Irradiated B7-1 transduced primary acute myelogenous leukemia (AML) cells can be used as therapeutic vaccines in murine AML

Recent studies have shown that tumor cells genetically modified by transduction of B7-1, a natural ligand for the T-cell costimulatory molecules CD28 and CTLA-4, are rejected in syngeneic hosts. In these reports, transformed cell lines and drug-selected cells have been used for vaccinations. To determine the effectiveness of B7-1-transduced primary acute myelogenous leukemia (AML) cells on the induction of antitumor immunity, we have studied a murine AML model in which primary AML cells were retrovirally transduced with the murine B7-1 cDNA. A defective retroviral producer clone expressing B7-1 and secreting a high titer of virus was used for infection of AML cells. Unselected transduced AML cells, expressing a high level of B7-1, were used for in vivo vaccinations. Our results show that one intravenous (IV) injection of irradiated B7-1-positive (B7-1+) AML cells can provide long-lasting (5 to 6 months) systemic immunity against subsequent challenge with wild-type AML cells. Furthermore, one exposure to irradiated B7-1+ AML cells results in rejection of leukemia by leukemic mice when the vaccination occurs in the early stages of the disease. The antileukemia immunity is CD8+ T-cell-dependent and B7/CD28-mediated, since in vivo treatment of mice with anti-CD8 monoclonal antibody or CTLA-4 Ig leads to abrogation of the specific antileukemia immune response. These results emphasize that B7-1 vaccines may have therapeutic usefulness for patients with AML.     

Expression of costimulatory molecules, B7-1 and B7-2 on human gastric carcinoma

Costimulation of T cells via B7-1 and B7-2 molecules on a tumor has been shown to be important for eliciting cell-mediated antitumor immunity. We studied the surface expression of B7-1 and B7-2 in 24 cases of gastric carcinoma from the primary locus, 20 cases of metastatic carcinoma from malignant ascites, 20 cases of benign gastric mucosa and 7 gastric carcinoma cell lines by two-color flow cytometry with mAb CD80 and CD86. The B7-1 and B7-2 molecules were expressed by 6 cell lines, and 1 cell line showed the predominant expression of B7-2 but not B7-1. Almost all patients with primary gastric carcinoma and benign gastric mucosa showed high levels of expression of the B7-1 and B7-2, revealing approximately 40%–60% positive cells. However, the percentage of B7-1-positive cells of poorly differentiated primary carcinomas was significantly lower than that of well-differentiated carcinoma and normal mucosa (P<0.01). Furthermore, all of the metastatic carcinoma cells revealed consistently very low or undetectable levels of expression of the B7-1 molecule, only 8% (mean) of cells being positive, despite showing higher levels of B7-2 expression. Thus, it seems likely that decreased or deleted expression of B7-1 correlates with the grade of tumor differentiation, tumor progression and metastasis. These results suggest that the B7-1 molecule on the gastric carcinoma bearing CD80⁺CD86⁺ is abrogated during tumor invasion and/or metastasis, and the tumor finally acquires the CD80⁻CD86⁺ phenotype. Consequently, inadequate B7-1 costimulation may contribute to the escape of tumors from destruction by the host's immune system. Received: 26 December 1997 / Accepted: 2 March 1998     

B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function

B7-H1 is a recently described B7-like molecule that costimulates T-cell growth and cytokine secretion without binding to CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and inducible costimulator (ICOS). In this report, a mouse homologue of human B7-H1 is identified, and its immunologic functions are studied in vitro and in vivo. Mouse B7-H1 shares 69% amino acid homology to the human counterpart. Similar to human B7-H1, mouse B7-H1 can be induced to express on macrophages, T cells, and B cells and to enhance T-cell proliferation and secretion of interleukin-10 (IL-10), interferon-gamma, and granulocyte-macrophage colony-stimulating factor but not IL-2 and IL-4. Furthermore, B7-H1 preferentially costimulates CD4+ T cells independently of CD28 and enhances mixed lymphocyte responses to allogeneic antigens. In contrast to B7-1, expression of B7-H1 on murine P815 tumor cells by transfection fails to increase allogeneic and syngeneic cytolytic T-cell responses in vitro and in vivo. Administration of B7-H1Ig fusion protein, however, enhances keyhole limpet hemocyanin- specific T-cell proliferation and 2,4,6-trinitrophenyl-specific immunoglobulin G2a antibody production. The study thus identifies a unique costimulatory pathway that preferentially affects T-helper cell functions.     

CTLA-4 dysregulation of self/tumor-reactive CD8+ T-cell function is CD4+ T-cell dependent

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) maintains peripheral tolerance by suppressing T-cell activation and proliferation but its precise role in vivo remains unclear. We sought to elucidate the impact of CTLA-4 expression on self/tumor-reactive CD8(+) T cells by using the glycoprotein (gp) 100-specific T-cell receptor (TCR) transgenic mouse, pmel-1. pmel-1 CLTA-4(-/-) mice developed profound, accelerated autoimmune vitiligo. This enhanced autoimmunity was associated with a small but highly activated CD8(+) T-cell population and large numbers of CD4(+) T cells not expressing the transgenic TCR. Adoptive transfer of pmel-1 CLTA-4(-/-) CD8(+) T cells did not mediate superior antitumor immunity in the settings of either large established tumors or tumor challenge, suggesting that the mere absence of CTLA-4-mediated inhibition on CD8(+) T cells did not directly promote enhancement of their effector functions. Removal of CD4(+) T cells by crossing the pmel-1 CLTA-4(-/-) mouse onto a Rag-1(-/-) background resulted in the complete abrogation of CD8(+) T-cell activation and autoimmune manifestations. The effects of CD4(+) CLTA-4(-/-) T cells were dependent on the absence of CTLA-4 on CD8(+) T cells. These results indicated that CD8(+) CLTA-4(-/-) T-cell-mediated autoimmunity and tumor immunity required CD4(+) T cells in which the function was dysregulated by the absence of CTLA-4-mediated negative costimulation.     

协同刺激分子B7-CD28/CTLA-4与自身免疫病

T细胞需要两组信号刺激才能充分活化,一组由MHC抗原肽和TCR结合后提供;另一组由抗原递呈细胞提供的协同刺激信号.T细胞上的CD28/细胞毒性T淋巴细胞相关抗原4[(Cytotoxic T lym-phocyte-associated antigen 4,CTLA-4(CD152)]与抗原递呈细胞上的B7(B7-1,B7-2)是目前发现的最为重要的协同刺激分子,它们在自身免疫病患者T细胞及抗原递呈细胞上有异常表达,且参与致病过程.     

B7-1 (CD80)-gene transfer combined with interleukin-12 administration elicits protective and therapeutic immunity against mouse hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) frequently recurs after primary therapy, resulting in poor prognosis. To try to find a way to prevent this, we examined the combined effectiveness of B7-1 (CD80)-gene transfer and interleukin-12 (IL-12) on the induction of protective antitumor immunity against poorly immunogenic BNL1ME A.7R. 1 (BNL) mouse HCC cells. We introduced mouse B7-1 gene into BNL1ME A. 7R.1 cells. Overexpression of B7-1 on BNL1ME A.7R.1 cells resulted in significant inhibititon of subcutaneous tumor development in syngeneic BALB/c mice, but not in complete rejection, suggesting that strong expression of B7-1 molecules may enhance the immunogenicity of BNL1ME A.7R.1 cells in immunocompetent mice. Lymphocyte study revealed that the cytolytic activity generated by immunization with B7-1 transfectants against BNL1ME A.7R.1 cells was mediated mainly by CD8(+) cytotoxic T lymphocytes (CTL). We examined the synergistic effect of IL-12 and immunization with B7-1 transfectants. The combination led to rejection of BNL1ME A.7R.1 cells in 6 of 10 tested mice and delayed tumor development in the remaining mice. Furthermore, the combined treatment against pre-established BNL1ME A.7R.1 tumors resulted in rejection in 3 of 8 tested mice or in significant inhibition of tumor growth in the remaining mice. In vivo lymphocyte subset depletion study indicated that the combined antitumor effect was dependent on the presence of both CD8(+) and CD4(+) T cells. In conclusion, the combination of immunization of B7-1-transfected HCC cells and IL-12 could induce protective and therapeutic immunity against parental HCC cells, and this combination may be therapeutically useful for suppressing recurrence of HCC.     

B7-CTLA4 interaction promotes cognate destruction of tumor cells by cytotoxic T lymphocytes in vivo

Costimulatory molecules B7-1 and B7-2 (hereby collectively called B7) interact with CD28 and CTLA4 on T cells and promote antitumor immunity. The function of B7-CTLA4 interaction in antitumor CTL response remains controversial. Here we used CD28(-/-) and CD28(+/-) or CD28(+/+) transgenic mice that express the T-cell receptor specific for an unmutated tumor antigen, P1A, and for tumor cells expressing a CTLA4-specific B7 mutant to evaluate the function of CD28-B7 and CTLA4-B7 interactions in induction and effector phases of antitumor immunity. We report that B7-CD28 and B7-CTLA4 interactions promote tumor rejection. However, this is achieved by distinct mechanisms. B7-CD28 interaction enhances T-cell clonal expansion, though a role for this interaction in the effector phase cannot be ruled out. In contrast, B7-CTLA4 interaction enhances the CTL-mediated destruction of tumors, but not T-cell clonal expansion.     

In a model of tumor dormancy, long-term persistent leukemic cells have increased B7-H1 and B7.1 expression and resist CTL-mediated lysis

In tumor dormancy, tumor cells persist in the host over a long period of time but do not grow. We investigated in the DA1-3b mouse model of acute myeloid leukemia how leukemic cells could persist for months in spite of an effective antileukemic immune response. Mice were immunized with irradiated interleukin 12 (IL12)- or CD154-transduced DA1-3b cells, challenged with wild-type DA1-3b cells, and randomly killed during 1-year follow-up. Quantification of residual disease 1 year after challenge showed that persistent leukemic cells represented less than 0.02% of spleen cells in most animals. These residual cells were still able to kill naive hosts, even when isolated after 1 year of persistence. Persistent leukemic cells were more resistant to specific cytotoxic T-cell (CTL)-mediated killing and had enhanced B7-H1 and B7.1 expression proportional to the time they had persisted in the host. Blocking B7-H1 or B7.1/cytotoxic T-lymphocyte-associated antigen (CTLA-4) interaction enhanced CTL-mediated killing of the persistent cells, and blocking B7-H1, B7.1, or CTLA-4 in vivo prolonged survival of naive mice injected with persistent leukemic cells. Thus, escape of leukemic cells from tumor immunity via overexpression of B7-H1 or B7.1 might represent a new mechanism of tumor dormancy in acute leukemia.     

CD28 disruption exacerbates inflammation in Tgf-beta1-/- mice: in vivo suppression by CD4+CD25+ regulatory T cells independent of autocrine TGF-beta1

Tgf-beta1-/- mice develop a progressive, lethal, inflammatory syndrome, but mechanisms leading to the spontaneous activation of Tgf-beta1-/- T cells remain unclear. Here we show the disruption of CD28 gene expression accelerates disease in Tgf-beta1-/- mice, and we link this increase in severity to a reduction in the number of CD4+CD25+ regulatory T cells. CD4+CD25+ T cells develop normally in Tgf-beta1-/- mice and display characteristic expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), alpha(E)beta7 integrin, and Foxp3. Adoptive transfer of Tgf-beta1-/- splenocytes to Tgf-beta1+/+/Rag2-/- mice induced an autoimmune inflammatory disease with features similar to those of the Tgf-beta1-/- phenotype, and disease transfer was accelerated by the depletion of Tgf-beta1-/- CD4+CD25+ T cells from donor splenocytes. Cotransfer of Tgf- beta1-/- CD4+CD25+ T cells clearly attenuated disease in Rag2-/- recipients of CD25+-depleted Tgf-beta1-/- spleen and lymph node cells, but suppression was incomplete when compared with Tgf-beta1+/+ CD4+CD25+ T cells. These data demonstrate that CD4+CD25+ regulatory T cells develop in complete absence of endogenous transforming growth factor-beta1 (TGF-beta1) expression and that autocrine TGF-beta1 expression is not essential for these cells to suppress inflammation in vivo.     

Different cell surface oligomeric states of B7-1 and B7-2: implications for signaling

The costimulatory ligands B7-1 and B7-2 are expressed on the surface of antigen-presenting cells and interact with the costimulatory receptors CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on T cells. Although B7-1 and B7-2 are homologous ligands having common receptors, they exhibit distinct biochemical features and roles in immune regulation. Several biochemical and structural studies have indicated differences in the oligomeric state of B7-1 and B7-2. However, the organization of B7 ligands on the cell surface has not been examined. By using photobleaching-based FRET (pbFRET), we demonstrate that B7-1 and B7-2 adopt different oligomeric states on the cell surface. Our study shows that B7-2 exists as a monomer on the cell surface whereas B7-1 exists predominantly as dimers on the cell surface. A series of mutations in B7-1 result in the expression of a predominantly monomeric species on the cell surface and validate the dimer interface proposed by prior crystallographic analysis. The difference in the oligomeric states of B7-1 and B7-2 provides insight into the geometric organization of the costimulatory receptor-ligand complexes in the immunological synapse and suggests constraints on signal transduction mechanisms involved in T cell activation.     

CD28/CTLA-4:B7在特发性血小板减少性紫癜患者外周血淋巴细胞中的表达及意义

目的:探讨特发性血小板减少性紫癜(ITP)患者外周血淋巴细胞CD28、CTLA-4(CD152)、B7-1(CD80)及B7-2(CD86)的表达及意义。方法:采用免疫荧光标记和流式细胞术检测41例ITP患者和40例健康对照者外周血CD3+CD28+细胞、CD3+CD152+细胞、CD80+CD19+细胞和CD86+CD19+细胞分别占淋巴细胞的比例及血小板表面相关抗体水平,进行2组对比、分析。结果:与正常对照组相比,急性ITP患者外周血CD3+CD28+细胞和CD3+CD152+细胞差异无统计学意义(P0.05),CD80+CD19+细胞增多(P0.05),CD86+CD19+细胞显著增多(P0.01),慢性ITP患者CD86+CD19+细胞增多(P0.05);急性ITP患者外周血CD86+CD19+细胞较慢性ITP患者增多(P0.05);与正常对照组相比,急性ITP患者PAIg's、PAIgG和PAIgM水平显著增高,慢性ITP患者PAIgG水平增高;CD80、CD86表达与PAIgG水平之间存在显著的相关性(均P0.01)。结论:ITP患者外周血B淋巴细胞上CD86和CD80表达均异常,可能与其发病相关。     

Dependency on intercellular adhesion molecule recognition and local interleukin-2 provision in generation of an in vivo CD8+ T-cell immune response to murine myeloid leukemia

The immune response to a murine myeloid leukemia (cell line C1498) was studied in vitro and in vivo. Natural killer (NK) cells and CD8+ cytotoxic T lymphocytes (CTL) were shown to lyse C1498 in vitro through the binding of leukocyte function antigen-1 (LFA-1) on effectors and intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on C1498 target cells. However, the ability of nonimmunized mice to resist an in vivo challenge of a low dose (10(4)) of C1498 was NK-cell, but not T-cell dependent. The failure of T cells to participate in the immune surveillance of a low leukemia burden appeared, in part, because of a lack of expansion of leukemia reactive CTL precursors (CTLp). Leukemia reactive CTLp frequency estimations in naive and leukemia bearing mice were not significantly different (range, 1:20,600 to 1:74,000) in contrast to immunized mice (range, 1:1,400 to 1:4,400). Leukemia reactive CTLp could be expanded to a level that could apparently mediate in vivo immune surveillance of 10(5) leukemia cells by injection of irradiated leukemia cells intraperitoneally (IP) or subcutaneously (SC), but not intravenously (IV). However, IV injection of 10(5) live leukemia cells engineered to secrete interleukin-2 (IL-2) resulted in systemic immunity mediated primarily by CD8+ T cells. We conclude that NK cells can mediate immune surveillance of a low leukemia burden. CD8+ CTL-mediated immune surveillance can eliminate a higher leukemia burden than NK cells, but requires T-cell help, which can be delivered by local IL-2. Both NK and CTL-mediated immune surveillance of C1498 murine myeloid leukemia is dependent on recognition through the LFA-1:ICAM adhesion pathway.     

B7x: a widely expressed B7 family member that inhibits T cell activation

B7 family proteins provide costimulatory signals that regulate T cell responses. Here we report the third set of B7 family-related T cell inhibitory molecules with the identification of a homolog of the B7 family, B7x. It is expressed in immune cells, nonlymphoid tissues, and some tumor cell lines. B7x inhibits cell-cycle progression, proliferation, and cytokine production of both CD4+ and CD8+ T cells. B7x binds a receptor that is expressed on activated, but not resting T cells that is distinct from known CD28 family members. Its receptor may be a recently identified inhibitory molecule, B and T lymphocyte attenuator. These studies identify a costimulatory pathway that may have a unique function in downregulation of tissue-specific autoimmunity and antitumor responses.     

Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation.

Signaling via the T-cell receptor complex is necessary but not sufficient to induce antigen-specific T lymphocytes to expand clonally. To proliferate, T cells must receive one or more costimulatory signals provided by antigen presenting cells (APCs). One such critical costimulatory signal is delivered by the CD28/CTLA-4 counterreceptor, B7, expressed on APCs. B7 costimulation induces CD28 signaling, resulting in interleukin 2 (IL-2) secretion, and T-cell proliferation. Conversely, T-cell receptor signaling in the absence of B7 costimulation results in induction of antigen-specific tolerance. Here, we show that activated human B lymphocytes express two additional CTLA-4 counterreceptors also capable of providing T-cell costimulation. At 24 hr postactivation, B cells express a CTLA-4 counterreceptor not recognized by anti-B7 or -BB-1 monoclonal antibodies (mAbs), which induces detectable IL-2 secretion and T-cell proliferation. At 48 and 72 hr postactivation, B cells express both B7 and a third CTLA-4 counterreceptor identified by the anti-BB-1 mAb. BB-1 appears to be a molecule distinct from B7 by its expression on B7- cells and its capacity to induce T cells to proliferate without significant accumulation of IL-2. As observed for B7, costimulatory signals mediated by these alternative CTLA-4/CD28 counterreceptors are likely to be essential for generation of an immune response and their absence may result in antigen-specific tolerance. We propose the following terminology for these CTLA-4 counterreceptors: (i) B7, B7-1; (ii) early CTLA-4 binding counterreceptor, B7-2; and (iii) BB-1, B7-3.     

Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules.

The inability of the autologous host to reject resident tumor cells is frequently the result of inadequate generation of tumor-specific T cells. Specific activation of T cells occurs after delivery of two signals by the antigen-presenting cell. The first signal is antigen-specific and is the engagement of the T-cell antigen receptor by a specific major histocompatibility complex antigen-peptide complex. For some T cells, the second or costimulatory signal is the interaction of the T-cell CD28 receptor with the B7 activation molecule of the antigen-presenting cell. In the present study, we demonstrate that mouse sarcoma cells genetically engineered to provide both T-cell activation signals stimulate potent tumor-specific CD4+ T cells that cause rejection of both engineered and wild-type neoplastic cells. Two other recent studies have also demonstrated that costimulation via B7 can improve tumor immunity. However, our study differs from these reports by two important observations. (i) One of these studies utilized mouse tumor cells expressing xenogenic viral antigens, and hence, the results are not applicable to wild-type resident tumors. Our study, however, demonstrates that coexpression of B7 by major histocompatibility complex class II+ tumor cells induces immunity in the autologous host that is specific for naturally occurring tumor antigens of poorly immunogenic tumors. (ii) In both earlier studies, only CD8+ T cells were activated after coexpression of B7, whereas in the present report, tumor-specific CD4+ T cells are generated. This report therefore illustrates the role of B7 activation molecule in stimulating potent tumor-specific CD4+ T cells that mediate rejection of wild-type tumors and provides a theoretical basis for immunotherapy of established tumors.     

Crystal structure of the receptor-binding domain of human B7-2: insights into organization and signaling

B7-1 and B7-2 are homologous costimulatory ligands expressed on the surfaces of antigen-presenting cells. Their interactions with CD28/CTLA-4 receptors expressed on T cell surfaces are crucial for the proper regulation of T cell activity. B7-1 and B7-2 display distinct roles in immune regulation, although they are usually considered to have redundant functions. Here, we report the crystal structure of the receptor-binding (Ig V-type) domain of human B7-2 at 2.7-A resolution. Structures of unliganded and liganded B7-1 and B7-2 suggest a physical-chemical basis for the observed functional similarities and differences between these two costimulatory ligands. Of particular note, whereas the majority of the residues mediating B7-1 dimerization are hydrophobic, the B7-2 dimer observed in the B7-2/CTLA-4 complex displays a very hydrophilic dimer interface. These differences provide a mechanism for preventing the formation of B7-1/B7-2 heterodimers. The divergence at the putative dimer interface is also consistent with the lower tendency of B7-2 to dimerize, as shown by the monomeric state of unliganded B7-2 both in solution and crystalline form, and may result in detailed differences in signaling mechanisms associated with B7-1 and B7-2.     

B7-1 and 4-1BB ligand expression on a myeloma cell line makes it possible to expand autologous tumor-specific cytotoxic T cells in vitro

OBJECTIVE: The aim of this study was to confer an antigen-presenting cell (APC) ability on multiple myeloma cell lines (HMCLs) using B7-1 and/or 4-1BBL gene transfer. MATERIALS AND METHODS: HMCLs were retrovirally transduced with B7-1 and/or 4-1BBL cDNAs. Allogeneic or autologous T cells were stimulated by coculture with B7-1- and/or 4-1BBL-transduced HMCLs in the presence of interleukin-2. T cell clones were obtained by limiting dilution. T-cell activation was assessed by interferon-gamma Elispot assays and cytotoxicity by (51)Cr release assays. RESULTS: Neither primary multiple myeloma cells (MMCs) nor HMCLs expressed B7-1 or 4-1BBL, and these molecules could not be induced by CD40 triggering. HMCLs failed to stimulate allogeneic or autologous T cells. Transduction of HMCLs with B7-1 and/or 4-1BBL retroviruses induced a high expression of B7-1 and 4-1BBL molecules and a strong T-cell activation ability. Long-term cultured CD8(+) T-cell lines could be obtained by stimulation with the autologous B7-1/4-1BBL XG-19 HMCL. These cytotoxic T lymphocytes (CTL) efficiently killed the autologous parental XG-19 HMCL as well as autologous primary MMCs and allogeneic HMCLs. They did not kill autologous CD34 cells and autologous EBV cell line or natural killer target K562 cells. Cloned CTL could recognize allogeneic HMCLs, demonstrating that a shared anti-MMC repertoire was expanded. CONCLUSION: Transduction with B7-1 and 4-1BBL retroviruses turned HMCLs into efficient APCs. It permitted the long-term expansion of autologous anti-tumor CTL with a shared anti-MMC repertoire, for one HMCL. These data suggest developing an immunotherapy using modified tumor cells in patients with multiple myeloma.     

Selective blockade of CD28 and not CTLA-4 with a single-chain Fv-alpha1-antitrypsin fusion antibody

B7-1 and B7-2 are costimulatory molecules expressed on antigen-presenting cells. The CD28/B7 costimulation pathway is critical for T-cell activation, proliferation, and Th polarization. Blocking both cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and CD28 interactions with a CTLA-4/Ig fusion protein inhibits various immune-mediated processes in vivo, such as allograft rejection and autoimmunity. However, selective blockade of CD28 may represent a better strategy for immunosuppression than B7 blockade, because CTLA-4/B7 interactions have been shown to participate in the extinction of the T-cell receptor-mediated activation signal and to be required for the induction of immunologic tolerance. In addition, selective CD28 inhibition specifically decreases the activation of alloreactive and autoreactive T cells, but not the activation of T cells stimulated by exogenous antigens presented in the context of self major histocompatibility complex (MHC) molecules. CD28 blockade cannot be obtained with anti-CD28 dimeric antibodies, which cluster their target and promote T-cell costimulation, whereas monovalent Fab fragments can block CD28 and reduce alloreactivity. In this study, we report the construction of a monovalent single-chain Fv antibody fragment from a high-affinity antihuman CD28 antibody (CD28.3) that blocked adhesion of T cells to cells expressing the CD28 receptor CD80. Genetic fusion with the long-lived serum protein alpha1-antitrypsin led to an extended half-life without altering its binding characteristics. The anti-CD28 fusion molecule showed biologic activity as an immuno-suppressant by inhibiting T-cell activation and proliferation in a mixed lymphocyte reaction.