Changing epidemiology of methicillin-resistant Staphylococcus aureus colonization in paediatric intensive-care units.

Community-associated methicillin-resistant S. aureus (CA-MRSA) accounts for a growing proportion of hospital-onset infections, and colonization is a risk factor. This study aimed to determine changes in the prevalence of CA-MRSA colonization in paediatric intensive-care units (ICUs). A total of 495 paediatric patients colonized with MRSA from neonatal, medical, surgical, and cardiac ICUs between 2001 and 2009 were identified. Isolates were characterized by spa type, staphylococcal cassette chromosome (SCC) mec type and the presence of the genes encoding Panton­Valentine leukocidin (PVL). The proportion of patients colonized with MRSA remained stable (average 3·2%). The proportion of isolates with spa type 1, SCCmec type IV and PVL increased over time to maximums in 2009 of 36·1% (P < 0·001), 54·2% (P = 0·03) and 28·9% (P = 0·003), respectively. Antibiotic susceptibility patterns showed increasing proportions susceptible to clindamycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole (P values <0·001). In conclusion, the proportion of MRSA-colonized children in ICUs with CA-MRSA increased significantly over time.

B7-1 or B7-2 is required to produce the lymphoproliferative phenotype in mice lacking cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

The costimulatory molecules B7-1 and B7-2 regulate T lymphocyte activation by delivering activating signals through CD28 and inhibitory signals through cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The importance of CTLA-4-mediated inhibition was demonstrated by the uncontrolled T cell activation and lymphoproliferative disease that develops in CTLA-4-deficient (-/-) mice. To examine the role of B7 signaling in the activation of CTLA-4-deficient T cells, we bred CTLA-4(-/-) mice with mice lacking B7-1, B7-2, or both B7 molecules. The CTLA-4/B7-1(-/-) and the CTLA-4/B7-2(-/-) mice develop lymphoproliferation and enhanced T cell activation. Mice lacking CTLA-4, B7-1, and B7-2 have a normal life-span, and do not have lymphocytic infiltrates in any organs, or increased T cell activation. Therefore, the two B7 molecules have overlapping functions, since either B7-1 or B7-2 alone can cause the CTLA-4(-/-) phenotype. Elimination of both B7-1 and B7-2 from the CTLA-4- deficient mouse abrogates the lymphocyte activation and disease, and does not reveal evidence for additional stimulatory CD28 ligands. The CTLA-4(-/-) phenotype can be reproduced with anti-CD28 antibody in mice lacking CTLA-4, B7-1, and B7-2, but wild-type mice are unaffected by the same treatment. This suggests that the inhibitory function of CTLA-4 can overcome strong CD28-mediated signaling in vivo.

Interleukin 3 enhances development of tumor-reactive cytotoxic cells by a CD4-dependent mechanism.

We investigated the effects that mouse interleukin 3 (IL-3), in comparison to mouse IL-2, has on the generation of cytotoxic effectors capable of killing line 1 tumor cells. These potent immunological mediators were delivered locally using gene transfection, rather than systemically, to the tumor site. We created line 1 transfectants that express high levels of IL-3 (3750 units/ml) or IL-2 (200 units/ml) by driving transcription from the beta-actin promoter. These levels of expression significantly enhanced tumor rejection in syngeneic mice. Tumor-infiltrating lymphocytes purified from IL-3 or IL-2 transfected tumors showed a dramatically enhanced cytotoxic response to parental line 1 targets. Also, IL-2, but not IL-3, expression enhanced the nonspecific lysis of YAC-1 cells. In vivo depletion of CD8+ cells with monoclonal antibody 2.43 abrogated the generation of cytotoxic effectors in both cases. Interestingly, depletion of CD4+ cells with monoclonal antibody GK1.5 abrogated the IL-3-mediated cytotoxic response but not the IL-2-mediated response. In vivo depletion of CD4+ or CD8+ cells abrogated the effect IL-3 had on reducing tumorigenicity. Reverse polymerase chain reaction analysis demonstrates that IL-3 transfected tumors, when compared to untransfected tumors, express increased levels of IL-2 and IL-4 mRNA. These results strongly suggest that IL-3, unlike IL-2, works to generate cytotoxic effectors by a mechanism that requires CD4+ cells.

The role of B7 co-stimulation in activation and differentiation of CD4+ and CD8+ T cells.

The functional significance of B7 co-stimulation in T-cell activation was described first in the context of preventing the induction of anergy. The functions of this pathway are far more complex than initially appreciated in view of the existence of two B7 molecules which have specificities for both CD28 and CTLA-4, which serve to amplify and terminate T-cell responses respectively. Mice lacking B7 co-stimulators and CD28 and CTLA-4 co-stimulatory receptors are helping to clarify the functions of this key immunoregulatory pathway. In this review we will focus on the role of B7 co-stimulation in the activation and differentiation of CD4+ helper cells and CD8+ cytotoxic cells. The contribution of B7 co-stimulation to CD4+ responses depends upon the activation history of the T-cell and the strength of the T-cell antigen receptor signal. B7 co-stimulation contributes to interleukin (IL)-2 production by both naive and previously activated CD4+ T cells. B7 co-stimulation is most critical for the differentiation of naive CD4+ T cells to IL-4 producers, but predominately influences IL-2 production by previously activated CD4+ cells. B7 co-stimulation is important in development of cytotoxic T cells through both effects on T-helper cells and by direct co-stimulation of CD8+ cells.

Either B7 costimulation or IL-2 can elicit generation of primary alloreactive CTL.

B7-1 and B7-2 are important costimulatory molecules in the activation of T cell immunity. We have used mice made genetically deficient in either or both B7 molecules to determine the role of B7 molecules in activation of primary alloreactive CTL. The absence of either B7-1 or B7-2 did not alter generation of CTL from unfractionated lymphocytes, but the absence of B7-2 greatly decreased CTL generation from purified CD8+ responder cells. However, if B7-1 was induced on the stimulating cells then CTL generation was restored to wild-type levels. Absence of both B7-1 and B7-2 from MLR using whole splenocytes resulted in a profound reduction in generation of CTL. This could completely be reversed by the addition of IL-2. B7 molecules could directly costimulate CD8+ cells, as purified CD8+ cells developed into mature CTL when stimulated with wild-type APC, but not with B7-deficient APC. Again, IL-2 could drive CTL generation from purified CD8+ cells, even in the absence of B7 molecules. Taken together, these results demonstrate an important role for B7 costimulation in CTL generation.

Mechanism of cytolytic T lymphocyte killing of a low class I-expressing tumor.

Many tumors have been shown to express minimal levels of class I MHC Ag, which makes them more resistant to recognition and lysis by cytolytic T lymphocytes. Line 1, a BALB/c spontaneous lung carcinoma, normally expresses very low levels of class I Ag, but expression can be increased 50-fold by treatment with agents such as DMSO or IFN-gamma. Because class I Ag serve as restricting elements for cytolytic T cell recognition of tumor Ag, we wished to determine if cytotoxic T lymphocytes could play a role in the immune response to this type of class I low, but inducible, tumor. After immunization in vivo and restimulation of splenic cells in vitro we were able to generate T cell clones that lysed line 1 cells induced to express high levels of class I, but did not lyse uninduced, low class I expressing line 1 cells in short term (6-h) 51Cr release assays. Paradoxically, incubation of the T cells with uninduced class I low line 1 cells for a few days resulted in complete destruction of the tumor cells. We demonstrate that the T cells, stimulated by the tumor cells, produce IFN-gamma, which in turn induces class I expression on the line 1 cells making them susceptible to lysis by the T cell clone. This suggests that a positive feedback reaction can occur in generating a response to this and perhaps other inducible tumor cell lines.

CD4-class II major histocompatibility complex interaction does not enhance killing by a class I-restricted CD4+CD8+ cytotoxic T cell clone.

As unusual tumor-specific cytotoxic T lymphocyte (CTL) clone was isolated which expressed both CD4 and CD8 molecules. The target cells for this CTL can be induced to express either class I major histocompatibility complex (MHC) alone (with dimethylsulfoxide) or both class I and class II MHC (with interferon-gamma). Lysis of the tumor target depends on expression of class I MHC molecules, but does not require expression of class II MHC molecules. Furthermore, the lysis of target cells expressing both class I and class II is inhibited only by antibodies to class I (Kd), and not by antibodies to class II, demonstrating that the T cell receptor is class I restricted. We have used this CTL to assess the role of the interaction between CD4 and class II MHC in the absence of a class II-restricted T cell receptor. Our data indicate that CD4-class II interaction does not contribute to recognition by T cells in the absence of binding of the T cell receptor to class II molecules.

B7 costimulation is critical for antibody class switching and CD8(+) cytotoxic T-lymphocyte generation in the host response to vesicular stomatitis virus.

Antibody and cytotoxic T-lymphocyte (CTL) responses have critical roles in eliminating many viral infections. In addition to stimulation of the T-cell receptor, T cells require costimulatory signals to respond optimally. We evaluated the role of B7 costimulatory molecules (B7-1 and B7-2) in the immune response to viral infection using vesicular stomatitis virus (VSV) and mice lacking either B7-1 or B7-2 or both molecules. Mice lacking both B7-1 and B7-2 had essentially no anti-VSV immunoglobulin G1 (IgG1) response, decreased IgG2a responses, and normal IgM responses, while mice lacking either B7-1 or B7-2 had unaltered anti-VSV antibody responses compared to wild-type mice. Depletion of CD4(+) cells further reduced the IgG2a response in mice lacking both B7 molecules, suggesting that CD4(-) cells may supply help for IgG2a in the absence of B7 costimulation. The absence of both B7 molecules profoundly reduced generation of both primary and secondary VSV-specific class I major histocompatibility complex (MHC)-restricted CTL, whereas VSV-specific CTL responses in mice lacking either B7-1 or B7-2 were similar to those of wild-type animals. Class I MHC-restricted CTL in wild-type mice were not dependent on CD4(+) cells, suggesting that the failure of CTL in the absence of B7s is due to a lack of B7 costimulation directly to the CD8(+) CTL. These data demonstrate that B7-1 and B7-2 have critical, overlapping functions in the antibody and CTL responses to this viral infection.

Synergistic effects of co-expression of the TH1 cytokines IL-2 and IFN-gamma on generation of murine tumor-reactive cytotoxic cells.

While the effect of cytokines on the generation of tumor-reactive cytotoxic cells has been a topic of active investigation, the effect of physiological cytokine combinations has not been determined. We have investigated the effect of co-expression of IL-2 and IFN-gamma on the generation of cytotoxic cells against the murine line 1 tumor in vivo. These cytokines were selected because they are normally produced in concert by a subset of T-helper cells called T-helper 1 (Th1). We transfected the line 1 murine carcinoma with cDNA for IL-2 and IFN-gamma, alone or combined. IFN-gamma alone does not elicit rejection of the transfectant, but IL-2 increases the tumorigenic dose by 10,000-fold above the parental cells. Co-expression of IFN-gamma and IL-2 increases this rejection to at least 100,000-fold above parental line 1. Unlike IL-2 transfectants, tumor cells expressing both IFN-gamma and IL-2 can also elicit rejection of admixed parental tumor cells. Finally, the IFN-gamma/IL-2 transfectants are more effective at generating memory cells that are cytolytic for the parental tumor. Our results show that synergistic interactions of Th1 cytokines can remarkably enhance the cytotoxic response to tumors.

Interleukin-3 inhibits the generation of nonspecific killers by interleukin-2.

Interleukin (IL)-3 has effects on a wide variety of cell types, including immature cells of the immune system, as well as mature cells such as granulocytes. We have investigated the effects of IL-3 on generation of cytolytic cells that can kill tumor cells. Previously, we have shown that IL-3 can enhance the development of cytolytic T cells (CTL) reactive with the line 1 tumor in a CD4-dependent manner. It is of interest that we found the development of CTL in response to IL-3 was not accompanied by increased development of nonspecific killer cells. This was in contrast to IL-2, which enhanced development of both CTL and nonspecific cells. To determine if IL-3 could inhibit the development of nonspecific killers, we added IL-3 to cultures of fresh spleen cells stimulated with high levels of IL-2. In this assay, IL-3 showed very potent inhibitory activity against the generation of nonspecific killers. To determine if IL-3 could inhibit the IL-2-driven generation of non-specific killers in vivo, we injected a mixture of IL-2- and IL-3-producing line 1 cells. Each cytokine-producing transfectant was also injected alone. The cytotoxicity of tumor-infiltrating lymphocytes (TIL) from these tumors confirmed that the presence of IL-3 inhibits the generation of nonspecific killer cells in vivo.

Class-I MHC expression in the mouse lung carcinoma, line 1: a model for class-I inducible tumors.

We have examined the expression and biological effects of class-I MHC molecules on the immune response to the line I lung carcinoma. The line I system is of interest because these tumor cells have very low constitutive levels of class-I molecules but can be induced to express levels found on spleen cells, by culturing the cells with agents such as dimethylsulfoxide (DMSO) or interferon gamma (IFN-gamma). This induction is significant immunologically, since induced cells can be lysed very effectively by cytotoxic T lymphocytes (CTL), whereas the uninduced cells cannot. CTL clones that are reactive with line I cells have been generated and used in vitro and in vivo, to examine the interactions of T cells with line I. We have shown that the expression of class I on tumor cells is induced in vivo by IFN-gamma, and that this induction is associated with the ability to reject the tumor. We will also introduce preliminary data concerning the mechanism of induction in which CTL appear to induce class-I MHC both in vitro and in vivo. The results are discussed in terms of a model which may be important generally for class-I inducible tumors.

Identification of a dopamine-binding protein on the membrane of the human platelet.

The binding of [3H]dopamine to platelet membranes has been examined in an attempt to identify the putative dopamine-uptake mechanism of the platelet. [3H]Dopamine has been shown to bind to a 42,000 Da glycoprotein in platelet membrane with high affinity (Kd = 22.6 nM) and binding of [3H]dopamine was competed for by dopamine, molecules with catechol moieties, 5-hydroxytryptamine, GSH and ascorbic acid. Differences in pharmacological profile and molecular mass suggest that [3H]dopamine does not bind to a known receptor, a neuronal-type dopamine transporter or the platelet 5-hydroxytryptamine-uptake site. It is proposed that this novel binding site for dopamine, which has been purified 1000-fold from particulate platelet membrane, is likely to be a component of the dopamine-uptake mechanism of the human platelet.

IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes.

Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.14 (B3Z) restricted to Ova/Kb, was used as an indicator for presentation of particulate Ag with class I MHC. When activated, this hybridoma expresses lacZ, allowing a simple colorimetric measurement of Ag-specific T cell stimulation. We demonstrated that bone marrow cells stimulated by IL-3 in vivo and in vitro exhibited significantly increased presentation of exogenous OVA linked to beads. Lysate from OVA-transfected line 1 murine lung adenocarcinoma cells (line 1/OVA) was also presented by IL-3-stimulated bone marrow cells, suggesting that these APC can process tumor fragments or debris. Studies using TAP1/2-deficient mice and Ag presentation inhibitors indicate that this exogenous Ag presentation is mediated via the conventional class I MHC pathway. Adoptive transfer of IL-3-stimulated bone marrow cells pulsed with lysate from line 1/OVA tumor cells into naive recipient mice led to the generation of a potent CTL response. These observations indicate that use of such cells may provide a new avenue for development of tumor vaccines.

Transfection of transforming growth factor-beta producing tumor EMT6 with interleukin-2 elicits tumor rejection and tumor reactive cytotoxic T-lymphocytes.

One mechanism by which potentially immunogenic tumors evade the immune response is production of immunosuppressive factors. The murine mammary sarcoma EMT6 has previously been demonstrated to inhibit the proliferation of B-cells, suggesting that this tumor produces immunosuppressive factors. Here, we show that supernatant from EMT6 inhibits the development of cytotoxic T-lymphocytes (CTLs) and that this inhibition can be reversed by addition of recombinant interleukin (IL)-2. Furthermore, we show that EMT6 produces high levels of the immunosuppressant factor transforming growth factor (TGF)-beta. To determine if the T-cell growth factor IL-2 within the tumor microenvironment could reverse the immunosuppressive effect of EMT6, we transfected EMT6 with an expression vector containing the cDNA for murine IL-2 under the control of the beta-actin promoter. These transfectants produce significant levels of IL-2 (26 U/ml). EMT6/IL-2 is rejected by mice at 100-fold higher challenge than are parental cells or control transfectants (neomycin resistance only). Thy-1-expressing cells purified from EMT6/IL-2 tumors show greater cytotoxicity against the parental EMT6 cells than do those from the control transfectant. Thus, IL-2 can reverse the effects of TGF-beta on development and/or proliferation of CTL reactive with EMT6, allowing the establishment of mature effectors in vivo. This has significant implications for the development of CTL and immunotherapy for immunosuppressive tumors.

Analysis of the effect of cytokines (interleukins 2, 3, 4, and 6, granulocyte-monocyte colony-stimulating factor, and interferon-gamma) on generation of primary cytotoxic T lymphocytes against a weakly immunogenic tumor.

We have investigated the role of cytokines (IL-2, IL-3, IL-4, IL-6, IFN-gamma, and GM-CSF) in the generation of primary cytotoxic T lymphocytes (CTL), within a single tumor system. The murine carcinoma line 1 was transfected with expression vectors with cDNA for these cytokines. Line 1 expresses low levels of class I MHC molecules, but can be induced with dimethyl sulfoxide or IFN-gamma to express high levels of class I. Class I low line 1 cells are not susceptible to CTL lysis, while class I high line 1 are lysed by CTL, which allows us to assay for CTL activity. To isolate primary CTL, tumor-infiltrating lymphocytes were isolated from cytokine-expressing or control tumors, growing in vivo. Most cytokines stimulated nonspecific killers, but IL-2 and IL-3 stimulated primary CTL. While IFN-gamma alone did not generate primary CTL, coexpression of IFN-gamma with IL-2 resulted in CTL generation. This is the first comparison of the effects of a series of cytokines on primary CTL development, and has important implications for vaccine development and immunotherapy.

B7-1 enhances natural killer cell-mediated cytotoxicity and inhibits tumor growth of a poorly immunogenic murine carcinoma.

The B7-1 molecule expressed on antigen presenting cells is an important costimulatory molecule for T cell activation. It has been demonstrated that murine B7-1 can enhance host immunity and lead to tumor rejection via its costimulatory function. Here, we investigate how transfection of B7-1 into line 1, a poorly immunogenic murine lung carcinoma, affects the generation and function of different immune effector cells. Line 1 cells expressing B7-1 form tumors that grow at a slower rate than the parental line 1. Our studies have shown that tumor infiltrating lymphocytes present within the B7-1 expressing tumors are primarily composed of nonspecific killer cells with no specific cytotoxic T cells present. To determine if increased nonspecific killer cells could inhibit the tumor growth of line 1 in the presence of B7-1, we examined the cytotoxicity of natural killer (NK) cells and lymphokine-activated killer (LAK) cells on the B7-1-transfected line 1 and the parental line 1. We found that B7-1 augments the NK- but not LAK-mediated killing against line 1 as measured in an in vitro 51Cr-release cytotoxicity assay. This enhancement could be blocked by CTLA-4 Ig. In vivo depletion of NK cells led to growth of the B7-1-transfected line 1 at the same rate as the parental line 1. These results suggest that in addition to its costimulatory role for T cell activation B7-1 could be an accessory molecule that intensifies NK-mediated cytotoxicity. This novel finding may provide a mechanism for the effect of B7-1 on tumors of low immunogenicity.

Expression of B7 molecules in recipient, not donor, mice determines the survival of cardiac allografts.

Blockade of the CD28/CTLA4/B7 costimulatory pathway using CTLA4-Ig has great therapeutic potential, and has been shown to prolong allograft survival in a variety of animal models. To gain further insight into the mechanism by which costimulatory blockade prevents allograft rejection, we studied cardiac allograft survival in the complete absence of B7 costimulation using mice lacking B7-1 and B7-2 (B7-1/B7-2-/- mice). To determine the role of B7 on donor vs recipient cells, we used B7-1/B7-2-/- mice as either donors or recipients of allografts. Wild-type (WT) recipients acutely reject fully allogeneic hearts from both WT and B7-1/B7-2-/- mice. In contrast, B7-1/B7-2-/- recipients allow long-term survival of grafts from both WT and B7-1/B7-2-/- mice, with minimal histologic evidence of either acute or chronic rejection in grafts harvested after 90 days. The B7-1/B7-2-/- mice acutely reject B7-1/B7-2-/- allografts if CD28 stimulation is restored by the administration of Ab to CD28 and can mount an alloresponse in mixed lymphocyte reactions. Therefore, B7-1/B7-2-/- mice are capable of generating alloresponses both in vivo and in vitro. Our results demonstrate that in the alloresponse to mouse heterotopic cardiac transplantation, B7 molecules on recipient cells rather than donor cells provide the critical costimulatory signals. The indefinite survival of allografts into B7-1/B7-2-/- recipients further shows that the absence of B7 costimulation alone is sufficient to prevent rejection.

ICOS is critical for CD40-mediated antibody class switching.

The inducible co-stimulatory molecule (ICOS) is a CD28 homologue implicated in regulating T-cell differentiation. Because co-stimulatory signals are critical for regulating T-cell activation, an understanding of co-stimulatory signals may enable the design of rational therapies for immune-mediated diseases. According to the two-signal model for T-cell activation, T cells require an antigen-specific signal and a second, co-stimulatory, signal for optimal T-cell activation. The co-stimulatory signal promotes T-cell proliferation, lymphokine secretion and effector function. The B7-CD28 pathway provides essential signals for T-cell activation, but does not account for all co-stimulation. We have generated mice lacking ICOS (ICOS-/- ) to determine the essential functions of ICOS. Here we report that ICOS-/- mice exhibit profound deficits in immunoglobulin isotype class switching, accompanied by impaired germinal centre formation. Class switching was restored in ICOS-/- mice by CD40 stimulation, showing that ICOS promotes T-cell/B-cell collaboration through the CD40/CD40L pathway.

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes.

Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans.