Markus Mohrs

Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function

Natural killer (NK) and NK T cells are tissue lymphocytes that secrete cytokines rapidly upon stimulation. Here, we show that these cells maintain distinct patterns of constitutive cytokine mRNAs. Unlike conventional T cells, NK T cells activate interleukin (IL)-4 and interferon (IFN)-γ transcription during thymic development and populate the periphery with both cytokine loci previously modified by histone acetylation. Similarly, NK cells transcribe and modify the IFN-γ gene, but not IL-4, during developmental maturation in the bone marrow. Lineage-specific patterns of cytokine transcripts predate infection and suggest evolutionary selection for invariant but distinct types of effector responses among the earliest responding lymphocytes.

Early Transcription and Silencing of Cytokine Genes Underlie Polarization of T Helper Cell Subsets

Naive CD4+ T cells activated through TCR/CD28 under Th1 or Th2 conditions expressed canonical cytokine patterns irrespective of cell division. Only cells that had divided fewer than four times were capable of reexpressing alternative cytokines when restimulated under opposing conditions. Although T cells transcribed both IFN-gamma and IL-4 within hours in a Stat4-/Stat6-independent manner, neither T-bet nor GATA-3 was induced optimally without Stat signals, and polarized cytokine expression was not sustained. Cytokine genes were positioned apart from heterochromatin in resting T cell nuclei, consistent with rapid expression. After polarization, the majority of silenced cytokine alleles were repositioned to heterochromatin. Naive T cells transit through sequential stages of cytokine activation, commitment, silencing, and physical stabilization during polarization into differentiated effector subsets.

Analysis of Type 2 Immunity In Vivo with a Bicistronic IL-4 Reporter

Effector T cells mediate adaptive immunity and immunopathology, but methods for tracking such cells in vivo are limited. We engineered knockin mice expressing IL-4 linked via a viral IRES element with enhanced green fluorescent protein (EGFP). Reporter T cells primed under Th2 conditions showed sensitive and faithful EGFP expression and maintained endogenous IL-4. After Nippostrongylus infection, reporter expression demonstrated the evolution of type 2 immunity from tissue lymphocytes and thence to lymph node CD4(+) T cells, which subsequently migrated into tissue. The appearance of EGFP(+) CD4(+) T cells in tissue, but not in lymph nodes, was Stat6-dependent. Transferred EGFP(+) CD4(+) T cells from infected animals conferred protection against Nippostrongylus to immunodeficient mice. These mice will provide a valuable reagent for assessing immunity in vivo.

T follicular helper cells differentiate from Th2 cells in response to helminth antigens

The relationship of T follicular helper (TFH) cells to other T helper (Th) subsets is controversial. We find that after helminth infection, or immunization with helminth antigens, reactive lymphoid organs of 4get IL-4/GFP reporter mice contain populations of IL-4/GFP-expressing CD4+ T cells that display the TFH markers CXCR5, PD-1, and ICOS. These TFH cells express the canonical TFH markers BCL6 and IL-21, but also GATA3, the master regulator of Th2 cell differentiation. Consistent with a relationship between Th2 and TFH cells, IL-4 protein production, reported by expression of huCD2 in IL-4 dual reporter (4get/KN2) mice, was a robust marker of TFH cells in LNs responding to helminth antigens. Moreover, the majority of huCD2/IL-4–producing Th cells were found within B cell follicles, consistent with their definition as TFH cells. TFH cell development after immunization failed to occur in mice lacking B cells or CD154. The relationship of TFH cells to the Th2 lineage was confirmed when TFH cells were found to develop from CXCR5− PD-1− IL-4/GFP+ CD4+ T cells after their transfer into naive mice and antigen challenge in vivo.

IL-4–producing CD4+ T cells in reactive lymph nodes during helminth infection are T follicular helper cells

Interleukin (IL)-4 is the quintessential T helper type 2 (Th2) cytokine produced by CD4+ T cells in response to helminth infection. IL-4 not only promotes the differentiation of Th2 cells but is also critical for immunoglobulin (Ig) G1 and IgE isotype-switched antibody responses. Despite the IL-4–mediated link between Th2 cells and B lymphocytes, the location of IL-4–producing T cells in the lymph nodes is currently unclear. Using IL-4 dual reporter mice, we examined the Th2 response and IL-4 production in the draining mesenteric lymph nodes during infection with the enteric nematode Heligmosomoides polygyrus. We show that although IL-4–competent Th2 cells are found throughout the B and T cell areas, IL-4–producing Th2 cells are restricted to the B cell follicles and associate with germinal centers. Consistent with their localization, IL-4 producers express high levels of CXCR5, ICOS, PD-1, IL-21, and BCL-6, a phenotype characteristic of T follicular helper (Tfh) cells. Although IL-4 was dispensable for the generation of Th2 and Tfh cells, its deletion resulted in defective B cell expansion and maturation. Our report reveals the compartmentalization of Th2 priming and IL-4 production in the lymph nodes during infection, and identifies Tfh cells as the dominant source of IL-4 in vivo.

Mast Cells, Basophils, and Eosinophils Acquire Constitutive IL-4 and IL-13 Transcripts during Lineage Differentiation That Are Sufficient for Rapid Cytokine Production

Mast cells, basophils, and eosinophils are myeloid cells that are distinguished by their capability to produce IL-4 and IL-13. However, it is not clear how this potential is related to the lineage differentiation of these subsets. In the present study we used bicistronic IL-4 reporter (4get) mice to directly visualize IL-4 expression by nonlymphoid cells in vitro and in vivo at the single-cell level. Our data show that frequent expression of both Il4 alleles is initiated and maintained during ontogeny by an IL-4Rα- or Stat6-independent mechanism. Despite the constitutive presence of cytokine transcripts in differentiated cells under steady state conditions, cytokine production is not detectable in the absence of stimulation. Moreover, mature mast cells, basophils, and eosinophils also constitutively express IL-13. Both preformed IL-4 and IL-13 mRNAs are sufficient for rapid cytokine production upon stimulation. Our data show that mast cells, basophils, and eosinophils are programmed for IL-4 and IL-13 expression early in ontogeny. These novel findings have important implications for the prevention and therapeutic intervention of allergic and asthmatic diseases.

Mouse Vα14i natural killer T cells are resistant to cytokine polarization in vivo

Under different circumstances, natural killer T (NKT) cells can cause a T helper (Th) 1 or a Th2 polarization of immune responses. We show here, however, that mouse NKT cells with an invariant Vα14 rearrangement (Vα14i NKT cells) rapidly produce both IL-4 and IFN-γ, and this pattern could not be altered by methods that polarize naive CD4+ T cells. Surprisingly, although cytokine protein was detected only after activation, resting Vα14i NKT cells contained IL-4 and IFN-γ mRNAs. Despite this finding, in vivo priming of mice with the glycolipid antigen recognized by Vα14i NKT cells resulted in a more Th2-oriented response upon antigen re-exposure. The Vα14i NKT cells from primed mice retain the ability to produce IL-4 and IFN-γ, but they are less effective at activating NK cells to produce IFN-γ. Our data therefore indicate that Vα14i NKT cells have a relatively inflexible immediate cytokine response, but that changes in their ability to induce IFN-γ secretion by NK cells may determine the extent to which they promote Th1 responses.

Alternative activation is an innate response to injury that requires CD4+ T cells to be sustained during chronic infection.

Alternatively activated macrophages (AAMΦ) are found in abundance during chronic Th2 inflammatory responses to metazoan parasites. Important roles for these macrophages are being defined, particularly in the context of Th2-mediated pathology and fibrosis. However, a full understanding of the requirements for alternative activation, particularly at the innate level, is lacking. We present evidence that alternative activation by the Th2 cytokines IL-4 and IL-13 is an innate and rapid response to tissue injury that takes place even in the absence of an infectious agent. This early response does not require CD4+ Th2 cells because it occurred in RAG-deficient mice. However, class II-restricted CD4+ T cell help is essential to maintain AAMΦ in response to infection, because AAMΦ were absent in RAG-deficient and MHC class II-deficient, but not B cell-deficient mice after chronic exposure to the nematode parasite, Brugia malayi. The absence of AAMΦ was associated with increased neutrophilia and reduced eosinophilia, suggesting that AAMΦ are involved in the clearance of neutrophils as well as the recruitment of eosinophils. Consistent with this hypothesis, AAMΦ show enhanced phagocytosis of apoptotic neutrophils, but not latex beads. Our data demonstrate that alternative activation by type 2 cytokines is an innate response to injury that can occur in the absence of an adaptive response. However, analogous to classical activation by microbial pathogens, Th2 cells are required for maintenance and full activation during the ongoing response to metazoan parasites.

Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses

Soluble egg antigens of the parasitic helminth Schistosoma mansoni (S. mansoni egg antigen [SEA]) induce strong Th2 responses both in vitro and in vivo. However, the specific molecules that prime the development of Th2 responses have not been identified. We report that omega-1, a glycoprotein which is secreted from S. mansoni eggs and present in SEA, is capable of conditioning human monocyte-derived dendritic cells in vitro to drive T helper 2 (Th2) polarization with similar characteristics as whole SEA. Furthermore, using IL-4 dual reporter mice, we show that both natural and recombinant omega-1 alone are sufficient to generate Th2 responses in vivo, even in the absence of IL-4R signaling. Finally, omega-1–depleted SEA displays an impaired capacity for Th2 priming in vitro, but not in vivo, suggesting the existence of additional factors within SEA that can compensate for the omega-1–mediated effects. Collectively, we identify omega-1, a single component of SEA, as a potent inducer of Th2 responses.

Nonredundant Roles for B Cell-Derived IL-10 in Immune Counter-Regulation

IL-10 plays a central role in restraining the vigor of inflammatory responses, but the critical cellular sources of this counter-regulatory cytokine remain speculative in many disease models. Using a novel IL-10 transcriptional reporter mouse, we found an unexpected predominance of B cells (including plasma cells) among IL-10-expressing cells in peripheral lymphoid tissues at baseline and during diverse models of in vivo immunological challenge. Use of a novel B cell-specific IL-10 knockout mouse revealed that B cell-derived IL-10 nonredundantly decreases virus-specific CD8+ T cell responses and plasma cell expansion during murine cytomegalovirus infection and modestly restrains immune activation after challenge with foreign Abs to IgD. In contrast, no role for B cell-derived IL-10 was evident during endotoxemia; however, although B cells dominated lymphoid tissue IL-10 production in this model, myeloid cells were dominant in blood and liver. These data suggest that B cells are an underappreciated source of counter-regulatory IL-10 production in lymphoid tissues, provide a clear rationale for testing the biological role of B cell-derived IL-10 in infectious and inflammatory disease, and underscore the utility of cell type-specific knockouts for mechanistic limning of immune counter-regulation.