T-cell Receptor Monoclonal Antibody Research Articles

T-cell inhibition does not aggravate bacterial translocation from rat small bowel

Background T-cell mediated immunity has been proposed to have an important function in the defence against translocating microbes from the gastrointestinal tract. After small bowel transplantation massive T-cell immunosuppression is necessary to avoid rejection. As a consequence, infections with intestinal bacteria are the main contributors to mortality in this setting. This could further imply that T cells are important in limiting bacterial translocation. In a model for bacterial translocation from small bowel in the rat we examined the outcome of T-cell inactivation. Methods The studies were performed in a model of bacterial translocation from a Thiry-Vella loop of small bowel in the rat. The animals were treated with an anti-α/β T-cell receptor monoclonal antibody (R73). Inhibition of T-cell activation was also made using the immunosuppressive drug cyclosporin A. All animals were sacrificed on day 3 postoperatively and translocation to the mesenteric lymph nodes, liver, spleen, lung and blood was evaluated. Results Treatment with R73 resulted in an almost complete labelling of T cells but did not result in any increased bacterial translocation compared to animals treated with saline. Neither did immunosuppression with cyclosporin A. Conclusions In the model of bacterial translocation from a defunctionalised loop of small bowel the inhibition of T cells does not increase bacterial translocation to mesenteric lymph nodes or promote the systemic spread of the translocating bacteria. This indicates that T cells do not have any important protective function against translocating microbes from defunctionalised small bowel.

Applications of Bilateral Vascularized Femoral Bone Marrow Transplantation for Chimerism Induction Across the Major Histocompatibility (MHC) Barrier

Bilateral vascularized bone marrow transplant (VBMT) model was designed to induce chimerism across the major histocompatibility (MHC) barrier under combined alphabeta T-cell receptor monoclonal antibody and cyclosporine A (alphabeta-TCRmAb/CsA) protocol. Seventeen transplants were performed between BN(RT1) donors and Lewis(RTI) recipients. Group I, isograft controls; Group II, allografts rejection controls; Group III, allografts under 7-day protocol of alphabeta-TCRmAb/CsA. Donor bilateral femoral bones were bilaterally anastomosed to the abdominal aorta and inferior vena cava of recipient. At day 7 posttransplantation, all bone flaps were viable. Groups I and III survived without signs of rejection. In Group III, peak level of chimerism in peripheral blood was evaluated at day 21 (24.2%), at day 63 declined to 1.5%, and was maintained at this level thereafter. Donor-derived cells were present in the bone marrow of recipients at 28.2% at day 21 posttransplant. Histology confirmed viability of bone marrow cells in isograft during the entire follow-up and up to 35 days in treatment Group III. Bilateral VBMT induced donor-specific chimerism across the MHC barrier under the immunomodulatory protocol of alphabeta-TCRmAb/CsA.

Immunomodulation with anti-αβ T-cell receptor monoclonal antibodies in combination with cyclosporine a improves regeneration in nerve allografts

To investigate the use of nerve allografts in animals treated with a short-term combined protocol of anti-alpha/beta T-cell receptor monoclonal antibodies and Cyclosporine A (CsA) to induce tolerance and allow for nerve regeneration. An established rat sciatic nerve model was used. A total of 76 rats were used in this experiment (Lew RT1L n=44, Lewis-Brown-Norway (LBN RT1L+N, n=22), Brown-Norway (BN RT1N, n=10). Sciatic nerve (1.5 cm) deficits were created in the Lewis rats and the animals were randomized to seven treatment groups to allow for repair with isograft controls (LEW-LEW) and with both semiallogeneic (LBN-LEW) and full major histocompatability (MHC) mismatched (BN-Lew) allografts. Nerve regeneration was evaluated at 6 and 12 weeks by somatosensory evoked potentials (SSEP) and standardized pin-prick and toe-spread tests. Nerve samples were harvested at 12 weeks and stained with toluidine blue to assess the total number of myelinated axons, axon area, and myelin sheath thickness. Muscle denervation atrophy was evaluated by gastrocnemius weights. Immunocompetence was investigated through skin grafting and fluorescent activated cell sorting (FACS) analysis. Improved functional, electrophysiologic, and histomorphometric outcomes were observed in animals treated with anti-alpha/betaTCR mAbs and CsA after nerve allograft transplantation when compared to animals receiving no treatment and CsA alone. The immunomodulating protocol of combination anti-alpha/beta TCR mAbs and CsA for a 5 week period altered the rejection process, affording nerve regeneration. It may provide for an expanded source of nerve tissue to alleviate the morbidity of harvesting peripheral nerves from multiple sites for those afflicted with extensive peripheral nerve injuries.

Short-term therapy with TCR and FK-506 leads to long-term laryngeal transplant function

Problem: A mouse anti-rat T-cell receptor monoclonal antibody (TCR) has great potential in the development of long-term immunologic tolerance. Combination therapy with FK-506 and TCR may enable the establishment of long-term immunotolerance while reducing dose-related toxicities. The use of combined TCR and FK-506 in a completely allogeneic model permits a rigorous evaluation of potential synergism. Methods: Nineteen Brown Norway larynges were transplanted to Lewis recipients. Treatment consisted of 7 days of FK-506 (1.2 mg/kg/day) alone and in combination with TCR (0.5 mL/day). Control groups consisted of untreated animals, as well as 11 Lewis-Brown Norway larynges transplanted to Lewis recipients and treated with FK-506 monotherapy. Each group consisted of 6–11 rats. Median duration of engraftment was 44 days (range, 14–106 days). Graft histopathology was graded according to an established 31-point scale in a blinded fashion. Long-term grafts (>75 days) were followed with serum PTH levels. Results: Untreated controls experienced almost complete rejection (mean score, 27, SD 0). Allogeneic transplants treated with FK-506 monotherapy experienced near-complete rejection (mean score, 25.2, SD 2.1). Allogeneic transplants treated with combination therapy and followed for a median duration of 100 days demonstrated significantly better rejection scores than controls (mean score, 11.1, SD 1.7; P = 0.003). Combination therapy was also significantly more effective in preventing acute rejection than monotherapy with FK-506 in a semi-allogeneic model (mean score, 22.1, SD 4.6; P < 0.04). Conclusion: Short-term combination therapy with FK-506 and TCR prevents rejection in an allogeneic model for up to 100 days. This regimen was associated with significantly better histopathologic rejection scores than monotherapy with either agent, or monotherapy with FK-506 in a semi-allogeneic model. Significance: The blockade of primary antigen recognition with TCR may prevent the need for daily immunosuppressive therapy, thereby limiting treatment-associated toxicities. Pulsing protocols may also provide a mechanism for intermittent immunomodulation with this regimen. Support: None reported.

Development of donor‐specific chimerism and tolerance in composite tissue allografts under αβ‐T‐cell receptor monoclonal antibody and cyclosporine a treatment protocols

Recently, we induced donor-specific tolerance to rat hindlimb allografts under a 35-day course of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCR mAb) and cyclosporine A (CsA). In this report, we investigated the role of shorter alphabeta-TCR/CsA protocols on tolerance induction. We performed 52 hindlimb transplantations, between Lewis-Brown-Norway (LBN, F1) donors and Lewis recipients to test the impact of 21-, 7-, and 5-day protocols of combined alphabeta-TCR/CsA treatment on tolerance induction. Donor-specific tolerance and immunocompetence were tested by mixed lymphocyte reaction (MLR) in vitro and by standard skin grafting in vivo. The efficacy of immunosuppressive protocol and donor-specific chimerism was assessed by flow cytometry. All transplants under 5, 7, and 21 days of combined alphabeta-TCR/CsA therapy survived over 350 days. Clinical tolerance and immunocompetence were confirmed by skin grafting in vivo and MLR in vitro. Flow cytometry revealed a high level of donor chimerism in the peripheral blood of long-term survivors. The extension of survival of limb allografts and allo-unresponsiveness were directly associated with the development of stable chimerism in the tolerant recipients.

Donor-specific tolerance in fully major histocompatibility major histocompatibility complex-mismatched limb allograft transplants under an anti-alphabeta T-cell receptor monoclonal antibody and cyclosporine A protocol.

Recent studies have demonstrated that treatment with alphabeta-T-cell receptor (TCR) monoclonal antibody and cyclosporine A (CsA) can extend survival in composite tissue allografts (CTA). The purpose of this study was to induce tolerance in fully major histocompatibility complex (MHC)-mismatched rat limb allografts under 7 days of a combined alphabeta-TCR-CsA protocol. The authors performed 30 hind-limb allotransplantations across the MHC barrier between Brown Norway donors (BN; RT1n) and Lewis recipients (LEW; RT1l). Isograft and allograft controls received no treatment. The experimental groups received monotherapy of alphabeta-TCR and CsA or a combination of alphabeta-TCR and CsA for 7 days only. Donor-specific tolerance and immunocompetence were determined by standard skin grafting in vivo and mixed lymphocyte reaction (MLR) in vitro. The efficacy of immunosuppressive therapy and the level of donor-specific chimerism were determined by flow cytometry. Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined alphabeta-TCR-CsA. Donor-specific tolerance and immunocompetence of long-term chimeras were confirmed by acceptance of skin grafts from the donors and rejection of the third-party alloantigens (AxC Irish). At day 120, MLR demonstrated unresponsiveness to the host and donor antigens but strong reactivity against third-party alloantigens. Flow cytometry confirmed the high efficacy of immunosuppressive treatment and the development of donor-specific chimerism (7.6% of CD4+-RT1n+ cells, 1.3% of CD8+-RT1n+ cells, and 16.5% of CD45RA+-RT1n+ cells) in the periphery of tolerated recipients. Combined therapy of alphabeta-TCR-CsA for 7 days resulted in tolerance induction in fully MHC-mismatched rat hind-limb allografts. Tolerance was directly associated with stable, donor-specific chimerism.

Dendritic epidermal gamma/delta T cells (DETC) activated in vivo proliferate in vitro in response to Mycobacterium leprae antigens.

gamma/delta T-cell receptor (TCR)+ dendritic epidermal T cells (DETC) are part of a primitive defense system in the skin; they are capable of responding only to a limited number of antigens. The aim of the present study was to test whether DETC can proliferate in vitro in response to antigens of Mycobacterium leprae. DETC were obtained from CBA mouse ear skin by trypsinization and Histopaque gradient centrifugation. The resulting epidermal cell suspension contained up to 20% DETC, as analyzed by the fluorescence activated cell sorter (FACS) after staining with anti-Thy-1 or anti-gamma/delta TCR monoclonal antibodies (mAbs). The freshly isolated cells, or DETC cultured up to 4 weeks with interleukin-2 (IL-2), were exposed in vitro for up to 6 days to varying doses of the following M. leprae antigens: (1) integral (live) M. leprae bacilli; (2) Dharmendra antigen; and (3) PGL-1 (phenolic glycolipid of M. leprae). The DETC response was assessed by tritiated thymidine (3H-TdR) incorporation. The freshly isolated DETC, or DETC cultured up to 4 weeks with IL-2, did not respond significantly to any of the M. leprae antigens, although at the same time they were able to respond vigorously to concanavalin A (Con A), as positive control. If, however, DETC were isolated from skin, painted 7 days before with croton oil (10 microL/cm2 to cause irritant dermatitis, they were able to respond to all M. leprae antigens by a 3-4-fold incrase in the 3H-TdR uptake. The most effective stimulator was a 1 : 1 mixture of Dharmendra and PGL-1 (0. 01 microg/mL), which was as effective as 10-fold higher doses of either antigen alone. Cell counts confirmed that increased DNA synthesis was associated with cell proliferation. Experiments employing alpha/beta-TCR CBA murine spleen cells and epidermal cell suspension treated with anti-gamma/delta or antialpha/beta mAbs + C' proved that only the gamma/delta DETC were the responder cells to M. leprae antigens. The results suggest that activation of DETC in vivo may make them responsive to M. leprae antigens. A significant increase in the number of class II major histocompatibility complex (MHC) positive, nondendritic cells was observed in the croton oil-treated epidermis. We hypothesize that croson oil-induced upregulation of class II MHC expression, which endows epidermal cells with antigen-presenting capabilities, might be an important factor in vivo in delivering an immunogenic signal to resident DETC in the skin.

The role of intrahepatic gammadelta-T cells for liver injury induced by Salmonella infection in mouse.

Liver injury was induced after infection with Salmonella choleraesuis 31N-1. In T-cell receptor-delta knockout mice, serum alanine transferase level was significantly decreased in comparison with normal control mice after Salmonella infection. On the contrary, in vivo administration of anti-gammadelta T-cell receptor monoclonal antibody (UC7-13D5) to stimulate gammadelta-T cells in infected mice significantly increased serum alanine transferase level but decreased bacterial growth compared with infected mice given control antibody (UC8; hamster IgG). These data suggest that gammadelta-T cells have effector activities not only for protection but also for liver injury during Salmonella infection.

Insufficient resistance of trehalose-6,6-dimycolate-treated T-cell receptor delta gene mutant (TCR delta-/-) mice against influenza virus infection.

Normal mice inoculated intravenously with 50 microg trehalose-6,6'-dimycolate, a glycolipid component of the cell wall of Mycobacterium, in an oil-in-water emulsion (TDM emulsion) acquired a high resistance to intranasal lethal infection of an influenza virus. In contrast, TDM emulsion-treated T-cell receptor delta gene mutant (TCR delta-/-) mice acquired insufficient resistance against the lethal influenza virus infection. The patterns of insufficient resistance were identical to the results obtained previously with mice which were depleted of T-lymphocytes bearing gammadelta T-cell receptors (gammadelta T-cells) by in vivo administration of anti-gammadelta T-cell receptor monoclonal antibody (Hoq et al, J. Gen. Virol. 78: 1597-1603, 1997). These results strongly suggest that the gammadelta T-cells play an important non-specific role in resistance against influenza virus infection.

Immunophenotypic identification of Sezary cells in peripheral blood.

Despite anecdotal literature that Sezary cells express the CD4+ CD7- immunophenotype, no formal validation has been published establishing the use of this immunophenotype for clinical or experimental purposes. Consequently, the only method presently available for Sezary cell identification is nuclear contour analysis, a labor-intensive procedure not generally available at most major medical centers. In this study, the accuracy of CD4+ CD7- subset quantitation for the identification of Sezary cells was examined. The study found that the percentage of CD4+ CD7- cells is elevated in many Sezary syndrome/MF patients relative to normal, healthy individuals. In addition, CD4+ CD7- enumeration correlates with enumeration by nuclear contour analysis in most patients (11 of 15) with elevated CD4/CD8 ratios. The CD4+ CD7- subset also correlates with the expression of other aberrant immunophenotypes, such as CD3low or CD4low. Lastly, CD4+ CD7- subset quantitation correlates with the number of clonal T lymphocytes, as measured using V beta-specific T-cell receptor monoclonal antibodies. The study found this method to be exceptionally accurate, with two caveats: (1) the absence of an expanded CD4+ CD7- subset in patients with a normal CD4/CD8 ratio is uninformative; and (2) in approximately 25% of patients with an elevated CD4/CD8 ratio, the Sezary cells are CD7+.

Treatment of Donor Mice With an αβ T-Cell Receptor Monoclonal Antibody Induces Prolonged T-Cell Nonresponsiveness and Effectively Prevents Lethal Graft-Versus-Host Disease in Murine Recipients of Major Histocompatibility Complex (MHC)-Matched and MHC-Mismatched Donor Marrow Grafts

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57-597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H-2d]) marrow grafts. Transplantation of H57-597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57-597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57-597. Donor T cells exposed to H57-597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.

Expression of T cell receptor gamma delta antigens in human brain tissue.

The human gamma delta T cell receptor (TCR) is normally expressed on lymphoid tissue. Since expression of different molecules of the T cell system has been described for human brain cells, we examined the expression of CD4 and TCR gamma delta antigens with a panel of various anti-gamma delta TCR monoclonal antibodies (mAbs) and an anti-CD4 mAb using immunohistochemistry on different regions of frozen human postmortem tissue from five different brains. We could confirm the expression of CD4 antigens. gamma delta TCR expression on brain tissue was found in different regions of the brain by immunohistochemistry. Double staining with anti-gamma delta TCR and antineuronal enolase (NSE) mAbs showed that gamma delta TCR+ cells were not stained by anti-NSE, although they were sometimes located near neurons and showed dendritic forms; they are possibly tissue-resident gamma delta T cells, as described in the skin. Polymerase chain reaction analysis using a highly sensitive primer sequence against the constant-region delta sequence supports, combined with immunohistochemistry findings, the notion that the gamma delta TCR is expressed in human brain.

Crosstalk between alpha/beta T cells and gamma/delta T cells in vivo: activation of alpha/beta T-cell responses after gamma/delta T-cell modulation with the monoclonal antibody GL3.

Although gamma/delta T cells express numerous in vitro functions similar to alpha/beta T cells, little is known about their biological functioning in vivo. Furthermore, it is unclear whether alpha/beta T cells and gamma/delta T cells act independently or in a coordinated way. In the present study, gamma/delta T cells were modulated in vivo by i.p. injection of the anti-gamma/delta T-cell receptor (TCR) monoclonal antibody GL3. GL3 administration caused disappearance of the gamma/delta TCR in spleen and lymph node cells and the gamma/delta TCR was reexpressed after in vitro cultivation for a few days. When cultured in vitro for 4 days, in the absence of foreign antigens, spleen and lymph node alpha/beta T cells from GL3-modulated mice showed vigorous proliferative responses. CD4 T lymphocytes from GL3-modulated mice produced interleukin 2, and CD8 T cells developed into cytolytic T lymphocytes in vitro capable of lysing syngeneic and allogeneic targets. Treatment with heat-inactivated GL3 or with normal hamster immunoglobulin did not cause any of these effects. These findings suggest that the anti-gamma/delta TCR monoclonal antibody GL3 modulates gamma/delta T cells in vivo and that this modulation has profound effects on alpha/beta T-cell reactivity. Hence, the data suggest a role for gamma/delta T cells in the regulation of alpha/beta T-cell activation in vivo.

Helper activity of CD4+ alpha beta T cells is required for the avian gamma delta T cell response.

We have studied the in vitro activation of chicken gamma delta T cells. Both splenic alpha beta and gamma delta T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4+ cells or alpha beta T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of gamma delta T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or alpha beta TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the gamma delta T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-gamma delta TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-gamma delta TcR monoclonal antibody, a strong proliferation of gamma delta T cells was observed. In contrast, both V beta 1- and V beta 2-expressing alpha beta T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either V beta 1+ or V beta 2+ T cell subset alone had no negative effect on the proliferation or blast formation of gamma delta T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4+ alpha beta T cells (both V beta 1- and V beta 2-expressing) play a role in the activation and response of chicken gamma delta T cells.

Listeria monocytogenes-induced gamma interferon secretion by intestinal intraepithelial gamma/delta T lymphocytes

gamma/delta T cells represent a major proportion of intestinal intraepithelial lymphocytes (IEL), and it has been suggested that these IEL serve as a first immune barrier against microbial invasion and that they do so by destroying infected epithelial cells. In the present study, we confirm that both alpha/beta and gamma/delta IEL from naive mice express potent cytotoxicity and produce gamma interferon (IFN-gamma) after T-cell receptor (TCR) engagement by specific monoclonal antibodies (MAb). Intraperitoneal administration of the anti-gamma/delta TCR MAb GL3 caused downregulation of the gamma/delta TCR in IEL, and IEL from gamma/delta TCR-modulated mice failed to express cytotoxic activity and to secrete IFN-gamma after gamma/delta TCR engagement. In contrast, alpha/beta IEL from such mice were still cytolytic and secreted IFN-gamma. Mice were infected orally with virulent Listeria monocytogenes at doses which caused bacterial invasion through the intestinal epithelia. Although alpha/beta and gamma/delta IEL from these mice expressed high cytolytic activities in antibody-redirected killer assays, target cells pulsed with listerial antigens were not lysed. In contrast, IFN-gamma secretion by IEL from L. monocytogenes-infected mice was induced not only by anti-TCR MAb but also by target cells pulsed with listerial antigens, whereas irrelevant antigens, including heat shock protein 60, did not induce IFN-gamma secretion. Furthermore, the number of IFN-gamma-secreting IEL, as assessed by the enzyme-linked immunospot technique, was increased during listeriosis. gamma/delta TCR modulation by GL3 administration abrogated antigen-induced IFN-gamma secretion by IEL from infected mice. These findings suggest that L. monocytogenes induced IFN-gamma secretion by gamma/delta IEL from mice suffering from intestinal L. monocytogenes infection and invasion. Thus, the data provide evidence for a role of IFN-gamma-secreting IEL in local resistance against listeriosis and perhaps other food-borne diseases.

Co-expression of human T cell receptor chains with mouse CD3 on the cell surface of a mouse T cell hybridoma

In this study we demonstrate that human T cell receptor (TcR) chains can be co-expressed with murine CD3 on the cell surface of a murine T cell hybridoma. Human TcR alpha and beta genes from the Jurkat leukaemic cell line were transfected into a TcR-negative mouse T cell hybridoma, TG40. The Jurkat TcR was successfully co-expressed at the cell surface with mouse CD3 components. Brightly staining transfectants were selected by fluorescence-activated cell sorting, and levels of expression comparable to a normal T cell line were achieved suggesting that the human TcR dimer assembled efficiently with the mouse CD3 complex. Northern blot analysis demonstrated similar levels of TcR messenger RNA to those found in the parental Jurkat line. Although we have not formally demonstrated surface expression of the Jurkat TcR alpha chain, the residual alpha gene transcript which is present in murine TG40 line is non-expressible. In order to test the signalling capacity of this human/mouse complex, the transfectants were stimulated with an anti-V beta 8 monoclonal antibody. This stimulus led to interleukin-2 production by the transfectants, demonstrating the delivery of a transmembrane signal. In addition, B10.A mice were immunised with the transfectants, and the antisera from these mice stained the transfectant and the Jurkat cell line, but not the parental T cell hybridoma. This interspecies transfection approach should now permit us to explore the requirements for T cell activation, the constraints on TcR alpha beta chain pairing, and creates ideal reagents for inducing a mouse anti-human TcR-specific response with a view to producing panels of anti-human TcR monoclonal antibodies.

A major fraction of human intraepithelial lymphocytes simultaneously expresses the γ/δ T cell receptor, the CD8 accessory molecule and preferentially uses the Vδ1 gene segment

The frequency of T cell receptor (TcR) type and the variable gene segment expression in human intraepithelial lymphocytes (IEL) from the large intestinal mucosa were studied by flow cytometry and immunohistochemistry, and compared to those in peripheral blood lymphocytes (PBL) - or lamina propria lymphocytes (LPL). Employing anti-gamma/delta TcR and anti-alpha/beta TcR monoclonal antibodies (mAb), flow cytometric analysis revealed that a large fraction of IEL (37%) are gamma/delta T cells, whereas within LPL and PBL gamma/delta T cells comprise a minor population (4.6% and 3.8% respectively). At these sites the number of gamma/delta T cells labeled with anti-CD8 mAb were 58.3% (IEL), 43.3% (LPL) and 24.4% (PBL). In situ staining of serial sections of large intestine confirmed these results. Hence, these data suggest a selective accumulation of CD8+ gamma/delta T cells in the human epithelium of the large intestine. Furthermore, analysis of gamma/delta TcR bearing IEL+ disclosed a marked preponderance of cells using the V delta 1 gene segment, whereas gamma/delta TcR+ PBL preferentially express V delta 2. Strikingly, the majority of these V delta 1+ IEL bear the CD8 molecule on their surface. These results are taken as evidence for a selective localization of V delta 1+ CD8+ gamma/delta T cells in the epithelium of the large intestine.

T-cell receptor Vβ5 usage defines reactivity to a human T-cell receptor monoclonal antibody

The monoclonal antibody 3D6 reacts with the T-cell receptor (Tcr) on the T-leukemic line HPBALL and with 2-13% of human peripheral blood T lymphocytes. In this study V alpha and V beta expression in a panel of T-cell populations and clones expressing the 3D6 epitope was determined by Southern and northern hybridization analysis. The results demonstrate that these 3D6-positive T cells, regardless of CD4/CD8 phenotype or function, express a gene of the V beta 5 family, also expressed by HPBALL. No correlation was found between 3D6 reactivity and expression of the HPBALL V alpha gene. The results demonstrate that 3D6 recognizes an epitope solely on the Tcr beta chain and that the use of this beta chain, together with an appropriate V alpha, can impart a diverse pattern of reactivity to a T cell.