Elizabeth H. Field

TLR-9 Activation of Marginal Zone B Cells in Lupus Mice Regulates Immunity Through Increased IL-10 Production

Bacterial DNA triggers B-cell proliferation and induces immunoglobulin secretion. Chromatin–IgG complexes activate autoreactive B cells by co-engaging B-cell receptor (BCR) and TLR-9, thus suggesting a role for innate signaling in systemic autoimmunity. Spleen cells from lupus prone Palmerston North (PN) mice produce several fold less IL-12p40 than controls in response to CpG–oligodeoxynucleotides (ODNs). Here we show that B cells are primarily responsible for this abnormality. The removal of B cells from PN cultures markedly increased IL-12p40. Moreover, the addition of purified B cells back to PN splenocyte cultures resulted in a B-cell number dependent/ IL-10-mediated suppression of IL-12p40. The B cells were the major source of IL-10. In response to CpG, B cells from several lupus strains produced twice as a much IL-10 as controls, but failed to produce IL-10 when stimulated through BCR or CD40. PN and control mice expressed IL-10R similarly, and the difference in IL-10 secretion remained when anti-IL-10R blocking antibodies were used. IFN-γ and IL-4 regulated CpG-induced IL-10 secretion in opposite directions. The abnormal IL-10 response in lupus mice was derived from B cells with the marginal zone phenotype, and could be downregulated with inhibitory ODNs. We hypothesize that TLR-9 activated lupus B cells can modulate T-cell mediated inflammatory responses through IL-10 production. Therefore, B cells may contribute to the lupus pathogenesis in many different ways: as antigen-presenting cells for self antigens, as effector cells for autoantibody production, and as IL-10 secreting regulatory cells.

Enhanced type 2 and diminished type 1 cytokines in neonatal tolerance.

We examined the cytokine profiles associated with tolerance and rejection using the mouse model of neonatal tolerance. BALB/c mice primed with CAF1 splenocytes during the neonatal stage showed increased A/J skin graft survival of > 60 days and failed to develop anti-A/J cytotoxic responses, but rejected third-party C57BL/6 grafts. Lymph node cells that drained A/J grafts on neonatal-primed mice produced allospecific immune cytokine responses characterized by high IL-4 and low IFN-gamma levels. In contrast, lymph node cells that drained either rejected third-party grafts or rejected A/J grafts placed on adult controls produced less IL-4 and more IFN-gamma. Tolerogen-specific immune responses from neonatal-primed mice made up to 100 times higher IL-4 to IFN-gamma ratios than did controls. Alloantigen priming during the immediate neonatal stage induced constitutive expression of IL-4 mRNA in the spleen without IFN-gamma mRNA, whereas alloantigen stimulation during adulthood induced the opposite pattern. IL-4 production from neonatal primed mice was confined to the CD4 population. The altered cytokine profile of enhanced IL-4/IFN-gamma in neonatal primed mice persisted for up to 12 weeks after priming in in vitro secondary MLR assays, which suggests that the initial timing of antigen stimulation critically influenced CD4 maturation. The results support a model of immunoredirection as a mechanism of tolerance and provide rationale for examining the therapeutic use of cytokines in transplantation.

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.

Downstream of Tyrosine Kinases-1 and Src Homology 2-Containing Inositol 5′-Phosphatase Are Required for Regulation of CD4 + CD25 + T Cell Development

The adaptor protein, downstream of tyrosine kinases-1 (Dok-1), and the phosphatase SHIP are both tyrosine phosphorylated in response to T cell stimulation. However, a function for these molecules in T cell development has not been defined. To clarify the role of Dok-1 and SHIP in T cell development in vivo, we compared the T cell phenotype of wild-type, Dok-1 knockout (KO), SHIP KO, and Dok-1/SHIP double-knockout (DKO) mice. Dok-1/SHIP DKO mice were runted and had a shorter life span compared with either Dok-1 KO or SHIP KO mice. Thymocyte numbers from Dok-1/SHIP DKO mice were reduced by 90%. Surface expression of both CD25 and CD69 was elevated on freshly isolated splenic CD4+ T cells from SHIP KO and Dok-1/SHIP DKO, suggesting these cells were constitutively activated. However, these T cells did not proliferate or produce IL-2 after stimulation. Interestingly, the CD4+ T cells from SHIP KO and Dok-1/SHIP DKO mice produced higher levels of TGF-β, expressed Foxp3, and inhibited IL-2 production by CD3-stimulated CD4+CD25− T cells in vitro. These findings suggest Dok-1 and SHIP function in pathways that influence regulatory T cell development.

Alloantigen priming after total lymphoid irradiation alters alloimmune cytokine responses.

Total lymphoid irradiation (TLI) treatment represents a model of acquired tolerance, as TLI-treated mice develop donor-specific tolerance when exposed to alloantigens shortly after completing TLI. To determine whether immunoredirection plays a role in immunologic tolerance in TLI, we examined whether antigen priming in the immediate post-TLI stage altered the cytokine profile toward Th2. Compared with splenocytes isolated from control primed mice, the splenocytes from TLI-primed mice failed to proliferate to immunogen in mixed leukocyte reaction cultures but proliferated normally to third-party alloantigen. Whole spleen and purified CD4 cells isolated from TLI-primed mice produced more interleukin (IL)-4 and less gamma-interferon (IFN-gamma) in response to immunogen-bearing stimulator cells than controls, resulting in higher IL-4 to IFN-gamma ratios. The CD4 subset from TLI-primed mice contained more IL-4-producing and fewer IFN-gamma-producing cells, suggesting that priming after TLI shifted CD4 maturation toward Th2 cells. Surprisingly, TLI-primed mice contained no immunogen-responsive, IFN-gamma-producing CD8 cells, indicating that priming after TLI abrogated development of this CD8 subset. In summary, the data show that priming in the immediate post-TLI phase shifts the allospecific memory cytokine pattern toward Th2 cytokines by enhancing IL-4-producing CD4 cells and preventing maturation of IFN-gamma-producing CD8 cells. We speculate that the cytokine milieu at the time of antigen priming drives differentiation of the tolerogen-specific immune response toward Th2 cells, because splenocytes isolated immediately after TLI produced high levels of IL-4 and little IL-2. The enhanced Th2 pattern that developed in the TLI-primed mice suggests that immunoredirection may also occur in the TLI model of tolerance.

Maternal-fetal HLA sharing and preeclampsia: variation in effects by seminal fluid exposure in a case-control study of nulliparous women in Iowa.

Whereas histocompatibility is critical for transplantation, HLA histoincompatibility is associated with successful pregnancy. Literature on HLA sharing and preeclampsia has been inconsistent; most studies focused on maternal-paternal rather than maternal-fetal sharing. This study examines whether maternal-fetal histocompatibility is associated with preeclampsia, and whether effects vary by semen exposure history. This case-control study of nulliparous women was designed to examine associations among HLA sharing, semen exposure, and preeclampsia. 258 preeclampsia cases and 182 normotensive controls met the eligibility criteria. HLA typing for mother and baby was performed for HLA-A, -B, -C, -DRB1, and -DQB1. We further restricted our study sample to 224 mother-baby pairs who had complete HLA typing for all five genes. Seminal fluid exposure indexes incorporated information on type of practice, frequency, contraceptive use (for vaginal exposure) and ingestion practices (for oral exposure). Multivariate models were adjusted for BMI and education. HLA-A matching, Class I matching, and combined Class I and II matching were associated with increased odds of preeclampsia. Among women with low semen exposure, effects of Class I matching were amplified (HLA-A matching, OR=6.27, 95%CI=1.04, 37.97; Class I matching, OR=4.49 per one-match increase, 95%CI=1.89, 14.50). With moderate to high semen exposure, Class II matching effects predominated (HLA-DQB1, OR=3.22, 95%CI=1.04, 9.99; Class II, OR=1.76 per one-match increase, 95%CI=1.05, 2.98; and total matches, OR=1.45 per one-match increase, 95%CI=1.02, 2.06). We found consistent evidence that maternal-fetal HLA sharing was associated with preeclampsia in a pattern influenced by prior vaginal exposure to paternal seminal fluid.

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4+CD25+Foxp3+ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4+ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.