Qinglin Gao

Prevention of Th1 response is critical for tolerance.

We investigated the role of Th1 ad Th2 cytokines in rejection and tolerance using the neonatal tolerance model. We reported previously that lymph nodes that drained immunogen-bearing tolerant grafts produced a 10- to 100-fold higher ratio of interleukin (IL)-4 to interferon (IFN)-gamma compared with lymph node cells from rejected grafts. Moreover, because neonatal antigen exposure triggers allospecific Th2 CD4 memory cells, whereas antigen exposure during adulthood triggers Th1 CD4 memory cells, we speculated that immunoredirection toward Th2 and away from Th1 functions as another mechanism of tolerance. To test the immunoredirection hypothesis, we examined whether recovery of Th1 cytokine responses abrogates tolerance. We now show that treatment with exogenous IFN-gamma at the time of neonatal priming recovered mixed lymphocyte reaction hypoproliferation and restored the ability of mice to reject skin grafts. Mice that received IFN-gamma at the time of neonatal priming produced more IFN-gamma and contained more A/J-reactive IFN-gamma producing CD4 cells compared with untreated neonatal primed mice, but failed to recover A/J-specific INF-gamma-producing CD8 cells or CTL responses, which suggests that graft rejection occurred via Th1 CD4 cells. Interestingly, draining lymph node cells from rejected grafts in IFN-gamma-treated neonatal primed mice also produced more IL-4, compared with cells from healthy grafts on untreated neonatal primed mice. Nonetheless, lower IL-4 to IFN-gamma ratio predicted graft rejection and higher ratios predicted acceptance. We conclude that neonatal tolerance depends on the ability to block generation of allospecific Th1 responses that lead to rejection. Thus, immunoredirection involves both the inhibition of Th1 and expansion of Th2 immune responses.

Balancing the immune system for tolerance: a case for regulatory CD4 cells.

In the past, tolerance mechanisms have focused on processes that involve elimination (deletion) or paralysis (anergy) of immune responses. It is now becoming clearer that peripheral tolerance to antigen depends on the generation of regulatory cells that function to maintain the tolerant state. The development of peripheral tolerance may require that the immune system utilize several strategies, including deletion, anergy, and immunoregulatory pathways, and these strategies may overlap. Recent investigations using animal models of transplantation tolerance have demonstrated that immunoregulatory CD4 mechanisms may play a central role in limiting organ-destructive immune responses. In this Overview, we discuss the rationale behind the need for invoking active regulatory mechanisms in peripheral immunologic tolerance and summarize the data that support or refute a CD4 regulatory mechanism.

The role of interleukin-4 in the induction phase of allogeneic neonatal tolerance

We previously reported that prolonged graft survival in neonatally tolerant mice was associated with enhanced Th2/Th1 cytokines. To determine whether Th2 CD4 cells function in tolerance, we examined whether we could prevent tolerance by blocking Th2 CD4 maturation, using anti-interleukin (IL)-4 monoclonal antibody treatment during neonatal antigen exposure. Anti-IL-4 treatment restored the ability BALB/c of mice to reject A/J skin grafts and blocked the induction of tolerance through multiple mechanisms. Anti-IL-4 treatment blocked the development of donor microchimerism and recovered the ability of mice to proliferate and to generate appropriate delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses against A/J in a dose-dependent manner. Low-dose anti-IL-4 recovered DTH responses and interferon (IFN)-gamma production, but failed to completely prevent IL-4 production or to recover the CTL activity. No A/J-reactive IFN-gamma-producing CD8 cells were detected in these mice. In contrast, mice treated with higher doses of anti-IL-4 generated normal CTL responses against A/J, and contained A/J-reactive IFN-gamma-producing CD8 cells. The recovery of CTL responses and IFN-gamma-producing CD8 cells was associated with a more complete blocking of Th2 cytokine production. Therefore, the presence of IL-4 may play an important role in the induction of neonatal tolerance by shifting maturation of CD4 cells toward Th2 cells and away from Th1 cells, and also by preventing maturation of alloreactive CD8 CTL cells.

CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice

BACKGROUND Injection of neonatal BALB/c mice with semi-allogeneic splenocytes leads to antigen-specific tolerance lasting into adulthood. Tolerant mice accept A/J skin grafts and fail to generate CD8 cytotoxic T lymphocyte (CTL) activity against A/J targets. Anergic CD8 T cells are present in tolerant mice, and CD4 regulatory cells function to maintain CD8 cell anergy. METHODS Neonatal BALB/c mice were injected with 108 live CAF, splenocytes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD8 cell proliferation was measured by in vitro incorporation of bromodeoxyuridine coupled with fluorescence-activated cell sorter analysis. Alloantigen-specific cytotoxicity was tested using 51Cr release assays of A/J or third-party targets. RESULTS We demonstrate that A/J-specific anergic CD8 cells are present in neonatal primed mice that develop tolerance but not in neonatal primed mice that reject A/J skin grafts. Anergic CD8 cells show decreased proliferation and no CTL activity against A/J targets. Addition of interleukin-2 (IL-2) to unfractionated cultures fails to restore CTL activity against A/J targets. However, addition of IL-2 to CD4-depleted cultures restores A/J-specific CD8 CTL activity. Removal of CD4+/CD25+ cells, but not CD4+/CD25- cells, also restores CD8 CTL activity against A/J in the presence, but not the absence, of IL-2. Moreover, when added back into cultures, purified CD4+/CD25+ cells from tolerant mice inhibit the generation of CD8 CTL against A/J targets. CONCLUSION These data indicate that CD8 anergy is associated with the state of tolerance, and that CD4+CD25+ cells from tolerant mice function to maintain A/J-specific CD8 cell anergy in vitro.

Expansion of memory TH2 cells over TH1 cells in neonatal primed mice

BALB/c mice primed with CAF1 splenocytes during the neonatal stage developed A/J-specific tolerance with prolonged survival (> 60 days) of A/J skin grafts. Mice failed to develop A/J-specific cytotoxicity, but rejected third-party skin grafts and generated appropriate third-party cytotoxic T cell responses. We demonstrated previously that graft acceptance was associated with enhanced interleukin (IL)-4 and diminished interferon [IFN]-gamma tolerogen-specific cytokine production, whereas third-party graft rejection was associated with the opposite pattern of cytokine production. We now report that neonatal mice do not mount mixed lymphocyte reaction responses against A/J, but the mice contain a higher percentage of IL-4-producing cells that were characterized as CD4+Mel-14lo cells. Although alloantigen priming of both neonatal and adult control mice expands the CD4+Mel-14lo subset, CD4+Mel-14lo cells from neonatal primed mice produce significantly higher levels of IL-4 and IL-10 and lower IFN-gamma, whereas CD4+Mel-14lo cells from adult primed mice produce mainly IFN-gamma. Moreover, enzyme-linked spot immunosorption analysis demonstrates that, compared with adult primed mice, neonatal primed mice contain more IL-4-producing CD4 cells and less IFN-gamma-producing cells, which indicates that neonatal antigen exposure induces and expands alloreactive Th2 memory CD4 cells. The addition of neutralizing antibodies against IL-4 and IL-10 to primary MLR failed to recover IFN-gamma by CD4+Mel-14lo cells, but cells secreted IFN-gamma after a second in vitro restimulation with tolerogen, which indicates that CD4 cells from neonatal tolerant mice have the capacity to differentiate into Th1 cells. In summary, neonatal tolerant mice contain higher ratios of Th2/Th1 CD4 cells, and the Th2 cytokines function to maintain the ratio by inhibiting Th1 differentiation.

Association between enhanced TH2/TH1 cytokine profile and donor T-cell chimerism following total lymphoid irradiation*

Total lymphoid irradiated (TLI) mice develop antigen specific tolerance if the initial antigen exposure occurs shortly after the completion of TLI. We injected TLI-treated mice with semiallogeneic donor cells at 2, 7, or 28 days after completing TLI and determined the levels of donor CD4 and CD8 cells 5 to 7 weeks after TLI treatment. The level of chimerism correlated with the timing of the initial alloantigen exposure. Donor CD4 and CD8 cells were noted only in day 2 or 7 injected mice. Because donor cell chimerism suggested increased in vivo survival of donor cells, we used the level of donor cell chimerism as a surrogate marker for tolerance to examine the relationship between the development of tolerance and enhanced Th2/Th1 cytokine responses to donor antigen. Increased levels of donor CD4 and CD8 cells in the TLI-treated mice was associated with increased Th2/Th1 cytokine production and decreased CTL activity to donor antigen in vitro. Higher Th2/Th1 cytokine levels also correlated with lower CTL activity. The results indicate that the increased production of Th2/Th1 may function to enhance survival of donor cells in TLI-treated mice and suggest that tolerance induction after TLI treatment involves immunoredirection.

CD4 TH2 cells do not functionally suppress CTL generation in neonatal tolerant mice

Injecting semiallogeneic CAF, spleen into BALB/c newborn mice renders mice tolerant, and the majority of mice show prolonged survival of tolerogen-bearing A/J skin grafts. Moreover, graft survival is associated with enhanced Th2 cytokine responses and graft rejection with Th1 cytokine responses. To further delineate the mechanisms of tolerance, we evaluated CTL responses and found that 74% of neonatal primed mice failed to generate A/J-specific CTL responses, as determined by standard CTL assays and pTc3 frequency analyses. CTL unresponsiveness coexisted with an enhanced tolerogen-specific Th2 memory cytokine profile; spleen cells from neonatal primed mice secreted more interleukin (IL)-4 and less IL-2 and interferon (IFN)–γ in MLR cultures compared with either adult primed or naive controls. We therefore examined the hypothesis that enhanced Th2 cytokine levels prevent the generation of tolerogen-specific CTL. Adding neutralizing antibodies to IL-4 and IL-10 recovered IFN-γ production in vitro but not A/J-specific CTL response. In addition, CD4 cells from neonatal primed mice provided help for primary or secondary CD8 CTL generation, which suggests that the enhanced Th2 cytokine profile does not actively suppress CTL generation. Furthermore, CD4 cells from adult primed mice failed to restore the A/J-specific CD8 CTL generation of neonatal primed mice. The results show that failure to develop A/J-specific CTL reaction occurs without suppression by the enhanced Th2-type responses and imply that either deletion or anergy mechanisms block CTL generation. Therefore, neonatal alloantigen exposure not only shifts the development of alloreactive CD4 cells toward Th2, but also blocks development of alloreactive CD8 CTL in this strain combination.