The second touch hypothesis: T cell activation, homing and polarization

The role of innate immune cells

Most animals (including all invertebrates) do not have adaptive immune systems, yet are well protected from pathogens. Yet all these animals have macrophages or macrophage-like cells. These macrophages directly sense pathogens and mount an appropriate defensive response. In vertebrates with an adaptive immune system, macrophages (and other innate immune cells) drive the polarization of the adaptive immune response. This was first shown in mice: certain inbred mouse strains have a Th1 bias (like C57BL/6) and others have a Th2 bias (like Balb/c). The underlying reason for this bias is not in the T cell compartment, but is found in the innate immune system¹. This was shown by using immunodeficient mice that do not have an adaptive immune system, yet still preserve the Th1/Th2 bias. This finding gave rise to the concept of M1 and M2 macrophages¹, where M1 macrophages metabolize arginine to nitric oxide (NO) and citrulline through iNOS and M2 macrophages metabolize arginine to ornithine and urea through arginase. NO promotes bacterial killing and inflammation, whereas ornithine promotes wound healing and collagen biosynthesis.

M2 macrophages produce TGF-β, which is secreted in an inactive form that requires activation by a process that depends on αVβ8 integrin^2–4. The activating integrin must be expressed on the same cell that presents the antigen, suggesting that only TGF-β secreted by αVβ8 integrin-expressing macrophages and DCs is relevant for iTreg induction.

Macrophage activation

Macrophages express a large number of cell surface and cytosolic receptors that allow them to recognize bacteria, fungi, parasites, viruses, but also altered self molecules. The macrophage activating receptors fall into five families:

Engagement of many of these receptors activates members of the transcription factor family NF-kB, which in turn is responsible for production of many inflammatory cytokines. How the engagement of specific pattern recognition receptors is related to the cytokines that drive CD4 T cell differentiation is an area of active investigation. Macrophages can secrete IL-1α and β, IL-18, IL-6, IL-12, IL-23, α and β interferons, IL-4, IL-10 and TGF-β. Activated macrophages express a range of molecules from the TNF and TNF receptor superfamilies.

Second touch hypothesis

The second touch hypothesis as formulated here states that T cell activation, proliferation, induction of homing receptors, and polarization are distinguishable and at least in part sequential. The second touch hypothesis maintains that full CD4 T cell polarization requires CD4 T cell interaction with antigen-presenting cells (DCs, macrophages, B cells and certain activated stromal cells) in the inflamed non-lymphoid tissue where the antigen was first encountered (Figure 1).

Figure 1. The second touch hypothesis.

Naïve T cells (light blue) enter peripheral lymph nodes (PLN) through high endothelial venules (HEV), where they encounter antigen (Ag) presented by a migratory dendritic cell (DC) or by a PLN-resident B cell (brown). If signals induce Bcl6 and CXCR5, the T cells may enter the germinal center and become follicular helper T cells (TFH, red). Upon encountering antigen in the context of co-stimulatory molecules like CD80 and CD86, most T cells will express homing receptors, downregulate the sphingosine-1-phosphate receptor S1P1 and leave the PLN. These cells (orange) are only partially programmed and referred to as pre-Th1, pre-Th2, pre-Th17 and pre-iTreg. These cells circulate and reach various tissues, including the inflamed tissue from which the antigen came. There, they encounter DCs, macrophages and other antigen-presenting cells (APCs) such as endothelial cells. These cells present antigen in the context of different co-stimulatory molecules such as TNFSF members, and in the context of inflammatory cytokines found in the inflamed tissue. If the prevailing signal is IL-12, the T cell will commit to Th1, if IL-4, 5 and 13, to Th2, if IL-1, TGF-β, IL-6, IL-21, to Th17. If the cytokine environment is dominated by TGF-β, an M2 cytokine, the T cell will become an iTreg.

This hypothesis holds that homing receptors are induced first, soon after activation of naïve T cells. Expression of homing receptors capacitates these T cells to leave the lymphatic system and enter specific tissues, including those where the antigen was first encountered.

The second touch hypothesis also holds that antigen-experienced T cells require a “second touch” by seeing antigen presented on a tissue macrophage or DC in the context of co-stimulatory molecules and in the inflammatory cytokine environment of the inflamed tissue (Figure 2). This “second touch” appears to be required for full differentiation to Th1, Th2, Th17, Treg or TFH. This may involve chromatin remodeling of the loci needed for the respective differentiation (see below).

Figure 2. Pathogen sensors and cytokine environment.

Tissue macrophages have a natural bias toward M2 (express arginase, produce TGF-β). T cells in this environment are likely to commit to the iTreg lineage. If tissue macrophages sense the presence of pathogens through their TLRs, NLRs, RLRs (see definitions in text) or C-type lectin receptors, they convert to M1 (express iNOS, produce IL-12, IL-23, upregulate MHC-II and CD86). M1 differentiation also induces expression of chemokines that attract more monocytes from the circulation, which have a propensity to become M1-polarized after transmigration. T cells receiving a second touch in this environment are likely to commit to Th1 if IL-12 dominates or Th17 if IL-23 dominates. The tissue environment producing Th2 cells is not well understood, but may involve M2 macrophages.

Naïve αβ T cells are activated by contacting antigen-presenting cells in secondary lymphoid organs⁵. Dendritic cells (DCs) are the most effective antigen-presenting cells for naïve T cells. T cells must receive two signals, one from T cell receptor (TCR) engagement with antigenic peptide in the context of MHC-II for CD4 or MHC-I for CD8 T cells, and the other one through CD28 engagement by CD80 and/or CD86⁶. Both the MHC and co-stimulatory signals are provided by the same dendritic cell⁷. The cytokine milieu at the time of antigen presentation drives T cell polarization. Here, I will only consider helper (CD4) T cells, but similar lineages, subsets and polarizations have also been described for CD8 T cells.

In the traditional view, naïve T cell activation, proliferation and differentiation are all considered as concomitant, simultaneous processes. The second touch hypothesis holds that full differentiation is only achieved after these T cells return to the site of inflammation and undergo a recall response.

Antigen transport to lymph nodes

Antigenic proteins reach draining lymph nodes either as soluble antigens or by way of migratory DCs that acquired antigen in the inflamed tissue. Soluble antigen is presented by at least 3 types of lymph node-resident DCs to naïve T cells. Multiple short (3 minutes) interactions allow the T cell to become activated. A little later, migratory DCs enter the draining lymph node through afferent lymphatics and present higher doses of antigen, or more highly processed antigen, to naïve T cells, resulting in long-term (~50 min) interactions⁶. Whether these longer-term interactions activate T cells more fully than repeated 3 min interactions is unclear. Many, if not all, migratory DCs are monocyte-derived, and only migrate to the regional lymph nodes under conditions of inflammation. These DCs must undergo a “maturation”, which involves expression of the chemokine receptor CCR7, and upregulation of the co-stimulatory molecule CD86. Maturation can be achieved by TLR ligands such as LPS or by engagement of CD40. Only mature DCs acquire the ability to migrate to lymph nodes⁸. Mature DCs share phenotypic characteristics and gene expression with M1 macrophages. M1 macrophages are characterized by the expression of iNOS, producing NO from arginine¹. M2 macrophages express arginase, an enzyme that converts arginine to ornithine. The environment (M1, M2) during antigen encounter is likely to influence the ensuing T cell response.

T cell activation

Naïve T cell activation in peripheral lymph nodes is a dramatic and violent process⁹. Most activated T cells die by apoptosis in the lymph node where they were generated. The resulting apoptotic bodies are phagocytosed by local macrophages, triggering an anti-inflammatory signal^10,11 that induces iTregs¹². iTregs are a natural by-product of any ongoing immune response, whether in infections or autoimmune diseases¹³. Some activated T cells survive and acquire activation markers including:

Antigen-experienced T cells proliferate in the lymph node for 3–7 days and acquire peripheral homing receptors that allow them to return to the tissue where the antigen resides.

Homing receptors

Homing receptors are defined as cell surface molecules that allow T cells to attach to endothelial cells in specific tissues and organs, transmigrate through the endothelial cell monolayer and the basement membrane and infiltrate the inflamed tissues. One category of homing receptors are chemokine receptors; heptahelical cell surface receptors that are coupled through heterotrimeric G-proteins. These include:

Other homing receptors are adhesion molecules.

The Th1 chemokine receptor CXCR3 is induced soon after a naïve T cell sees antigen¹⁷. Interestingly, induction of most homing receptors is T-bet dependent¹⁸.

T cell polarization

CD4 T cells differentiate to Th1, Th2, Th17, iTreg and TFH cells. All CD4 T cell expansion and activation is IL-2-dependent. It is useful to emphasize that IL-2 deficient mice suffer from autoimmune disease and not from immunodeficiency. The autoimmune phenotype is driven by a lack of regulatory T cells. This suggests that the primary function of T cell activation and expansion is the production of iTregs, which appears to be necessary because central tolerance (elimination of self-reactive T cell clones in the thymus) is ineffective^19,20. Although T cell clones are eliminated in the thymus by negative selection, other T cell clones differentiate to natural regulatory T cells (nTregs). nTregs show chromatin remodeling that allows stable FoxP3 expression. nTregs are produced in the thymus and not in secondary lymphoid organs and thus are not subject to the second touch hypothesis and therefore not considered further.

CD4 T cells produce memory cells that can be divided into effector- (TEM), central memory- (TCM)²⁷, and into resident memory cells (TRM) that stay in the skin and intestinal epithelium²⁸. TEM are CD45RB^low, CD44^high and LFA-1^high. TCM express CCR7 and CD62L. TRM express CD103 and CD69. It is currently controversial whether long-lived memory T cells originate from effector T cells (the cells expanding during the acute insult) or represent a separate lineage²⁹. There is some evidence that memory T cells retain memory of the phenotype (Th1, Th2, Th17) they had during the initial encounter of antigen.

Antigen presentation in non-lymphoid tissues

Antigen presentation to antigen-experienced T cells in non-lymphoid tissues is not well studied. Only a handful of reports touch on this subject.

Flugel’s group tracked antigen-specific CD4 effector T cells in the mouse brain under conditions of experimental autoimmune encephalitis (EAE)³⁰. Although the antigen presenting cells (APCs) were not directly visualized, 35% of the T cells stopped and appeared to make an immunologic synapse suggested by polarized expression of LFA-1 and TCR. This was antigen-specific, because ovalbumin-specific T cells were only tethered after intrathecal injection of antigen. Carbone’s group studied memory T cell activation using reactivation by herpes simplex virus in dorsal root ganglia of mice³¹. Although no imaging was performed, the key finding is that resident T cells can expand locally in an antigen-specific manner. Bousso’s group studied Leishmania major antigen recognition in the skin and found that CD4 T cells can activate nearby cells by IFN-γ, which can diffuse as far as 80 μm³².

Steinman’s group isolated vascular dendritic cells from CD11c-YFP mouse aortas and aortic valves and incubated them with transgenic T cells³³. They found that these CD11c+ DCs expressed MHC-II, CD80 and CD86, but not CD40. These DCs were able to cross-present protein antigens on MHC-I to CD8 T cells. In a second study, the same group showed that these vascular DCs are dependent on the growth factor receptor Flt3, have tolerogenic properties and some of them express the integrin CD103³⁴. In a study of antigen presentation in the atherosclerotic aorta, my lab showed that antigen-experienced (CD44^hiCD62L^-), but not naïve CD4 T cells interact productively with APCs in the adventitia and the atherosclerotic plaque³⁵. The APCs were visualized by yellow fluorescent protein (YFP) driven by the CD11c promoter³⁶ and the T cells were labeled ex vivo to directly study interactions in the aorta. Three lines of evidence showed that the interactions were productive. First, long interactions between CD4 T cells and APCs were only observed when antigen was present. Second, the migration velocity of the T cells was drastically reduced during these interactions, from about 10 to about 3 μm/min. Third, cytokine production (IFN-γ, TNF) was observed when explanted aortas were incubated with antigen-experienced, but not naïve T cells. Taken together, these data show that a productive recall response is supported in the non-lymphoid tissue³⁵. We did not investigate which co-stimulatory molecules were involved in these interactions of CD4 T cells with APCs. We also did not identify the precise nature and phenotype of the DCs. Also, we did not retrieve the T cells from the aortas to study their commitment to Th1 or Th17 or other subsets. Finally, we did not study chromatin remodeling in these T cells.

The second touch hypothesis, if correct, allows several testable predictions:

An interesting example of CD4 T cell interaction with APCs in a non-lymphoid tissue is “licensing” of encephalitogenic T cells in the lung as detailed in³⁷. The details of this “licensing” process are not known. The licensing process is not antigen-specific.

Chromatin remodeling and phenotypic stability

CD4 T-helper cells can interconvert between phenotypes. In general, iTregs and Th17 cells are thought to be less stable and Th1 and Th2 cells are thought to be more stable. A more stable phenotype is associated with chromatin remodeling in the region of key cytokine and transcription factor genes. This is best described for Th1 and Th2 cells.

The histone 3 lysine 4 trimethylation (H3K4me3) mark is associated with a permissive chromatin state, whereas H3K27me3 is suppressive. The H3K27me3 repressive tag is effectively removed by the enzyme encoded by Jmjd3, which is induced by T-bet, the defining transcription factor of Th1 cells. This includes removal of H3K27me3 from the Ifng locus^38,39. To some extent, T-bet also represses genes that would be expressed in alternative T-helper fates, like Bcl6, the defining transcription factor of TFH cells¹⁸. However, FOXP3 and T-bet can be co-expressed in the same CD4 T cell⁴⁰. Similarly, some FOXP3+ T cells secrete IL-17A⁴¹. There is no accepted nomenclature for these “Th1 Tregs” and Th17 Tregs” yet.

The genes encoding the Th2 cytokines IL-4, IL-5 and IL-13 are found on mouse chromosome 11 very close to each other. This locus is extensively remodeled in Th2 cells. A 3’ enhancer of IL-4 expression called HSV is required for stable activation of the IL-4 promoter⁴², especially in TFH cells. The epigenomic modifications leading to iTreg⁴³, Th17 and TFH are under investigation.

In the context of the second touch hypothesis, it would seem reasonable to postulate that the second touch is required for epigenomic modifications and stable T-helper phenotype commitment.

Evidence supporting the second touch hypothesis

The finding that induction of most homing receptors is T-bet dependent¹⁸ supports the hypothesis that acquisition of homing receptors may precede full T cell differentiation: T-bet is the defining transcription factor of the Th1 lineage, yet the homing T cells can be of any lineage. As a specific example, the Th1 chemokine receptor CXCR3 is induced rapidly, and the peak of its expression precedes maximal T cell expansion¹⁷.

The expression profile of co-stimulating receptors (TNF superfamily members TNFSF and TNF receptor superfamily members TNFRSF) and cytokines (IL-12, IL-23, TGF-β) is different in macrophages and DCs in inflamed tissues compared to DCs in secondary lymphoid tissues⁴⁴. This finding supports the idea that the signals received by antigen-experienced CD4 T cells cruising through inflamed tissues are different from those received in secondary lymphoid organs. Myeloid cells in tissues are mostly macrophages, which are usually M2 polarized, secrete active TGF-β and thus promote Treg induction. In inflamed tissues, most macrophages are M1-polarized, secrete IL-12 and IL-23 and thus support Th1 and Th17 cell development.

Only a very small fraction of DCs are migratory. These few cells are unlikely to fully represent the entire cytokine environment of the inflamed tissue. Certainly, resident tissue macrophages, a cell type known to be M2 polarized and anti-inflammatory, are not migratory⁴⁵. It follows that it is likely that antigen-experienced T cells would encounter a different cytokine milieu and different co-stimulating receptors in the inflamed tissue itself rather than in the secondary lymphoid organs.

Tissue macrophages are M2-polarized and produce TGF-β. However, TGF-β is secreted in an inactive form and requires αVβ8 integrin for activation^2–4. The activating integrin must be expressed on the same cell that presents the antigen, suggesting that only TGF-β secreted by αVβ8 integrin-expressing macrophages and DCs is relevant for iTreg induction. Therefore, only M2 resident macrophages (which cannot migrate to secondary lymphoid organs) in tissues would be expected to be effective at producing iTregs. There is good evidence that iTregs from non-lymphoid tissues are the ones that are relevant in curbing excessive inflammation¹³. Therefore, one of the most striking consequences of the second touch hypothesis would be that only iTregs that circulate back into the tissue are effective at curbing inflammation, because it is only in the context of M2 tissue macrophages that they become effectively committed to the iTreg lineage.

In inflamed tissues, pro-inflammatory M1 macrophages, both converted from resting tissue macrophages and derived from newly recruited monocytes, express their own characteristic cytokines including IL-12 and IL-23. The vast majority of M1 macrophages remain in the inflamed tissue, but some leave and enter the draining lymph node. Under inflammatory conditions, the difference between dendritic cells and M1 macrophages blurs, because both are monocyte-derived^46,47.

Consequences of the second touch hypothesis

If the second touch hypothesis is correct, it follows that activated antigen-experienced CD4 T cells are “immature” effector T cells when they first leave the priming lymph node. They are immature, because they are not fully committed to a differentiation program yet. To fully commit, they would require the second touch of seeing antigen presented by APCs in the inflamed tissue, providing different co-stimulators, perhaps in the form of interactions between TNF superfamily members with their receptors, or via soluble cytokines, or both. The “immature” effector T cell may be on the path to differentiation, but may not have undergone chromatin remodeling, which is needed for stable T helper phenotypes.

In addition, the nature and location of the second touch should also influence memory T cell polarization. This is well documented in two cases. Memory cells derived from TFH cells have the phenotype of central memory (TCM) cells⁴⁸, because they reside in the secondary lymphoid organs and home back there (through CCR7, CD62L and other homing mechanisms). Conversely, effector T cells recirculating through an inflamed tissue (via CXCR3, CCR4, CCR9, α4β7 integrin or PSGL-1) would be expected to have a phenotype of effector memory T cells (TEM). They may show Th1, Th2 or perhaps Th17 polarization. Specifically, T cells that encountered antigen in the intestinal tract acquire expression of the α4β7 integrin and preferentially home back to the intestine^15,49.

Co-stimulation by TNF and TNF receptor superfamily members

Co-stimulation occurs not only through CD28 binding CD80 and CD86, but also through TNF and TNF receptor superfamily molecules on T cells binding their partners on APCs⁵⁰ (Figure 3). The main co-stimulator molecules of this class are listed in Table 1.

Figure 3. First and second touch.

The first touch occurs in the lymph node, where the naïve T cell (light blue) is exposed to antigen (green circle) presented by MHC-II (purple) in the context of CD80 and CD86 (orange) co-stimulation, which bind CD28 (pink). The MHC-II-peptide complex is recognized by the T cell receptor (TCR, yellow) with CD4 (green). The second touch occurs in the tissue, where antigen is presented to antigen-experienced (orange) T cells by M1 (pink) and M2 (green) macrophages. Co-stimulatory molecules are likely from the TNFSF (red) on the T cell and the TNFRSF (blue) on the APC (see table for list of molecules). Characteristic M1 cytokines like IL-12 and IL-23 commit the CD4 T cell to Th1 and Th17, respectively. The M2 cytokine TGF-β commits T cells to the iTreg lineage.

OX40, 4-1BB, CD27, and DR3 co-stimulatory molecules are constitutively expressed on Tregs and are either constitutive on conventional CD4 and CD8 T cells or induced usually within 24 hours after antigen presentation. Engagement of these receptors increases IL-4 and IFN- production, promotes division, and enhances survival. OX40 promotes effector T cells and blocks the development of iTregs. Inhibiting any of the listed pairs modifies many models of autoimmune disease. Taken together, the TNFSF and TNFRSF molecules are possible candidates for the second touch signal that T cells may receive in the inflamed tissue.

Conclusion

In conclusion, the second touch hypothesis accommodates the plasticity of CD4 T cell phenotypes. It fills in the disconnect between the location of naïve T cell activation (PLN) and T cell effector functions (inflamed tissues). The concept of the second touch hypothesis generates many sub-hypotheses that can be tested by manipulating the mouse genome in a cell-specific fashion. Finally, the second touch hypothesis generates a conceptual framework for successful translation of immunological concepts to medical applications. This would include protective and tolerogenic vaccines and biologics that manipulate cytokines and their receptors.