Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte–macrophage progenitors (GMPs), whereas differentiation into megakaryocyte–erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1⁺Mac-1⁺ myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca²⁺ chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte–monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines.     

Influenza virus-specific human cytotoxic T cell clones: Heterogeneity in antigenic specificity and restriction by class II MHC products

Human cytotoxic T lymphocytes specific for A/JAP/57 (H2N2) influenza virus were cloned from in vitro stimulations of peripheral blood lymphocytes. Analysis of the viral specificity in cytotoxic function revealed one clone that killed all type A influenza-infected targets, another clone that was specific for the hemagglutinin subtype of the immunizing influenza virus, and the third clone that demonstrated cytotoxicity restricted to the hemagglutinin of A/JAP/57 and A/JAP/62 (H2N2) and not other type A influenza strains with the H2N2 subtypes. The phenotype of these three clones was Leu 2^?, Leu 3⁺, Leu 4⁺; MHC restriction of their cytotoxic function was mapped to HLA-DR by a panel of target cells as well as by inhibition of cytotoxicity with monoclonal antibodies. Proliferation of these clones, examined in a tritiated thymidine incorporation assay, was found to be driven by antigen in the absence of exogenous lymphokines. For all three clones antigen-dependent production and secretion of lymphokines with IL-2 activity was demonstrated. The antigen specificity of proliferation and factor production was shown to be identical to the pattern that each clone revealed in its cytotoxic function.     

Clonotype and repertoire changes drive the functional improvement of HIV-specific CD8 T cell populations under conditions of limited antigenic stimulation

Persistent exposure to cognate Ag leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Ag withdrawal, attributable either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. In this study, we conducted a longitudinal analysis of clonality, phenotype, and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Ag decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of programmed death-1, and the emergence of polyfunctional HIV-specific CD8 T cells. High-definition analysis of individual clonotypes revealed that the Ag loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two nonexclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes endowed with superior functional capabilities.     

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.     

Stability of a diverse immunological memory is determined by T cell population dynamics

The correlation between properties of the T cell memory pool and the two regulatory mechanisms of cell death (apoptosis) and memory entry (differentiation) is investigated mathematically. Apoptosis of T cells occurs at the end of an immune response, removing unwanted activated T cells. T cells escaping apoptosis enter the memory pool composed of T cells specific for previously encountered antigens. We find that the relative efficiencies of these two pathways determine the clonal distribution and the long-term stability of the memory pool by regulating the number of new entries. The main result presented in this paper is that immunological memory of previously encountered pathogens cannot be erased by either severe or repeat infections with a particular pathogen (the diversity of the memory pool is ensured) only if apoptosis and/or memory differentiation are regulated by population dependent processes. Furthermore, vaccination properties are improved significantly by population dependent mechanisms and our mathematical analysis reveals that the T cell population must communicate with other parts of the immune system to ensure optimal performance of immunological memory.     

Increased expression of the NK cell receptor KLRG1 by virus-specific CD8 T cells during persistent antigen stimulation

The killer cell lectin-like receptor G1 (KLRG1) is a natural killer cell receptor expressed by T cells that exhibit impaired proliferative capacity. Here, we determined the KLRG1 expression by virus-specific T cells. We found that repetitive and persistent antigen stimulation leads to an increase in KLRG1 expression of virus-specific CD8+ T cells in mice and that virus-specific CD8+ T cells are mostly KLRG1+ in chronic human viral infections (human immunodeficiency virus, cytomegalovirus, and Epstein-Barr virus) but not in resolved infection (influenza virus). Thus, by using KLRG1 as a T-cell marker, our results suggest that the differentiation status and function of virus-specific CD8+ T cells are directly influenced by persistent antigen stimulation.     

Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors.

For the pathogenic yeast Candida albicans, secreted aspartyl proteinase (Sap) activity has been correlated with virulence. A family consisting of at least eight SAP genes can be drawn upon to produce Sap enzymatic activity. In this study, the levels of Sap1, Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several strains, including strain WO-1, which alternates between two switch phenotypes, white (W) and opaque (O). When cultured under proteinase-inducing conditions, most strains and W cells produce Sap2, while O cells produce Sap1, Sap2, and Sap3. Both W and O cells of strain WO-1 produce Saps in enriched and defined media that do not induce Saps from other strains. The specific Sap isoenzyme that is produced is determined by the cell type, while the level of Sap production is determined by environmental factors. The levels and temporal regulation of the SAP mRNAs as determined by Northern (RNA) analysis were consistent with Sap protein levels and with previous results. S1 analysis showed that SAP6 is the predominant SAP gene transcribed during hyphal induction at neutral pH. These studies define the culture conditions which control the levels of SAP mRNAs and Sap proteins, and they indicate that both the yeast/hyphal transition and phenotypic switching can determine which of the Sap isoenzymes is produced.     

The balance between donor T cell anergy and suppression versus lethal graft-versus-host disease is determined by host conditioning

Graft-vs-host disease (GVHD) remains the most life-threatening complication following the transfer of allogeneic bone marrow into immunocompromised hosts. Transferred alloreactive T cells respond in a complex manner. While massive T cell expansion is observed upon entry into an allogeneic environment, anergy, apoptosis, and repertoire selection are also observed. The study presented here shows that alloreactive T cell expansion and differentiation vs anergy and suppression are dramatically influenced by host conditioning. Using alloreactive CD4(+) and CD8(+) TCR transgenic (Tg) T cells, a novel GVHD model is presented that allows for the visualization of how alloreactive T cells behave when host conditioning is manipulated. Following the transfer of alloreactive CD4(+) and CD8(+) TCR Tg T cells into sublethally irradiated hosts, both Tg T cells populations expand, develop effector function, and cause GVHD. In contrast, when Tg T cells are transferred in non-irradiated hosts, expansion is observed, but there is no development of effector function or disease. Assessment of CD4(+) Tg T cell function following transfer into non-irradiated hosts reveals that these CD4(+) Tg cells are profoundly anergic and have acquired a regulatory function, as manifested in their ability to suppress the expansion of naive TCR Tg T cells in vitro and in vivo as well as the development of GVHD. These findings underscore the decisive effect of the inflammatory environment created by irradiation in determining the ultimate fate and function of alloreactive T cells in vivo     

Ex vivo phenotype and frequency of influenza virus-specific CD4 memory T cells

Recent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonpersistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct "central memory" CD62L(+) phenotype.     

HCMV spread and cell tropism are determined by distinct virus populations

Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.     

The T lymphocyte response to cytochrome c. V. Determination of the minimal peptide size required for stimulation of T cell clones and assessment of the contribution of each residue beyond this size to antigenic potency

The B10.A T cell proliferative response to pigeon cytochrome c is mainly directed against a single antigenic determinant located at the carboxy-terminal end of the molecule. In the present experiments, we used synthetic peptide analogs of the carboxy-terminal sequence of moth cytochrome c to explore the structural requirements for antigenic potency. The minimum-sized peptide capable of stimulating a full response varied with the T cell clone, but within the limits of the biological systems studied, was shown to be moth fragment 97-103. Addition of more amino acids at the amino terminal end increased the antigenic potency in uneven increments, with a large contribution being made at residue 95. Analysis of amino acid substitutions at this position provided no evidence that it contained a residue that directly contacted the T cell receptor. Instead, good agreement with an analysis that made use of helix-coil transition theory suggested that this residue, as well as others, increased antigenic potency by contributing to the stabilization of the secondary structure of the molecule in an alpha-helical configuration. The maximum effect of chain length on antigenic potency appeared to stop at residue 93, in agreement with the theoretical analysis. However, addition of several more amino-terminal residues to residue 93 showed one additional significant increment of increased potency. This was almost entirely accounted for by a single lysine located four amino acids beyond the glutamic acid at residue 93 (approximately one turn of an alpha-helix away). To experimentally test whether alpha-helix-forming tendencies could account for the increased potency of the larger analogs, the degree of helix formation in trifluoroethanol was assessed by circular dichroism measurements. A good correlation was found between antigenic potency and percentage of alpha-helix for peptides of increasing chain length from moth 95-103 up to moth 86-90; 94-103. These results suggest that secondary structure may play an important role in determining the potency of antigenic determinants involved in the activation of T lymphocytes.     

Inhibition of influenza-immune T cell effector function by virus-specific hybridoma antibody.

The in vitro activity of influenza-specific cytotoxic T cells can be inhibited by incubation of the target cells with monoclonal anti-influenza antibodies. Hybridoma antibodies that bind to the virus HA inhibit the cytotoxic activity of TDL for the virus-infected target by as much as 80%, whereas these same antibodies never reduce splenic T cell function by more than 40%. This reflects the fact that TDL from anti-influenza strain A/WSN/33 (HON1) are highly subtype-specific, whereas splenic effector cells from the same mice are cross-reactive for target cells infected with heterologous influenza A viruses. These findings are discussed in the light of previous failures to block virus-immune T cell effector function with heterogeneous antisera produced in vivo, and are considered to favor the idea that at least some of the "virus-immune" T cells are indeed recognizing viral antigens.     

Regulation of influenza virus-specific cytotoxic T cell responses by monoclonal antibody to a human T cell differentiation antigen

The results in this report indicate that the OKT3 monoclonal antibody, which is specific for a human T cell differentiation antigen present on 90 to 95% of peripheral T cells, can exert several effects that regulate the generation and expression of human influenza virus-immune cytotoxic T lymphocytes (CTL). The OKT3 antibody, but not OKT1 or OKT11 (which bind to all peripheral T cells), is able to inhibit anti-influenza CTL effector cell activity. An F(ab')2 preparation of OKT3 IgG were as effective as whole IgG for the inhibition of CTL effectors, indicating that the inhibitory activity of the antibody was not a function of the Fc portion of the molecule. OKT3 IgG and OKT3 F(ab')2 fragments (but not OKT4, OKT8, or OKI were able to inhibit the generation of anti-influenza CTL. The culture of human lymphoid cells with OKT3 in the presence or absence of influenza virus induced radioresistant cells that could suppress the CTL response of fresh autologous lymphocytes to influenza. These results suggest that T cell functions can be regulated by signals that are initiated by the binding of antibody to cell surface molecules that may not be related to the T cell antigen-specific receptor(s).     

The contraction phase of virus-specific CD8+ T cells is unaffected by a pan-caspase inhibitor

The effectiveness of protection conferred by CD8(+) memory T cells is determined by both their quality and their quantity, which suggests that vaccine efficacy might be improved if it were possible to increase the size of the memory pool. Approximately 90% of virus-specific CD8(+) T cells die during the contraction phase and, herein, we have attempted to increase the memory pool by reducing CD8(+) T cell death. CD8(+) T cell contraction has been attributed to apoptosis, or programmed cell death (PCD), which, classically, is dependent on caspases. Caspase-dependent PCD can be prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD), and here we evaluate the effect of this compound on virus-specific T cell responses in mice. zVAD prevented caspase-dependent PCD of freshly isolated virus-specific T cells in tissue culture, and a fluorescent analog, FITC-VAD, entered CD8(+) T cells following in vivo injection. However, despite using 11 different regimens of zVAD administration in vivo, no significant effects on CD8(+) or CD4(+) memory T cell numbers were observed. Furthermore, the CD8(+) memory T cell responses to secondary virus infection were indistinguishable, both qualitatively and quantitatively, in zVAD-treated and normal mice. The absence of effect cannot be attributed to a technical flaw, because identical doses of zVAD were able to rescue mice from hepatocyte apoptosis and lethal intrahepatic hemorrhage, induced by inoculation of anti-Fas Ab. We conclude that the contraction phase of the virus-specific T cell response is unlikely to require caspase-dependent PCD. We propose that contraction can be mediated by an alternative, caspase-independent pathway(s).     

The growth of the very large CD8+ T cell clones in older mice is controlled by cytokines

Older humans and mice frequently contain very large clones of CD8(+) T cells. In mice these cells are phenotypically very similar to memory CD8(+) T cells. Like memory CD8(+) T cells, most members of the clones are in continuous slow division, apparently independently of Ag stimulation. Proliferation of the CD8(+) clonal T cells is inhibited in mice treated with Ab to the IL-2R beta-chain that blocks signaling by either IL-2 or IL-15. However, inhibition of IL-2 increases the numbers of dividing clonal cells. Therefore, like normal memory CD8(+) T cells, expansion of the clones is driven by IL-15 and inhibited by IL-2 and is probably limited by the amounts of IL-15 and IL-2 present in the host. Control by these two cytokines may account for the fact that, although the clones can be very large, they do not overwhelm or kill their hosts. Nevertheless the clonal cells compete successfully with normal memory CD8(+) T cells for growth. Perhaps the clonal cells use IL-15 more effectively or are more resistant to the inhibitory effects of IL-2. Thus they might affect the immune response of their hosts by competing for factors that stimulate and inhibit normal CD8(+) memory T cells.     

Macrophage-derived nitric oxide inhibits the proliferation of activated T helper cells and is induced during antigenic stimulation of resting T cells

To examine how macrophage-derived nitric oxide (NO) affects T helper (Th) cell activity, T cell clones representing Th1 and Th2 subsets were activated before exposure to stimulated peritoneal macrophages or microglia. Both Th subsets were similarly sensitive to inhibition by NO, indicating that macrophage-derived NO regulates the proliferation of activated Th1 and Th2 cells equally well. Since IFN-gamma production remained intact in NO-treated Th1 cells, we studied whether NO was produced during antigen-specific activation of Th1 cells by unstimulated macrophages. Indeed, T cell proliferation only occurred when a NO synthase inhibitor was included, while IFN-gamma was essential for the induction of NO. These studies demonstrate that macrophages produce NO following antigen presentation to Th1 cells and that macrophage-derived NO inhibits Th1 and Th2 cell proliferation without inhibiting cytokine production.     

Presence of an antigen-specific T cell subset that forms IgE-suppressive factor and IgG-suppressive factor on antigenic stimulation

B6D2F1 mice were given three i.v. injections of ovalbumin (OA), and antigen-specific T cell clones were established from their spleen cells. One of the FcR+ T cell clones formed IgE-binding factors on incubation with OA-pulsed syngeneic macrophages. Neither soluble antigen nor macrophages alone induced factor formation. T cell hybridomas were constructed by fusion of the antigen-specific T cell clone with BW 5147 cells. Among 11 T cell hybridomas established, six clones produced IgE-binding factors on incubation with OA-pulsed BDF1 macrophages. Mouse IgE also induced the same hybridoma to form IgE-binding factors. The majority of IgE-binding factors formed by two T hybridomas and by those produced by the parent T cell clone had affinity for peanut agglutinin but for neither lentil lectin nor Con A. These hybridomas and the original T cell clone spontaneously released glycosylation-inhibiting factor, which inhibits the assembly of N-linked oligosaccharide(s) on IgE-binding factors. On antigenic stimulation, the T cell hybridomas produced both IgE-binding factors and IgG-binding factors. The IgE-binding factors consisted of three species with m.w. of 60,000, 30,000, and 15,000. Both the 60K and 15K IgE-binding factors selectively suppressed the IgE response of DNP-OA-primed rat mesenteric lymph node cells, whereas IgG-binding factors selectively suppressed the IgG response. The results indicate that antigen-primed FcR+ T cells produced IgE-suppressive factors and IgG-suppressive factors on antigenic stimulation. However, the T cell hybridomas were not committed to suppressive activity. When the hybridomas were stimulated by antigen in the presence of glycosylation-enhancing factor (GEF), the 60K, 30K, and 15K IgE-binding factors formed by the cells selectively potentiated the IgE response. IgG-binding factors formed by the cells in the presence of GEF failed to suppress the IgG response. It appears that antigen-specific FcR+ T cells regulate the antibody response through the formation of Ig-binding factors, but that the function of the cells could be switched from suppression to enhancement, depending on the environment of the cells.     

Antigen presentation by resting B cells. Effectiveness at inducing T cell proliferation is determined by costimulatory signals, not T cell receptor occupancy

Resting B cells stimulated the proliferation of two T cell clones much less efficiently than T cell-depleted low-density APC. In contrast, low-density cells and resting B cells stimulated the clones to produce similar levels of inositol phosphates, a rapid biochemical event dependent only on occupancy of the TCR. The inefficient stimulation of T cell proliferation by resting B cell APC was dramatically improved by the addition of allogeneic low-density accessory cells incapable of being recognized by the TCR on the responding T cells. The results are most consistent with a model where low-density and resting B cell APC display similar amounts of Ag/Ia molecule complexes capable of being recognized by the TCR on the responding T cells but differ in the provision of costimulatory signals that, together with TCR occupancy, are required for IL-2 production.     

The cell cycle time of CD8+ T cells responding in vivo is controlled by the type of antigenic stimulus

A hallmark of cells comprising the mammalian adaptive immune system is the requirement for these rare na?ve T (and B) lymphocytes directed to a specific microorganism to undergo proliferative expansion upon first encounter with this antigen. In the case of na?ve CD8(+) T cells the ability of these rare quiescent lymphocytes to rapidly activate and expand into effector T cells in numbers sufficient to control viral and certain bacterial infections can be essential for survival. In this report we examined the activation, cell cycle time and initial proliferative response of na?ve murine CD8(+) T cells responding in vivo to Influenza and Vaccinia virus infection or vaccination with viral antigens. Remarkably, we observed that CD8(+) T cells could divide and proliferate with an initial cell division time of as short as 2 hours. The initial cell cycle time of responding CD8(+) T cells is not fixed but is controlled by the antigenic stimulus provided by the APC in vivo. Initial cell cycle time influences the rate of T cell expansion and the numbers of effector T cells subsequently accumulating at the site of infection. The T cell cycle time varies with duration of the G(1) phase of the cell cycle. The duration of G(1) is inversely correlated with the phosphorylation state of the retinoblastoma (Rb) protein in the responding T cells. The implication of these findings for the development of adaptive immune responses and the regulation of cell cycle in higher eukaryotic cells is discussed.     

The maternal DDK syndrome phenotype is determined by modifier genes that are not linked to Om

The DDK syndrome is a polar, early embryonic lethal phenotype caused by incompatibility between a maternal factor of DDK origin and a paternal gene of non-DDK origin. Both maternal factor and paternal gene have been mapped to the Om locus on mouse Chromosome (Chr) 11. The paternal contribution to the syndrome has been shown to segregate as a single locus. Although the inheritance of the maternal contribution has not been characterized in depth, it as been assumed to segregate as a single locus. We have now characterized the segregation of the DDK fertility phenotype in over 240 females. Our results demonstrate that females require at least one DDK allele at Om to manifest the syndrome. However, the DDK syndrome inter-strain cross-fertility phenotype of heterozygous females is highly variable and spans the gamut from completely infertile to completely fertile. Our results indicate that this phenotypic variability has a genetic basis and that the modifiers of the DDK syndrome segregate independently of Om. Received: 24 November 1998 / Accepted: 19 January 1999