Chimeric monoclonal anti-CD20 antibody (rituximab)—an effective treatment for a patient with relapsing hairy cell leukaemia

A case story is presented, describing a 46 y old man, with a relapsing hairy cell leukaemia. After treatment with monoclonal anti CD-20 antibodies (rituximab) 375 mg/week, four times, a complete remission was obtained which has lasted >9 months. The rituximab treatment produced a better remission than earlier treatments with alpha-interferon and chlorodeoxyadenosine. In addition, in contrast to other treatments, no initial worsening of the pancytopenia was observed.     

T cell immunity to lymphoma following treatment with anti-CD40 monoclonal antibody

In this study we demonstrate that treatment with anti-CD40 mAb eradicates a range of mouse lymphomas (BCL(1), A31, A20, and EL4), but only when used against i.v. tumor doses in excess of 10(7) cells. Only partial protection was seen against smaller tumor loads. We saw no evidence that anti-CD40 mAb changed the phenotype of the lymphomas or inhibited their growth in the initial period following treatment, but it did result in a rapid expansion of cytotoxic CD8(+) cells that was able to clear the neoplastic disease and provide long-term protection against tumor rechallenge. The CTL responses were blocked by mAb against a range of coreceptors and cytokines, including CD8, B7-1, B7-2, LFA-1, and IFN-gamma, but not CD4 or CTLA-4, indicating the presence of a conventional cellular Th1 response. Furthermore, we found evidence of cross-recognition between lymphomas (BCL(1) and A20) as measured by cytotoxicity and IFN-gamma responses in vitro and using tumor rechallenge experiments, suggesting common target Ags. Finally, although anti-CD40 was shown to stimulate NK cell killing, we could find no role for these cells in controlling tumor growth. These data underline the ability of anti-CD40 mAb to potentiate CTL responses and the potency of cellular immunity in eradicating large quantities of syngeneic tumor.     

Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody

The effect of stimulating normal type 1 murine T cell clones with anti-CD3 antibody was examined in vitro. In the absence of accessory cells, anti-CD3 antibody immobilized on plastic plates stimulated inositol phosphate production, suboptimal proliferation, IL-2 and IL-3 production, and maximal IFN-gamma production. Addition of accessory cells augmented lymphokine production and proliferation when the effects of "high-dose suppression" were relieved by removing the T cells from the antibody-coated plates. Exposure of type 1 T cell clones to immobilized anti-CD3 antibody alone rapidly induced long-lasting proliferative unresponsiveness (anergy) to Ag stimulation that could be prevented by accessory cells. This anergic state was characterized by a lymphokine production defect, not a failure of the T cells to respond to exogenous IL-2 or to express surface Ti/CD3 complexes. In addition, anergy could not be induced in the presence of cyclosporine A. These results suggest that under certain conditions anti-CD3 antibodies may have potent immunosuppressive effects independent of Ti/CD3 modulation. Furthermore, our results support a two-signal model of type 1 T cell activation in which Ti/CD3 occupancy alone (signal 1) induces anergy, whereas Ti/CD3 occupancy in conjunction with a costimulatory signal (signal 2) induces a proliferative response.     

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.     

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO? and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.     

Growth factor production and requirements during the proliferative response of human T lymphocytes to anti-CD3 monoclonal antibody

Anti-CD3 was administered with three different accessory stimuli to purified populations of human T cells. Sepharose conjugated anti-CD3, monocytes, and PMA each could induce the p55 component of the IL-2R as well as responsiveness to exogenous IL-2. Sepharose anti-CD3 did not induce IL-2, although the levels of IL-2 protein and mRNA were 10 to 30 times higher with PMA than with monocytes. Despite these differences in IL-2 production, the amount of DNA synthesis and the number of lymphoblasts were comparable when monocytes or PMA were used as the accessory stimulus, and the responses were equally sensitive to inhibition by an anti-IL-2R antibody. To pursue the functional relevance of the "supraoptimal" levels of IL-2 that are induced by PMA, anti-CD3-induced lymphoblasts were isolated free of monocytes and challenged with lymphokines. It could be shown that 1) the small amounts of IL-2 in the monocyte-T cell conditioned medium would drive DNA synthesis, but that 2) higher levels of IL-2 (20 to 100 U/ml) were needed to induce IFN-gamma, as well as the mRNA for IL-4 and the p55 IL-2R. We suggest that the capacity to produce high levels of IL-2, as seen with PMA, is required under physiologic conditions for two reasons: to up-regulate the IL-2R when small amounts of Ag rather than large amounts of anti-CD3 are ligands for the T cell, or to induce the release of lymphokines like IL-4 and IFN-gamma from sensitized lymphoblasts.     

Increased frequency of islet cell antibodies in unaffected brothers of children with type 1 diabetes

AIM: To assess the relation between islet cell antibody (ICA) positivity and demographic characteristics in an extensive series of first-degree relatives of children with type 1 diabetes (T1D). METHODS: Family members of children diagnosed with T1D before the age of 16 years and attending one of 27 participating paediatric units in Finland taking care of children with diabetes were invited to volunteer for an ICA screening program aimed at identifying individuals eligible for inclusion in the European Nicotinamide Diabetes Intervention Trial (ENDIT). The final series comprised 2,522 first-degree relatives (1,107 males) with a mean age of 20.4 (range 0.1-51.9) years, out of whom 390 were fathers, 622 mothers, 717 brothers, and 793 sisters of affected cases. RESULTS: Two hundred and four family members (8.1%) tested positive for ICA with levels ranging from 3 to 564 (median 18) Juvenile Diabetes Foundation (JDF) units. One hundred and five relatives (4.2%) had an ICA level of 18 JDF units or more. Males had detectable ICA more often than females (9.6 vs. 6.9%; p = 0.02). Antibody-positive family members under the age of 20 years had higher ICA levels than the older ones [median 18 (range 3-514) JDF units vs. 10 (range 3-564) JDF units; p = 0.008]. Among the adult relatives (>or=20 years of age) antibody-positive females had higher ICA levels than the males [median 10 (range 5-564) JDF units vs. 9 (range 3-130) JDF units; p = 0.04]. Siblings had an increased frequency of high-titre ICA (>or=18 JDF units) when compared to the parents (4.8 vs. 3.2%; p = 0.05). Among siblings, we found a higher frequency of ICA positivity in brothers than in sisters (10.8 vs. 6.9%; p = 0.01), and this was also true for high-titre ICA (6.0 vs. 3.8 %; p = 0.04). Geographically, the highest ICA prevalence was seen among relatives living in the middle of Finland (10.4 vs. 7.2% in the other parts of Finland; p = 0.01). CONCLUSIONS: These results imply that male gender and young age favour positive ICA reactivity among family members of children with T1D. Siblings test positive for high ICA titres (>or=18 JDF units) more frequently than parents. Accordingly, judged from demographic characteristics, the yield of ICA screening in first-degree relatives would be maximized by targeting young brothers of affected cases.     

A requirement for nonspecific T cell factors in antibody responses to "T cell independent" antigens

Using murine splenic B cell preparations depleted of macrophages and rigorously depleted of T cells, we studied the role of nonspecific helper factors in in vitro antibody responses to T cell-independent (TI) type 1 and type 2 antigens. TNP-lipopolysaccharide, TNP-Brucella abortus, and DNP-liposomes containing lipid A were chosen as examples of TI type 1 antigens. DNP-Ficoll and DNP-liposomes without lipid A were chosen as TI type 2 antigens. Only the type 1 antigens were able to elicit significant, albeit very weak, responses without added helper factors. Both type 1 and 2 antigens required factors present in supernatants from concanavalin A-stimulated spleen cells (Con A SN) to stimulate optimum antibody responses. Interleukin 2- (IL 2) containing supernatant from the T cell hybridoma FS6-14.13 supported suboptimal responses to varying degrees with each TI antigen, in contrast to its lack of effect on responses to sheep red blood cells in the absence of additional factors. This activity of the FS6-14.13 supernatant was removed by absorption with the IL 2-dependent T cell line HT-2, suggesting that IL 2 was the active component. Another factor, IL-X, which is distinct from both IL 1 and IL 2 and is also found in Con A SN, was required in addition to IL 2 to achieve optimal responses with both types of TI antigens. These results clearly establish a role for factors derived from T cells in the activation of B cells by both type 1 and type 2 TI antigens.     

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.     

Antibody blockade of Thy-1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti-CD3 monoclonal antibody

Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.     

Regulation of influenza virus-specific cytotoxic T cell responses by monoclonal antibody to a human T cell differentiation antigen

The results in this report indicate that the OKT3 monoclonal antibody, which is specific for a human T cell differentiation antigen present on 90 to 95% of peripheral T cells, can exert several effects that regulate the generation and expression of human influenza virus-immune cytotoxic T lymphocytes (CTL). The OKT3 antibody, but not OKT1 or OKT11 (which bind to all peripheral T cells), is able to inhibit anti-influenza CTL effector cell activity. An F(ab')2 preparation of OKT3 IgG were as effective as whole IgG for the inhibition of CTL effectors, indicating that the inhibitory activity of the antibody was not a function of the Fc portion of the molecule. OKT3 IgG and OKT3 F(ab')2 fragments (but not OKT4, OKT8, or OKI were able to inhibit the generation of anti-influenza CTL. The culture of human lymphoid cells with OKT3 in the presence or absence of influenza virus induced radioresistant cells that could suppress the CTL response of fresh autologous lymphocytes to influenza. These results suggest that T cell functions can be regulated by signals that are initiated by the binding of antibody to cell surface molecules that may not be related to the T cell antigen-specific receptor(s).     

Murine anti-CD3 monoclonal antibody induces potent cytolytic activity in both T and NK cell populations

Antibodies specific for the CD3 complex have the capacity to both stimulate and inhibit a variety of T cell functions. We show here that a monoclonal antibody to the epsilon chain of CD3 can induce efficient non-MHC-restricted cytolytic activity in murine lymphocytes with peak activity occurring after 48 hr of incubation. In a panel of targets, the anti-CD3-activated effectors lysed tumor cells but not normal lymphoblasts. Cytolysis was not dependent on the presence of the antibody in the cytolytic assay. Moderate to high cytolytic activity was elicited from lymph nodes, spleen, and thymus by anti-CD3 treatment in vitro, whereas only low activity was apparent in bone marrow. The precursors of anti-CD3-activated cells consisted largely of mature T cells, although a smaller component of immature T cells was also involved. Thus, separation of thymocytes based on adhesion to peanut agglutinin revealed that both positive (immature) and negative (mature) fractions could be activated, while cytotoxic pretreatment of spleen cells with an antibody (J11d) to immature T cells before anti-CD3 activation significantly decreased the resulting cytotoxicity. The majority of precursors in spleen were Thy 1+ and CD8+ and/or AGM1+. Antibody depletion studies showed that the effector cells have both a T and a NK component consisting of Thy 1+, CD5+, CD8+, CD4-, and AGM1- cells and Thy 1-, CD5-, CD8-, CD4-, and AGM1+ cells, respectively. The production of significant amounts of IL-2 and TNF in culture following anti-CD3 treatment, along with the synergistic effect of exogenously added IL-2, suggests that one or both of the effector cell types could be induced by lymphokines. The intraperitoneal administration of the anti-CD3 antibody induces cytolytic activity in vivo. Therefore, the direct activation of cytolysis by anti-CD3 antibody and the additional effects, both direct and synergistic, of lymphokines produced by the activated lymphocytes could conceivably provide a potent anti-tumor therapy.     

Tumor-specific targeting of T helper type 1 (Th1) cells by anti-CD3 × anti-c-ErbB-2 bispecific antibody

T helper type1 (Th1) or type2 (Th2) cells were induced from naive Th cells obtained from ovalbumin-specific T cell receptor (TCR) transgenic mice. Th1 cells producing interferon γ (IFNγ) exhibited stronger antigen-specific cytotoxicity against ovalbumin-(323–339)-peptide-pulsed A20 tumor cells than did Th2 cells. To develop a general method for applying antigen-nonspecific Th1 cells to tumor immunotherapy, we examined the targeting of Th1 cells to tumor cells using a bispecific antibody (bsAb) consisting of anti-(mouse CD3) mAb and anti-(human c-ErbB-2) mAb. When ovalbumin-specific Th1 or Th2 cells were cocultured with c-erbB-2-positive transfectants (CMS7HE), neither type of cell showed significant cytotoxicity or cytokine production in response to tumor cells. However, addition of bsAb resulted in the triggering of both Th1 and Th2 cells. Th1 cells showed higher levels of bsAb-dependent cytotoxicity against CMS7HE tumor cells than did Th2 cells. The targeting of Th1 cells to CMS7HE tumor cells by bsAb also triggered the production of cytokines such as IFNγ, interleukin-2 and tumor necrosis factor α (TNFα). The released TNFα was demonstrated to be a critical cytolytic factor in bsAb-mediated cytotoxicity by Th1 cells. Finally, Th1 cells were demonstrated to show antitumor activity in vivo against human c-erbB-2-positive tumor cells implanted in nude mice. These results suggest that Th1 cells are useful effector cells for the application to adoptive tumor immunotherapy in conjunction with bsAb. Received: 22 April 1999 / Accepted: 2 July 1999     

Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens: evidence from studies of proliferative responses to SB antigens

The T4 molecule has been identified as a marker of human T cell differentiation, but the function of this molecule remains to be defined. We have investigated its possible functional involvement in T cell proliferative responses to class II HLA antigens encoded by the recently described SB locus. The responses of SB-primed cells (specific for each of four different SB antigens) were studied with the use of two proliferation-inducing stimuli, SB antigen or TCGF. The proliferative responses to both stimuli were found to be mediated by T4+, T8- cells. Monoclonal antibodies against some epitopes on the T4 molecule (OKT4A and OKT4B) substantially blocked antigen-stimulated proliferative responses; antibodies against other epitopes of the T4 molecule (OKT4, T4C, T4D) blocked less well. Inhibition of SB-specific proliferation by antibodies to the T4 molecule was maximal only when the antibodies were incubated with the responder cells before the addition of stimulator cells. Proliferative responses of SB-primed cells stimulated with TCGF alone were not inhibited by any of the OKT4-related antibodies, but were completely inhibited by the anti-Tac monoclonal antibody, which reacts with the TCGF receptor. These results lend further support for the hypothesis that the T4 molecule is involved in T cell recognition of and/or activation by class II HLA antigens. We suggest that 1) the T4 molecule binds a nonpolymorphic epitope on class II HLA molecules, and 2) this interaction may facilitate, but not be an obligate requirement for, T cell activation by class II antigens.     

Inverse Ir gene control of the antibody and T cell proliferative responses to human basement membrane collagen

The immune response to pepsin-soluble human basement membrane-derived type IV collagen in mice has been characterized. Both T cell proliferative and antibody responses have been shown to be under major histocompatibility complex (MHC)-linked Ir gene control in inbred and MHC congenic mice. However, unlike previous examples studied, this response shows a separation of these two types of immunologic responsiveness. Only mice having I-As give potent in vitro T cell proliferative responses to type IV collagen whereas all mice except those having I-As give high antibody responses to this antigen. In (I-As X I-Anon-s) F1 mice, the T cell proliferative response was dominant, whereas antibody responses were markedly reduced compared with the responder parent. Given the recent demonstration that class II MHC-restricted, L3T4+ T cells can be divided into two sets, one of which helps for antibody responses and the other of which produces interleukin 2 and can also suppress such responses, it seems likely that these data can be accounted for on the basis of differential activation by this antigen of these two cell sets in mice of different MHC genotypes.     

Persistent T cell anergy in human type 1 diabetes

An anergic phenotype has been observed in nonobese diabetic (NOD) mice and some autoreactive T cells from patients with type I diabetes. To better understand this phenomenon, we measured T cell proliferative responses to 10 diabetes-associated and up to 9 control Ags/peptides in 148 new diabetic children, 51 age- and MHC (DQ)-matched siblings (sibs), 31 patients with longstanding diabetes, and 40 healthy controls. Most (78-91%) patient and sib responses to glutamate decarboxylase of 65 kDa (GAD65), islet cell cytoplasmic autoantibody (ICA) 69, diabetes-associated T cell epitopes in ICA69 (Tep69), and heat shock protein (Hsp) 60 involved anergic T cells that required exogenous IL-2 to proliferate. Responses to proinsulin, IA-2 (and tetanus toxoid) required no IL-2 and generated sufficient cytokine to rescue anergic T cell responses. Most new patients (85%) had autoreactive T cells, three quarters targeting more than half of the diabetes Ags. Only 7.8% of the sibs and none of the controls had such multiple T cell autoreactivities, which thus characterize overt disease. Multiple anergic and nonanergic T cell autoreactivities were sustained during 2 yr follow-up after onset and in patients with longstanding (3-26 yr) diabetes. Activated patient T cells survived severe IL-2 deprivation, requiring 20-100 times less IL-2 than normal T cells to escape apoptosis. Diabetic T cell anergy thus persists for decades and is Ag and host specific but not related to disease course. Rescue by IL-2 from bystander T cells and high resistance to apoptosis may contribute to this persistence. These data explain some of the difficulties in the routine detection of disease-associated T cells, and they emphasize challenges for immunotherapy and islet transplantation.