Botanical drug clinical trial: Common issues and future options.

In order to understand this disparity between human use and drugs approved by regulatory agencies, we analyzed botanical drug clinical trials registered at ClinicalTrial.gov to detect trends in current trials and guide future trials. A total of 195 botanical drug clinical trials were registered from 2016 to 2019, of which 81 are phase II or phase II/III. 95% of all phase II and II/III studies were designed with 100 or less participants per arm, indicating a more observational nature due to the limited power to detect differences in outcomes between treatment and control groups. Due to the limited number of participants, efficacy outcome from results may be highly subjective. 14% of the total trials were phase I studies. For botanical drugs with well-documented or extensive history of human use, phase I may not provide significant additional information, and may, therefore, not be necessary. For the trial design, we suggest added-on studies when botanical drugs are used as part of a combination treatment. Additionally, we believe standardized data collection methods and criteria are critical to utilizing the vast collection of human experience as quality evidence to support regulatory approval.

The Efficacy of Traditional Chinese Medicine Shoutai Pill Combined with Western Medicine in the First Trimester of Pregnancy in Women with Unexplained Recurrent Spontaneous Abortion: A Systematic Review and Meta-Analysis.

BACKGROUND: Shoutai Pill (STP), a famous classic herbal formula documented in traditional Chinese medicine (TCM), is widely available in China for treating unexplained recurrent spontaneous abortion (URSA). This systematic review and meta-analysis aims at evaluating the efficacy and safety of STP in the first trimester of pregnancy in women with a history of unexplained recurrent spontaneous abortion. METHODS: The following eight databases were searched from their establishment to Dec 31, 2019, for randomized controlled trials (RCTs): PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure (CNKI), the Chinese BioMedical database (CBM), Chinese Scientific Journal Database (VIP), and the Wanfang database. The quality of evidence was estimated by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE). RESULTS: A total of 12 studies (916 patients) with URSA were contained in this meta-analysis. The forest plot showed that patients treated with Shoutai Pill and western medicine had a significantly lower incidence of early pregnancy loss (RR: 0.42; 95% CI: 0.34-0.52; P < 0.01, I 2 = 0%). Subgroup analysis revealed that different types of TCM syndrome differentiation had the similar results. Also, in the combined group, patients had a lower TCM syndromes and symptoms and serum D-dimer level, while higher incidence of live birth. CONCLUSIONS: Our findings suggest that cotreatment with STP and western medicine might be superior to western medicine alone in the first trimester of pregnancy to prevent miscarriage in women with unexplained recurrent spontaneous abortion, and there was no adverse event in the experimental group reported. However, the methodological quality of included RCTs was unsatisfactory; it is necessary to verify its effectiveness with further more standardized researches of rigorous design.

Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-ß1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

Glutamic acid decarboxylase-derived epitopes with specific domains expand CD4(+)CD25(+) regulatory T cells.

BACKGROUND: CD4(+)CD25(+) regulatory T cell (Treg)-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used. METHODOLOGY/PRINCIPAL FINDINGS: Splenic lymphocytes from nonobese diabetic (NOD) mice were stimulated with different glutamic acid decarboxylase (GAD)-derived epitopes for 7-10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524-538)-expanded CD4(+)CD25(+) T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509-528)- or p530 (GAD530-543)-expanded CD4(+)CD25(+) T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes) between the above-mentioned epitopes and MHC class II I-A(g7) were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-A(g7), while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-A(g7). Furthermore, p524 bound to I-A(g7) more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570-585, p570), which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4(+)CD25(+) T cells suppressed the onset of diabetes in NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of protective epitopes to expand functional Tregs.

Natural killer cells modulate overt autoimmunity to homeostasis in nonobese diabetic mice after anti-CD3 F(ab')2 antibody treatment through secreting transforming growth factor-beta.

Recently, the anti-CD3 antibody has been shown to be a promising candidate for the efficient treatment of overt autoimmunity. However, the mechanisms underlying this effect remain unclear. Our previous studies demonstrated that natural killer (NK)T cells and transforming growth factor (TGF)-beta were key elements in anti-CD3 F(ab')(2)-mediated re-establishment of glucose homeostasis and restoration of self tolerance to islets in type 1 diabetes. In this report, we further investigate the regulatory pathways involved, especially the cellular source of TGF-beta production. The treatment of new-onset nonobese diabetic mice with anti-CD3 F(ab')(2) resulted in a significant increase in the numbers of NK cells in spleen and pancreatic lymph nodes that secrete TGF-beta. Depletion of this cell population with a specific anti-AsGM1 antibody abrogated anti-CD3 F(ab')(2) therapeutic effects and splenic TGF-beta production. When fractionated from recovered mice after CD3 antibody therapy, these NK cells actively suppressed diabetogenic cell proliferation and prevented the cotransfer of diabetes into nonobese diabetic-severe combined immunodeficient mice in a TGF-beta-dependent manner. In addition, the regulatory NKT cells from remitting mice were capable of causing NK cells to exhibit a TGF-beta-producing phenotype by the secretion of the T helper 2 cytokines interleukins 4 and 10. Overall, these data indicate that NK cells are the main source of TGF-beta production after anti-CD3 F(ab')(2) treatment, which are controlled by a population of T helper 2-like NKT cells.

CD8+ regulatory T cells are responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.

Our previous studies demonstrated that lipopolysaccharide (LPS)-stimulated splenocytes, retrovirally transduced with a glutamate decarboxylate 65 (GAD) and immunoglobulin G (IgG) fusion construct, can protect non-obese diabetic (NOD) mice from diabetes by inducing GAD-specific tolerance, and also that there are increased numbers of CD4(+) regulatory T cells (Tregs) in GAD-IgG-treated NOD mice. However, little is known about the role of CD8(+) Tregs in GAD-IgG gene-transferred tolerance induction in NOD mice. Here, we found that GAD-IgG-transduced splenocytes induced an increase in the number of CD8(+) Foxp3(+) Tregs in vitro. Using a T-cell depletion assay, we found that, compared with undepleted groups, NOD recipients transfused with CD8(-) or CD8(-) CD25(-) GAD-IgG-transduced splenocytes showed a decrease in the percentage of CD8(+) Foxp3(+) T cells, a high incidence of diabetes, serious insulitis, GAD-specific hyperresponsiveness at both the cellular and humoral levels, and changes in cytokine expression. These results indicate that CD8(+) Tregs, which were induced in vitro by GAD-IgG-transduced splenocytes, were also responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.

Induction of active tolerance and involvement of CD1d-restricted natural killer T cells in anti-CD3 F(ab')2 treatment-reversed new-onset diabetes in nonobese diabetic mice.

The application of anti-CD3 F(ab')(2) monoclonal antibodies has recently been expanded to treat established autoimmune diseases, including type 1 diabetes. However, the mechanism underlying their effect remains largely unclear. We report that short-phase administration of anti-CD3 F(ab')(2) antibodies efficiently allowed 80% of new-onset, nonobese diabetic (NOD) mice to significantly regain both normoglycemia and pancreatic beta cell-specific autoantigen (ie, glutamic acid decarboxylase and insulin) tolerance, with both effects lasting more than 40 weeks. The responsible mechanism appears to involve the induction and maintenance of a population of immunoregulatory CD1d-restricted natural killer T (NKT) cells, which were marked by an enhanced Th2 response and secretion of elevated levels of interleukin-10. In vivo neutralization of interleukin-4 and/or interleukin-10 bioactivity abrogated this anti-CD3-mediated effect. Importantly, when the cotransfer of NKT cells from the livers of anti-CD3-treated mice and splenocytes from untreated, acutely diabetic NOD mice was performed in NOD-severe combined immunodeficient mice, the NKT cells were sufficient to either delay or prevent the onset of diabetes compared with controls where only splenocytes were introduced. These data suggest that CD1d-restricted NKT cells may play a critical role in anti-CD3 antibody-induced diabetes remission and the restoration of immune tolerance.

GAD-IgG-inducing CD4+Foxp3+Treg cells suppressing diabetes are involved in the increasing ratio of CD80+:CD86+ CELLS in NOD mice.

BACKGROUND: Our previous studies have demonstrated that GAD-IgG-transduced splenocytes protect non-obese diabetic (NOD) mice from diabetes by in vitro-inducing CD4(+)Foxp3(+)Treg cells. However, the underlying mechanisms by which CD4(+)Foxp3(+)Treg cells suppress diabetes remain unclear. METHODS: Seven-week-old female NOD mice were intravenously injected with GAD-IgG-transduced splenocytes. The ratio of CD80(+):CD86(+) cells in splenocytes was analyzed by flow cytometry. The effect of the ratio of CD80(+):CD86(+) cells on tolerance, diabetes prevention, and Foxp3 expression in GAD-IgG-treated NOD mice was tested by in vitro proliferation, in vivo antibody block, and semi-quantified RT-PCR, respectively. RESULTS: We found that the ratio of CD80(+):CD86(+) cells increased in GAD-IgG-treated NOD mice. After CD4(+)Treg cells were depleted from GAD-IgG-transduced splenocytes before transfer, the ratio of CD80(+):CD86(+) cells decreased in NOD mice recipients. The increasing ratio of CD80(+):CD86(+) cells was positively associated with tolerance, diabetes prevention, and the high level of Foxp3 in GAD-IgG-treated NOD mice. CONCLUSIONS: These findings suggest that the high ratio of CD80(+):CD86(+) cells is required for the suppressive function of GAD-IgG-inducing CD4(+)Foxp3(+)Treg cells in NOD mice.

Gene delivery GAD 500 autoantigen by AAV serotype 1 prevented diabetes in NOD mice: transduction efficiency do not play important roles.

We previously found that adeno-associated viral vector serotype 2 (AAV-2) muscle gene delivery of GAD 500-585 autoantigen efficiently prevented autoimmune diabetes in NOD mice. Recent reports suggest that AAV vectors based on serotype 1 (AAV-1) transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2 to 1000-fold. To determine whether this increased efficacy of AAV-1 could result in increased therapeutic effects in mice, we constructed rAAV1/GAD 500-585 vectors and compared their effects in preventing autoimmune diabetes in NOD mice with those of rAAV2/GAD 500-585 after muscle injection. rAAV(1)/GAD(500-585) gene therapy prevented diabetes in NOD mice. However, although much higher level of GAD 500-585 expression was found in mice using AAV-1 as gene delivery vector than those using AAV-2, no increased efficiency of AAV-1 vectors were found in their capability to prevent autoimmune diabetes, as higher titers of rAAV1/GAD 500-585 virus (3x10(11)v.g./mouse) were needed to obtain therapeutic effects in NOD mice, a titer not different from that of AAV-2. Protection resulted from rAAV1/GAD 500-585 gene therapy were marked by enhanced Th2 immune response and up-regulated CD4+ Foxp3+T regulatory cells, which might actively suppress effector T cells in NOD mice. As here we found that the therapeutic effects of AAV1 were not positively correlated to it's transduction efficiency, our data suggested that the safety and other factors besides efficiency should be considered when use different AAV serotype to treat autoimmune disease.

Essential roles of TGF-beta in anti-CD3 antibody therapy: reversal of diabetes in nonobese diabetic mice independent of Foxp3+CD4+ regulatory T cells.

Anti-CD3 mAb have potentials to treat overt autoimmunity as reported recently. However, the underlying mechanisms remain unclear. In this report, using an animal model of type 1 diabetes, we found that TGF-beta1, an important immunoregulatory cytokine, plays a critical role in anti-CD3-mediated diabetes reversion and immune tolerance. Anti-CD3 treatment increased the TGF-beta1 production, lasting for a long period of time, which contributed to maintaining peripheral tolerance by controlling pathogenic cells. Furthermore, we found that anti-CD3 treatment did not increase the forkhead box p3+ (Foxp3+)CD4+ regulatory T cells (Tregs). When fractionated from anti-CD3-treated, remitting mice and cotransferred with splenic cells from diabetic NOD mice, these Tregs failed to inhibit diabetes development in NOD.scid mice. Moreover, we found that the depletion of these Tregs did not affect an anti-CD3-mediated, therapeutic effect and the level of TGF-beta1 production, which suggested that an increased level of TGF-beta1 may not derive from these Tregs. Thus, our data showed a dispensable role of Foxp3+CD4+ Tregs in anti-CD3 antibody-reversed diabetes in NOD mice. These findings may have an important implication for understanding the involved mechanisms responsible for immunomodulatory function of anti-CD3 antibody on autoimmune diseases.

The role of STAT3 in antigen-IgG inducing regulatory CD4(+)Foxp3(+)T cells.

Unraveling the events that control the suppressive function of regulatory T (Treg) cells is extremely important because it will enable investigators to manipulate these cells to inhibit or enhance their functions as necessary. One of the members of the Signal Transducer and Activators of Transcription (STATs) family, STAT3, has emerged as a negative regulator of inflammatory responses. Here, we study the role of STAT3 in Treg cell induction. We found that GAD-IgG-transduced splenocytes induce a CD4(+)Foxp3(+)Treg cell increase in NOD mice. In parallel with the Treg cell increase, an IL-6-STAT3 signal pathway is activated. When STAT3 activation is blocked, GAD-specific tolerance disappears, the percentage of Treg cells decreases and IL-10 secretion is reduced in the splenocytes of NOD mice recipients of GAD-IgG-transduced splenocytes. Our findings indicate that transcription factor STAT3 plays an important role in immune tolerance.

The combination of GM-CSF and IL-2 as local adjuvant shows synergy in enhancing peptide vaccines and provides long term tumor protection.

Many strategies have been used to enhance the peptide vaccine immune response and to establish therapeutic benefits. This includes the utilization of cytokines to improve antigen presentation or enhance T cell response. Here, we have tested the combination of GM-CSF and IL-2 as locally administered adjuvant to enhance the immune response to the HPV16 E7 peptide. Female C57BL/6 mice were immunized intradermally with a 9-mer HPV16 E7 peptide (aa: 49-57) alone, or in combination with GM-CSF, IL-2, or both cytokines. Specific immune responses were measured by ELISA and Chromium-Release Assays. Furthermore, therapeutic effects of these vaccines and long term tumor protection were assessed in mice bearing established tumors. We showed that GM-CSF and IL-2, when co-administered locally in an emulsion with peptide, exert a synergistic effect in enhancing the immune response to the antigen. This combination induced higher CTL and cytokine release responses and did not increase the T(reg) population. Therapeutic intervention with this synergistic combination led to a complete response of established tumors. Furthermore, this combination induced a memory response which protected mice against subsequent additional tumor challenge. We identified a new vaccine adjuvant, a local combination of GM-CSF and IL-2, which is synergistic in enhancing peptide specific immune response through local effect without increasing T(reg) cells. This immune response was found to be long lasting and protective in tumor bearing mice.

Foxp3-expressing CD4(+)T cells under the control of INF-gamma promoter prevent diabetes in NOD mice.

Foxp3-transduced CD4(+)T-cells have been used for treating autoimmune diseases such as type I diabetes. However, while suppressing the activity of pathogenic T cells, they could suppress the activity of bystander T cells as well. Therefore more specific strategies need to be developed. We designed and tested a new strategy that involves converting pathogenic CD4(+)Th1 cells into regulatory T-cells by lentiviral transduction with Foxp3 under the control of interferon-gamma (IFN-gamma) promoter (IgammaP-Foxp3). After transduction under the IgammaP control, Foxp3 expression in diabetic CD4(+)Th1 cells was favored. IgammaP-Foxp3-transduced CD4(+)T cells were anergic in vitro to stimulation by antigen. The process of IgammaP-Foxp3-transduced CD4(+)T cells differentiating into Treg cells and Treg cells losing their phenotype and functions has the effect of significantly suppressing incidence and onset of diabetes and autoantigen-specific T cell response, while increasing/maintaining endogenous Tregs in nonobese diabetic (NOD) mice recipients. In this manner, CD4(+)T cells of greater specificity were developed by transducing pathogenic CD4(+)Th1 cells with Foxp3 under the control of IgammaP, in order to prevent diabetes in NOD mice. The findings of this study provide a basis for more reasonable regulatory T cells (Tregs)-based therapy, with autoimmunity being suppressed through indirect means known as "infectious tolerance".

Combined prophylactic and therapeutic cancer vaccine: enhancing CTL responses to HPV16 E2 using a chimeric VLP in HLA-A2 mice.

We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8(+) T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and anti-CD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8(+) T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogenous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c(+) and CD11c(+) CD40(+) double positive dendritic cells in mice that received immune-modulators, GM-CSF and anti-CD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.

Identification of H-2Db-specific CD8+ T-cell epitopes from mouse VEGFR2 that can inhibit angiogenesis and tumor growth.

Vascular endothelial growth factor receptor 2 (VEGFR2/KDR) plays a crucial role in tumor-associated angiogenesis and vascularization. It has been established that monoclonal antibodies against VEGFR2 can inhibit angiogenesis. In this study, two naturally processed CD8 T-cell epitopes (VILTNPISM and FSNSTNDILI) were identified from murine KDR. Cytotoxic T lymphocytes targeting endothelial cells could be directly monitored by KDR2 and KDR3 Elispots or major histocompatibility complex class I tetramer staining. Immunization with these two peptides effectively reduced angiogenesis and inhibited tumor growth in mouse models. Thus, vaccination with KDR peptides alone or in combination with other anti-angiogenesis agents may afford a novel immunotherapy for inhibition of tumor growth.

Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD(500-585).

Tolerance induction of autoreactive T cells against pancreatic beta cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD(500-585) could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD(500-585) (3 x 10(11) IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD(500-585)-responsive T lymphocytes, the enhanced GAD(500-585)-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-gamma production; especially elevated anti-GAD(500-585) IgG1 titer; and relatively unchanged anti-GAD(500-585) IgG2b titer), the increased secretion of TGF-beta, and the production of protective regulatory cells. Our studies also revealed that peptides 509-528, 570-585, and 554-546 in the region of GAD(500-585) played important roles in rAAV/GAD(500-585) immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD(500-585), can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.

Immunodominant T-cell epitopes in the factor VIII C2 domain are located within an inhibitory antibody binding site.

Formation of inhibitor antibodies to factor VIII (FVIII) is a major complication of FVIII replacement therapy for hemophilia A patients, and it occurs through a T-cell dependent process. The C2 domain of FVIII contains epitopes that are recognized by antibody inhibitors. We have examined regions of the C2 domain that form epitopes for T cells in mice congenitally deficient in FVIII. We obtained CD4(+)T cells from mice immunized by intravenous infusion of therapeutic doses of recombinant human FVIII (rFVIII), or by subcutaneous injections of rFVIII or recombinant human C2 domain in adjuvant. In all cases, the T cells recognized most strongly and consistently two overlapping peptides that spanned residues 2191 to 2220 of the C2 domain. Analysis of the crystal structure of human factor VIII C2 bound to a human monoclonal antibody, BO2C11, showed these residues also constitute part of a human alloimmune B-cell epitope (Spiegel et al., Blood 2001; 98: 13-19). This region includes one of the "hydrophobic spike" protrusions, consisting of M2199 and F2200, as well as the basic residues R2215 and R2220. These residues contribute to membrane binding and to association with von Willebrand factor (vWF). These findings suggest that a major T-cell epitope in the C2 domain recognized by hemophilic mice is located within the same region that binds to inhibitors, vWF, and activated membranes.

Gene transfer of Ig-fusion proteins into B cells prevents and treats autoimmune diseases.

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PLxSJL F(1) mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7-10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9-23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.