Transfer Of Wild-type Research Articles

Regulatory T cells crosstalk with tumor cells and endothelium through lymphotoxin signaling

Regulatory T cells (Tregs) with multifaceted functions suppress anti-tumor immunity by signaling surrounding cells. Here we report Tregs use the surface lymphotoxin (LT)α1β2 to preferentially stimulate LT beta receptor (LTβR) nonclassical NFκB signaling on both tumor cells and lymphatic endothelial cells (LECs) to accelerate tumor growth and metastasis. Selectively targeting LTβR nonclassical NFκB pathway inhibits tumor growth and migration in vitro. Leveraging in vivo Treg LTα1β2 interactions with LTβR on tumor cells and LECs, transfer of wild type but not LTα-/- Tregs promotes B16F10 melanoma growth and tumor cell-derived chemokines in LTβR-/- mice; and increases SOX18 and FLRT2 in lymphatic vessels of LTβR-/- melanoma. Blocking the nonclassical pathway suppresses tumor growth and lymphatic metastasis by reducing chemokine production, restricting Treg recruitment to tumors, and retaining intratumoral IFNγ+ CD8 T cells. Our data reveals that Treg LTα1β2 promotes LTβR nonclassical NFκB signaling in tumor cells and LECs providing a rational strategy to prevent Treg promoted tumor growth and metastasis.

Investigating the synaptic mechanisms of the spread of wild type and P301S 1N4R human tau in in vitro and in vivo models

AbstractBackgroundTau aggregates propagate across functionally connected neuronal networks in tauopathies as Alzheimer’s disease. However, the mechanisms underlying this process are poorly understood. Different studies showed that tau release is dependent on neuronal activity and that pathological tau can be found in the extracellular space confined in extracellular vesicles or in a free form. We recently showed that metabotropic glutamate receptors and SNAP25 modulate the release of pathologic tau from human and mouse synaptosomes. Here, with the use of blocking agents such as botulinum neurotoxins (BoNTs), we plan to test the effect on the synaptic release of tau in vitro and in vivo. BoNTs enter synapses and cleave specific synaptic SNARE proteins, impairing synaptic vesicle fusion and release. We aim at demonstrating the potential of BoNTs as tools to study neurodegenerative diseases.MethodsPrimary mouse hippocampal neurons were cultured in microfluidic chambers, transduced with lentiviruses expressing human tau (hTau) isoforms, and treated with BoNTs and stimulators. ELISA was used to quantify hTau in the culture media. AAVs expressing hTau were injected in the vitreous of wild‐type mice, and BoNT type A (BoNT/A) was injected in the superior colliculus (SC). Retina, SC and lateral geniculate nucleus (LGN) were analysed through immunohistochemistry.ResultsOverexpression of hTau in primary neurons increases the expression of synaptic markers as compared to non‐transduced cells, while any difference is detected between wild‐type and P301S hTau. Neuronal stimulation significantly increases the release of P301S hTau, whereas BoNT/A blocks it. Preliminary in vivo data show no difference in the expression and transfer of wild type and P301S hTau in the SC and LGN. The ongoing analysis of BoNT/A injected mice will reveal any effect on tau spread in the brain.ConclusionsThe release of hTau is modulated by specific SNARE proteins and differs depending on the isoform in vitro. The use of BoNT/A in vivo will help us to clarify the mechanisms modulating tau release in a complex system. Moreover, with the use of other BoNTs, we will provide novel insights into the mechanisms of synaptic tau release and identify novel molecular targets for the development of therapeutic interventions to treat tauopathies.

P-Selectin is a Critical Factor for Platelet-Mediated Protection on Restraint Stress-Induced Gastrointestinal Injury in Mice.

Psychological stress is associated with increased risk of gastrointestinal (GI) tract diseases. Evidence indicated that platelets facilitate GI tissue repair in intestinal anastomosis models. However, whether platelets are involved in native mechanism of the rescue of stress-induced GI injury for maintaining the GI homeostasis remains elusive. Because P-selectin-deficient (Selp−/−) mice displayed higher stress-induced GI injury compared to the wild-type (Selp+/+) mice, and P-selectin is specifically expressed in platelets, we hypothesize that P-selectin-expressing platelets play a protective role in the rescue of stress-induced GI injury. Our goal is to clarify the putative protective role of platelets in a GI system, thereby develop a feasible intervention strategy, such as platelet transfer, to overcome stress-induced GI injury. Through monitoring the plasma levels of GI-nonabsorbable Evans blue dye to reveal the progression course of GI injury in live mice, we found that intravenous treatments of purified platelets ameliorated stress-induced GI leakage. The transfer of platelets from wild-type mice was more potent than from Selp−/− mice in the rescue of stress-induced-GI leakage in the recipients. As such, platelet transfer-mediated rescue was conducted in a P-selectin dependent manner. Additionally, platelet-mediated protection is associated with corrections of stress-induced aberrant GI mRNA expressions, including tight junctions claudin 3 and occludin, as well as stress-induced genes activating transcription factor 3 and AMP-activated protein kinase, after the transfer of wild-type platelets into wild-type and Selp−/− mice. Furthermore, the stress-induced apoptosis of CD326+ GI epithelial cells was rescued by the transfer of wild type, but not P-selectin-deficient platelets. These results suggest that platelet plays a protective role for maintaining the GI homeostasis during stress in vivo, and that P-selectin is a molecular target for managing stress-induced GI tract injury.

Abstract 098: Foxp3+ T Regulatory Lymphocytes Counteract Angiotensin Ii-induced Vascular Injury

Objective: T effector lymphocytes contribute to vascular injury in angiotensin (Ang) II-induced hypertension, but the role of T regulatory lymphocytes (Tregs) is unclear. Ang II-induced hypertension is blunted in T and B lymphocyte-deficient (Rag1-/-) mice, and restored with reconstitution of T cells. We hypothesized that adoptive transfer of FOXP3-deficient Scurfy (Sf) vs. wild-type (WT) T cells will exacerbate Ang II-induced vascular damage in Rag1-/- mice. Methods: Eleven-week old male Rag1-/- mice were injected IV with vehicle, 10 million WT or Sf T cells, 1 million CD4+CD25+ Tregs alone or with Sf T cells, and 2 weeks later were infused or not with Ang II (490 ng/kg/min, SC) for 14 days (n=3-8). Telemetric BP, vascular function and structure, and reactive oxygen species (ROS) production and fibronectin expression in mesenteric arteries (MA) were determined. Results: Ang II induced a 40 mmHg systolic BP rise in all the groups, but diastolic BP rise was ~10 mmHg greater in WT and Sf T cell-injected mice than in controls (P<0.01). Treg injection alone or with Sf T cells prevented or delayed by 7 days the BP rise, respectively (P<0.05). Ang II did not induce endothelial dysfunction in vehicle or Treg only-injected mice. Adoptive transfer of WT T cells restored Ang II induced-endothelial dysfunction (60±5% vs. 83±4%, P<0.05), which was exaggerated in Sf T cell-injected mice (56±6% vs. 97±7%, P<0.01), but reduced by Treg co-injection (74±4%, P<0.05). Ang II increased ROS production in MA wall (239±32% vs. 119±20%) and perivascular fat (369±39% vs. 84±8%) in Sf T cell-injected mice (P<0.01), but not when co-injected with Tregs. Ang II induced increased vascular stiffness (P<0.01) and media/lumen (M/L, P<0.05) in vehicle (strain at 140 mmHg: 0.60±0.02 vs. 0.80±0.02; M/L: 4.1±0.2 vs. 2.9±0.2%) and Sf T cell-injected mice (strain at 140 mmHg: 0.63±0.01 vs. 0.89±0.04; M/L: 4.7±0.3 vs. 2.9±0.1%). Ang II increased MA fibronectin expression (P<0.01) in vehicle (113±12 vs. 51±14 RFU/μm2) and Sf T cell-injected mice (85±7 vs. 36±9 RFU/μm2). Conclusion: These results demonstrate that Foxp3+ Tregs have a protective role against Ang II-induced vascular dysfunction, remodeling and oxidative stress.

Elucidating Sources and Roles of Granzymes A and B during Bacterial Infection and Sepsis

During bacterial sepsis, proinflammatory cytokines contribute to multiorgan failure and death in a process regulated in part by cytolytic cell granzymes. When challenged with a sublethal dose of the identified mouse pathogen Brucella microti, wild-type (WT) and granzyme A (gzmA)(-/-) mice eliminate the organism from liver and spleen in 2 or 3 weeks, whereas the bacteria persist in mice lacking perforin or granzyme B as well as in mice depleted of Tc cells. In comparison, after a fatal challenge, only gzmA(-/-) mice exhibit increased survival, which correlated with reduced proinflammatory cytokines. Depletion of natural killer (NK) cells protects WT mice from sepsis without influencing bacterial clearance and the transfer of WT, but not gzmA(-/-) NK, cells into gzmA(-/-) recipients restores the susceptibility to sepsis. Therefore, infection-related pathology, but not bacterial clearance, appears to require gzmA, suggesting the protease may be a therapeutic target for the prevention of bacterial sepsis without affecting immune control of the pathogen.

Interleukin-10 Receptor Signaling in Innate Immune Cells Regulates Mucosal Immune Tolerance and Anti-Inflammatory Macrophage Function

Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4(+) T cell transfer, Rag2(-/-)Il10rb(-/-) mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb(-/-) anti-inflammatory macrophages ameliorated colitis induction by WT CD4(+) T cells in Rag2(-/-)Il10rb(-/-) mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.

Abstract 46: FOXP3+ T Regulatory Lymphocytes Counteract Angiotensin II-Induced Vascular Injury

Introduction: T lymphocytes participate in the low-grade inflammatory response that causes vascular injury in angiotensin (Ang) II-induced hypertension. Ang II-induced hypertension and endothelial dysfunction are blunted in T and B lymphocyte-deficient ( Rag1 -/- ) mice, and restored with reconstitution of T cells. However, the role of T regulatory lymphocytes (Treg) in Ang II-induced vascular injury is unclear. We hypothesized that adoptive transfer of FOXP3-deficient (Scurfy) T lymphocytes vs. wild-type (WT) cells will exacerbate Ang II-induced vascular damage in Rag1 -/- mice. Methods: Eleven-week old male Rag1 -/- mice were injected IV with PBS/2% FBS (control), 10 7 WT or Scurfy T lymphocytes, and 2 weeks later underwent sham surgery or were infused with Ang II (490 ng/kg/min, s.c.) using mini-osmotic pumps for 14 days (n=3-8). Systolic (SBP) and diastolic (DBP) blood pressure were measured by telemetry. Vascular function and structure were assessed in second order mesenteric arteries by pressurized myography. Reactive oxygen species (ROS) production and fibronectin and collagen I and III expression were determined in aorta. Results: Ang II induced a 40 mmHg SBP rise in Rag1 -/- mice for all treatment groups, but DBP rise was ~10 mmHg greater for WT and Scurfy T cell-injected mice than for control mice ( P <0.01). Adoptive transfer of WT T cells restored Ang II induced-endothelial dysfunction in mesenteric arteries ( P <0.05), which was exaggerated in Scurfy T cell-injected mice ( P <0.01). Ang II induced a greater increase in ROS production in aortic perivascular fat of Scurfy T cell-injected mice compared to WT T cell-injected mice ( P <0.05). Ang II induced mesenteric artery stiffness ( P <0.01) and hypertrophic remodeling ( P <0.05) in control and Scurfy T cell-injected mice, but not in WT T cell-injected mice. Ang II increased fibronectin expression to a greater extent in the aorta of control and Scurfy T cell-injected mice compared to WT T cell-injected mice ( P <0.01). Collagen I and III content was greater in the aorta of control and Scurfy T cell-injected mice than in WT T cell-injected mice ( P <0.01), but expression was unaltered by Ang II treatment. Conclusion: Foxp3+ T regulatory lymphocytes have a protective role against Ang II-induced vascular remodeling.

Macrophage A2A adenosinergic receptor modulates oxygen-induced augmentation of murine lung injury.

Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality. Exacerbating factors increasing the risk of ARDS remain unknown. Supplemental oxygen is often necessary in both mild and severe lung disease. The potential effects of supplemental oxygen may include augmentation of lung inflammation by inhibiting anti-inflammatory pathways in alveolar macrophages. We sought to determine oxygen-derived effects on the anti-inflammatory A2A adenosinergic (ADORA2A) receptor in macrophages, and the role of the ADORA2A receptor in lung injury. Wild-type (WT) and ADORA2A(-/-) mice received intratracheal lipopolysaccharide (IT LPS), followed 12 hours later by continuous exposure to 21% oxygen (control mice) or 60% oxygen for 1 to 3 days. We measured the phenotypic endpoints of lung injury and the alveolar macrophage inflammatory state. We tested an ADORA2A-specific agonist, CGS-21680 hydrochloride, in LPS plus oxygen-exposed WT and ADORA2A(-/-) mice. We determined the specific effects of myeloid ADORA2A, using chimera experiments. Compared with WT mice, ADORA2A(-/-) mice exposed to IT LPS and 60% oxygen demonstrated significantly more histologic lung injury, alveolar neutrophils, and protein. Macrophages from ADORA2A(-/-) mice exposed to LPS plus oxygen expressed higher concentrations of proinflammatory cytokines and cosignaling molecules. CGS-21680 prevented the oxygen-induced augmentation of lung injury after LPS only in WT mice. Chimera experiments demonstrated that the transfer of WT but not ADORA2A(-/-) bone marrow cells into irradiated ADORA2A(-/-) mice reduced lung injury after LPS plus oxygen, demonstrating myeloid ADORA2A protection. ADORA2A is protective against lung injury after LPS and oxygen. Oxygen after LPS increases macrophage activation to augment lung injury by inhibiting the ADORA2A pathway.

B cell development and immunoglobulin production are altered in the absence of IκBNS (109.4)

Abstract NF-κB transcription factors are key mediators of immune function as well as inflammatory responses. The activation of NF-κB is regulated by several IκB proteins, a family comprising classical cytoplasmic as well as nuclear IκBs. In this study, we focused on the role of IκBNS, a nuclear IκB protein, in B lymphocyte development and function. Analysis of lymphoid tissues from IκBNS knockout (KO) mice revealed that a lack of IκBNS affects the development of peritoneal B-1 cells and splenic marginal zone B cells. Development of B-2 cells was essentially normal. We also found that B cell proliferative activity and immunoglobulin production were altered in the absence of IκBNS. These defects were confirmed by the analysis of B cells in bone marrow chimeric mice constructed by the transfer of wild type or IκBNS KO bone marrow cells into B cell-deficient mice. Furthermore, histological analysis of spleens from chimeric mice revealed that re-constitution of B cell areas, marginal zones and germinal centers were markedly reduced in IκBNS KO cell recipient mice. Examination of the functions of IκBNS KO B cell subsets suggested that IκBNS is important for antibody production by both marginal zone and follicular B cells. These results indicate that IκBNS plays a key role in peripheral B cell development and function.

Role of LFA-1 in the activation and trafficking of T cells: implications in the induction of chronic colitis.

We have previously demonstrated that adoptive transfer of naïve CD4(+) T cells devoid of lymphocyte function-associated antigen-1-deficient (LFA-1; CD11a/CD18) into recombination activating gene-1 (RAG-1) deficient (RAG(-/-) ) mice fails to induce chronic colitis whereas transfer of wild type (WT) T-cells induces unrelenting and chronic disease. The objectives of this study were to assess the role of lymphocyte function-associated antigen-1 (LFA-1) in enteric antigen (EAg)-induced activation of T cells in vitro and in vivo and to define the importance of this integrin in promoting trafficking of T cells to the mesenteric lymph nodes (MLNs) and colon. We found that EAg-pulsed dendritic cells (DCs) induced proliferation of LFA-1-deficient (CD11a(-/-) ) CD4(+) T cells that was very similar to that induced using WT T cells, suggesting that LFA-1 is not required for activation/proliferation of T cells in vitro. Coculture of WT or CD11a(-/-) T cells with EAg-pulsed DCs induced the generation of similar amounts of interferon-gamma, interleukin (IL)-4, and IL-10, whereas IL-17A production was reduced ≈ 2-fold in cocultures with CD11a(-/-) T cells. Short-term (20-22 hours) trafficking studies demonstrated that while both WT and CD11a(-/-) T cells migrated equally well into the spleen, liver, lungs, small intestine, cecum, and colon, trafficking of CD11a(-/-) T cells to the MLNs was reduced by 50% when compared to WT T cells. When the observation period was extended to 3-7 days posttransfer, we observed ≈ 2-3-fold more WT T cells within the MLNs and colon than CD11a(-/-) T cells, whereas T-cell proliferation (as measured by CFSE dilution) was comparable in both populations. Taken together, our data suggest that LFA-1 is not required for EAg-induced activation of CD4(+) T cells in vitro or in vivo but is required for trafficking of T cells to the MLNs and homing of colitogenic effector cells to the colon where they initiate chronic gut inflammation.

α7 Nicotinic Acetylcholine Receptor (α7nAChR) Expression in Bone Marrow-Derived Non-T Cells Is Required for the Inflammatory Reflex

The immune response to infection or injury coordinates host defense and tissue repair, but also has the capacity to damage host tissues. Recent advances in understanding protective mechanisms have found neural circuits that suppress release of damaging cytokines. Stimulation of the vagus nerve protects from excessive cytokine production and ameliorates experimental inflammatory disease. This mechanism, the inflammatory reflex, requires the α7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel expressed on macrophages, lymphocytes, neurons and other cells. To investigate cell-specific function of α7nAChR in the inflammatory reflex, we created chimeric mice by cross-transferring bone marrow between wild-type (WT) and α7nAChR-deficient mice. Deficiency of α7nAChR in bone marrow-derived cells significantly impaired vagus nerve-mediated regulation of tumor necrosis factor (TNF), whereas α7nAChR deficiency in neurons and other cells had no significant effect. In agreement with recent work, the inflammatory reflex was not functional in nude mice, because functional T cells are required for the integrity of the pathway. To investigate the role of T-cell α7nAChR, we adoptively transferred α7nAChR-deficient or WT T cells to nude mice. Transfer of WT and α7nAChR-deficient T cells restored function, indicating that α7nAChR expression on T cells is not necessary for this pathway. Together, these results indicate that α7nAChR expression in bone marrow-derived non-T cells is required for the integrity of the inflammatory reflex.

Meningeal Immunity, Learning and Memory, and IL-4: Do T cells Make You Smart? (87.35)

Abstract T cells were recently shown to support learning and memory, although the underlying mechanisms for this phenomenon are yet unknown. Here, we show that severe combined immune deficient (SCID) mice, shown to be impaired in assays of learning and memory, display a pronounced pro-inflammatory (M1) skew of meningeal myeloid cells. Acute lymphopenia induced by FTY720, a sphingosine-phosphate receptor agonist, or blockade of T cell migration into the meninges using anti-VLA4 antibodies, results in similar cognitive impairment and M1-skewed meningeal myeloid phenotype in wild type mice. The role of IL-4 is critical: we show accumulation of IL-4+ CD4+ T cells in the meninges of wild type mice after MWM training. Moreover, IL-4 knockout mice exhibit profound cognitive impairment and M1 skew. Transfer of IL-4 knockout bone marrow to wild type mice results in meningeal myeloid inflammation and impaired learning, while transfer of wild type T lymphocytes to IL-4 knockout mice is sufficient to rescue both behavioral phenotype and M1 skew. Our results point to a critical role for meningeal T cells, and for IL-4 in particular, in the regulation of meningeal myeloid cells and concomitant cognitive function. These results might lead to development of new immune-based therapies for cognitive conditions associated with immune decline, such as HIV- and age-associated dementia.

A key role for Fas ligand expressed on B cells in initiation of autoimmune diabetes (99.17)

Abstract Type-1 diabetes (T1D) is an organ specific autoimmune disease that affects children. T, B and dendritic cells infiltrate islets in the early stage of disease leading to insulitis followed by islet destruction and hyperglycemia. Development of T1D in non-obese diabetic (NOD) mice is completely inhibited by gld or lpr mutations of Fas and FasL, respectively, even though mutant mice develop T cell lymphoproliferation and the Fas pathway is not essential for T cell activation. Recently, we have shown heterozygous gld mutation (gld/+) provides complete protection without causing lymphoproliferation. Using NOD-gld/+ mouse as a model, we found that the protection is not due to deletion of autoreactive T cells or switching of T helper polarization. Islet-reactive BDC2.5 TCR transgenic CD4 T cells transferred into NOD-gld/+ mice were efficiently primed in the pancreatic lymph nodes but failed to accumulate in the pancreas. FasL deficiency selectively reduced expression of CD40 and frequency of B cells in the pancreas. Transfer of wild type B cells into NOD-gld/+ mice results in mobilization of endogenous T cells particularly CD8 T cells and of co-transferred BDC2.5 T cells resulting in severe insulitis. These results provide the first evidence that B cells play an essential role in gld-mediated protection from T1D and that expression of functional FasL on B cells is key for initiation insulitis. Supported by grants from AHA and JDRF.

CD40/CD40L contributes to hypercholesterolemia-induced microvascular inflammation

Hypercholesterolemia is associated with phenotypic changes in endothelial cell function that lead to a proinflammatory and prothrombogenic state in different segments of the microvasculature. CD40 ligand (CD40L) and its receptor CD40 are ubiquitously expressed and mediate inflammatory responses and platelet activation. The objective of this study was to determine whether CD40/CD40L, in particular T-cell CD40L, contributes to microvascular dysfunction induced by hypercholesterolemia. Intravital microscopy was used to quantify blood cell adhesion in cremasteric postcapillary venules, endothelium-dependent vasodilation responses in arterioles, and microvascular oxidative stress in wild-type (WT) C57BL/6, CD40-deficient ((-/-)), CD40L(-/-), or severe combined immune deficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 wk. WT-HC mice exhibited an exaggerated leukocyte and platelet recruitment in venules and impaired vasodilation responses in arterioles compared with ND counterparts. A deficiency of CD40, CD40L, or lymphocytes attenuated these responses to HC. The HC phenotype was rescued in CD40L(-/-) and SCID mice by a transfer of WT T cells. Bone marrow chimeras revealed roles for both vascular- and blood cell-derived CD40 and CD40L in the HC-induced vascular responses. Hypercholesterolemia induced an oxidative stress in both arterioles and venules of WT mice, which was abrogated by either CD40 or CD40L deficiency. The transfer of WT T cells into CD40L(-/-) mice restored the oxidative stress. These results implicate CD40/CD40L interactions between circulating cells and the vascular wall in both the arteriolar and venular dysfunction elicited by hypercholesterolemia and identify T-cell-associated CD40L as a key mediator of these responses.

TNF Receptor-Associated Factor 1 Expressed in Resident Lung Cells Is Required for the Development of Allergic Lung Inflammation

TNF is a major therapeutic target in a range of chronic inflammatory disorders, including asthma. TNFR-associated factor (TRAF)1 is an intracellular adaptor molecule important for signaling by TNFR. In this study, we investigated the role of TRAF1 in an adoptive transfer model of allergic lung inflammation. Mice deficient in TRAF1 (TRAF1(-/-)) and wild-type (WT) control animals were adoptively transferred with WT OVA-immune CD4(+) T cells, exposed to an aerosol of LPS-free OVA, and analyzed for the development of allergic lung inflammation. In contrast to WT mice, TRAF1(-/-) recipients failed to display goblet cell hyperplasia, eosinophilic inflammation, and airway hyperresponsiveness in this model of asthma. Neither T cell recruitment nor expression of the proinflammatory cytokines IL-4, IL-5, IL-13, or TNF occurred in the lungs of TRAF1(-/-) mice. Although purified myeloid TRAF1(-/-) dendritic cells (DCs) exhibited normal Ag-presenting function and transmigratory capacity in vitro and were able to induce OVA-specific immune responses in the lung draining lymph nodes (LNs) following adoptive transfer in vivo, CD11c(+)CD11b(+) DCs from airways of TRAF1(-/-) recipients were not activated, and purified draining LN cells did not proliferate in vitro. Moreover, transfer of WT or TRAF1(-/-) DCs failed to restore T cell recruitment and DC activation in the airways of TRAF1(-/-) mice, suggesting that the expression of TRAF1 in resident lung cells is required for the development of asthma. Finally, we demonstrate that T cell-transfused TRAF1(-/-) recipient mice demonstrated impaired up-regulation of ICAM-1 expression on lung cells in response to OVA exposure.

Abstract 1128: The Leukocyte Integrin Mac-1 Promotes Acute Cardiac Allograft Rejection

Background: In allograft rejection, donor endothelial cells (EC) are the principal early targets of host leukocytes and alloantibodies. Although the leukocyte integrin Mac-1(αmβ2, CD11b/ CD18) facilitates leukocyte-leukocyte as well as leukocyte-EC interactions, its roles in allograft survival and vasculopathy remains unclear. This study used murine cardiac transplantation to test the hypothesis that Mac-1 deficiency in the recipient reduces accumulation of graft infiltrating cells and attenuates cardiac allograft rejection. Results: In total-allomismatched murine cardiac allografts (BALB/c donor hearts and C57BL/6 recipients), survival averaged 13.8 ± 2.3 days (n=6) for allografts in Mac-1-deficient (Mac-1−/−) recipients compared to 8.3 ± 1.3 days (n = 18, p<0.0001) in wild-type (WT) recipients. At 7 d post-transplantation, allografts in Mac-1−/− recipients had significantly lower parenchymal rejection (PR) scores (2.3 ± 0.4) than WT recipients (3.0 ± 0.4, p<0.01). Allograft accumulation of neutrophils, T cells, and macrophages (Mϕ) significantly diminished in Mac-1−/− compared to WT recipients. To determine the cell type involved in acute graft dysfunction, we performed adoptive transfer experiments. Adoptive transfer of WT, but not Mac-1−/−, Mϕ exacerbated parenchymal rejection of 7-day allografts placed into Mac-1−/− recipients (PR score: 2.3 ± 0.4 for WT vs. 1.5 ± 0.5 for Mac1−/− Mϕ; p < 0.05), and significantly reduced graft survival (8.0 ± 1.4 vs. 13.5 ± 2.6 days, p<0.01). Neither WT nor Mac-1−/− neutrophil adoptive transfer affected graft survival in Mac-1−/− recipients. Mac-1−/− Mϕ co-cultured with allo-EC (BALB/c) expressed significantly lower levels of the co-stimulatory molecules CD40 and CD80, and mixed leukocyte reaction using allo-EC-primed Mac-1−/− Mϕ showed significantly lower antigen-presenting function than WT Mϕ. Despite attenuating acute rejection, recipient Mac-1-deficiency did not prevent graft arterial disease. Conclusion: These studies demonstrate that Mac-1 contributes to acute allograft rejection by modulating macrophage recruitment, antigen presentation, and T cell priming. These observations establish a molecular target for modulating recipient responses to prolong graft survival.

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-α-deficient (LTα −/−) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer’s patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF. Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LTα −/− mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.

T-Cell–Derived Interferon-γ Contributes to Arteriolar Dysfunction During Acute Hypercholesterolemia

T-lymphocytes and interferon-gamma (IFN-gamma) contribute to leukocyte recruitment in postcapillary venules during hypercholesterolemia. Our objectives were to determine whether: (1) T-lymphocytes are the source of this IFN-gamma, and (2) whether T-cell-derived IFN-gamma also mediates the accompanying arteriolar dysfunction and platelet adhesion. Intravital videomicroscopy was used to quantify arteriolar responses to acetylcholine, and leukocyte and platelet adhesion in postcapillary venules of wild-type (WT), immunodeficient (SCID), and IFN-gamma(-/-) mice on a normal (ND) or high-cholesterol (HC) diet. Acetylcholine-induced arteriolar dilation was impaired in WT-HC, compared with WT-ND. This endothelial dysfunction was absent in SCID-HC or IFN-gamma(-/-)-HC mice. Vasodilation was impaired by transfer of WT, but not IFN-gamma(-/-), T-cells to these immunodeficient mice. WT-HC mice exhibited elevated leukocyte and platelet adhesion in venules, versus WT-ND. This blood cell recruitment was attenuated to ND levels in SCID-HC and IFN-gamma(-/-)-HC mice, but restored to WT-HC levels by transfer of WT, but not IFN-gamma(-/-), T-lymphocytes. These data reveal a novel role of T-lymphocyte-derived IFN-gamma in the development of endothelial dysfunction in arterioles during hypercholesterolemia and extend our previous observations that IFN-gamma mediates both inflammatory and thrombogenic responses to hypercholesterolemia in postcapillary venules.

Identification of a novel subset of activation induced deaminase (AID)-dependent B-1 cells that mediate Initiation of Contact Sensitivity (37.10)

Abstract Contact sensitivity (CS) to haptens is related to delayed type hypersensitivity (DTH) and is a well characterized prototype of T cell mediated inflammation. CS is additionally dependent on a parallel immune pathway. In response to sensitization, a population of B-1 cells produces antigen-specific IgM which mediates a rapid inflammatory response to hapten re-exposure. Interference with this pathway prevents the full development of the classic delayed response and is therefore termed the “CS-Initiation” pathway. The identity of the B-1 cell subset responsible for CS-Initiation is not known. Here, we show that CS-Initiation is dependent on activation induced deaminase (AID), an enzyme central to the process of somatic hypermutation (SHM). Using adoptive transfer experiments, we demonstrate that the defect is specific to B-1 cells as transfer of wild type B-1 cells reconstitutes CS-Initiation in deficient recipients. We went on to identify and characterize a unique subpopulation of B-1 cells in the spleens of sensitized mice that possess initiation activity. Detailed analysis of B cell receptor genes isolated from these cells revealed evidence of AID-mediated SHM. The sensitivity of CS-Initiation to very low amounts of hapten and the dependence on AID suggests that these cells are selected to generate antibodies capable of mediating CS-Initiation. This work funded by the Canadian Institutes of Health Research, the American Academy of Allergy, Asthma and Immunology and the National Institutes of Health.

Transmembrane TNF Is Sufficient to Initiate Cell Migration and Granuloma Formation and Provide Acute, but Not Long-Term, Control ofMycobacterium tuberculosisInfection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.