Expression of class II antigens by subsets of activated T cells

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.     

Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients.

We identified circulating CD8+ T-cell populations specific for the tumor-associated antigens (TAAs) MART-1 (27-35) or tyrosinase (368-376) in six of eleven patients with metastatic melanoma using peptide/HLA-A*0201 tetramers. These TAA-specific populations were of two phenotypically distinct types: one, typical for memory/effector T cells; the other, a previously undescribed phenotype expressing both naive and effector cell markers. This latter type represented more than 2% of the total CD8+ T cells in one patient, permitting detailed phenotypic and functional analysis. Although these cells have many of the hallmarks of effector T cells, they were functionally unresponsive, unable to directly lyse melanoma target cells or produce cytokines in response to mitogens. In contrast, CD8+ T cells from the same patient were able to lyse EBV-pulsed target cells and showed robust allogeneic responses. Thus, the clonally expanded TAA-specific population seems to have been selectively rendered anergic in vivo. Peptide stimulation of the TAA-specific T-cell populations in other patients failed to induce substantial upregulation of CD69 expression, indicating that these cells may also have functional defects, leading to blunted activation responses. These data demonstrate that systemic TAA-specific T-cell responses can develop de novo in cancer patients, but that antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth.     

Intravenous and intradermal TriMix-dendritic cell therapy results in a broad T-cell response and durable tumor response in a chemorefractory stage IV-M1c melanoma patient

Dendritic cells (DCs) electroporated with mRNA encoding CD70, CD40L and a constitutively active toll-like receptor 4 (TriMix-DC) have an increased T-cell stimulatory capacity. In a prospective phase IB clinical trial, we treated melanoma patients with intradermal and intravenous injections of autologous TriMix-DC co-electroporated with mRNA encoding full-length MAGE-A3, MAGE-C2, tyrosinase and gp100. We report here the immunological and clinical results obtained in one patient with a particularly favorable outcome. This patient had stage IV-M1c melanoma with documented progression during dacarbazine chemotherapy and received 5 TriMix-DC injections. Following DC therapy, a broad CD8(+) T-cell response against multiple epitopes derived from all four treatment antigens was found in the blood and among T cells derived from DTH biopsy. In addition, CD4(+) T cells recognizing different MAGE-A3-derived epitopes were detected in DTH-derived cells. A spontaneous anti-MAGE-C2 CD8(+) T-cell response was present prior to TriMix-DC therapy and increased during treatment. The tumor response was assessed with 18-fluorodeoxyglucose-positron emission/computed tomography. We documented a partial tumor response according to RECIST criteria with a marked reduction in (18)F-FDG-uptake by lung, lymph node and bone metastases. The patient remains free from progression after 12 months of follow-up. This case report indicates that administration of autologous TriMix-DC by the combined intradermal and intravenous route can mediate a durable objective tumor response accompanied by a broad T-cell response in a chemorefractory stage IV-M1c melanoma patient.     

Holoendemic Malaria Exposure Is Associated with Altered Epstein-Barr Virus-Specific CD8+ T-Cell Differentiation

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8⁺ T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8⁺ T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8⁺ T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8⁺ T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8⁺ T cells or the global CD8⁺ memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8⁺ T-cell compartment that illuminates the etiology of eBL.     

Circulating CD4+ T lymphocytes with intracellular but no surface CD3 antigen in five of seven patients consecutively diagnosed with angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) accounts for less than 1% of all lymphatic malignancies. Oligoclonality or monoclonality for any of the T-cell receptor (TCR) chain genes can be demonstrated in the majority of the cases. During systematic screening for the presence of circulating lymphocytes with atypical coexpression of differentiation antigens in patients with T-cell lymphomas, we have discovered a minor population (accounting for 0.2% to 10.% of all lymphocytes) of atypical lymphocytes in the blood of five of seven patients consecutively diagnosed in 1997/1998 by lymph node histology to have AITL. The major distinguishing feature of these cells consists of the lack of the surface expression of the CD3 antigen, but not of the intracellular expression. These cells express the T-cell antigens CD2 and CD5 on their surface, but not CD7, and they express CD4 and CD45 at numbers of molecules per cell typical for T lymphocytes. Gene scan analyses for the TCR gamma chain revealed oligoclonality of these flow-sorted cells in one patient and monoclonality in two patients, the same patterns of TCR gamma chain gene as determined processing the respective diagnostic lymph nodes. Circulating CD4-expressing T lymphocytes with exclusively cytoplasmic expression of CD3 appear to represent the malignant population in patients with histologically diagnosed AITL.     

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8⁺ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 10⁶). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1_126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1_950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.     

Subcellular antigen location influences T-cell activation during acute infection with Toxoplasma gondii

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8(+) T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8(+) T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8(+) T-cell activation was assayed using 'B3Z' reporter cells in vitro, or adoptively-transferred OVA-specific 'OT-I' CD8(+) T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8(+) T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8(+) T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.     

Dendritic cells crosspresent antigens from live B16 cells more efficiently than from apoptotic cells and protect from melanoma in a therapeutic model

Dendritic cells (DC) are able to elicit anti-tumoral CD8(+) T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8(+) T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8(+) T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy.     

Immunomodulation of T-cell responses with Vibrio cholerae O135 capsular polysaccharide and its protein conjugate, novel cholera vaccine study models

We studied T-cell immune responses to surface capsular polysaccharide (CPS) of Vibrio cholerae O135 and its protein conjugate. CPS and CPS-bovine serum albumin (BSA) activation and presentation are characterized with induced alterations in expression and upregulation of membrane antigens CD25, CD11b, CD16/32, MHCII and CD45 on blood- and spleen-derived T cells. Expression of the early activation marker CD25 revealed efficient CPS-BSA conjugate activation especially of CD4(+) CD3(+) and CD8(+) CD3(+) cells. Specific CPS-BSA-induced CD25(+) T-cell subsets in blood were observed after the first application, i.e. a 4.2-fold increase of CD4(+) CD25(+) and 7.6-fold increase of CD8(+) CD25(+) vs. preimmune levels was determined. The upregulation of surface antigens MHCII and CD45 involved in antigen presentation and cell activation of CD3(+) cells and their significant reciprocal correlation (R(2) =?0.92) observed only with CPS-BSA conjugate suggested efficient T-cell dependency and presentation. The pattern of accelerated T-cell activation and engagement of T cells as antigen-presenting cells throughout CPS-BSA immunization contrary to CPS alone was also confirmed in CD4(+) /CD8(+) /CD3(+) splenic cells. The results revealed different T-cell antigen presentation and activation following administration of CPS and CPS-BSA conjugates, as supported also by evaluation of CD45, MHCII and CD25 expression on CD19(+) B cells.     

Compartmentalization of human immunodeficiency virus type 1 between blood monocytes and CD4+ T cells during infection

Distinct sequences of human immunodeficiency virus type 1 (HIV-1) have been found between different tissue compartments or subcompartments within a given tissue. Whether such compartmentalization of HIV-1 occurs between different cell populations is still unknown. Here we address this issue by comparing HIV-1 sequences in the second constant region through the fifth hypervariable region (C2 to V5) of the surface envelope glycoprotein (Env) between viruses in purified blood CD14(+) monocytes and CD4(+) T cells obtained longitudinally from five infected patients over a time period ranging from 117 to 3,409 days postseroconversion. Viral populations in both cell types at early infection time points appeared relatively homogeneous. However, later in infections, all five patients showed heterogeneous populations in both CD14(+) monocytes and CD4(+) T cells. Three of the five patients had CD14(+) monocyte populations with significantly more genetic diversity than the CD4(+) T-cell population, while the other two patients had more genetic diversity in CD4(+) T cells. The cellular compartmentalization of HIV-1 between CD14(+) monocytes and CD4(+) T cells was not seen early during infections but was evident at the later time points for all five patients, indicating an association of viral compartmentalization with the time course of HIV-1 infection. The majority of HIV-1 V3 sequences indicated a macrophage-tropic phenotype, while a V3 sequence-predicted T-cell tropic virus was found in the CD4(+) T cells and CD14(+) monocytes of two patients. These findings suggest that HIV-1 in CD14(+) monocytes could disseminate and evolve independently from that in CD4(+) T cells over the course of HIV-1 infection, which may have implications on the development of new therapeutic strategies.     

An approach to diagnosis of T-cell lymphoproliferative disorders by flow cytometry

T-cell lymphoproliferative disorders are among the most challenging diagnoses in hematopathology. Unlike the more common B-cell disorders, in which clonality is often readily discernible by surface immunoglobulin light chain restriction, there is no specific immunophenotypic signature that is diagnostic of a clonal T-cell population. Immunophenotypic criteria that are helpful in the diagnosis of T-cell neoplasms include T-cell subset antigen restriction, anomalous T-cell subset antigen expression, deletion or diminution of one of the pan T-cell antigens, a precursor T-cell phenotype, and expression of additional markers (e.g., CD30, CD20, major myeloid antigens, and TCRgammadelta). Analysis of the inherent forward and orthogonal light scatter properties of the cell can also provide important diagnostic clues. None of these features is 100% specific, however, for aberrant expression of pan-T antigens may be seen in viral infections, B-cell malignancies, or in reactive changes following administration of certain medications. An increased CD4:CD8 ratio is often observed in Hodgkin's lymphoma. Based on the analysis of 87 neoplastic and 80 control cases, we conclude that flow cytometric features that are most suspicious for malignancy include the loss or markedly dim expression of CD45; complete loss of one or more pan-T antigens; diminished expression of more than two pan-T antigens in conjunction with altered light scatter properties; and CD4/CD8 dual-positive or dual-negative expression (except thymic lesions).     

Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune responses

Hepatitis C virus (HCV) chronic infection is characterized by low or undetectable cellular immune responses against HCV antigens. Some studies have suggested that HCV proteins manipulate the immune system by suppressing the specific antiviral T-cell immunity. We have previously reported that the expression of HCV core and E1 proteins (CE1) in dendritic cells (DC) impairs their ability to prime T cells in vitro. We show here that immunization of mice with immature DC transduced with an adenovirus encoding HCV core and E1 antigens (AdCE1) induced lower CD4(+)- and CD8(+)-T-cell responses than immunization with DC transduced with an adenovirus encoding NS3 (AdNS3). However, no differences in the strength of the immune response were detected when animals were immunized with mature DC subsequently transduced with AdCE1 or AdNS3. According to these findings, we observed that the expression of CE1 in DC inhibited the maturation caused by tumor necrosis factor alpha or CD40L but not that induced by lipopolysaccharide. Blockade of DC maturation by CE1 was manifested by a lower expression of maturation surface markers and was associated with a reduced ability of AdCE1-transduced DC to activate CD4(+)- and CD8(+)-T-cell responses in vivo. Our results suggest that HCV CE1 proteins modulate T-cell responses by decreasing the stimulatory ability of DC in vivo via inhibition of their physiological maturation pathways. These findings are relevant for the design of therapeutic vaccination strategies in HCV-infected patients.     

Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI.

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.     

A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs

Latently infected cells form the major obstacle to HIV eradication. Studies of HIV latency have been generally hindered by the lack of a robust and rapidly deployable cell model that involves primary human CD4 T lymphocytes. Latently infected cell lines have proven useful, but it is unclear how closely these proliferating cells recapitulate the conditions of viral latency in non-dividing CD4 T lymphocytes in vivo. Current primary lymphocyte models more closely reflect the in vivo state of HIV latency, but they are limited by protracted culture periods and often low cell yields. Additionally, these models are always established in a single latently infected cell type that may not reflect the heterogeneous nature of the latent reservoir. Here we describe a rapid, sensitive, and quantitative primary cell model of HIV-1 latency with replication competent proviruses and multiple reporters to enhance the flexibility of the system. In this model, post-integration HIV-1 latency can be established in all populations of CD4 T cells, and reactivation of latent provirus assessed within 7 days. The kinetics and magnitude of reactivation were evaluated after stimulation with various cytokines, small molecules, and T-cell receptor agonists. Reactivation of latent HIV proviruses was readily detected in the presence of strong activators of NF-κB. Latently infected transitional memory CD4 T cells proved more responsive to these T-cell activators than latently infected central memory cells. These findings reveal potentially important biological differences within the latently infected pool of memory CD4 T cells and describe a flexible primary CD4 T-cell system to evaluate novel antagonists of HIV latency.     

Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins

Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4(+) T cells responding to HCV core in patients with chronic HCV infection. However, CD4(+) response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4(+) response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.     

Beta-adrenoceptor blockade alters thymocyte differentiation in aged mice.

The thymus is the primary site for generation of naive T-lymphocytes in the young animal. With age, the thymus progressively involutes and fewer mature T-cells are produced and migrate to the periphery. With thymic involution, increased density of sympathetic noradrenergic (NA) innervation and concentration of norepinephrine (NE) have been observed. To determine if the age-related changes in thymocyte differentiation are modified by NE signaling through beta-adrenergic receptors, 2-month (mo) and 18-mo old BALB/c mice were implanted subcutaneously with pellets containing the non-selective beta-adrenoceptor antagonist nadolol. Four and one-half weeks later, thymus and peripheral blood were collected to assess changes in thymocyte differentiation and naive T-cell output by flow cytometric analysis of T-cell subpopulations. In old mice, but not in young mice, thymocyte CD4/CD8 co-expression was altered by beta-adrenoceptor blockade. In nadolol-treated old mice, the frequency of the immature CD4-8- population was increased, and the intermediate CD4+8+ population was reduced. A corresponding increase in the frequency of mature CD4-8+, but not CD4+8- cells was observed. The increase in CD4-8+ cells is most likely not mediated by more CD4-8+ cells undergoing positive selection, because CD3hi expression in the CD4+8+ population was not altered by nadolol. The percentage of CD8+44low naive cells in peripheral blood increased in nadolol-treated mice, suggesting that more CD4-8+ cells were exported from the thymus to the periphery. These results indicate that the age-associated increase in sympathetic NA innervation of the thymus modulates thymocyte maturation. Pharmacological manipulation of NA innervation may provide a novel means of increasing naive T-cell output and improving T-cell reactivity to novel antigens with age.     

Lung T-cell subset composition at the time of surgical resection is a prognostic indicator in non-small cell lung cancer

NSCLC arises in the complex environment of chronic inflammation. Depending on lung immune polarization, infiltrating immune cells may either promote or suppress tumor growth. Despite the importance of the immune microenvironment, current staging techniques for NSCLC do not take into consideration the immune milieu in which the neoplasms arise. T-cell subset content was compared between paired tumor-bearing and contralateral lungs, patient and control peripheral blood. The relationship between T-cell subset distribution and survival were evaluated. CD4 and CD8+ T cells were subsetted by CD45RA/CD27 and analyzed for expression of activation, adhesion, and homing markers. Strikingly, T-cell content was indistinguishable between lungs. Compared with peripheral blood, naïve CD4 and CD8 T cells were rare in BAL. CD4+ BAL T cells showed increased CD95 (higher apoptotic potential) and CD103 expression (epithelial adhesion), but decreased CD38 (activation) and CCR7 expression (lymph node homing). CD8+ BAL T cells showed increased CD103 expression and decreased CD28 expression (co-stimulation). Differences in CD28, CD95, and CCR7 expression were more pronounced within memory cells, while differences in CD4+ CD103 expression were more prominent in effector/memory cells. Of these populations, the absence of lung CD4 T cells with an effector-like phenotype (CD45RA+/CD27?) emerged as a predictor of favorable outcome. Patients with a low proportion (≤0.44%) had 90% 5-year survival (n = 10, median survival 2,343 days), compared with 0% (n = 9, median survival 516 days) of patients with a higher proportion. Further study is required to confirm this association prospectively and define the function of this subpopulation.     

Reconstitution of Human Thymic Implants Is Limited by Human Immunodeficiency Virus Breakthrough during Antiretroviral Therapy

Human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu thymic implants depleted of CD4(+) cells can support renewed thymopoiesis derived from both endogenous and exogenous T-cell progenitors after combination antiretroviral therapy. However, successful production of new thymocytes occurs transiently. Possible explanations for the temporary nature of this thymic reconstitution include cessation of the thymic stromal support function, exhaustion of T-cell progenitors, and viral resurgence. Distinguishing between these processes is important for the development of therapeutic strategies aimed at reconstituting the CD4(+) T-cell compartment in HIV-1 infection. Using an HIV-1 strain engineered to express the murine HSA heat-stable antigen surface marker, we explored the relationship between HIV-1 expression and CD4(+) cell resurgence kinetics in HIV-1-depleted SCID-hu implants following drug therapy. Antiviral therapy significantly suppressed HIV-1 expression in double-positive (DP) CD4/CD8 thymocytes, and the eventual secondary decline of DP thymocytes following therapy was associated with renewed viral expression in this cell subset. Thymocytes derived from exogenous T-cell progenitors induced to differentiate in HIV-1-depleted, drug-treated thymic implants also became infected. These results indicate that in this model, suppression of viral replication occurs transiently and that, in spite of drug therapy, virus resurgence contributes to the transient nature of the renewed thymic function.     

Antigenic Profile of Osteoblasts Present in Human Bone Tissue Sections

The antigenic profile of human osteoblasts was previously analyzed by our group using primary cultures as study samples. These studies suggested a novel functional approach to this cell population. Osteoblasts have a characteristic antigenic profile and share antigens in common with other cell populations that also originate in the bone marrow. Some of the detected antigens are constitutively expressed, while others are modulated by different factors and/or cytokines. The aim of the present study was to analyze the antigens present in osteoblasts in vivo, since the presence of certain biomolecules in fetal bovine serum may modulate the antigenic expression, compromising the results. For this purpose, human bone tissue sections were analyzed with a wide panel of mAbs and using the immunoperoxidase technique. CD10, CD44 and alkaline phosphatase antigens and IL-12, IL-18 and IFNγ cytokines were detected in osteoblasts in the bone tissue. However, CD80 and HLA-DR antigens were not found in all samples and when present their expression was weak. The expression of CD54 antigen was moderate or weak. These results allow data obtained by the primary culture of osteoblast-like cells to be endorsed.     

Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process

We have studied the effects of prostaglandin E2 (PGE2) on in vitro human T-cell activation induced by crosslinking of the CD3-Ti complex with the monoclonal anti-CD3 antibodies OKT3 and UCHT-1. PGE2 (greater than or equal to 3 X 10(-9) M) when added simultaneously with anti-CD3 to cultures of peripheral blood mononuclear cells (PBMC), significantly suppressed, in a dose-dependent way, T-cell proliferation (P less than 0.002). However, when T cells were first preactivated with OKT3 for 3 days, subsequent proliferation driven by recombinant interleukin 2 (IL-2) was not inhibited by addition of PGE2. This indicates that PGE2 affects the activation step resulting from crosslinking of CD3-Ti, but not the IL-2-driven proliferative phase. Other manifestations of T-cell activation were therefore examined. Both IL-2 production and the expression of receptors for IL-2 (as detected with the anti-Tac monoclonal antibody) were inhibited by PGE2. The addition of purified interleukin 1 (IL-1) or recombinant IL-2 to the cultures did not reverse the inhibiting effect of PGE2 on IL-2-receptor expression. PGE2, added at the time of culture initiation, also inhibited T-cell proliferation in cultures which were supplemented with exogenous IL-1 or IL-2. Proof for a direct effect of PGE2 on T cells was obtained in experiments in which monocyte-depleted T cells were stimulated, in the presence of IL-1, with solid-phase-bound anti-CD3 antibody. Proliferation of T cells in this system is accessory cell independent and still was strongly inhibited by PGE2. Finally, preincubation of PBMC with PGE2 (3 X 10(-6) M) for 48 hr did not result in the generation of suppressor cells for anti-CD3-induced T-cell proliferation or for IL-2 production. Our results demonstrate that PGE2 has a direct inhibitory effect on an early step of T-cell activation, resulting in decreased IL-2 production, decreased IL-2-receptor expression, decreased responsiveness to exogenous IL-2, and decreased proliferation. However, PGE2 does not affect IL-2-driven proliferation of activated T cells. The inhibitory effect on T-cell activation is not mediated through suppressor T cells, nor through inhibition of accessory cell function.