anti-T8 Antibodies Research Articles

Efficacy profile of a bivalent Staphylococcus aureus glycoconjugated vaccine in adults on hemodialysis: Phase III randomized study

In a previous study in end-stage renal disease (ESRD) hemodialysis patients, a single dose of Staphylococcus aureus type 5 and 8 capsular polysaccharides (T5/T8) conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A investigational vaccine showed no efficacy against S. aureus bacteremia 1 year post-vaccination, but a trend for efficacy was observed over the first 40 weeks post-vaccination. Vaccine efficacy (VE) of 2 vaccine doses was therefore evaluated. In a double-blind trial 3359 ESRD patients were randomized (1:1) to receive vaccine or placebo at week 0 and 35. VE in preventing S. aureus bacteremia was assessed between 3–35 weeks and 3–60 weeks post-dose-1. Anti-T5 and anti-T8 antibodies were measured. Serious adverse events (SAEs) were recorded for 42 days post-vaccination and deaths until study end. No significant difference in the incidence of S. aureus bacteremia was observed between vaccine and placebo groups between weeks 3–35 weeks post-dose 1 (VE -23%, 95%CI: -98;23, p = 0.39) or at 3–60 weeks post-dose-1 (VE -8%, 95%CI: -57;26, p = 0.70). Day 42 geometric mean antibody concentrations were 272.4 μg/ml and 242.0 μg/ml (T5 and T8, respectively) in vaccinees. SAEs were reported by 24%/25.3% of vaccinees/placebo recipients. These data do not show a protective effect of either 1 or 2 vaccine doses against S. aureus bacteremia in ESRD patients. The vaccine induced a robust immune response and had an acceptable safety profile. Further investigation suggested possible suboptimal vaccine quality (manufacturing) and a need to expand the antigen composition of the vaccine. This study is registered at www.clinicaltrials.gov NCT00071214.

Immunomagnetic Purging of T8 Cells from Peripheral Blood and Bone Marrow: A Preclinical Study for Allogeneic Bone Marrow Transplantation

Methodology for T-cell subset depletion in allogeneic BMT was developed using anti-T8 antibody and magnetic microspheres. Anti-T8 antibodies bound effectively to the cytotoxic/suppressor T-cell subset and were subsequently removed by anti-mouse IgG coated magnetic microspheres. Following optimization of antibodyxell binding, microsphere:cell ratios and flow rate, T8-cells in peripheral blood were effectively removed (95-99% depletion), with an overall recovery of 37.8% ± 5.8%, and with little, if any, differential damage to either stem cells or to NK-cells. When T8 cells were removed from bone marrow mononuclear cells, an average recovery of 76% ± 8.5% was observed, with 90% ± 4.3% removal of T8 cells. This method was developed both for peripheral blood and bone marrow and could therefore be readily used in a clinical purge setting for allogeneic BMT.

Implications for the role of cognate interactions in in vitro human B cell activation by Staphylococcus aureus Cowan I and pokeweed mitogen.

Human B cell-triggering mechanisms were investigated using the polyclonal activators Staphylococcus aureus Cowan I (SAC) and pokeweed mitogen (PWM). When the cultures of B cells, T cells, and monocytes were stimulated for 5 d by SAC or PWM, B cells could be activated by both mitogens to proliferate and secrete Ig. Even when T cells were substituted by T cell-derived soluble factors, SAC-stimulated B cells could differentiate into Ig-secreting cells. In contrast, interactions of B and T cells for at least the first 6 h of culture were necessary for the B cell triggering by PWM. Experiments that allow a more precise delineation of the B cell-triggering mechanisms by PWM demonstrated that interactions of B cells with T4+ but not T8+ cells are required for the B cell triggering; anti-Ia or anti-T4 antibody can block this triggering; in contrast, anti-T3 or anti-T8 antibody do not exert any effects on the B cell triggering. However, all these monoclonal antibodies could not modulate the ability of B cells that had been already activated by PWM to respond to T cell-derived factors. These data suggest that SAC can directly activate B cells, while cognate interactions between Ia-like antigens on B cells and T4+ cells are essential for B cell triggering by PWM. Furthermore, once B cells are triggered, they will proliferate, differentiate, and secrete Ig in response to T cell-derived factors; Ia-like antigens or T cell differentiation antigens may not be involved in the processes in this cascade.

Discrete stages of human thymocyte activation and maturation in vitro: correlation between phenotype and function.

Using light scatter, flow cytometric and functional analyses, three major stages in the in vitro activation of human thymocytes were defined. The earliest stage (day 0-2) was characterized by the induction of the interleukin 2 (IL2) and transferrin receptors, the T cell lineage specific Ta1 antigen, and increased reactivity with anti-T8 antibody. At this time, major changes in nuclear morphology but not cell size were observed. Early-stage thymocytes were immature with regard to T3 and cytotoxic capacities. The second stage (day 3-4) of in vitro culture was distinguished by loss of T6, maximal activation (both in cell size and nuclear morphology) and maximal expression of both transferrin and IL2 receptors. At this stage, nearly all thymocytes expressed T3, 30-70% of thymocytes were T4+T8+, and functionally, only small increases in cytotoxic capacities were observed. The third stage of maturation (day 5-7) represented thymocytes with reduced levels of activation as measured by forward and right angle light scatter analysis and declining IL2 and transferrin receptor expression. However, these thymocytes exhibited high levels of T3 antigen density, loss of T4, T8 coexpression and pronounced cytotoxic and detectable inducer function capabilities.

Characterization of T8 epitopes by enzymatic digestion, cross-blocking, and involvement in cell-mediated lympholysis

Eleven monoclonal antibodies, directed versus the T8 glycoprotein, were compared using enzyme digestion, phylogenetic comparisons, cross-blocking of antibody binding, and blocking of specific cell-mediated lympholysis (CML). It was found that none of the 11 anti-T8 antibodies tested define the same epitope on the T8 glycoprotein. Some of these antibodies react, however, with closely related structures, as shown by cross-blocking of antibody binding and similar enzyme sensitivity of the epitopes. Moreoever, these structural related epitopes show a similar involvement in the effector phase of CML reactions, since the antibodies to these neighboring epitopes inhibit the same CML reactions. Thus, it is possible to apply structural and functional criteria to define “regions” on the T8 glycoprotein, some of which are consistently involved in CML reactions, some never, and some of these regions appear to be involved in specific effector-target cell combinations only.

One allogeneic cytolytic T lymphocyte clone distinguishes three different HLA-B27 subtypes: identification of amino acid residues influencing the specificity and avidity of recognition.

The HLA-B27 antigen may be divided into at least three subgroups, designated HLA-B27.1, -B27.2, and -B27.3, by specific cytolytic T lymphocytes. In an attempt to explore the functional relevance of HLA polymorphism, an alloimmune cytolytic T cell clone T3+, T8+, T4- has been characterized, which displays a distinct reactivity pattern with each one of the three HLA-B27 subtypes. This cell kills both B27.1- and B27.2- but not B27.3-positive targets. Its lytic efficiency is greater with B27.1 than with B27.2 cells. The clone does not recognize either B7-positive targets or most B27-negative cells. But HLA-B40-bearing cells are lysed, albeit with significantly less efficiency than any B27-positive targets. The differences in killing ability for B27.1, B27.2, and B40 are also evident in cold-target inhibition studies, indicating that a) B27.1 cells can efficiently inhibit lysis of B27.2 and B40 targets, b) B27.2 cells inhibit the lysis of B40 but not of B27.1 targets, and c) B40 cells do not inhibit B27.1 or B27.2 target lysis. In addition, anti-T3 and anti-T8 antibodies are much more effective in inhibiting the lysis of B27.2 targets than that of B27.1-positive cells, suggesting that the observed differences in killing efficiency of the various targets are due to the fact that the tightness of the effector-target interaction is affected by the structural changes between the different HLA antigens. A correlation of the reactivity pattern of this T cell clone with the known amino acid sequences of the HLA-B27, HLA-B40, and HLA-B7 antigens suggests that the clone recognizes a conformational determinant contributed to by residues within the segments 149-156 and 67-83. Those in the former segment appear to be an essential portion of this determinant, whereas polymorphism in the region 67-83 has a modulating effect on the reactivity of the effector but does not abrogate recognition.

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens.

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.

Comparison of the Inhibitory Activity of Seven Anti-T8 Antibodies on Specific Cellular Cytotoxicity

To evaluate the functional significance of the T-cell membrane structure recognized by OKT8 or Leu2a, seven different monoclonal antibodies, reacting with different epitopes of this 76 KD big membrane structure, were evaluated for their ability to alter specific cell mediated lympholysis after a mixed leukocyte reaction. The stimulated effector cells were first incubated with a monoclonal antibody, washed and then tested for their cytotoxicity. Those antibodies, which react with a highly trypsin-sensitive region of the T8 glycoprotein, were the most effective inhibitors of the cytotoxicity (T811, FK18). Those antibodies, which react with a relative trypsin-resistant region (OKT8, Leu2a, WT82, WT85) or with a trypsin-sensitive epitope (WT81), which is in a close neighborhood to WT82 or WT85, appear to be able to block the cytotoxicity only in certain effector/target cell combinations. The data suggest that different regions of the T8 glycoprotein are involved in the specific cytotoxic reactions, and that the involvement of certain epitopes may depend on the effector/target cell combination.

Surface molecules involved in self-recognition and T cell activation in the autologous mixed lymphocyte reaction.

Although the importance of T cell lineage-specific surface glycoproteins and MHC gene products in soluble-, viral-, and alloantigen-stimulated immune responses has been well characterized, those involved in autoreactivity are less defined. To address this issue, we examined the ability of monoclonal antibodies to influence the autologous mixed lymphocyte reaction (AMLR). Here we show that both monoclonal anti-T3 and several monoclonal anti-T4 antibodies profoundly inhibited the autoreactivity of unseparated T cells, isolated T4+ cells, and isolated T8+ cells (cultured in the presence of irradiated T4 cells). Furthermore, monoclonal anti-T8 antibodies markedly inhibited the reactivity of the T8 cells co-cultured with irradiated T4 cells, only partially inhibited the proliferative response of unseparated T cells, and had no effect on the T4 cell AMLR responsiveness. Monoclonal antibodies against class II MHC (Ia) antigenic determinants blocked the AMLR between non-T cells and any of the above responder populations; in contrast, monoclonal anti-class I (HLA-A, B, C) antibodies inhibited the response of T8+ cells but not of isolated T4+ cells. These data support the notion that T4 and T8 antigens serve, respectively, as associative recognition elements for class II and class I MHC antigens during the AMLR, and further suggest that interactions involving non-T cell determinants and the T3-Ti- T cell antigen receptor complex are important in autologous reactivity.

Distinct epitopes on the T8 molecule are differentially involved in cytotoxic T cell function

The present report attempts to determine if there are distinct epitopes on the T8 molecule that are involved in class I-restricted cytotoxic T lymphocyte (CTL) function. A panel of 9 monoclonal antibodies (OKT8A,B,C,D,E,F,G,H,I, and OKT5) was produced and all antibodies were shown to bind to the T8 molecule. This panel of antibodies was employed to characterize the distribution of distinct epitopes on the T8 molecule and to block the activity of class I-specific influenza virus-immune and allo-immune CTL effector function. Significant differences in the ability of the anti-T8 antibodies to block CTL function were observed: OKT8C and T8F blocked best (49 and 55% respectively); OKT8A,E,G,H,I, and OKT5 blocked less well (24–31%); and OKT8B blocked marginally (11%). There was no correlation between the capacity of the antibodies to block CTL function and their heavy chain isotype. Competitive binding of the different OKT8 antibodies to the cell surface and differential trypsin sensitivity of the epitopes recognized by the antibodies indicated that OKT8C and T8F were located on topographically distinct regions of the T8 molecule. These results indicate that specific epitopes on the T8 molecule are involved in CTL function, and that there could be more than one functional site on the molecule.

Identification of a clonally restricted 90 kD heterodimer on two human cloned natural killer cell lines. Its role in cytotoxic effector function.

The present studies were carried out to identify surface molecules involved in the cytotoxic effector function of a human natural killer (NK) clone termed JT9. This clone represents a mature T lymphocyte (T3+T8+T11+) mediating NK-like activity. Using JT9 for immunization, one monoclonal antibody termed anti-NKTa was selected that blocked the cytotoxicity of the clone towards K562 cells. Reactivity of anti-NKTa antibody was assessed using a large panel of lymphoid and nonlymphoid cells including a variety of cloned cell lines with either cytotoxic T lymphocyte (CTL) or NK-like activity. Among all cells tested, only two individual clones, JT9 and JT10, were found to express NKTa antigen. JT10 was derived independently from the same individual as JT9 and also represents a mature T cell (T3+T8+T11+) mediating NK-like activity. Like the Ti structure on CTL clones, the molecule defined by anti-NKTa was shown to be membrane associated with T3 in co-modulation experiments. Moreover, anti-NKTa precipitated a 90 kD heterodimeric structure in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 125I surface-labeled JT9 cells. The blocking capacity of anti-NKTa was evaluated in cytotoxicity assays using a panel of target cells. The influence of anti-T3 was tested in parallel and it was found that both anti-NKTa and anti-T3 blocked the cytotoxicity of the cloned cells against all targets. Given the potential role of 90 kD molecules as antigen-receptor structures, the specificity of the two NKTa+ NK clones was compared and found superimposable when assessed using 15 in vitro established cell lines. However, in contrast to conventional CTL clones, the expression of cytotoxicity by JT9 and JT10 was not dependent upon recognition of class I or class II major histocompatibility complex gene products on the target cells. In addition, the cytotoxicity of these T8+ NK active clones could not be blocked by anti-T8 antibodies. Taken together, the present data suggest that the specificity of one population of human NK active lymphocytes is determined by clonotypic structures. The NKTa determinant identified here appears to belong to the same family of molecules as Ti structures, previously identified on antigen-specific T lymphocytes.

Antigen-like effects of monoclonal antibodies directed at receptors on human T cell clones.

Recent studies suggested that the clonally unique Ti epitopes defined by non-cross-reactive monoclonal antibodies might represent the variable regions of the antigen receptor. Here we determine whether such anti-Ti antibodies could trigger clonal T cell activation. Anticlonotypic monoclonal antibodies to the 49/43-kdalton heterodimer of a given clone or antibodies to the 20/25-kdalton membrane associated monomorphic T3 molecule selectively induce proliferation and IL-2 secretion when linked to a solid support. In contrast, anti-T4 and anti-T8 antibodies under the same conditions have no effect. In conclusion, these results imply that anticlonotypic antibody functions in a fashion analogous to antigen and further support the notion that the T3-Ti molecular complex represents the antigen receptor on human T lymphocytes.

T-Cell Subsets and Langerhans Cells in Normal and Diseased Conjunctiva

AbstractTo identify the infiltrating mononuclear cells in conjunctival lesions, we stained frozen tissue sections of conjunctiva with a series of monoclonal antibodies against T cells and T-cell subsets with an immunoperoxidase method. In vernal conjunctivitis and ocular pemphigoid, most lymphocytes reacted with the anti-T4 antibodies that define helper/inducer T cells (T4+); a minority reacted with the anti-T8 antibody that defines suppressor/cytotoxic T cells (T8+). However, T8+ cells predominated in a conjunctival specimen from a patient with chronic graft-vs-host disease. A few T8+ cells and rare T4+ cells were also found in normal conjunctiva. In some diseased conjunctivas, there was an increase in cells stained with anti-T6 antibody, which stains Langerhans cells in normal conjunctiva and skin. T lymphocyte subsets and Langerhans cells probably play an important role in the pathogenesis of inflammatory conjunctival lesions

In situ immunologic characterization of cellular constituents in lymph nodes and spleens involved by Hodgkin's disease

The cellular constituents in lymph nodes and spleens of patients with Hodgkin's disease were studied with a series of monoclonal antibodies directed against human thymocyte, peripheral T-cell, and la antigens. Utilizing both an immunoperoxidase technique on frozen tissue sections and indirect immunofluorescence on cell suspensions, wer found that a majority of lymphocytes were T cells, since they stained with anti-T1 and anti-T3 antibodies, which react with all peripheral T cells. In addition, most of these cells were reactive with anti-T4 antibody, which defines the helper/inducer T-cell population, whereas only a minority of cells stained with anti-T5 and anti-T8 antibodies, which are reactive with suppressor/cytotoxic T cells. Moreover, a large proportion of T cells expressed T10 antigen, which is found on activated T cells. A minority of the T cells also expressed la antigen(s), again suggesting that some of the T cells are activated. In contrast, the Reed-sternberg cells did not react with any of these anti- T-cell antibodies or with anti-IgM antiserum, but displayed strong membrane and cytoplasmic staining with anti-la antibody. Taken together, these findings suggest that Reed-Sternberg cells are not of T- cell lineage but may be derived from antigen-presenting reticulum cells in the thymus-dependent areas of lymphoid tissues; these cells are normally associated with T4+ cells.

In situ immunologic characterization of cellular constituents in lymph nodes and spleens involved by Hodgkin's disease

The cellular constituents in lymph nodes and spleens of patients with Hodgkin's disease were studied with a series of monoclonal antibodies directed against human thymocyte, peripheral T-cell, and la antigens. Utilizing both an immunoperoxidase technique on frozen tissue sections and indirect immunofluorescence on cell suspensions, wer found that a majority of lymphocytes were T cells, since they stained with anti-T1 and anti-T3 antibodies, which react with all peripheral T cells. In addition, most of these cells were reactive with anti-T4 antibody, which defines the helper/inducer T-cell population, whereas only a minority of cells stained with anti-T5 and anti-T8 antibodies, which are reactive with suppressor/cytotoxic T cells. Moreover, a large proportion of T cells expressed T10 antigen, which is found on activated T cells. A minority of the T cells also expressed la antigen(s), again suggesting that some of the T cells are activated. In contrast, the Reed-sternberg cells did not react with any of these anti-T-cell antibodies or with anti-IgM antiserum, but displayed strong membrane and cytoplasmic staining with anti-la antibody. Taken together, these findings suggest that Reed-Sternberg cells are not of T-cell lineage but may be derived from antigen-presenting reticulum cells in the thymus-dependent areas of lymphoid tissues; these cells are normally associated with T4+ cells.

T cell subsets and Langerhans cells in lichen planus: in situ characterization using monoclonal antibodies.

Skin biopsies from four patients with lichen planus were studied using monoclonal antibodies directed against T lymphocytes. Anti-TI and anti-T3 antibodies, which react with all peripheral T cells, stained most cells in the dermal infiltrates. The majority of infiltrating cells also stained with anti-T4 and anti-T4b antibodies, which react with helper/inducer cells, whereas a minority of cells stained with anti-T8 antibody, which reacts with cytotoxic/suppressor cells. Surface IgM was not identified on any infiltrating cells, providing evidence against B cell participation. Intraepidermal and dermal cells with long cytoplasmic extensions stained with any anti-T6 antibody in all cases, defining them as Langerhans cells or their precursors. T6-positive cells were seen in greater number than in normal control epidermis and dermis. The results indicate that well-developed lesions of lichen planus are characterized by an influx of helper/inducer T lymphocytes and increased numbers of Langerhans cells. These observations support the contention that cellular immunity is important in the pathogenesis of this disorder.

Distribution of T cell subsets in human lymph nodes.

A series of T cell-specific monoclonal antibodies was used to determine the location of T lymphocyte subpopulations in frozen sections of human lymph nodes by means of an immunoperoxidase technique. The majority of cells in the paracortical regions were reactive with anti-T1 and anti-T3 antibodies, which define all mature peripheral T cells. In contrast, the majority of cells within primary follicles were unreactive with anti-T1 and anti-T3 antibodies, but were reactive with anti-Ia and anti-IgM antibodies. In addition, a substantial number of T1+, T3+ cells were found in the germinal centers of secondary follicles on the capsular side. The vast majority of T1+, T3+ cells in the paracortex and the follicles were reactive with anti-T4 antibody, which defines inducer/helper T cells. Only a minority of cells in these areas were reactive with anti-T5 and anti-T8 antibodies, which define cytotoxic/suppressor cells. No lymphocytes were stained with anti-T6 antibody, which reacts with a majority of thymocytes but not with peripheral T cells. Scattered cells in the paracortex showed staining for Ia antigen in an irregular dendritic pattern. The findings demonstrate that the major T cell population found within human lymph node bears the mature T1+, T3+, T4+ phenotype characteristic of inducer T cells. Moreover, the location of this population indicates that they play a role in the induction of B cell differentiation in vivo.