Addition Of anti-CD3 Research Articles

Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice.

ObjectiveThe benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels in vivo and in vitro on T cells, response and also the effects on T cells subsets in normal mice.MethodsA plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28 in vitro. T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR.ResultsWestern blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibit CD4+ and CD8+ T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8+ T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4+ CD25+ Tregs and Th2 enhancement. The in vitro study provided similar results.ConclusionThe results indicated Th1 and CD8+ T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.

Adoptive T Cell Therapy for Progressive Multifocal Leukoencephalopathy Using Sequential Ex-Vivo Stimulation with JCV Peptide Pulsed Dendritic Cells and Anti-CD3/CD28

Abstract▪2175▪This icon denotes a clinically relevant abstract Background:Progressive multifocal leukoencephalopathy (PML) is a rare but deadly demyelinating disease of the central nervous system caused by reactivation of the polyomavirus JCV. It develops in the setting of severe cellular immune deficiency such AIDS, hematologic malignancies, recipients of hematopoietic stem cell transplants, and following administration of immunodepleting monoclonal antibodies such as rituximab. Patients who demonstrate an antiviral response characterized by the presence of JCV-specific CD8+ cytotoxic T lymphocytes (CTL) exhibit improved outcomes. However, efforts to elicit the expansion of JCV specific T cells using dendritic cells (DCs) pulsed with JCV peptides have been frustrated by the lack of effector cell responses in this setting. We have demonstrated that sequential stimulation with DC based vaccines and ligation of the costimulatory complex using anti-CD3/CD28 results in the dramatic expansion of activated T cells. In this study we sought to examine whether ex vivo stimulation with anti-CD3/CD28 facilitated JCV responses that might be used as adoptive immunotherapy. Methods: Dendritic cells (DCs) were generated from adherent mononuclear cells isolated from normal donors and cultured with rhIL-4 and GM-CSF for 5 days and then matured with TNF for 2 days. The non-adherent cells were cultured with IL-2 as a source of autologous T cells. DCs were pulsed with the VP1p36 and VP1p100 peptides and co-cultured with autologous T cells in the presence of low-dose IL-2. After 10 days, an aliquot of the stimulated T cells were exposed to antiCD3/CD28 antibody coated plates for 2 days. The expression of cell surface molecules, intracellular cytokines and VP1p36/p100 tetramers were assessed by flow cytometry. Results: Sequential stimulation with DCs pulsed with JCV derived peptides and anti-CD3/CD28 resulted in a 3 fold expansion of T cells specific for the JCV derived peptides as compared to that observed with peptide pulsed DCs alone. The addition of anti-CD3/CD28 also resulted in polarization of the T cell population towards a Th1 phenotype as evidenced by an increase in the expression of IFNγ by CD4+(4.1 to 8%, p=0.04) and CD8+ cells (2.72 to 7.7%,p=0.035) T cells. In contrast, expression of IL-10 on CD4+ and CD8+ T cells decreased following anti-CD3/CD28 stimulation. Consistent with these findings, sequential stimulation with DCs pulsed with peptides and anti-CD3/CD28 resulted in an increase in CD4/CD25/CD69+ cells (5 to 14.9%; p=0.01). Conversely, sequential stimulation with the DC-JCV peptide vaccine and anti-CD3/CD28 did not result in an increase in regulatory T cells as represented by CD4/CD25/FoxP3+ cells (7.0% vs. 5.9%, respectively (p>0.5)). Discussion: These findings suggest that sequential stimulation with a DC-peptide based vaccine for PML and anti-CD3/CD28 ligation results in a significant increase in JCV specific T cells that exhibit an activated phenotype. The stimulated T cell population is characterized by the expression of Th1 cytokines, activation markers, and a lack of concurrent regulatory T cell expansion. This approach provides a potential strategy for the development of adoptive immunotherapy for PML. Disclosures:No relevant conflicts of interest to declare.

Triggered human mucosal T cells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cytotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unseparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18 h at 37 degrees C. Cytokines were measured by cytotoxicity assays using L929 (murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatants derived from unseparated, CD4+ (greater than 95% pure) and CD8+ (greater than 90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the cytotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was detected). These same supernatants were cytotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h 111In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target cells. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.

Antigenic cross-reactivity between human T lymphotropic virus type I (HTLV-I) and retinal antigens recognized by T cells.

To investigate the relationship between uveitis and HTLV-I infection, we examined the cross-reactivity between HTLV-I antigens and retinal antigens recognized by T cells in B10.BR mice immunized with human HTLV-I-infected MT-2 cells. We found that T cells obtained from MT-2 immune mouse spleen responded not only to HTLV-I antigens but also to retinal antigens of various species. However, they did not respond to HTLV-I-negative lymphoid cell lines. Furthermore, established T cell lines from MT-2 immune spleen cells also responded to both HTLV-I and retinal antigens. The phenotype of the immune cells that responded to both HTLV-I and retinal antigens was CD4+, CD8-, and CD3+. The proliferative response of T cell lines to HTLV-I as well as various retinal antigens, was clearly blocked by addition of anti-CD3, anti-CD4, or anti-I-Ak MoAbs, but not by anti-CD8 antibody. The established T cell lines from HTLV-I immune spleen cells were all found to be CD3+, TCR beta +, CD4+, CD8- cells by flow cytometric analysis. These results indicate that an epitope of HTLV-I antigens is cross-reactive to an epitope of retinal antigens extracted from either human retinoblastoma or normal murine, rat, and bovine retinae at a T cell recognition level.

The Important Role of T Regulatory Cells in Bone Marrow Failure Syndromes.

Autoreactive T lymphocytes are implicated in the immune mechanisms involved in the bone marrow failure syndromes (BMFS), including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndrome (MDS). CD4+CD25highFoxP3+ T regulatory cells (Treg) play crucial roles in regulating immune responses to autoreactive T lymphocytes via interferon (IFN)-gamma- and interleukin (IL)-10-dependent pathways. Recently, it has also been reported that a stimulation of CD4+ T cells by anti-CD3/CD46 antibodies induces Treg that kill autoreactive T lymphocytes activated (Grossman WJ et al, Blood, 2004). Then, we hypothesized that some distributional and/or functional abnormalities of Treg may lead to an inappropriate proliferation or reduction of autoreactive T lymphocytes in BMFS. In this study, to clarify some roles of Treg in an immune pathophysiology of BMFS, we examined the proportions of Treg in CD4+ T cells from BMFS patients using flow cytometry. Subsequently, we evaluated autologous cytotoxicity of cultured CD4+ T cells (target cells), which were stimulated by the addition of anti-CD3/CD28 antibodies, by cultured CD4+ T cells (effector cells), which were activated by the addition of anti-CD3/CD46 antibodies, at an effector versus target ratio of 1:1 by flow cytometry using carboxyfluorescein diacetate succinimidyl ester and 7-amino-actinomycin D and then calculated the percentages of specific lysis of the targeted CD4+ T cells from BMFS patients (Lecoeur H et al., J Immunol Methods, 2001). We also measured the concentrations of IFN-gamma and IL-10 in aliquots of supernatant of cultured CD4+ T cells from BMFS patients, stimulated by the addition of anti-CD3/CD46 antibodies, using enzyme-linked immunosorbent assay. We found significantly higher proportions of Treg in CD4+ T cells from MDS patients (n=10; mean ± standard deviation; 5.66 ± 2.70%; p<0.05), but not from AA (n=5; 3.88 ± 1.37%) and PNH patients (n=5; 2.81 ± 1.20%), than those from healthy individuals (n=10; 3.36 ± 1.39%). We also found that cytotoxicity of anti-CD3/CD46 antibodies-stimulated Treg from MDS patients (n=3; 10.82 ± 3.52%) and AA patients (n=3; 1.06 ± 0.89%) were significantly higher and lower, respectively, than those from healthy individuals (n=5; 4.35 ± 1.92%; p<0.01 and p<0.05, respectively). In contrast, the cytotoxicity of them from PNH patients (n=3; 1.61 ± 1.16%) was not different from that from healthy individuals. The concentrations of IFN-gamma in aliquots of supernatant of anti-CD3/CD46 antibodies-stimulated Treg from 3 MDS (1290 ± 380 IU/ml) and 3 PNH (1058 ± 173 IU/ml) patients, but not from 3 AA patients (326 ± 366 IU/ml), were higher than those from 5 healthy individuals (410 ± 175 IU/ml). In addition, the concentrations of IL-10 in aliquots of supernatant of anti-CD3/CD46 antibodies-stimulated Treg from 3 MDS (2250 ± 420 IU/ml) and 3 PNH (1440 ± 1340 IU/ml) patients, but not from 3 AA patients (428 ± 110 IU/ml), were higher than those from 5 healthy individuals (659 ± 282 IU/ml). Our results suggest that insufficiency or excess in function and/or distribution of Treg may partly contribute to marrow injury of AA or immunologic selection of PNH and MDS clones, respectively. In conclusion, some abnormalities in Treg may play a crucial role in an immunologic pathophysiology of BMFS.

Immune regulatory activity of CD34+ progenitor cells: evidence for a deletion-based mechanism mediated by TNF-α

AbstractPrevious studies suggest that cells within the CD34+ hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti–IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti–tumor necrosis factor-α (anti–TNF-α) and not by anti–transforming growth factor-β (anti–TGF-β), suggesting a potential role for TNF-α in the regulatory activity of CD34+ cells.

Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood.

A subpopulation of peripheral human CD4(+)CD25(+) T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte-associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4(+)CD25(+) T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-4, or interferon (IFN)-gamma on either protein or mRNA levels. The anergic state of CD4(+)CD25(+) T cells is not reversible by the addition of anti-CD28, anti-CTLA-4, anti-transforming growth factor beta, or anti-IL-10 antibody. However, the refractory state of CD4(+)CD25(+) T cells was partially reversible by the addition of IL-2 or IL-4. These data demonstrate that human blood contains a resident T cell population with potent regulatory properties.

In vitro analysis of CD40-CD154 and CD28-CD80/86 interactions in the primary T-cell response to allogeneic "nonprofessional" antigen presenting cells.

Recently, several ligand interactions have been examined in detail as potential mediators of costimulatory signaling. The CD154/CD40 and CD28/B7 interactions have been highlighted as being among the more-significant contributors to proper activation of unprimed T lymphocytes. Human keratinocytes (HK) and human dermal fibroblasts (HF) are capable of expressing Class II HLA and CD40 antigens after interferon-gamma exposure, yet neither express significant levels of B7. HK and HF have been characterized as "nonprofessional" antigen presenting cells (APC) and their poor APC function has been partially attributed to deficient costimulatory activity. In this study, we examined whether substituting for costimulatory signaling events through the addition of cross-linked monoclonal antibodies against the T-cell ligand/s (CD28 and/or CD154) could restore allostimulation. Mixed lymphocyte reactions were performed combining enriched human peripheral blood T cells and allogeneic HK or HF with or without stimulatory anti-CD28 and/or anti-CD154 antibodies. The results show that the addition of anti-CD28 alone permitted HF but not HK to present alloantigen effectively. In contrast, addition of both anti-CD154 and anti-CD28 was required to generate even a moderate proliferative response to allogeneic HK. Further, adding a monomorphic anti-HLA-DR antibody substantially inhibited these responses. Additional experiments suggest that signaling through CD40/CD154 directs HK to produce TGF-beta, which would adversely affect T-cell activation. The data presented highlight significant differences in signaling capacities for HK versus HF and provide evidence for a partial mechanism by which allogeneic human skin equivalents might be immunologically null upon engraftment.

Distinct requirements for C-C chemokine and IL-2 production by naive, previously activated, and anergic T cells.

Ag presented by activated APCs promote immunogenic responses whereas Ag presented by resting APCs leads to tolerance. In such a model, the regulation of cytokine release by the presence or absence of costimulation might potentially play a critical role in dictating the ultimate outcome of Ag recognition. C-C chemokines are a structurally defined family of chemoattractants that have diverse effects on inflammation. We were interested in determining the activation requirements for chemokine production by CD4+ T cells. Our data demonstrate for T cell clones and previously activated T cells from TCR-transgenic mice that stimulation with anti-TCR alone results in the production of copious amounts of macrophage-inflammatory protein-1alpha (MIP-1alpha) and other C-C chemokines, and that addition of anti-CD28 gives very little augmentation. Furthermore, MIP-1alpha production is nearly equivalent from both anergic and nonanergic cells. For naive T cells, anti-CD3 stimulation alone led to as much MIP-1alpha production as Ag + APC stimulation. The addition of costimulation gave a 3-10-fold enhancement, but this was 70-fold less than the effect of costimulation on IL-2 production. Thus, although C-C chemokines play a broad role in influencing inflammation, their production by signal 1 alone makes them unlikely to play a critical role in the decision between a tolerogenic and an immunogenic response. Furthermore, the production of MIP-1alpha by anergic T cells, as well as following signal 1 alone, raises the possibility that in vivo this chemokine serves to recruit activated T cells to become tolerant.

Immunotherapy of B-Cell Lymphoma With CD3x19 Bispecific Antibodies: Costimulation via CD28 Prevents “Veto” Apoptosis of Antibody-Targeted Cytotoxic T Cells

AbstractBispecific antibodies (CD3x19) against the CD3ɛ-chain of the T-cell–receptor/CD3 complex and the CD19 antigen on B cells can target polyclonal, nontumor-specific T cells to B lymphoma cells. This induces T-cell activation, and generation of cytotoxic T cells (CTLs). These polyclonal CTLs, targeted by the CD3x19 bispecific antibodies, can lyse CD19+ B-lymphoma cells. In a xenotransplant model in severe combined immunodeficiency deficient (SCID) mice, we and others observed that CD28 triggering is required for efficient elimination of B-lymphoma cells and cure from the tumor in addition to CD3x19 administration. We also showed that the activation and targeting of CTLs to the target cell by signal one alone, ie, the CD3x19 mab, induces T-cell death by apoptosis. In blocking experiments we showed that this “veto” apoptosis is mediated by the CD95/Fas ligand. Addition of anti-CD28 (signal 2) renders the T cells resistant for veto apoptosis both in vitro and in vivo. We therefore conclude that the role of costimulation in immunotherapy with bispecific antibodies or other T-cell–based immune strategies is not only to facilitate T-cell activation but also to prevent T-cell deletion by apoptosis.

Immunotherapy of B-Cell Lymphoma With CD3x19 Bispecific Antibodies: Costimulation via CD28 Prevents “Veto” Apoptosis of Antibody-Targeted Cytotoxic T Cells

Bispecific antibodies (CD3x19) against the CD3ɛ-chain of the T-cell–receptor/CD3 complex and the CD19 antigen on B cells can target polyclonal, nontumor-specific T cells to B lymphoma cells. This induces T-cell activation, and generation of cytotoxic T cells (CTLs). These polyclonal CTLs, targeted by the CD3x19 bispecific antibodies, can lyse CD19+ B-lymphoma cells. In a xenotransplant model in severe combined immunodeficiency deficient (SCID) mice, we and others observed that CD28 triggering is required for efficient elimination of B-lymphoma cells and cure from the tumor in addition to CD3x19 administration. We also showed that the activation and targeting of CTLs to the target cell by signal one alone, ie, the CD3x19 mab, induces T-cell death by apoptosis. In blocking experiments we showed that this “veto” apoptosis is mediated by the CD95/Fas ligand. Addition of anti-CD28 (signal 2) renders the T cells resistant for veto apoptosis both in vitro and in vivo. We therefore conclude that the role of costimulation in immunotherapy with bispecific antibodies or other T-cell–based immune strategies is not only to facilitate T-cell activation but also to prevent T-cell deletion by apoptosis.

Cytokine production by subsets of CD4 memory T cells differing in P-glycoprotein expression: effects of aging.

We have shown previously that the mouse CD4 memory cell subset can be divided into two subpopulations based on differential expression of P-glycoprotein. Cells with high levels of P-glycoprotein can be detected by extrusion of the fluorochrome Rhodamine 123; they are referred to as R123lo cells. These R123lo T cells increase with age and have been shown not to respond to anti-CD3 and IL-2 by proliferation or IL-4 production. We report here (a) that the failure of the R123lo CD4 memory population to respond to anti-CD3/IL-2 stimulation cannot be overcome by addition of anti-CD28, PMA, IL-4 or IL-12, alone or in various combinations, and (b) that this age-dependent subset exhibits impaired production of IL-5 and IL-10 as well as decreased proliferation. R123lo CD4 memory cells from young mice are also deficient in IFN gamma secretion by this subset. Although the R123lo cells respond poorly to receptor-dependent agonists, they can be triggered to proliferate and produce IFN gamma by the combination of PMA and ionomycin. In addition to increasing the proportion of R123lo cells in the memory CD4 pool, aging also leads to a decline in the ability of R123hi cells to produce IL-5 and IL-10. Thus, the accumulation of R123lo cells cannot by itself account for the poor proliferation and Th2 cytokine production of aged T cells in cytokine-supplemented culture conditions.

Induction of Hapten-Specific Tolerance of Human CD8+ Urushiol (Poison Ivy)–Reactive T Lymphocytes

The interaction of CD28 with B7 molecules (CD80 or CD86) is an essential second signal for both the activation of CD4+ T cells through the T-cell receptor and the prevention of anergy. We studied the requirement of hapten-specific human CD8+ cells for CD28 co-stimulation in recognition of hapten, and anergy induction. Urushiol, the immunogenic hapten of poison ivy (Toxicodendron radicans), elicits a predominantly CD8+ T-cell response. Autologous PBMC were pre-incubated with urushiol prior to fixation by paraformaldehyde. Fixed antigen-presenting cells were unable to present urushiol to human CD8+ urushiol-specific T cells. Addition of anti-CD28, however, overcame this antigen-presenting defect, enabling CD8+ cells to proliferate. Fixation of antigen-presenting cells prevents upregulation of B7, and addition of anti-CD28 substitutes for this signal. Proliferation of CD8+ T cells in response to urushiol was blocked by CTLA4Ig, a recombinant fusion protein that blocks CD28/B7 interactions. Preincubation of urushiol-specific CD8+ cells with fixed PBMC + urushiol for 7 d induced anergy. Anergic CD8+ cells were viable and able to proliferate in response to IL-2, but not in response to urushiol. Induction of anergy required the presence of urushiol, and pre-incubation with irradiated PBMC + urushiol did not have this effect. It is proposed that anergy was induced by presentation of urushiol by fixed PBMC, in the absence of adequate co-stimulation signals. Induction of anergy by blocking of co-stimulation could potentially induce clinical hyposensitization to haptens.

Tumor-associated glycoantigen, sialyl Lewis a as a target for bispecific antibody-directed adoptive tumor immunotherapy

The KM231 mAb recognizing sialyl Lewis a (sLe a) epitope of glycoprotein or glycolipid expressed on various human cancers was used to prepare bispecific antibody (BSAb) containing anti-CD3 × anti-sLe a mAb. The effect of anti-CD3 × anti-sLe a BSAb on the induction of cytotoxicity by activated T cells was investigated. The activated CD3 + T cells expressing CD8 or CD4 were induced from human peripheral blood mononuclear cells by culture with recombinant IL-2 plus immobilized anti-CD3 mAb. The activated CD8 + and CD4 + T cells showed marginal cytotoxicity against tumor cells by themselves. However, addition of anti-CD3 × anti-sLe a BSAb resulted in a great augmentation of their cytotoxicity against gastrointestinal tumor cells. The BSAb also triggered IL-2 production of CD4 + helper/killer T cells during lysis of tumor cells. Moreover, the BSAb was demonstrated to have a potent in vivo antitumor activity against human colon cancer implanted in nude mice by combination with CD4 + helper/killer cells. These results demonstrated that sLe a antigen might be a good target molecule for BSAb-directed adoptive tumor immunotherapy.

Helper effector function of human T cells stimulated by anti‐CD3 mAb can be enhanced by co‐stimulatory signals and is partially dependent on CD40‐CD40 ligand interaction

In this study we have investigated whether anti-CD3-induced human T cell help for immunoglobulin production could be enhanced by co-stimulation of the T cells via other T cell surface molecules, and the contribution of CD40-CD40 ligand interaction to the execution of T helper effector function induced by these different stimulatory signals. In a system in which irradiated tonsillar T cells were stimulated with immobilized anti-CD3 monoclonal antibody (mAb), it was found that ligation of CD2 with a mitogenic pair of mAb considerably enhanced anti-CD3-induced T cell help for immunoglobulin production. Likewise, ligation of CD28 with mAb enhanced T helper activity, although to a lesser extent. Upon addition of anti-CD28 and anti-CD2 mAb together, an even higher immunoglobulin production was observed. This combination resulted in a four- to fivefold increase in immunoglobulin production as compared to cultures in which T cells were stimulated with anti-CD3 mAb alone. The effect of ligation with B7, the natural ligand of CD28, was studied in a system which utilizes the presentation of anti-CD3 mAb on human Fc gamma RII-expressing mouse fibroblasts which were co-transfected with human B7. It appeared that B7 could stimulate help for immunoglobulin production much more efficiently than ligation of CD28 with mAb did. Physical separation of B cells from T cells led to complete abrogation of immunoglobulin production. Blocking of CD40 with specific mAb, which have no intrinsic B cell stimulatory properties, or the CD40 ligand with a soluble CD40-human IgM fusion protein, resulted in dose-dependent, but only partial, inhibition of T cell-dependent immunoglobulin production with all modes of T cell activation tested. A clear correlation was found between the induction of CD40 ligand expression on the T cells by the different modes of co-stimulation and subsequent immunoglobulin production by the B cells. It is concluded that ligation of CD28 and/or CTLA-4, and of CD2 can generate co-stimulatory signals for T cell help for immunoglobulin production, which was found to be only partially dependent on the CD40-CD40 ligand interaction.

CD28 ligation by monoclonal antibodies or B7/BB1 provides an accessory signal for the cyclosporin A-resistant generation of cytotoxic T cell activity.

Ligation of the T cell membrane Ag CD28 with mAb 9.3 or with its natural ligand B7/BB1 on accessory cells has been shown to provide a helper signal for stimulation through the TCR/CD3 complex. The present study was undertaken to investigate whether CD28 could function as an accessory signal receptor in the generation and effector phase of CTL activity. Purified resting human T cells were activated for 3 to 4 days with immobilized anti-CD3 mAb as the primary stimulus, and CTL activity was then measured by an anti-CD3-redirected 4-hr 51Cr release assay on Fc gamma R-bearing P815 target cells. When the concentration of immobilized anti-CD3 mAb as the primary signal for CTL generation was below threshold, CTL activity could be generated by addition of mAb 9.3 to the cultures. At optimal concentrations of immobilized anti-CD3, the addition of anti-CD28 did not further enhance the generation of CTL activity, but under these conditions generation of CTL activity was almost completely resistant to cyclosporin A (CsA) as a result of CsA-resistant IL-2 production. When 3T6 mouse fibroblasts, transfected with Fc gamma RII and B7, were used as accessory cells, anti-CD3 and B7 were also found to generate cytotoxic activity. Cytotoxic T cell generation under these conditions could be blocked by anti-B7 mAb, but was totally resistant to CsA. CTL activity could be generated by CD3 and CD28 ligation in both CD4(+) and CD8(+) subpopulations. Finally, we found that the activity of CTL lines (isolated from ascitic fluid of a patient with ovarian carcinoma and cultured in IL-2) was higher on B7-transfected targets than on the B7(-) targets. We conclude that CD28 ligation provides a major accessory signal for the CsA-resistant generation of CTL activity and that CD28-B7 interaction also enhances cytotoxic effector functions of CTL. These findings might have important implications for immunotherapeutic interventions.

Soluble CD4 suppress the antigen driven proliferative response of certain T cell clones specific for mycobacteria and for peptides by mycobacterial heat shock proteins.

We have investigated the effect of soluble recombinant CD4 (sCD4) on the antigen specific (BCG, peptides of mycobacterial 65 kDa hsp) responses of T cell lines of T cell clones. The majority of the antigen specific clones could be suppressed in their antigen driven response by the addition of sCD4, while others, including the parental polyclonal T cell line, were not. The suppression of the specific T cell response was reversed by the addition of anti-CD3, did not affect the proliferative response to IL-2, and was independent of the amount of antigen. A decreased capacity to produce IFN-gamma in response to the antigen by the addition of sCD4 was seen only with those clones that were also inhibited in their specific proliferative response. This model may be used to delineate further the interaction between T cells and the antigen presenting cell, and the finding may limit the possible in vivo use of sCD4 in the therapy of human immunodeficiency virus (HIV) infections.

Changes in the Rate of Crypt Epithelial Cell Proliferation and Mucosal Morphology Induced by a T-Cell-Mediated Response in Human Small Intestine

The rate of crypt epithelial cell proliferation and mucosal morphology have been studied in vitro in explants of fetal human small intestine in organ culture in which a cell-mediated immune response has been elicited by stimulating lamina propria T cells with pokeweed mitogen or monoclonal anti-CD3 antibodies. Twelve hours after the addition of anti-CD3 or pokeweed mitogen, most lamina propria T cells expressed CD25. By 18 and 24 h after the addition of anti-CD3, there were significantly more crypt cells in the cell cycle than in controls, although villus height and crypt depth are the same in both groups. After 3 days, by dissecting microscopy, the villi appeared shorter in cultures in which the T cells were stimulated than in control cultures, and the mucosal surface was obscured by a layer of extruded enterocytes. Villus atrophy was confirmed by direct measurement of Feulgen-stained, microdissected villi. Crypts were longer in T-cell-stimulated cultures, and the rate of crypt epithelial cell proliferation measured by metaphase arrest was increased 10-fold. By electron microscopy, the microvilli and cellular morphology of the surface enterocytes were normal in T-cell-stimulated cultures. These experiments clearly show that a profound immune-mediated crypt epithelial cell hyperplasia can occur in the absence of damage to surface enterocytes.

Immune-Mediated Cytotoxicity in Inflammatory Bowel Disease

Thirty years of research on the role of immune-mediated cytotoxic activity in the tissue injury of inflammatory bowel disease (IBD) has yielded only inconclusive data on the relevance of cytotoxic mechanisms. Two hypotheses have been advanced. One is that the destruction of target cells is mediated by direct recognition of target antigens by cytotoxic cells which in turn triggers lysis. Another hypothesis is that lysis occurs via an indirect bystander mechanism in which cells do not recognize a specific antigen on the target, but upon nonspecific activation release cytokines which are capable of lysing the target. The authors have investigated both hypotheses and studied the role of cytotoxic T cells and cytotoxic rectors released from activated T cells in the destruction of epithelial cells. Elevated levels of cytotoxic T cells were found in peripheral blood lymphocytes of patients with IBD, particularly Crohn's disease. The cytotoxic T cells were contained within the Leu 7+, CD8+ T cell subset and were detected using anti-CD3-triggered lysis. Increased cytotoxic T cell activity was also present within inflamed mucosa of patients with both Crohn's disease and ulcerative colitis. The specific targets of this activity have yet to be determined. Activation of mucosal T cells by antibodies to the T cell receptor (anti-CD3) in organ culture of normal fetal jejunum induces an enteropathy characterized by villous atrophy and crypt cell hyperplasia. This same change is seen near aphthous ulcers in patients with Crohn's disease. Soluble cytokines produced by T cells might be responsible for the mucosa! damage observed in these two models of mucosal injury. The goal of this study was to establish in vitro if cytotoxic cytokines can be released by triggering isolated colonic T cells, and what cytokine interactions are required for killing of colonic epithelial cells. These results demonstrate that human lamina propria lymphocyte T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in mucosal damage in IBD. The development of animal models and long term cultures of epithelial cells will allow many advances in research of the role of immune-mediated cytotoxicity in IBD.