Thomas G. Forsthuber

Induction of TH1 and TH2 Immunity in Neonatal Mice

The neonatal period has been thought of as a window in ontogeny, during which the developing immune system is particularly susceptible to tolerization. In the present study, the classic system for induction of neonatal tolerance to protein antigens was reexamined in mice. The presumably tolerogenic protocol was found to trigger a vigorous T helper cell type 2 (TH2) immune response. Thus, neonatal “tolerization” induces immune deviation, not tolerance in the immunological sense. Neonates are not immune privileged but generate TH2 or TH1 responses, depending on the mode of immunization.

The p53-dependent effects of macrophage migration inhibitory factor revealed by gene targeting

Macrophage migration inhibitory factor (MIF) is a mediator of host immunity and functions as a high, upstream activator of cells within the innate and the adaptive immunological systems. Recent studies have suggested a potentially broader role for MIF in growth regulation because of its ability to antagonize p53-mediated gene activation and apoptosis. To better understand MIFs activity in growth control, we generated and characterized a strain of MIF-knockout (MIF-KO) mice in the inbred, C57BL/6 background. Embryonic fibroblasts from MIF-KO mice exhibit p53-dependent growth alterations, increased p53 transcriptional activity, and resistance to ras-mediated transformation. Concurrent deletion of the p53 gene in vivo reversed the observed phenotype of cells deficient in MIF. In vivo studies showed that fibrosarcomas induced by the carcinogen benzo[α]pyrene are smaller in size and have a lower mitotic index in MIF-KO mice relative to their WT counterparts. The data provide direct genetic evidence for a functional link between MIF and the p53 tumor suppressor and indicate an important and previously unappreciated role for MIF in carcinogenesis.

T cell subsets and their signature cytokines in autoimmune and inflammatory diseases

CD4(+) T helper (Th) cells are critical for proper immune cell homeostasis and host defense, but are also major contributors to pathology of autoimmune and inflammatory diseases. Since the discovery of the Th1/Th2 dichotomy, many additional Th subsets were discovered, each with a unique cytokine profile, functional properties, and presumed role in autoimmune tissue pathology. This includes Th1, Th2, Th17, Th22, Th9, and Treg cells which are characterized by specific cytokine profiles. Cytokines produced by these Th subsets play a critical role in immune cell differentiation, effector subset commitment, and in directing the effector response. Cytokines are often categorized into proinflammatory and anti-inflammatory cytokines and linked to Th subsets expressing them. This article reviews the different Th subsets in terms of cytokine profiles, how these cytokines influence and shape the immune response, and their relative roles in promoting pathology in autoimmune and inflammatory diseases. Furthermore, we will discuss whether Th cell pathogenicity can be defined solely based on their cytokine profiles and whether rigid definition of a Th cell subset by its cytokine profile is helpful.

Pertussis Toxin Modulates the Immune Response to Neuroantigens Injected in Incomplete Freund’s Adjuvant: Induction of Th1 Cells and Experimental Autoimmune Encephalomyelitis in the Presence of High Frequencies of Th2 Cells

Pertussis toxin (PT) has been widely used to facilitate the induction of experimental autoimmune encephalomyelitis (EAE) in rodents. It has been suggested that this microbial product promotes EAE by opening up the blood-brain barrier and thereby facilitates the migration of pathogenic T cells to the CNS. However, PT has other biological effects that could contribute to its activity in EAE, such as enhancing the cytokine production by T cells and induction of lymphocytosis. In this work, we investigated the effects of PT on the pathogenicity, cytokine differentiation, and clonal sizes of neuroantigen-reactive T cells in EAE in mice. Our results show that PT prevented the protection from EAE conferred by injection of PLPp139–151 in IFA and induced high frequencies of peptide-specific Th1 cells and disease. Interestingly, the mice developed EAE despite the simultaneous vigorous clonal expansion of PLPp139–151-specific Th2 cells. The data indicate that the Th2 cells in this model neither were protective against EAE nor promoted the disease. Furthermore, the results suggested that the effects of the toxin on neuroantigen-reactive T cells were promoted by the PT-induced activation of APCs in lymphoid tissues and the CNS. Together, the results suggest that microbial products, such as PT, could contribute to the initiation of autoimmune disease by modulating the interaction between the innate and adaptive immune system in the response to self Ags.

In Vivo Blockade of Macrophage Migration Inhibitory Factor Ameliorates Acute Experimental Autoimmune Encephalomyelitis by Impairing the Homing of Encephalitogenic T Cells to the Central Nervous System

Macrophage migration inhibitory factor (MIF) is a cytokine that plays a critical role in the regulation of macrophage effector functions and T cell activation. However, its role in the pathogenesis of T cell-mediated autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), has remained unresolved. In this study, we report that anti-MIF Ab treatment of SJL mice with acute EAE improved the disease severity and accelerated the recovery. Furthermore, the anti-MIF treatment impaired the homing of neuroantigen-reactive pathogenic T cells to the CNS in a VCAM-1-dependent fashion. Interestingly, MIF blockade also decreased the clonal size of the neuroantigen-specific Th1 cells and increased their activation threshold. Taken together, the results demonstrate an important role for MIF in the pathogenesis of EAE/multiple sclerosis and suggest that MIF blockade may be a promising new strategy for the treatment of multiple sclerosis.

T cell epitopes of human myelin oligodendrocyte glycoprotein identified in HLA-DR4 (DRB1*0401) transgenic mice are encephalitogenic and are presented by human B cells.

Myelin oligodendrocyte glycoprotein (MOG) is an Ag present in the myelin sheath of the CNS thought to be targeted by the autoimmune T cell response in multiple sclerosis (MS). In this study, we have for the first time characterized the T cell epitopes of human MOG restricted by HLA-DR4 (DRB1*0401), an MHC class II allele associated with MS in a subpopulation of patients. Using MHC binding algorithms, we have predicted MOG peptide binding to HLA-DR4 (DRB1*0401) and subsequently defined the in vivo T cell reactivity to overlapping MOG peptides by testing HLA-DR4 (DRB1*0401) transgenic mice immunized with recombinant human (rh)MOG. The data indicated that MOG peptide 97–108 (core 99–107, FFRDHSYQE) was the immunodominant HLA-DR4-restricted T cell epitope in vivo. This peptide has a high in vitro binding affinity for HLA-DR4 (DRB1*0401) and upon immunization induced severe experimental autoimmune encephalomyelitis in the HLA-DR4 transgenic mice. Interestingly, the same peptide was presented by human B cells expressing HLA-DR4 (DRB1*0401), suggesting a role for the identified MOG epitopes in the pathogenesis of human MS.

Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift.

A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-II binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase II (EcAII), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAII variant containing eight amino acid substitutions within computationally predicted T-cell epitopes—of which four were nonconservative—while still exhibiting kcat/KM = 106 M-1 s-1 for L-Asn hydrolysis. Further, immunization of HLA-transgenic mice expressing the ALL-associated DRB1*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAII IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAII may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins.

Tumor necrosis factor alpha production from CD8+ T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum Infection

ABSTRACT The immunopathogenesis of Chlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin−/− mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α−/− mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8+ T cells, we evaluated the role of CD8+ T cells during genital Chlamydia muridarum infection and oviduct sequelae. Following vaginal chlamydial challenge, (i) mice deficient in TAP I (and therefore the major histocompatibility complex [MHC] I pathway and CD8+ T cells), (ii) wild-type mice depleted of CD8+ T cells, and (iii) mice genetically deficient in CD8 (CD8−/− mice) all displayed similar levels of vaginal chlamydial clearance but significantly reduced hydrosalpinx, compared to those of wild-type C57BL/6 mice, suggesting a role for CD8+ T cells in chlamydial pathogenesis. Repletion of CD8−/− mice with wild-type or perforin−/−, but not TNF-α−/−, CD8+ T cells at the time of challenge restored hydrosalpinx to levels observed in wild-type C57BL/6 mice, suggesting that TNF-α production from CD8+ T cells is important for pathogenesis. Additionally, repletion of TNF-α−/− mice with TNF-α+/+ CD8+ T cells significantly enhanced the incidence of hydrosalpinx and oviduct dilatation compared to those of TNF-α−/− mice but not to the levels found in wild-type mice, suggesting that TNF-α production from CD8+ T cells and non-CD8+ cells cooperates to induce optimal oviduct pathology following genital chlamydial infection. These results provide compelling new evidence supporting the contribution of CD8+ T cells and TNF-α production to Chlamydia-induced reproductive tract sequelae.

Revisiting Tolerance Induced by Autoantigen in Incomplete Freund’s Adjuvant

Injection of autoantigens in IFA has been one of the most effective ways of preventing experimental, T cell-mediated, autoimmune disease in mice. The mechanism that underlies this protection has, however, remained controversial, with clonal deletion, induction of suppressor cells or of type 2 immunity being implicated at one time or another. Using high resolution enzyme-linked immunospot (ELISPOT) analysis, we have revisited this paradigm. As models of autoimmunity against sequestered and readily accessible autoantigens, we studied experimental allergic encephalomyelitis, induced by myelin oligodendrocyte glycoprotein, proteolipid protein, myelin basic protein, and renal tubular Ag-induced interstitial nephritis. We showed that the injection of each of these Ags in IFA was immunogenic and CD4 memory cells producing IL-2, IL-4, and IL-5, but essentially no IFN-γ. IgG1, but not IgG2a, autoantibodies were produced. The engaged T cells were not classic Th2 cells in that IL-4 and IL-5 were produced by different cells. The IFA-induced violation of self tolerance, including the deposition of specific autoantibodies in the respective target organs, occurred in the absence of detectable pathology. Exhaustion of the pool of naive precursor cells was shown to be one mechanism of the IFA-induced tolerance. In addition, while the IFA-primed T cells acted as suppressor cells, in that they adoptively transferred disease protection, they did not interfere with the emergence of a type 1 T cell response in the adoptive host. Both active and passive tolerance mechanisms, therefore, contribute to autoantigen:IFA-induced protection from autoimmune disease.

T-cell activation and receptor downmodulation precede deletion induced by mucosally administered antigen

The fate of antigen-specific T cells was characterized in myelin basic protein (MBP) T-cell receptor (TCR) transgenic (Tg) mice after oral administration of MBP. Peripheral Th cells are immediately activated in vivo, as indicated by upregulation of CD69 and increased cytokine responses (Th1 and Th2). Concurrently, surface TCR expression diminishes and internal TCR levels increase. When challenged for experimental autoimmune encephalomyelitis during TCR downmodulation, Tg mice are protected from disease. To characterize Th cells at later times after antigen feeding, it was necessary to prevent thymic release of naive Tg cells. Therefore, adult Tg mice were thymectomized before treatment. TCR expression returns in thymectomized Tg mice 3 days after MBP feeding and then ultimately declines in conjunction with MBP-specific proliferation and cytokine responses (Th1-type and Th2-type). The decline correlates with an increase in apoptosis. Collectively, these results demonstrate that a high dose of fed antigen induces early T-cell activation and TCR downmodulation, followed by an intermediate stage of anergy and subsequent deletion.