TM Cells Research Articles

Regulatory dendritic cells program generation of interleukin-4–producing alternative memory CD4 T cells with suppressive activity

AbstractThe heterogeneity and mechanisms for the generation of CD4 memory T (CD4 Tm) cells remain elusive. Distinct subsets of dendritic cells (DCs) have been found to regulate a distinct T-helper (Th)–cell subset differentiation by influencing cytokine cues around CD4 T cells; however, whether and how the regulatory DC subset can regulate Tm-cell differentiation remains unknown. Further, there is no ideal in vitro experimental system with which to mimic the 3 phases of the CD4 T-cell immune response (expansion, contraction, memory generation) and/or to culture CD4 Tm cells for more than a month. By analyzing CD4 T cells programmed by long-term coculture with regulatory DCs, we identified a population of long-lived CD4 T cells with a CD44hiCD62L−CCR7− effector memory phenotype and rapid, preferential secretion of the Th2 cytokines interleukin-4 (IL-4), IL-5, IL-10, and IL-13 after antigenic stimulation. These regulatory DC-programmed Tm cells suppress CD4 T-cell activation and proliferation in vitro via IL-10 and inhibit the delayed-type hypersensitivity response once infused in vivo. We also identify their natural counterpart, which is up-regulated by regulatory DC transfusion and negatively regulates the recall response in vivo. Different from interferon-γ–producing conventional Tm cells, these IL-4–producing CD4 Tm cells act as alternative Tm cells with a regulatory function, suggesting a new way of negative immune regulation by memory T cells.

LFA-1–specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.

Combined Application of Blocking Antibodies and MicroRNA Interference in Inhibiting CD44 Expression

Background We sought to explore the effect of CD44 targeting on the tolerance to memory cell-mediated graft rejection. Methods We developed a cardiac transplantation model in nude mice and administered anti-CD44 monoclonal antibodies (mAbs) to these mice. Then, we used anti-CD44 mAb and CD44-interfering microRNA (miRNA) to inhibit CD44 expression in vitro. Results The median survival time (MST) associated with multiple intraperitoneal injections was >100 days, whereas that associated with CD4 + Tm cells blocked CD44 and that associated with a single intraperitoneal injection of anti-CD44 mAb was 11 and 10.3 days, control group was 5.5 days. The inhibition effect of the anti-CD44 mAb in 3T3 cells significantly reduced with cell proliferation. Used CD44 miRNA in 3T3 cells, the most obvious inhibition effect of mRNA appeared at 48 hours after transfection and the inhibition decreased subsequently. In combination, antibody-mediated blocking and miRNA showed some synergistic effects. Conclusion The inhibition of CD44 can significantly prolong the MST in memory models. The inhibition effect of combined application showed limitations with regard to cell proliferation and duration of action, but the short-term synergistic effect of the combined approach was stronger than the effects of individual approaches.

Lowering TCR expression on naive CD8+ T cells does not affect memory T‐cell differentiation

The generation of long-lived memory T (Tm) cells is critical for the success of vaccination, but the factors controlling their differentiation are still poorly defined. We examined the hypothesis that the level of T-cell receptor (TCR) engagement contributed to memory CD8(+) T-cell generation. By manipulating TCR expression levels on murine, naive ovalbumin (OVA)-specific CD8(+) T cells, we showed that the expansion of antigen (Ag)-specific CD8(+) T cells is minimally affected by the level of TCR expression. Indeed, naive CD8(+) T cells expressing as little as a 1000 TCRs (30-fold less) show only a 2.5-fold reduction in the number of effectors generated. Furthermore, the TCR expression levels influenced neither the acquisition of effector functions nor the generation of functional Tm cells. Our data indicate that during an in vivo immune response, a threshold in the number of TCRs engaged by naive CD8(+) T cells is required for full T-cell expansion but not for their differentiation into effector and Tm cells.

Histone Acetylation at the Single-Cell Level: A Marker of Memory CD8+ T Cell Differentiation and Functionality

Following stimulation, memory T (T(M)) cells rapidly express many effector functions, a hallmark feature that allows them to provide protective immunity. Recent studies suggest that genes involved in this rapid recall response may maintain an open chromatin structure in resting T(M) cells via epigenetic modifications. However, these studies have mostly focused on a few loci, and the techniques used required a large number of cells. We have developed a flow cytometric assay measuring histone modifications in individual murine T cells in combination with lineage-specific markers. In this study, we show that the per-cell level of a marker of open chromatin, diacetylated histone H3 (diAcH3), increases as naive CD8(+) T cells develop into T(M) cells, demonstrating a novel correlation between the differentiation state of a CD8(+) T cell and its abundance of a specific histone modification. Furthermore, our results show that T(M) cells defective in rapid recall ability have less diAcH3 than their fully functional counterparts, indicating that the diAcH3 level of individual T(M) cells is a useful marker for assessing their functionality.

DN-Treg cells induce long-term cardiac allograft survival in fully MHC mismatched recipients but do not eliminate T memory cells (145.13)

Abstract Establishment of immune tolerance is a major goal in transplantation. Regulatory T (Treg) cells play an important role in the regulation of immune responses. Our previous studies have shown that TCRαβ+CD3+CD4-CD8-NK1.1- (double negative, DN) Treg cells suppress anti-donor T cell responses and prolong allograft survival in a single MHC-mismatched mouse model. In this study, we tested whether DN-Treg cells can induce long-term graft survival in the stringent BALB/c to C57BL/6 heart transplant model. Adoptive transfer of 107 DN-Treg cells in combination with rapamycin (RAPA) treatment (day 1- 9) induced long-term graft survival (101 vs. 39 days RAPA alone, p<0.01). DN-Treg treated recipients had few graft-infiltrating CD4+ and CD8+ T cells on day 40 compared to massive infiltration in RAPA treated controls. Memory T (Tm) cells (CD3+CD44+) were found to be resistant to DN-Treg cell-mediated suppression in vitro. The effect of DN-Treg on Tm in vivo was tested in B6-Rag1-/- mice. DN-Treg cells along with CD44+ Tm cells modestly prolonged BALB/c skin graft survival in contrast to DN-Treg with CD44- T cells (26 vs 43 days, P<0.001). In conclusion, DN-Treg cells combined with short term immunosuppression, can prolong allograft survival in the absence of tolerance. Furthermore, this may be related to the resistance of Tm cells to DN-Treg cell suppression, suggesting alternative strategies will be needed to control Tm cells and establish tolerance after transplantation.

Staphylococcus aureus specific memory CD8 T cells in dairy cows (40.12)

Abstract Staphylococcus aureus is a versatile pathogen that causes disease in both humans and animals. An increase in occurrence of nosocomial infections and chronic mastitis caused by antibiotic resistant strains warrants the need for an effective vaccine. This study assessed the presence of S. aureus specific memory T cells (Tm) in previously infected cows using CD14+ monocyte derived dendritic cells (DC). Results indicate that DC are able to uptake and recognize S. aureus antigens through various pattern recognition receptors (PRRs). Blocking of endocytic pathways using inhibitors confirmed S. aureus uptake by DC. S. aureus stimulated DC showed increased cell surface expression of MHC I and II and cytokine production suggesting activation and antigen presentation. An initial screening study for Tm in cows previously infected with S. aureus showed a significant increase in lymphocyte proliferation compared to control animals. To characterize the proliferating population of Tm, CFSE labeled lymphocytes were co-cultured with irradiated or live S. aureus stimulated autologous DC from previously infected cows identified as CD4, CD8 and WC1+ γδ T cells by flow cytometry. Proliferated CD4 and CD8 T cells were further characterized for memory markers, CD45RO and CD62L. Being the first study to show the presence of S. aureus specific CD8 Tm cells, our next step is to identify the S. aureus antigens that initiate T cell clonal expansion.

Memory CD8. + T Cells Specific for Minor Histocompatibility Antigen H60 Can Mediate the Graft-Versus-Leukemia Effect.

Abstract 1332Poster Board I-354In allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells mediate graft-vs-leukemia (GVL) but also cause graft-vs.-host disease (GVHD). Our previous studies indicate that memory T cells (TM) induce less GVHD than do naïve T cells (TN). Therefore the selective infusion of memory T cells could improve immune reconstitution with less GVHD. However, human memory T cells have a more restricted TCR repertoire than do TN and therefore may be less effective in mediating GVL. One approach for addressing this concern would be to vaccinate donors against a single miHA expressed by host leukemic cells. Then the selective transfer of memory T cells could improve both GVL and immune reconstitution with less GVHD. Questions remain about how best to apply this strategy. For example, differences in target antigen distribution could affect the re-expansion of transferred memory cells and determine whether vaccination augments GVHD. Also, different types of memory cells could behave differently. To begin to optimize this approach in mouse models we chose as our target antigen the H-2Kb-restricted minor histocompatibility antigen (miHA) H60. We cloned the H60 peptide sequence (LTFNYRNL) into a genetic construct encoding the heavy chain of a monoclonal antibody against DEC205. Donor C3H.SW (H-2b, H60-) mice were vaccinated with a single injection of anti-DEC205-H60 plus an agonist antibody against CD40 (FGK45). By two months post vaccination, H60-reactive memory cells were a stable population that comprised approximately 4-8% of splenic and peripheral blood CD8 cells. Approximately 50% of H60-tetramer+ cells had central memory phenotype, which could be ideal as central memory T cells mount strong anti-tumor responses. Similar results were obtained by using a prime/boost approach with H60-pulsed DCs. To test their function, CD44+ memory CD8 cells from H60-vaccinated mice were sort-purified and transferred into recipient B6 mice congenic for H60 (B6.H60; expression hematopoietically restricted ). By day 7 post transplant, H60-specific CD8+ TM from vaccinated mice comprised 70-90% of total splenic and blood CD8 cells, as compared to 1-5% in recipients of TM from unvaccinated mice. In contrast to anti-H60 responses by naïve CD8 cells, expansion of H60-tetramer+ cells from H60-vaccinated mice did not require CD4 help. To test whether target antigen distribution affects the re-expansion of H60-reactive cells from H60-vaccinated mice, expansion was compared in B6.H60 and B6.H60<right arrow>B6.actH60 (ubiquitous expression of H60 driven by an actin promoter) bone marrow chimeras. Approximately 60% of splenic and lymph node CD8 cells in both recipient groups were H60-tetramer+, though there was a trend towards increased overall numbers of tetramer+ cells in B6.H60 recipients. We are currently testing the GVL potency of TM from H60-vaccinated against mouse models of chronic phase (CP-CML) and blast crisis chronic myelogenous leukemia (BC-CML). In an ongoing experiment, very low numbers of CD8+ TM from H60-vaccinated mice mediate potent GVL against CP-CML relative to CD8+ TM from unvaccinated donors. Small numbers of CD8+ TM cells from vaccinated mice also mediated GVL against BC-CML, which is typically GVL resistant. Future studies will better define the potency of TM from H60-vaccinated mice and test these cells against CP-CML and BC-CML that do or do not express H60 but are otherwise identical. GVHD effect will also be tested. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry Detection of BCR-ABL Fusion Proteins in Leukemia Patients- An Indian Experience.

Abstract 4710 BackgroundThe BCR-ABL fusion gene results from the translocation t(9;22). It is the hallmark of chronic myeloid leukemia (CML) and is present in a poor risk subgroup of precursor B cell Acute Lymphoblastic Leukemia(ALL),which represents 25 to 30% of adult ALL and 3 to 5 % of childhood ALL. So far, the BCR-ABL aberration has been detected by cytogenetics, FISH or PCR, all techniques that are time consuming and require special facilities. We developed a simple flow cytometric bead assay for detection of the BCR-ABL fusion protein in cell lysates,using a bead bound catching antibody against one side of the fusion protein and a fluorochrome-conjugated detection antibody against the other side of the fusion protein. Flow cytometry allows for the discrimination of particles on the basis of attributes such as, size and fluroscence. BDTM Cytometric Bead. The aim of our study was to see the effectiveness of Flowcytometric detection of BCR-ABL and cost effectiveness of the method. Materials and MethodsTest Specimens: 50 patients, CML 37 and Adult ALL 10, Paediatric ALL.Reagents: BD BCR-ABL Protein Kit (BD Cat No: 643939) BD BCR-ABL buffer Kit (BD Cat No: 560272), Detection Reagent.BD BCR-ABL Protein Kit Protocol: Lysate preparation from samples (PBMCs) : Blood samples were processed through Ficoll-Paque TM medium to collect the mono nuclear cell layer as PBMCs. Cell collected were washed with PBS with 5% FBS and resuspended with PBS with 5% FBS before cell counting. PBMC s were resuspended in pretreatment buffer (PBS with 5% FBS plus IX pretreatment A and IX pretreatment B) at 10 million cells/ml and incubated for 10 mins. on ice. The pretreated cells were washed ones, then resuspended in lysing solution (BD Pharmingen TM cell lysis buffer plus IX BD lysate treatment reagent )at 25 million cells/ ml and incubated for 15 mins. on ice. The resulting supernatant (cell lysate) was Colleted after centrifugation.CBA Assay: to a BD Falcon TM 12 × 75mm polystyrene tube 50μl each of lysate, BCRABL Capture Beads and BCRABL detection reagent were added and mixed carefully. The tube was incubated for 2hrs at room temperature in the dark with constant mixing, and then contents were washed with 1 ml of BD CBA wash Buffer. Tube contents were resuspended with 300μl of BD CBA washed buffer. Samples were acquired with the BD FACS Canto II Flow Cytometer. The Cytometer flow rate was set to medium and data were acquired from 20 negative samples. The PE MFI for each sample was determining, and the mean of at least 20 values was calculated. The limit of detection was set at the mean plus two standard deviation.Karyotyping: The Kaiser Home-brew reagent was used and related protocol was followed.FISH: Abbott Visys LSI BCR/ABL, Dual Fusion Translocation Probe.QRT PCR Detection: The Kaiser Home-brew reagent was used and related protocol was followed. ResultDetection of all variants of the BCR ABL fusion protein. The anti BCR antibody was developed against a nonhomologus region of appox. 80 amino acids encoded by exon 1, which allowed us to detect all known BCR aBL variants, including p190, p 210, p230 as demonstrated by analysis of a series of well defined cell lines. The assay appeared to be specific and sufficiently sensitive to detect BCR ABl proteins in Lysates of Leukocyte samples of Leukemia patients at diagnosis.Protease inhibition needed in cell samples with many mature myeloid cells. Protein stability problems were encounter when cells samples contain high frequencies of mature myeloid cells with high levels of protease activity, such as CML cells and granulocyte fractions. This problem could be significantly encountered by adding protease inhibitors to several steps of the immunobead assay. ConclusionWe conclude that the flow cytometry immunobead assay is a fast and easy technique for specific detection of BCR ABL proteins in Leukemic cells.The main advantaged of the immunobead assay are :§ Not dependent on the breakpoint position in the BCR gene; does not need special laboratory facilities other than a Dual laser(4 colour) flow Cytometer and Provides results within few hours. The result is semiquantitative depending on the Mean Fluroscence Intensity. (MFI)§ Can be run in parallel to routine immunophenotyping§ Easy diagnosis and classification of leukemias with fusion genes. Disclosures:No relevant conflicts of interest to declare.

Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4 + memory T cells in mice

Donor-reactive memory T (Tm) cells undermine transplanted organs more readily than naive T cells. Rapamycin (RAPA) and tacrolimus (FK-506) are current mainstay immunosuppressants used for preventing acute allograft rejection. Although their efficacy in suppressing naive T cell is established, their suppressing effect on memory T cells is undefined. This study was conducted to investigate the inhibiting capability of RAPA or FK-506 against transferred alloreactive CD4 + Tm cells in a mouse cardiac transplant model. We found that these drugs alone prolonged the median survival time (MST) of allograft from 5 days to 9 days in recipient mice with CD4 + Tm infusion ( P < 0.01), which however was not significantly longer than that (8 days) in untreated recipient mice without CD4 + Tm infusion (naive control). Mean histologic rank of rejection activity in section of cardiac allograft on day 5 postgrafting was Grade 4 in the Tm control recipients versus Grade 3A in both of the immunosuppressant treatment recipients with CD4 + Tm infusion. RAPA or FK-506 alone failed to completely suppress proliferation and differentiation of the alloreactive CD4 + Tm, which was confirmed by in vitro mixed lymphocyte reaction (MLR) and by flow cytometry (FCM) of the splenocytes for detecting CD44 highCD62L − effector/memory as well as CD69 +/CD25 + activation phenotype cells from the respective recipients. Furthermore, the agent alone didn't completely inhibit the activation of CD4 + Tm, for serum level of IFN-γ and its gene expression at the cardiac allograft from the immunosuppressant-treated recipients were as still high as the untreated naive control. Thus, RAPA or FK-506 alone couldn't completely suppress the proliferation and activation of the alloantigen-primed CD4 + Tm cells responding to the alloantigen, indicating that alloreactive CD4 + Tm was insensitive to these immunosuppressants. The characteristics of alloreactive CD4 + Tm to resist immunosuppressants and its potency to initiate quick and vigorous rejection despite treatment with the immunosuppressant make it to be a critical barrier to prolongation of allograft survival and induction of transplant tolerance.

Pulmonary Eosinophilia Is Attenuated by Early Responding CD8+Memory T Cells in a Murine Model of RSV Vaccine-Enhanced Disease

Vaccination with formalin-inactivated respiratory syncytial virus (RSV) vaccine results in enhanced respiratory tract inflammation and injury following subsequent RSV infection. RSV vaccine-enhanced disease can also be produced in mice by prior vaccination with a vaccinia virus vector containing the RSV G protein, followed by intranasal infectious RSV challenge, a process characterized by induction of a potent memory CD4(+) T-cell response to challenge infection with some features characteristic of Th-2 CD4(+) T-cell responses, including increased eosinophil accumulation in pulmonary inflammatory infiltrates. The adaptive immune response to the RSV G protein in immunized BALB/c mice is characterized by a weak or absent primary and secondary recall CD8(+) T-cell response. These and related results have led to the hypothesis that the failure of the infected animals to mount an effective CD8(+) memory T-cell (CD8(+) Tm) response in this model could account for the pulmonary eosinophilia associated with the development of enhanced disease, and that CD8(+) T cells may control the development of eosinophilia. In this study, we investigated how and when the generation of a CD8(+) Tm response to RSV infection might affect the development of pulmonary eosinophilia in this model of vaccine-enhanced disease. By defining the CD8(+) T-cell response kinetics and monitoring lung parenchymal eosinophil accumulation, we show that the establishment of an RSV-specific CD8(+) Tm response in the infected lungs early after challenge infection (i.e., within the first 3 d of RSV infection) is necessary and sufficient to control pulmonary eosinophilia development. Additionally, our work suggests that the mechanism by which CD8(+) T cells regulate this process is not by modulating the differentiation or development of the CD4(+) Tm response. Rather, we demonstrate that IL-10 produced by early responding CD8(+) Tm cells may regulate the pulmonary eosinophilia development observed in RSV vaccine-enhanced disease.

Regulation of Cross-linked Actin Network (CLAN) Formation in Human Trabecular Meshwork (HTM) Cells by Convergence of Distinct β1 and β3 Integrin Pathways

To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.

Extracellular Release of ATP Mediated by Cyclic Mechanical Stress Leads to Mobilization of AA in Trabecular Meshwork Cells

To investigate the mechanisms that mediate the release of ATP induced by cyclic mechanical stress (CMS) and the role of extracellular ATP in the mobilization of arachidonic acid (AA) and prostaglandin secretion. Porcine trabecular meshwork (pTM) cells were subjected to CMS. Extracellular ATP was detected with a luciferin-luciferase assay in the presence or absence of transport inhibitors and a lipid raft disrupter. ATP vesicles were visualized with quinacrine. The release of AA (AA 1-14C) was measured with and without ATP, ATP inhibitors, and phospholipase-A and -C inhibitors. Prostaglandin E2 (PGE2) and viability were measured with ELISA and a lactate dehydrogenase assay, respectively. CMS induced ATP release that was inhibited by the vesicle inhibitors N-ethylmaleimide (NEM) and monensin. Lipid raft disruption significantly increased the extracellular ATP induced by CMS. CMS induced AA release (1-4-fold increase) and its metabolic product PGE2 (3.9-fold increase). The AA mobilization induced by CMS could be mimicked by the addition of extracellular ATP and was partially inhibited by a P2 antagonist, by an ATP inhibitor, and by inhibitors of phospholipase-A2 and -C. Addition of PGE2 (10 microM) to the media exerted cytoprotective effects against long-term CMS. Extracellular release of ATP induced by CMS in TM cells is mediated by exocytosis of ATP-enriched vesicles into lipid rafts. The resulting activation of purinergic receptors leads to mobilization of AA from the plasma membrane. The subsequent release of PGE could exert protective effects by preventing TM cell loss that may result from chronic exposure to CMS.

Cross-linked actin networks (CLANs) in bovine trabecular meshwork cells

A cytoskeletal feature of human trabecular meshwork (HTM) cells in vitro and ex vivo is the presence of cross-linked actin networks (CLANs) that are abundant in a proportion of TM cells exposed to dexamethasone (DEX) and also in cells from glaucoma patients. We wished to determine whether CLANs were present in the bovine trabecular meshwork (BTM), whether they were similarly induced by dexamethasone and whether the structures were comparable to CLANs in HTM cells. Cultures of HTM and BTM cells and ex vivo dissections of BTM tissue were stained with phalloidin (F-actin) and propidium iodide (nuclei) and imaged by confocal microscopy, thereafter being subjected to image analysis. Some CLAN-like structures were identified in ex vivo BTM tissue cultured with and without DEX. However we found that BTM cells in culture produced abundant CLANs when exposed to DEX; comparable to the best response from HTM cells. The CLANs were of similar dimensions and morphology to those found in human cells and they had a similar half life of 2 or 3 days following the removal of DEX. This work demonstrates that BTM cells provide a suitable model for future investigations of CLAN formation and function.BTM cultures are sufficiently hardy to thrive in low serum and serum-free conditions so we were able to show that aqueous humor stimulates CLAN formation in the target cells. Future research is directed at identifying the aqueous component(s) responsible for CLAN production.

Memory T-lymphocyte survival does not require T-cell receptor expression

The factors controlling memory T (Tm)-cell longevity are still poorly defined, and their identification is pivotal to the design of a vaccine conferring long-term protection against infection. Tm cells have the ability to survive in the absence of the T-cell receptor (TCR)-MHC interaction. This does not exclude a possible role for TCR-intrinsic ligand-independent constitutive signaling in Tm-cell homeostasis. Using a unique TCR tetracycline-inducible expression system, we show that the ablation of TCR expression, which abrogates any possible signaling via the TCR, did not influence the survival and self-renewal of antigen-specific CD8(+) Tm cells even when they have to compete with endogenous T cells for survival factors. Moreover, CD8(+) Tm-cell functionality was not altered even on prolonged maintenance in the absence of TCR-MHC interactions. Furthermore, our results show that a subset of CD4(+) Tm cells can survive in the absence of TCR expression in nonlymphopenic hosts.

Cells and mediators which participate in immunoglobulin synthesis by human mononuclear cells. III. Null cells secrete a factor(s) (human immunoglobulin synthesis/secretion-facilitating factor) that can replace the null cells in the synthesis of immunoglobulin by cultured B cells

In the accompanying communication, it was demonstrated that the null cells, the TM cells, monocytes and PWM are all obligatory participants in the synthesis and secretion of immunoglobulins by human B cells in culture. Here we demonstrate that the null cells secrete a factor, referred to as human immunoglobulin synthesis/secretion-facilitating factor (HISFF) that can replace the null cells in the cultures. HISFF is distinct from the known T cell-derived interleukins. HISFF functions in an HLA-unrestricted fashion since it can facilitate the synthesis and secretion of immunoglobulins by allogeneic B cells. The null cells cultured with TM helper cells and PWM required monocytes in the culture in order to secrete HISFF. Furthermore, B cells cultured with TM cells in medium containing HISFF, monocyte-derived factors and PWM nevertheless required monocytes in order to respond to the HISFF signal. Thus, the monocyte plays a pivotal role in the secretion of and response to HISFF. Normal levels of immunoglobulin were synthesized even when HISFF was added to the cultures of B cells, TM cells and monocytes, in the presence of PWM, as late as day 6 of the 7 day culture. We conclude that the null cells participate in immunoglobulin synthesis by the B cells by secreting a soluble mediator, HISFF, capable of replacing the null cells in the culture; and that the HISFF signal is the last signal received by the B cell before it begins to synthesize and secrete immunoglobulins.

Cells and mediators which participate in immunoglobulin synthesis by human mononuclear cells. II. The mechanism of null cell participation in immunoglobulin synthesis and secretion by B cells

Immunoglobulins were synthesized and secreted by human B cells cultured with T cells with receptors for FcM (TM) helper cells, monocytes, null cells and PWM for 7 days. Immunoglobulin synthesis did not take place if the null cells were omitted from the cultures irrespective of the duration of the culture period. Null cells incorporated into the cultures at only 25% of their optimal concentration did not affect immunoglobulin synthesis markedly by the cultured B cells. However, the number of B cells in the culture could not be diluted without an accompanying marked reduction in immunoglobulin synthesis. The B cells synthesized and secreted significant quantities of immunoglobulin even when the null cells were added as late as day 6 of the 7-day culture whereas no or very little immunoglobulin was synthesized if the B cells were not present from the beginning of the 7-day culture. It was demonstrated that cultured null cells do not transform into B cells and do not attain their immunoglobulin-synthesizing function. Furthermore, cultured B cells do not transform into null cells and do not attain their helper function. The null cells can also be distinguished from the B cells on the basis of cell-surface markers, receptors, and blastogenic responsiveness to phytomitogens. It is concluded that (i) the human circulating B cells require the null cells, in addition to the TM cells, monocytes and PWM, in culture in order to synthesize and secrete immunoglobulin; (ii) the null cell signal that stimulates immunoglobulin synthesis and secretion by the B cells is probably the last signal following the TM helper cell, monocyte and PWM signals received by the B cells; and (iii) the null cells and the B cells constitute distinct lineages of cells.

Defect of CD8+ Memory T Cells Developed in Absence of IL-12 Priming for Secondary Expansion

IL-12 priming plays an important role in stimulation of CD8+ effector T cells and development of CD8+ memory T (Tm) cells. However, the functional alteration of CD8+ Tm cells developed in the absence of IL-12 priming is elusive. In this study, we investigated the capacity of secondary expansion of CD8+ Tm cells developed from transgenic OT I CD8+ T cells. The latter cells were in vitro and in vivo stimulated by ovalbumin (OVA)-pulsed dendritic cells [DCOVA and (IL-12-/-)DCOVA] derived from wild-type C57BL/6 and IL-12 gene knockout mice, respectively. We demonstrated that IL-12 priming is important not only in CD8+ T cell clonal expansion, but also in generation of CD8+ Tm cells with the capacity of secondary expansion upon antigen re-encounter. However, IL-12 signaling is not involved in CD8+ Tm cell survival and recall responses. Therefore, this study provides useful information for vaccine design and development.

Defective capacity of CD8+ memory T cells developed from absence of IL‐12 priming for secondary expansion

In vivo CD8+ T cell responses consist of three main phases, namely: (i) a proliferation phase consisting of effector T cell clonal expansion, (ii) a contraction (transition) phase leading to the transition from the large population of effector T cells to a smaller population of long‐lived memory T (Tm) cells, and (iii) a maintenance phase involving Tm cell survival in the host. Recently, it has also been shown that IL‐12 priming during in vitro Ag stimulation changes properties of CD8+ T cells such as downregulation of CD69, alteration in the distribution of lipid rafts, a decrease in T cell receptor structural avidity and cytolytic activity. However, the potent functional property of CD8+ Tm cells without IL‐12 priming for secondary expansion upon Ag reencounter is still unclear.In this study, we attempted to clarify the role of IL‐12 by transferring the CD8+ effector T cells into wild‐type C57BL/6 and IL‐12 gene KO mice, respectively. Our findings showed that IL‐12 priming is important not only in CD8+ T cell clonal expansion in the proliferation phase, but also in generation of CD8+ Tm cells with the capacity of secondary expansion upon antigen re‐encounter. However, IL‐12 signaling is neither involved in CD8+ effector T cell survival in the transition phase nor in the CD8+ Tm cell's secondary expansion in recall responses.

FK506-Binding Protein 51 Regulates Nuclear Transport of the Glucocorticoid Receptor β and Glucocorticoid Responsiveness

A spliced variant of the human glucocorticoid receptor GRbeta has been implicated in glucocorticoid responsiveness in glaucoma. Over-expression of the FK506-binding immunophilin FKBP51 also causes a generalized state of glucocorticoid resistance. In the present study, the roles of FKBP51 in the nuclear transport of GRbeta and glucocorticoid responsiveness were investigated. Human trabecular meshwork cells (GTM3 and TM5) and HeLa cells were treated with dexamethasone (DEX) and FK506 and transfected with GRbeta and FKBP51 expression vectors. Coimmunoprecipitation and Western blot analyses were performed to study interactions of FKBP51 and FKBP52 with GRalpha, GRbeta, Hsp90, or dynein. The cells were transfected with a GRE-luciferase reporter to evaluate the effects of DEX and FK506 and the overexpression of GRbeta and FKBP51 on glucocorticoid-mediated gene expression. FKBP51 was involved in constitutive nuclear transport of both GRalpha and -beta in the absence of ligands. FKBP52 appeared to be solely responsible for the nuclear transport of ligand-activated GRalpha. DEX stimulated the translocation of GRalpha but not GRbeta. Overexpression of either GRbeta or FKBP51 stimulated GRbeta translocation and reduced DEX-induced luciferase in HeLa cells. FK506 did not alter DEX-induced translocation of GRalpha. However, FK506 increased the association of FKBP51 with GRbeta and stimulated DEX-induced translocation of GRbeta in normal TM cells, but not in glaucoma TM cells. Increased nuclear GRbeta significantly inhibited glucocorticoid responsiveness in TM cells. Nuclear transport of GRbeta represents a novel mechanism through which FKBP51 alters GC sensitivity. GRbeta and FKBP51 may be responsible for increased responsiveness in steroid-induced ocular hypertensive individuals as well as in patients with glaucoma.