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Biomedical subjects

M Croft

Publications and source records attributed to M Croft.

At least 19 recordsLinked to original sources
PubMed2001

Microchimerism does not induce tolerance and sustains immunity after in utero transplantation.

BACKGROUND: To date, over 40 in utero transplants have been performed in humans; the only successes were documented in the treatment of severe combined immunodeficiency syndromes. Hemoglobinopathies and metabolic disorders are candidate diseases for this approach; however, when applied clinically, the results have been discouraging. To address the role of the fetal immune system in the outcome of in utero transplantation, we have developed a murine model of in utero transplantation in immunologically intact murine recipients and have studied chimerism and tolerance/immunity to allogeneic donor cells through the lives of the animals. METHODS: We have performed experiments in which purified murine sca-1+/lin- cells and c-kit+/lin- cells of C57BL/6 (H2b) mice were injected into Balb/c (H2d) fetal recipients at early gestational ages. Chimerism was tested by highly sensitive semiquantitative polymerase chain reaction assay and tolerance/immunity to donor cells was studied by in vivo (skin grafts, responses to postnatal boosts) and in vitro (mixed lymphocyte culture, cytotoxicity, and cytokine release) assays. RESULTS: One hundred percent (10/10) of mice transplanted with c-kit+ cells and 44% (4/9) of mice transplanted with sca+ cells showed circulating donor cells within the first 6 months of life (P=0.031). Mice in the sca+ group rejected donor skin grafts at a mean time of 9.1+/-0.2 days, whereas mice in the c-kit+ group rejected donor skin grafts at a mean time of 15.1+/-0.7 days (P=0.001). The difference between the transplanted groups and non-transplanted controls was also significant (P<0.05). All mice transplanted with sca+/lin- cells showed greater response to donor cells than to third-party cells at all effector to target ratios (P=0.002). Differences in response to donor alloantigen between sca+ and c-kit+ groups were significant (P=0.003). Cytokine quantification demonstrated higher TH1 than TH2 cytokine release in all groups, and the response to donor cells was higher in the sca+ compared with c-kit+ mice (P=0.031). CONCLUSION: These results demonstrate a low level of chimerism and tolerance in mice transplanted in utero with sca+/lin- and c-kit+/lin- cells. The possibility of active in utero immunization to donor cells is supported by accelerated skin graft rejection in mice transplanted with sca+ cells and enhanced in vitro immune responses in mice with persistent microchimerism.

Animals↗
PubMed2001

Development of allergic inflammation in a murine model of asthma is dependent on the costimulatory receptor OX40.

Asthma is thought to result from an abnormal expansion of CD4 T cells reactive with airborne allergens, and pathology is controlled by several cytokines of the T helper type 2 (Th2) family. The exact molecules which are involved in generating allergen-reactive T cells are not clear. Studies with blocking reagents or knockout animals have shown that the CD28/B7 interaction partially controls development of allergic asthma in mouse models, but may not be the sole molecule involved. In this report, we have investigated the role of the tumor necrosis factor receptor family member OX40 in allergic inflammation using OX40-deficient mice. OX40 has been shown to participate in regulating clonal expansion and memory development of CD4 T cells and may synergize with CD28. Our studies demonstrate that OX40(-/)- mice, primed with the model allergen ovalbumin and challenged through the airways with aerosolized antigen, are severely impaired in their ability to generate a Th2 response characterized by high levels of interleukin (IL)-5, IL-4, and immunoglobulin E. Moreover, OX40(-/)- mice exhibit diminished lung inflammation, including an 80-90% reduction in eosinophilia and mucus production, less goblet cell hyperplasia, and significantly attenuated airway hyperreactivity. These studies highlight the potential importance of OX40 in development of allergic asthma and suggest that targeting OX40 may prove useful therapeutically.

Allergens↗
PubMed2001

Signaling through OX40 (CD134) breaks peripheral T-cell tolerance.

Peripheral T-cell tolerance is a mechanism to limit autoimmunity, but represents a major obstacle in diseases such as cancer. Tolerance is due to limited accumulation of antigen-specific T cells accompanied by functional hypo-responsiveness, and is induced by antigen encounter in a non-inflammatory environment. In contrast to advances in preventing induction of T-cell tolerance, there has been little progress in defining targets to reverse established tolerance. Here we show that signals from a single dose of an agonistic antibody against OX40 (CD134, a member of the tumor necrosis-factor family of receptors) can break an existing state of tolerance in the CD4+ T-cell compartment. OX40 signals promote T-cell expansion after the hypo-responsive phenotype is induced and restore normal functionality. These data highlight the potent costimulatory capacity of OX40, and indicate OX40 as a target for therapeutic intervention in a variety of related diseases.

Adaptation, Physiological↗
PubMed2000

Functional cooperation between T helper cell determinants.

The immune response to T helper (Th) cell determinants of a variety of antigens is often poor and limits severely the potential efficacy of current therapeutic measures through vaccination. Here, we report that an immunologically silent tumor determinant can be rendered immunogenic if linked with a dominant determinant of a parasite antigen, suggesting the existence of functional Th-Th cooperation in vivo. This phenomenon could be mimicked in part by signaling either through CD40 to the antigen-presenting cells or through OX40 to the tumor-determinant reactive T cells, with maximal effects obtained by combined anti-CD40 and anti-OX40 treatment in vivo. The data suggest that CD4 T cells reactive with a dominant determinant provide help to other CD4 T cells through up-regulating the costimulatory ability of antigen-presenting cells, in much the same way as help for CD8 cells. CD4 help for CD4 T cells represents a new immunological principle and offers new practical solutions for vaccine therapy against cancer and other diseases in which antigenic help is limiting.

Amino Acid Sequence↗
PubMed2000

The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion.

The costimulatory receptor OX40 has recently been shown to be involved in primary CD4 responses to several defined Ags. However, to date there has been little information regarding the mechanism of action of OX40, such as whether it regulates T cell numbers, reactivity, or both, and whether it contributes to induction of long-term T cell responses. With an agonist Ab to OX40, and by tracking Ag-specific TCR transgenic T cells in vivo, we show that ligation of OX40 induces clonal expansion and survival of CD4 cells during primary responses, and results in the accumulation of greater numbers of memory cells with time. Significantly, OX40-deficient T cells, from mice generated by gene targeting, secrete IL-2 and proliferate normally during the initial period of activation, but cannot sustain this during the latter phases of the primary response, exhibiting decreased survival over time. Mice lacking OX40 develop only low frequencies of Ag-specific CD4 cells late in primary responses in vivo and generate dramatically lower frequencies of surviving memory cells. These results demonstrate that OX40-OX40L interactions control primary T cell expansion and the ability to retain high numbers of Ag-specific T cells. In this way, OX40 signals promote survival of greater numbers of T cells with time and control the size of the memory T cell pool.

Animals↗
PubMed2000

CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen.

The involvement of specific accessory/costimulatory molecules in differentiation to Th1 and Th2 phenotypes is controversial. Reports suggest that molecules such as CD4, CD28, and Ox-40 support Th2 differentiation and suppress Th1 differentiation, whereas others such as LFA-1 support Th1 responses and suppress Th2 responses. We have previously defined an in vitro model of differentiation that is absolutely dependent on the initial dose and affinity of peptide presented to a naive CD4 cell. The dose and affinity of Ag regulate autocrine production of IL-2, IL-4, and IFN-gamma, which in turn govern differentiation to Th1 and Th2 phenotypes. We have used this system to confirm that CD4, CD28, and Ox-40 interactions can promote, and LFA-1 interactions can suppress, differentiation of cells secreting the Th2 cytokines IL-5 and IL-13. However, for CD4 and LFA-1, this is only seen over a certain range of peptide doses. In addition, CD28 and Ox-40 interactions also promote Th1 differentiation. In general, agonist Abs to accessory molecules shifted the response curves for IFN-gamma, IL-5, and IL-13 to lower doses, whereas antagonist reagents resulted in similar curves shifted toward the higher doses. We conclude that ligation of cell surface accessory receptors enables low doses of Ag to promote responses normally induced only by higher doses. Individual receptors do not intrinsically regulate one cytokine phenotype or another, suggesting that differentiation is controlled by the level of expression of multiple accessory molecule pairs integrated with the number and affinity of peptide/MHC complexes.

Adjuvants, Immunologic↗
PubMed2000

Co-stimulation of antigen-specific CD4 T cells by 4-1BB ligand.

4-1BB is a member of the TNF receptor family predominantly expressed on activated T cells, and binds an inducible ligand found on B cells, macrophages and dendritic cells. Whereas ligation of 4-1BB has been shown to enhance response of purified CD8 T cells to mitogens, and to augment NK activity and generation of cytotoxic T lymphocytes in vivo, there are little direct data on 4-1BB action during CD4 responses. Using pigeon cytochrome c-presenting fibroblast antigen-presenting cells transfected with 4-1BB ligand (4-1BBL), we show that engaging 4-1BB on naive CD4 cells promotes proliferation, cell cycle progression and IL-2 secretion, and suppresses cell death, all to a similar extent as B7-1 engagement of CD28. In addition, 4-1BBL synergizes with B7 and ICAM to enhance naive CD4 proliferation when antigen is limiting. 4-1BBL alone, and to a greater extent with B7, also augmented IL-2 secretion resting antigen-experienced CD4 cells, as typified by T helper clones, whereas short-term effector cells showed similar levels of proliferation and cytokine secretion regardless of whether 4-1BB was engaged. A major role in augmenting IFN-gamma, IL-4 or IL-5 was not demonstrated. Blocking studies with activated B cells presenting antigen showed that 4-1BB participates in promoting IL-2 production by resting CD4 cells, confirming that 4-1BBL can play a role in antigen-specific CD4 T cell responses.

4-1BB Ligand↗
PubMed2000

Epitope affinity for MHC class I determines helper requirement for CTL priming.

We show here that priming and memory generation of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) does not require help if the immunogen binds major histocompatibility complex (MHC) class I molecules with high affinity. This conclusion was based on the study of three chemically distinct optimal length CTL epitopes with high affinity for the restriction element Kb. In contrast, when two subdominant epitopes with intermediate MHC binding affinity were studied, either a class II MHC-restricted T helper cell epitope or administration of antibody to CD40 was required to obtain significant CTL priming. Depending on the epitope, one source of help was much more efficient than the other.

Animals↗
PubMed1999

Peptide dose, affinity, and time of differentiation can contribute to the Th1/Th2 cytokine balance.

Opposing viewpoints exist regarding how Ag dose and affinity modulate Th1/Th2 differentiation, with data suggesting that both high and low level stimulation favors Th2 responses. With transgenic T cells bearing a single TCR, we present novel data, using peptides differing in affinity for the TCR, that show that the time period of differentiation can determine whether Th1 or Th2 responses predominate as the level of initial stimulation is altered. Over the short term, IFN-gamma-producing cells were induced by lower levels of stimulation than IL-4-producing cells, although optimal induction of both was seen with the same high level of stimulation. Over the long term, however, high doses of high affinity peptides led selectively to IFN-gamma-secreting cells, whereas IL-4- and IL-5-secreting cells predominated with lower levels of initial signaling, brought about by moderate doses of high affinity peptides. In contrast, too low a level of stimulation at the naive T cell stage, with low affinity peptides at any concentration, promoted only IL-2-secreting effectors or was not sufficient for long term T cell survival. These results demonstrate that the level of signaling achieved through the TCR is intimately associated with the induction of distinct cytokine-secreting T cells. We show that dose, affinity, time over which differentiation occurs, and initial production of IL-4 and IFN-gamma all can contribute to which T cell subset will predominate. Furthermore, these data reconcile the two opposing views on the effects of dose and affinity and provide a unifying model of Th1/Th2 differentiation based on strength of signaling and length of response.

Amino Acid Sequence↗
PubMed1999

Activation of CD4 T cells by somatic transgenesis induces generalized immunity of uncommitted T cells and immunologic memory.

Cellular immune responses were analyzed in vivo after a single intraspleen inoculation of DNA coding for a 12-residue Th cell determinant associated with a 12-residue B cell epitope, a process termed somatic transgene immunization. We show that CD4 T cells are readily activated and produce IL-2, IFN-gamma and IL-4, characteristics of an uncommitted phenotype. Linked recognition of the two epitopes coded in the same transgene promoted IgM-IgG1 switch and enhanced the total Ab response but had no effect on IgG2a Abs. Although originating in the spleen, T cell responsiveness was found to spread immediately and with similar characteristics to all lymph nodes in the body. A single inoculation was also effective in establishing long term immunologic memory as determined by limiting dilution analysis, with memory T cells displaying a cytokine profile different from that of primary effector T cells. These studies provide evidence that by initiating immunity directly in secondary lymphoid organs, an immune response is generated with characteristics that differ from those using vaccines of conventional DNA or protein in adjuvant administered in peripheral sites. Somatic transgene immunization can therefore be used to probe T cell responsiveness in vivo and represents a tool to further understanding of the nature of the adaptive immune response.

Animals↗
PubMed1999

Lymphotoxin alphabeta is expressed on recently activated naive and Th1-like CD4 cells but is down-regulated by IL-4 during Th2 differentiation.

Lymphotoxin (LT) is a cytokine that orchestrates lymphoid neogenesis and formation of germinal center reactions. LT exists as a membrane heterotrimer of alpha and beta subunits and is secreted as a homotrimer, LTalpha3. Using LTbetaR.Fc, expression of LTalphabeta on CD4 T cell subsets was investigated in a TCR transgenic model. LTalphabeta was evident 24-72 h after activation of naive T cells with specific Ag, and declined thereafter. Early expression was independent of IFN-gamma and IL-12, however, IL-12 prolonged expression. LTalphabeta was reinduced within 2-4 h after Ag restimulation, but declined by 24 h regardless of IL-12 or IFN-gamma priming. Exposure of naive T cells to IL-4 did not affect early LTalphabeta expression at 24 h, but resulted in subsequent down-regulation. IL-4-differentiated Th2 effectors did not re-express LTalphabeta, and LTalphabeta was transiently found on Th1 clones but not Th2 clones. LTalpha3 and TNF were immunoprecipitated from supernatants and lysates of IL-12 primed cells but not IL-4 primed cells. These studies demonstrate that LTalphabeta is expressed by activated naive CD4 cells, unpolarized IL-2-secreting effectors, and Th1 effectors. In contrast, loss of surface LTalphabeta and a lack of LTalpha3 and TNF secretion is associated with prior exposure to IL-4 and a Th2 phenotype.

Animals↗
PubMed1998

Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses.

Ox-40 and Ox-40 ligand (Ox-40L) are thought to be involved in T cell-APC interactions. However, their exact role in T cell responses is undefined. Using fibroblast transfectants expressing Ox-40L and/or B7-1, and CD4 cells from TCR transgenic mice, we investigated the effect of Ox-40 signaling on primary responses to the Ag pigeon cytochrome c. Ox-40 expression on naive CD4 cells peaked 2 to 3 days after activation, and was lost by 4 to 5 days. APCs with Ox-40L promoted partial activation of naive T cells with some IL-2 secretion, but were unable to enhance proliferation, unlike those with B7-1. APCs coexpressing Ox-40L with B7-1 induced large quantities of IL-2 and promoted proliferative responses that persisted for several days. Effector cells taken 5 days after naive T cell activation reexpressed Ox-40 within 4 h and responded strongly to APCs expressing Ox-40L, whereas B7-1 had little effect. Synergy was also seen between Ox-40L and B7-1, with primarily IL-2 being elevated, although IL-4 and IL-5 were also up-regulated. The most striking action was on effector T cell proliferation, which continued at high levels for up to 4 days, with little proliferation evident at this time in the absence of Ox-40 signals. These data suggest that Ox-40/Ox-40L interactions act after initial activation events to prolong clonal expansion and enhance effector cytokine secretion, and may be involved in promoting long-lived primary CD4 responses.

Animals↗
PubMed1998

Generation, persistence, and modulation of Th0 effector cells: role of autocrine IL-4 and IFN-gamma.

Many studies have classified CD4 responses into either Th1-like or Th2-like, based on cytokine secretion profiles, but little significance has been placed on Th0 cells. This has largely resulted from studies that suggested that Th0 populations primarily comprise individual Th1 and Th2 cells. Here, we show that priming of Ag-specific naive CD4 cells with moderate dose IL-4 generates a Th0 population that is evident after 3 days in vitro and becomes prevalent after successive encounters with Ag over a 9-day period. By intracellular cytokine staining, the majority (>60%) of effector cells generated in this way produce either IL-4, IFN-gamma and IL-2, or IL-4 and IFN-gamma without IL-2. Endogenous IFN-gamma secreted over the initial 3 days of culture was critical for generating Th0 cells, since neutralization allowed IL-4 to induce differentiation into Th2-like cells. Successive encounters with Ag were required for generating Th0 cells, and their stability and persistence were governed by the balance of endogenous IL-4 and IFN-gamma secreted during the later stages of differentiation. Studies blocking Fas-induced cell death showed that this process played no role in Th0 cell generation, and differential death of committed Th1 or Th2 cells was not required for Th0 persistence. These data suggest that Th0 cells can be as prevalent as Th1- or Th2-like cells after naive CD4 activation, that the relative levels of autocrine IL-4 and IFN-gamma are important to the lack of commitment, and that not all cells are predestined to the Th1 or Th2 phenotypes early in the response.

Animals↗
PubMed1998

Modulation of naive CD4 T cell activation with altered peptide ligands: the nature of the peptide and presentation in the context of costimulation are critical for a sustained response.

Altered peptide ligands containing single amino acid substitutions have the potential to be used for modulating immune function. Using a panel of moth cytochrome c peptides, we demonstrate that different phases of naive CD4 T cell response are alternately modulated depending on altered peptide ligand dose and accessory molecule expression by APC. Weak agonists presented at high concentration, and with costimulation, efficiently induced early phase naive T cell activation as assessed by IL-2R/CD69 expression, but could only promote sufficient IL-2 for a short-lived proliferative response. In contrast, strong agonists and heteroclitic peptides induced early phase T cell activation even at low concentrations with costimulation, and allowed sustained IL-2 secretion and proliferation. In the absence of accessory molecule help, early and late phase activation was impaired with weak agonists, whereas strong agonists partially compensated for a lack of costimulation for early phase activation, and also promoted enhanced IL-2 with sustained proliferation. These studies support the hypothesis that the naive T cell response will be determined by the balance between provision of accessory molecule help and the affinity of peptide/MHC complexes for individual TCRs, and suggest that extended IL-2 production is the main facet of naive CD4 activation that is affected by altering the nature of the peptide.

Amino Acid Sequence↗
PubMed1998

Augmentation of naive, Th1 and Th2 effector CD4 responses by IL-6, IL-1 and TNF.

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-gamma, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-gamma and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.

Adjuvants, Immunologic↗
PubMed1998

OX-40: life beyond the effector T cell stage.

The OX-40 receptor (OX-40R) is a transmembrane protein found on the surface of activated CD4(+) T cells. When engaged by an agonist such as anti-OX-40 antibody or the OX-40 ligand (OX-40L) during antigen presentation to T cell lines, the OX-40R generates a costimulatory signal that is as potent as CD28 costimulation. Engagement of OX-40R enhances effector and memory-effector T cell function by up-regulating IL-2 production and increasing the life-span of effector T cells. We hypothesize that the signal generated by the OX-40R inhibits activation-induced T cell death (AICD) and thereby increases the number of cells differentiating from the effector to memory T cell stage. In experimental autoimmune encephalomyelitis (EAE) OX-40R+ T cells are found only within the inflammatory site [central nervous system (CNS)]. Sorting OX-40R+ T cells from the CNS of animals with EAE revealed that they are autoantigen-specific T cells. Therefore, OX-40R-specific therapies were devised to eliminate or inhibit autoreactive T cells, while sparing the remainder of the T cell repertoire. In contrast, in vivo costimulation through the OX-40R in animals with cancer generated enhanced tumor-specific immunity leading to improved tumor-free survival. Thus, manipulation of the OX-40R during inflammatory responses can alter effector CD4(+) T cell function by enhancing or limiting T cell activation and survival.

Animals↗
PubMed1997

Partial activation of naive CD4 T cells and tolerance induction in response to peptide presented by resting B cells.

Tolerance is thought to occur when Ag is presented to T cells in the absence of costimulatory interactions from APC accessory molecules. Of the professional APC, the resting B cell may be the main tolerizing cell in vivo. We have analyzed several aspects of activation of naive transgenic CD4 cells stimulated with resting or activated B cells presenting peptide Ag. Similar results were obtained with stimulation from peptide presenting fibroblast APC lacking or expressing B7-1 with intracellular adhesion molecule-1. TCR ligation with little or no accessory molecule coreceptor engagement induced efficient blastogenesis; up-regulation of CD25, CD44, CD69, CD95 and CD71; and down-regulation of CD62L over a 48-h period. Accessory molecule help enhanced the expression of CD25, CD44, CD69, and CD71, but to very modest degrees. Only two molecules, CD40 ligand and IL-2, were found to be extremely dependent on accessory molecule help, with little or no expression evident with peptide presented on resting B cells or class II-positive fibroblasts. T cells induced on resting B cells expanded minimally over 3 days, and this was followed by extensive cell death and hyporesponsiveness of the resulting cells. These studies suggest that under tolerizing conditions, such as Ag presentation by resting B cells, much of the naive CD4 response is induced efficiently. Partial activation, however, may be the overall result due to the lack of CD40 ligand expression, which may regulate costimulatory activity in APC and, in turn, may contribute to limiting the production of IL-2 required for T cell expansion and survival.

Abatacept↗