Blocking the CD28 or CD40 costimulatory pathways using soluble ligands to CD80/CD86 or antibodies to CD154 suppresses the development of T cell dependent but not T-independent humoral immunity. Therefore, it was not surprising that we found that anti-CD137 mAbs also suppressed T-dependent but not T cell-independent B cell responses. What was surprising was that suppression was not mediated by blocking receptor-ligand binding but appeared to require CD137-mediated signaling.
Classic T-dependent antigens include allogeneic cells and soluble proteins foreign to the host. In our studies we used sheep red blood cells (SRBC), human IgG (hulgG), or Keyhole Limpet hemocyanin (KLH) to immunize mice. We also employed a commonly used T-independent antigen, Ficoll-TNP. Mice injected with anti-CD137 mAbs were unable to generate CD4 T cell-dependent humoral immune responses to each of these three T-dependent antigens, and we found that as little as 50|g of anti-CD137 mAbs was sufficient to induce suppression. Pharmacokinetic analysis of circulating rat anti-CD137 mAb in mice revealed a serum half-life of 7.5 days, nevertheless, the mice remained non-responsive to further challenge with antigen for a period of 3-6 months (Mittler et al., 1999; Figure 5.3).
Characterization of the kinetics of anti-CD137-mediated suppression of T-dependent humoral immunity showed that the induction of suppression is restricted to an early phase of B cell activation. For example, we found that when mice were injected with anti-CD137 during antigen priming or within several days thereafter they failed to generate antibody responses to the immunizing antigen. However, when anti-CD137 injection was delayed until 10 days after vaccination,
+ Anti-CD137 + ami-CDl37
Figure 5.3. (A) Anti-CD137-mediated suppression of anti-SRBC humoral immunity. BALB/c mice were injected intravenously (I.V.) with 50 |l of a 1% suspension of washed sheep erythrocytes (SRBC) in PBS containing 200 |g of anti-CD137 (filled triangles) or an isotype matched control mAb (filled squares). Seven weeks later the mice were challenged with a second I.V injection of SRBC. The mice were bled on a weekly basis and anti-SRBC serum titers were measured by ELISA. (B) CD137-mediated suppression of anti-huIgG humoral immunity. BALB/c mice were injected with 20 |g of human IgG in 50 |l of PBS containing 200 |g of anti-CD137 mAbs I.V Six weeks later the mice were challenged with a second injection of huIgG. The mice were bled on a weekly basis and serum anti-huIgG titers were measured by ELISA.
no suppression was observed and the mice responded normally to subsequent antigen challenge almost two months later (Figure 5.4).
In later studies employing LCMV-infected mice, we found that anti-CD137 mAbs had to be injected within 48 hr of infection in order to exert maximal immune suppression. When anti-CD137 mAbs were injected into virus-infected mice after 72 hr, injection of anti-CD137 enhanced rather than suppressed anti-viral immunity. Besides defining the time limitation of anti-CD137 induced suppression, the LCMV infection studies resolved a longstanding paradox concerning the in vivo biological effects of anti-CD137 mAbs. We, and others, have found that injection of anti-CD137 mAbs into tumor-bearing and allografted mice induced potent CD8 T cell immunity. Yet, when these antibodies were injected into immunized mice they suppressed development of T-dependent humoral immunity. The reasons for this dichotomy were not known and led us to speculate that CD137-mediated signaling imparted different effects on CD4 and CD8 T cells. However, this notion was abandoned following the LCMV infection experiments in which we found that suppression was not restricted to CD4 T cells but that CD8 T cell mediated
Figure 5.4. Anti-CD137-mediated suppression of Ag-priming. Two groups (n = 5) of BALB/c mice were immunized with SRBC on day 0 as previously described. One group received a 200 |g I.V injection of anti-CD137 the same day (filled squares) and the second group on day 10 post-SRBC priming (filled triangles). Seven weeks later the mice were challenged with a second injection of SRBC and serum anti-SRBC titers measured as before.
immunity was equally suppressed. It seems from these studies that suppression is dependent upon antigen priming and not on MHC restriction. Mice bearing tumors as well as allografted mice, display tumor associated or alloantigens in MHC class I molecules, and T cells are most likely primed to these antigens long before anti-CD137 is administered. In contrast, this situation does not occur in immunized or virus infected mice.
A series of adoptive cell transfer experiments were then carried out in SRBC immunized mice to determine whether anti-CD137 treated T cells were deleting antigen-specific B cells. This proved not to be the case. SRBC-specific B cell function in anti-CD137 treated mice was unimpaired; instead T cell function was. This was shown in experiments in which mice were immunized to SRBC and injected with anti-CD137 mAbs. The mice were later challenged with SRBC and found to be non-responsive. Two weeks later splenic T cells or B cells from these mice were purified and mixed with B cells from naive mice or T cells from naive mice, respectively, and injected into C.B-17 SCID recipients. In addition, a group of SCID mice were reconstituted with B cells and T cells from naive mice. The SCID recipients were then injected with SRBC 4 weeks later and challenged 9 weeks after adoptive cell transfer. Ten weeks after cell transfer the SCID mice were immunized with KLH to measure their capacity to generate T-dependent humoral immunity to a second antigen (Figure 5.5). We then determined whether CD8
SRBC + Anti-CD137
Weeks Prior to Cell Transfer ■ Lymphocyte Donor Mice
Figure 5.5. Anti-CD137 mAbs suppress T cell help. T cells and naïve B cells (inverted triangles) or B cells and naïve T cells (filled triangles) from SRBC immunized, anti-CD137 treated mice, or T and B cells from non-vaccinated naïve BALB/c mice (filled squares) were adoptively transferred into naïve recipient C.B-17 SCID mice. 2 x 107 BALB/c B cells, or 1 x 107 T cells, or a combination of both were adoptively transferred into SCID mice two weeks following SRBC challenge. All recipients were bled on a weekly basis, and at weeks 4 and 9 post-adoptive transfer the mice were challenged with SRBC. Nine weeks post-adoptive transfer the recipients were immunized with KLH to show they could be immunized to a second antigen. Serum anti-SRBC and KLH IgG titers were measured by ELISA.
T cells mediated CD4 T cell suppression or deletion. CD8 T cell-deficient MHC class I deficient mice were immunized with SRBC and injected at the same time with anti-CD137 mAbs. Like wild type mice, in the absence of anti-CD137 SRBC immunized MHC class I deficient mice generated normal primary and secondary anti-SRBC antibody responses. In contrast, anti-SRBC humoral immunity was totally suppressed when the mice were injected with anti-CD137 mAbs during immunization (Mittler et al., 1999). Thus, CD8+ T cells are not required for the induction or maintenance of suppression. Rather, our results suggested that anti-CD137 treatment impaired CD4 T helper function. This could be caused by CD4 T cell death through antibody-dependent cell cytotoxicity (ADCC), AICD, induction of regulatory T cells, interference with T cell-APC costimulation or antigen priming of T cells. We found that rat IgG2A anti-CD137 mAbs do not deplete T cells and do not bindmouse Fc-receptors and cause ADCC. Furthermore, depleting rat anti-CD4 mAbs were found to suppress CD4 T cell-mediated help for a period of 4-5 weeks whereas anti-CD137 mAb treatment lasted 3-6 months. Since both anti-CD4 and anti-CD137 mAbs were of rat origin it is unlikely that they have very different clearance rates in mice. Therefore, we suspect that anti-CD137 mAbs function by inducing long-term anergy, and that CD4 regulatory
Figure 5.6. Anti-CD137 mAbs fail to block type II TI humoral responses. BALB/c mice were injected I.V with 50 |g of TNP-Ficoll in PBS on days 0 and 14. One group of mice was injected I.V. with 200 |gofanti-CD137 (filled triangles) on day 0, and a second group received an equivalent amount of isotype matched control mAb (filled squares). The mice were bled weekly and serum anti-TNP IgG titers were measured by ELISA.
T cells enforce this state of anergy. This subject will be addressed further later on in this chapter.
Although anti-CD137 treatment suppressed the induction of T-dependent humoral immune responses, it failed to suppress T-independent humoral immunity. When mice undergoing immunization to Ficoll-TNP were injected with anti-CD137 mAbs we failed to suppress the development of Type II T-independent humoral immune responses (Mittler et al., 1999; Figure 5.6). This is not altogether surprising since CTLA-4 Ig and anti-CD154 mAbs, reagents that block the CD28 and CD40 signaling pathways, respectively, do not suppress T independent humoral immunity and B cells do not express CD137. Although some APC can express CD137, B cell activation is not dependent upon APC function.
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