Unexpected role for MHC II-peptide complexes in shaping CD8 T-cell expansion and differentiation in vivo

Here we report a unique role for MHC II–peptide complexes in controlling immune responses of naïve CD8 T cells. Compared with CD8 T cells from WT mice, CD8 T cells isolated from MHC II^−/− mice hyperproliferated under lymphopenic conditions, differentiated into effector cells producing proinflammatory cytokines, and mediated more severe tissue inflammation. The elevated responses of MHC II^−/− CD8 T cells were due to the absence of MHC II, but not CD4, T cells. The hyperreactivity appeared to be a feature of mature T cells, given its absence in CD8 single positive thymocytes derived from MHC II^−/− mice. Expression of the MHC II ligand LAG3 was markedly enhanced during in vivo activation of MHC II^−/− CD8 T cells, and blockade of MHC II–LAG3 interactions further enhanced T-cell expansion. Importantly, CD8 T cells isolated from H-2M^−/− mice expressing WT levels of MHC II also displayed hyperresponsiveness similar to that of MHC II^−/− CD8 T cells, suggesting that peptides presented on MHC II are involved in the control of CD8 T-cell responses. Our results uncover a previously undefined MHC II-dependent regulation that tunes CD8 T-cell reactivity and may have implications for an improved understanding of CD8 T-cell homeostasis and functions.     

Monkeypox virus evades antiviral CD4+ and CD8+ T cell responses by suppressing cognate T cell activation

Monkeypox virus (MPV) is a virulent human pathogen that has gained increased attention because of its potential use as a bioterrorism agent and inadvertent introduction into North America in 2003. The US outbreak also provided an important opportunity to study MPV-specific T cell immunity. Although MPV-specific CD4⁺ and CD8⁺ T cells could recognize vaccinia virus (VV)-infected monocytes and produce inflammatory cytokines such as IFNγ and TNFα, they were largely incapable of responding to autologous MPV-infected cells. Further analysis revealed that, unlike cowpox virus (CPV), MPV did not interfere with MHC expression or intracellular transport of MHC molecules. Instead, MPV-infected cells were capable of preventing T cell receptor (TcR)-mediated T cell activation in trans. The ability to trigger a state of nonresponsiveness represents a unique MHC-independent mechanism for blocking antiviral T cell activation and inflammatory cytokine production and is likely an important attribute involved with viral dissemination in the infected host.     

CD4 T‐cell hyporesponsiveness induced by schistosome larvae is not dependent upon eosinophils but may involve connective tissue mast cells

In areas endemic for schistosomiasis, people can often be in contact with contaminated water resulting in repeated exposures to infective Schistosoma mansoni cercariae. Using a murine model, repeated infections result in IL‐10‐dependent CD4⁺ T‐cell hyporesponsiveness in the skin‐draining lymph nodes (sdLN), which could be caused by an abundance of eosinophils and connective tissue mast cells at the skin infection site. Here, we show that whilst the absence of eosinophils did not have a significant effect on cytokine production, MHC‐II⁺ cells were more numerous in the dermal cell exudate population. Nevertheless, the absence of dermal eosinophils did not lead to an increase in the responsiveness of CD4⁺ T cells in the sdLN, revealing that eosinophils in repeatedly exposed skin did not impact on the development of CD4⁺ T‐cell hyporesponsiveness. On the other hand, the absence of connective tissue mast cells led to a reduction in dermal IL‐10 and to an increase in the number of MHC‐II⁺ cells infiltrating the skin. There was also a small but significant alleviation of hyporesponsiveness in the sdLN, suggesting that mast cells may have a role in regulating immune responses after repeated exposures of the skin to S. mansoni cercariae.     

Batf3 deficiency proves the pivotal role of CD8α+ dendritic cells in protection induced by vaccination with attenuated Plasmodium sporozoites

Increasing evidence indicates that hepatic CD8α⁺ dendritic cells (DCs) are important antigen cross‐presenting cells (APC) involved in the priming of protective CD8⁺ T‐cell responses induced by live‐attenuated Plasmodium sporozoites. Experimental proof for a critical role of CD8α⁺ DCs in protective pre‐erythrocytic malaria immunizations has pivotal implications for vaccine development, including improved vectored subunit vaccines. Employing Batf3^?/? mice, which lack functional CD8α⁺ DCs, we demonstrate that deficiency of these particular APCs completely abolishes protection and corresponding signatures of vaccine‐induced immunity. We show that in wild‐type, but not in Batf3^?/?, mice CD8α⁺ DCs accumulate in the liver after immunization with live irradiation‐attenuated P. berghei sporozoites. IFN‐γ production by Plasmodium antigen‐specific CD8⁺ T cells is dependent on functional Batf3. In addition, our results demonstrate that the dysfunctional cDC‐CD8+ T‐cell axis correlates with MHC class II upregulation on splenic CD8α^? DCs. Collectively, these findings underscore the essential role of CD8α⁺ DCs in robust protection induced by experimental live‐attenuated malaria vaccines.     

Patients with high-risk myelodysplastic syndrome can have polyclonal or clonal haemopoiesis in complete haematological remission

The clonality of mature peripheral blood-derived myeloid and lymphoid cells and bone marrow haemopoietic progenitors from 18 females with myelodysplasia (MDS) (five refractory anaemia, RA; one RA with ringed sideroblasts, RARS; three chronic myelomonocytic leukaemia, CMML; four RA with excess of blasts, RAEB; five RAEB in transformation, RAEB-t) was studied by X-chromosome inactivation analysis. Using the human androgen-receptor (HUMARA) assay, we analysed the clonal patterns of highly purified immature CD34⁺38^? and committed CD34⁺38⁺ marrow-derived progenitors, and CD16⁺14^? granulocytes, CD14⁺ monocytes, CD3⁺ T and CD19⁺ B lymphocytes from peripheral blood. In high-risk patients (RAEB, RAEB-t), clonality analysis was performed before and after intensive remission-induction treatment. All patients, except one with RA, had predominance of a single clone in their granulocytes and monocytes. The same clonal pattern was found in CD34⁺ progenitor cells. In contrast, CD3⁺ T lymphocytes were polyclonal or oligoclonal in 14/18 patients. X-chromosome inactivation patterns of CD19⁺B cells were highly concordant with CD3⁺ T cells except for two patients (one RA, one CMML) with monoclonal B and polyclonal T lymphocytes, therefore suggesting a clonal mutation in a progenitor common to the myeloid and B-lymphoid lineages or the coexistence of MDS and a B-cell disorder in these particular patients. After high-dose non-myeloablative chemotherapy, polyclonal haemopoiesis was reinstalled in the mature myeloid cells and immature and committed marrow progenitors in three of four patients achieving complete haematological remission. Therefore we conclude that most haematological remissions in MDS are associated with restoration of polyclonal haemopoiesis.     

Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming

Although viral MHC class I inhibition is considered a classic immune-evasion strategy, its in vivo role is largely unclear. Mutant cowpox virus lacking its MHC class I inhibitors is markedly attenuated during acute infection because of CD8⁺ T-cell–dependent control, but it was not known how CD8⁺ T-cell responses are affected. Interestingly, we found no major effect of MHC class I down-regulation on priming of functional cowpox virus-specific CD8⁺ T cells. Instead, we demonstrate that, during acute infection in vivo, MHC class I down-regulation prevents primed virus-specific CD8⁺ T cells from recognizing infected cells and exerting effector responses to control the infection.     

Haemopoietic growth factor tyrosine kinase receptor expression profiles in normal haemopoiesis

Expression profiles were generated for the haemopoietic tyrosine kinase receptors (HGF-TKRs or class III TKRs) by PCR on cDNA samples (RT-PCR) using a degenerate primer set. Each profile consisted of primary and secondary, i.e. enriched for less-expressed sequences, fingerprints. This method was applied on FACS-purified haemopoietic CD34⁺ cells, both from bone marrow (BM) and umbilical cord blood (UCB), and on mature cells from peripheral blood. CD34⁺ BM cells showed expression of c-fms, flt3, whereas CD34⁺ UCB cells expressed c-fms and, to a lesser extent, c-kit and flt3. In mature blood cells, only c-fms was observed in monocytes and a weaker flt3 expression in monocytes and T lymphocytes, whereas no known class III TKRs were detected in B lymphocytes and polymorphonuclear cells (PMNs). In all fractions a novel band could be observed, which appeared to be RET. Expression of RET was confirmed by RT-PCR and showed the highest levels in monocytes, followed by PMNs and CD34⁺ cells. B lymphocytes revealed low levels of expression. RET is known to be essential in neural development. Our results suggest a possible role for this receptor in haemopoiesis.     

Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV‐coinfected patients

Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV‐infected and HIV/HCV‐coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy‐derived liver‐infiltrating lymphocytes from 26 HIV/HCV‐coinfected, 10 chronic HCV‐infected and 10 HIV‐infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T‐cell frequency by flow cytometry. CD8⁺ T cells expressing the exhaustion marker PD‐1 were increased in HCV‐infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4⁺CD25⁺Foxp3⁺ T cells, as well as CD4⁺CD25⁺PD1⁺ T cells, were more frequent in HIV/HCV‐coinfected than in HCV‐monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD‐1⁺ T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8⁺ expression was observed only in PD‐1⁺CD8⁺ T cells from HCV‐infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8⁺ T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.     

Schistosome infection aggravates HCV‐related liver disease and induces changes in the regulatory T‐cell phenotype

Schistosome infections are renowned for their ability to induce regulatory networks such as regulatory T cells (Treg) that control immune responses against homologous and heterologous antigens such as allergies. However, in the case of co‐infections with hepatitis C virus (HCV), schistosomes accentuate disease progression and we hypothesized that expanding schistosome‐induced Treg populations change their phenotype and could thereby suppress beneficial anti‐HCV responses. We therefore analysed effector T cells and n/iTreg subsets applying the markers Granzyme B (GrzB) and Helios in Egyptian cohorts of HCV mono‐infected (HCV), schistosome‐co‐infected (Sm/HCV) and infection‐free individuals. Interestingly, viral load and liver transaminases were significantly elevated in Sm/HCV individuals when compared to HCV patients. Moreover, overall Treg frequencies and Helios^posTreg were not elevated in Sm/HCV individuals, but frequencies of GrzB⁺Treg were significantly increased. Simultaneously, GrzB⁺ CD8⁺ T cells were not suppressed in co‐infected individuals. This study demonstrates that in Sm/HCV co‐infected cohorts, liver disease is aggravated with enhanced virus replication and Treg do not expand but rather change their phenotype with GrzB possibly being a more reliable marker than Helios for iTreg. Therefore, curing concurrent schistosome disease could be an important prerequisite for successful HCV treatment as co‐infected individuals respond poorly to interferon therapy.     

Targeted Antigen Delivery to DEC-205+ Dendritic Cells for Tolerogenic Vaccination

Dendritic cells (DCs) and Foxp3-expressing CD4⁺ regulatory T (Treg) cells play non-redundant roles in the maintenance of peripheral tolerance to self-antigens, thereby preventing fatal autoimmunity. A common hallmark of intra- and extra-thymic Treg cell lineage commitment is the induction of Foxp3 expression as a consequence of appropriate T cell receptor engagement with MHC class II:agonist ligand. It has now become increasingly clear that agonist ligand presentation by immature DCs in the steady state induces T cell tolerance by both recessive and dominant mechanisms, rather than promoting productive T helper cell responses. In this context, the ability of steady-state DCs to promote the extrathymic conversion of initially naïve CD4⁺Foxp3^- T cells into Foxp3⁺ Treg cells is of particular interest as it provides novel perspectives to enhance antigen-specific Treg cell function in clinical settings of unwanted immunity, such as β-cell autoimmunity.     

High antigen levels are the cause of T cell exhaustion during chronic viral infection

Many persistent viral infections induce dysfunctional T cell responses. Although a negative correlation exists between viral load and T cell responses during chronic infection, it is not known whether high antigen levels are the cause or just the consequence of T cell exhaustion. Furthermore, it is unclear what role antigen presentation by bone-marrow (BM) derived versus infected parenchymal cells has on T cell exhaustion. To address these issues, we examined the influence of antigen presentation by different cell types on CD8⁺ T cell responses during persistent infection of mice with lymphocytic choriomeningitis virus (LCMV) clone 13. We generated BM chimeric mice, in which non-BM derived cells were MHC class I deficient. Virus-specific CD8⁺ T cells in lymphoid and nonlymphoid tissues were increased in both number and ability to produce cytokines in these mice soon after infection. However, viral clearance from infected MHC I^−/− parenchyma was significantly impaired, despite increased populations of cytokine producing CTL. The CD8⁺ T cell response was overwhelmed by sustained antigen persistence, becoming increasingly exhausted within 4–6 weeks. Thus, we find that (i) sustained antigen presentation directly drives T cell exhaustion during a chronic viral infection, (ii) CTL require direct antigen-MHC interactions to clear virus-infected cells, and (iii) persistent interactions with antigen presented on both hematopoietic and nonhematopoietic cells negatively impacts virus-specific T cell responses during chronic infection.     

Plasma Exchange Downregulates Activated Monocytes and Restores Regulatory T Cells in Kawasaki Disease

In Kawasaki disease (KD), the effect of plasma exchange (PE) on immune cells has not been fully elucidated. Therefore, we examined the changes in the number of CD14⁺ CD16⁺ activated monocytes, regulatory T (T_reg), and T‐helper type 17 (Th17) cells in KD patients treated with PE. The percentage of total monocytes and subclasses of lymphocytes, including CD4⁺ and CD8⁺ T cells, and CD19⁺ B cells, showed no significant difference before and after PE. However, the percentage of CD14⁺ CD16⁺ monocytes in total leukocytes decreased significantly after PE (1.1% ± 1.5% vs. 2.1% ± 2.3%, P < 0.05). Furthermore, while the percentage of Th17 cells in CD4⁺ T cells did not change, the percentage of T_reg cells in CD4⁺ T cells increased significantly after PE (11.1% ± 5.1% vs. 8.0% ± 4.4%, P < 0.05). Therefore, PE downregulates activated monocytes and upregulates T_reg cells toward normal levels and thus attenuates inflammation in KD.     

Cellular responses during liver fluke infection in sheep and its evasion by the parasite

The cellular immune response in sheep to an acute and chronic primary and an acute secondary liver fluke infection were examined by immunohistology of liver tissue and flowcytometry of lymphocytes from the draining hepatic lymph nodes. Ten days after primary infection, portal tract areas surrounding migratory tunnels were infiltrated with CD4⁺ and CD8⁺ lymphocytes with fewer B cells and T19⁺ T cells. Micro abscesses were distributed sporadically in the liver parenchyma and young flukes could be easily observed in the liver tissue free from inflammatory cells. More intensive infiltration of the portal tract areas was observed during a secondary liver fluke infection characterized by a pronounced increase in eosinophils, B cells and CD4⁺ T cells. In addition, there was an increase in MHC class II⁺ fibroblastic-like cells surrounding the migratory tracts. In contrast to the primary infection, no young flukes were observed in the same tissue areas during the secondary infection. Chronic primary infections were characterized by perilobular fibrosis and a predominance of CD8⁺ and γδ-TCR⁺ T19^- T cells distributed within fibrotic strands. Distinct B cell follicles were observed in the fibrotic strands and near major bile ducts and necrotic patches. Pronounced lymphocyte infiltration could occasionally be observed surrounding liver fluke eggs lodged in liver tissue. A progressive increase in lymph node weight, cell number and CD4/CD8 ratio was observed in the acute and chronic primary infections. The role of the infiltrating cell populations and possible mechanisms of immune evasion by the parasite are discussed.     

Minor populations of paroxysmal nocturnal hemoglobinuria‐type cells in patients with chronic idiopathic neutropenia

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells under the influence of pro‐inflammatory mediators and oligoclonal/monoclonal T‐lymphocytes. Because patients with immune‐mediated BM failure display frequently paroxysmal nocturnal hemoglobinuria (PNH)‐type cells in the peripheral blood (PB), we investigated the possible existence of PNH‐type cells in 91 patients with CIN using flow cytometry. The patients displayed increased proportions of PNH‐type glycophorin A⁺/CD59^dim and glycophorin A⁺/CD59^? red blood cells (RBCs), FLAER^?/CD24^? granulocytes, and FLAER^?/CD14^? monocytes, compared to controls (n = 55). A positive correlation was found between the proportions of PNH‐type RBCs, granulocytes, and monocytes and an inverse correlation between the number of PB neutrophils and the proportions of PNH‐type cell populations. The number of patients, displaying percentages of PNH‐type cells above the highest percentage observed in the control group, was significantly increased among patients with skewed compared to those with normal T‐cell receptor repertoire suggesting that T‐cell‐mediated immune processes underlie the emergence of PNH‐type cells in CIN. Our findings suggest that patients with CIN display PNH‐type cells in the PB at a high frequency corroborating the hypothesis that CIN belongs to the immune‐mediated BM failure syndromes.     

Normal development is an integral part of tumorigenesis in T cell-specific PTEN-deficient mice

PTEN is a tumor suppressor gene but whether cancer can develop in all PTEN-deficient cells is not known. In T cell-specific PTEN-deficient (tPTEN^−/−) mice, which suffer from mature T cell lymphomas, we found that premalignancy, as defined by elevated AKT and senescence pathways, starts in immature T cell precursors and surprisingly not in mature T cells. Premalignancy only starts in 6-week-old mice and becomes much stronger in 9-week-old mice although PTEN is lost since birth. tPTEN^−/− immature T cells do not become tumors, and senescence has no role in this model because these cells exist in a novel cell cycle state, expressing proliferating proteins but not proliferating to any significant degree. Instead, the levels of p27^kip1, which is lower in tPTEN^−/− immature T cells and almost nonexistent in tPTEN^−/− mature T cells, correlate with the proliferation capability of these cells. Interestingly, transient reduction of these cancer precursor cells in adult tPTEN^−/− mice within a crucial time window significantly delayed lymphomas and mouse lethality. Thus, loss of PTEN alone is not sufficient for cells to become cancerous, therefore other developmental events are necessary for tumor formation.     

Quantification of lymph node transit times reveals differences in antigen surveillance strategies of na?ve CD4+ and CD8+ T cells

Naïve T cells continually recirculate between blood and secondary lymphoid organs, scanning dendritic cells (DC) for foreign antigen. Despite its importance for understanding how adaptive immune responses are efficiently initiated from rare precursors, a detailed quantitative analysis of this fundamental process has not been reported. Here we measure lymph node (LN) entry, transit, and exit rates for naïve CD4⁺ and CD8⁺ T cells, then use intravital imaging and mathematical modeling to relate cell–cell interaction dynamics to population behavior. Our studies reveal marked differences between CD4⁺ vs. CD8⁺ T cells. CD4⁺ T cells recirculate more rapidly, homing to LNs more efficiently, traversing LNs twice as quickly, and spending ∼1/3 of their transit time interacting with MHCII on DC. In contrast, adoptively transferred CD8⁺ T cells enter and leave the LN more slowly, with a transit time unaffected by the absence of MHCI molecules on host cells. Together, these data reveal an unexpectedly asymmetric role for MHC interactions in controlling CD4⁺ vs. CD8⁺ T lymphocyte recirculation, as well as distinct contributions of T cell receptor (TCR)-independent factors to the LN transit time, exposing the divergent surveillance strategies used by the two lymphocyte populations in scanning for foreign antigen.     

Induction of regulatory T cells by Opisthorchis viverrini

Opisthorchis viverrini causes public health problems in South‐East Asia. Recently, TGF‐β and IL‐10 have been reported to increase in O. viverrini‐infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4⁺ T cells in infected hamsters were investigated. It was demonstrated that IL‐4⁺CD4⁺ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL‐10⁺CD4⁺ T cells were also discovered at a significant level while Treg (T regulatory)‐like TGF‐ β⁺CD4⁺ T cells were in MLNs of infected hamsters. Moreover, the CD4⁺CD25⁺Foxp3⁺ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T‐cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF‐β⁺CD4⁺ while TCC35, TCC41 and TCC108 were IL‐4⁺CD4⁺ and TCC23 was IFN‐γ⁺CD4⁺. This TGF‐β⁺CD4⁺ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF‐β‐mediated mechanisms. This study indicated that O. viverrini‐infected hamsters could induce TGF‐β⁺ CD4⁺ Treg‐like cells. The CSAg‐specific Tregs secreted high TGF‐β, and limited immune cell proliferation.     

The antiapoptotic activity of Heligmosomoides polygyrus antigen fractions

Our study identified Heligmosomoides polygyrus antigen factors with potential activity for regulation of T‐cell proliferation and surviving of CD4⁺CD25^?, CD4⁺CD25^hi and CD3⁺CD8⁺ cell populations. The antiapoptotic activity of antigenic fractions separated by HPLC was evaluated in vitro after exposure of cells to DEX and rTNF‐α. Different populations of cells responded to antigen fractions in distinct pattern; the most sensitive population of cells to H. polygyrus products were CD4⁺CD25^hi after exposure to DEX and CD3⁺CD8⁺ T cells after exposure to rTNF‐α. H. polygyrus antigens may influence survival of CD8⁺ T cells by regulation of c‐FLIP rather than Bcl‐2, which affects survival of CD4⁺CD25^hi Treg cells and CD4⁺ T cells. Activation of NF‐κB subunits, for example, p50 and p65 was essential for resistance of cells to apoptosis, and antigenic fractions F9 and F17 exerted different effect to F13. The most active fraction in inhibition of apoptosis was F9, which includes Hsp‐60, calumenin, ferritin, galectin and thrombospondin. This study may provide new clues for recognition of factors that regulate the immune response during infection and which engage the TNF‐α receptor‐mediated and the mitochondria‐mediated death pathway.     

Rapid retroviral infection of human haemopoietic cells of different lineages: efficient transfer in fresh T cells

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP⁺ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP⁺ cells, all present within the CD3⁺ population, with CD4⁺ and CD8⁺ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34⁺ cells were also susceptible, varying from 6% to 10% GFP⁺ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP⁺ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP⁺ cells did not correlate with the percentage of S/G₂/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.     

Increased frequency and cell death of CD16+ monocytes with Mycobacterium tuberculosis infection

Monocytes from tuberculosis patients exhibit functional and phenotypical alterations compared with healthy controls. To determine whether these discrepancies can be explained by changes in monocyte subsets, the expression of CD14 and CD16 was evaluated in tuberculosis patients and healthy controls; additionally, some markers related to the mononuclear phagocytes maturation, differentiation and function, such as CD1a, CD1c, CD11b, CD11c, CD13, CD33, CD36, CD40, CD64, CD68, CD80, CD83, CD86, HLA-DR, CCR2, CCR5, and non-specific esterases (NSE) were determined in monocyte subsets. Patients had increased percentage of circulating CD14^HiCD16⁺ and CD14^LoCD16⁺ monocytes. The percentage of monocytes expressing CD11b, CD36, CD64, CD68, CD80, CD86, CCR2 and NSE was lower in CD14^HiCD16⁺ and CD14^LoCD16⁺ cells than in CD14^HiCD16⁻ monocytes. M. tuberculosis infected CD16⁺ monocytes produced more TNF-α and less IL-10 than CD16⁻ cells at 6 h post-infection. Isolated CD16⁺ monocytes spontaneously underwent apoptosis during differentiation into macrophages; in contrast to CD16⁻ monocytes that became differentiated into monocyte-derived macrophages (MDM) with a minimal induction of cell death. In addition, there were more Annexin V and propidium iodide positive monocytes in the CD16⁺ subset infected with live M. tuberculosis at 24 h than CD16⁻ monocytes. Under the culture conditions established for this study, the monocyte subsets did not differentiate into dendritic cells. These results show that tuberculosis patients have an augmented frequency of CD16⁺ circulating monocytes which are more prone to produce TNF-α and to undergo cell death in response to M. tuberculosis infection.