue analysis (p = 0.07, two-way analysis of variance). CONCLUSION: A breakdown of BBB was evident in young 4- to 10-month-old Tg2576 mice. Compromised barrier function could explain the mechanisms of Abeta entry into the brain observed in experimental Alzheimer disease vaccination models. Such structural changes to the BBB caused by elevated Abeta could play a central role in Alzheimer disease development and might define an early point of intervention for designing effective therapy against the disease.

Identification and functional characterization of voltage-dependent calcium channels in T lymphocytes.

In T lymphocytes, sustained calcium (Ca2+) influx through Ca2+ channels localized in the plasma membrane is critical for T cell activation and proliferation. Previous studies indicated that voltage-dependent Ca2+ channels (VDCCs) play a role in Ca2+ mobilization during T lymphocyte activation. However, the role of VDCCs in otherwise nonexcitable cells is still poorly understood. We used RT-PCR to identify a transcript encoding the pore-forming alpha1F-subunit of an L-type Ca2+ channel in T lymphocytes. Its identity was confirmed by DNA sequencing. To further investigate the contribution of Ca2+ influx through VDCCs, we assessed the effects of the 1,4-dihydropyridine L-type Ca2+ channel agonist, (+/-) Bay K 8644, and antagonist, nifedipine, on the human Jurkat T cell leukemia line, human peripheral blood T lymphocytes and mouse splenocytes. We found that treatment of T lymphocytes with (+/-) Bay K 8644 increased intracellular Ca2+ and induced the activation of phosphoextracellular-regulated kinase 1/2 (Erk1/2), whereas nifedipine blocked Ca2+ influx, the activity of Erk1/2 and nuclear factor of activated T cells (NFAT), interleukin-2 (IL-2) production, and IL-2 receptor expression. Nifedipine also significantly suppressed splenocyte proliferation in an in vitro mixed lymphocyte reaction and the proliferation of male antigen (H-Y)-specific T cell receptor-transgenic CD8+ T cells in transplanted male mice in vivo. Taken together these novel findings indicate that an L-type Ca2+ channel plays a significant role in the Ca2+ influx pathways mediating T lymphocyte activation and proliferation in vitro and in vivo.