Delayed-type Hypersensitivity 

Cell-mediated immunity (CMI) is a T-cell–mediated defense mechanism against microbes that survive within phagocytes or infect nonphagocytic cells. CMI functions to enhance antimicrobial actions of phagocytes to eliminate microbes. CMI manifests as delayed type cellular immune responses as typically seen in Mantaux test. This T-cell–mediated activation of phagocytes depends on interferon gamma (IFN-γ), a major cytokine produced by CD4⁺ T-helper (Th1) cells. However, anti-IFN-γ neutralizing antibodies (Abs) do not completely abrogate CMI. IFN-γ or IFN-γR knock out (KO) mice do reveal attenuated CMI. These results indicate that CMI cannot be solely attributed to IFN-γ.

The identification of Th17 cells, which produce interleukin (IL)-17A, IL-17F, and IL-22, shed a light on the previously observed CMI in the absence of IFN-γ actions. IL-17 KO mice did display attenuated delayed-type hypersensitivity (DTH) against bovine serum albumin and bacille Calmette-Guérin (BCG).^[1] The role of Th17 and Th1 cells in CMI may vary depending on stimulants.^[2]

Phagocytic cell activation and inflammation induced by CMI can cause tissue injury, typically called delayed-type hypersensitivity. In experimental animal models, delayed-type hypersensitivity responses are characterized by a granulomatous response consisting of macrophages, monocytes, and T lymphocytes. In skin, keratinocytes are also thought to have a role.

Delayed-type hypersensitivity responses in the skin have been used to assess CMI in vivo. An antigen (Ag) is introduced intradermally (ID), and induration and erythema at 48-72 hours postinjection indicate a positive reaction. Positive responses require the subject's exposure to the Ag at least 4-6 weeks prior to skin testing. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. In the absence of underlying diseases, anergy may indicate primary or secondary T-cell immunodeficiency. The prototype recall Ag is Mycobacterium tuberculosis; other commonly used Ags for delayed-type hypersensitivity responses in humans include tetanus, Candida and Trichophyton species, and mumps. Several fungi and streptococci Ags are no longer available or not recommended for clinical use.

Delayed-type hypersensitivity responses in the skin can also occur as contact hypersensitivity (CH) to certain chemicals, including nickel, dinitrochlorobenzene (DNCB), and picryl chloride. Delayed-type hypersensitivity reaction can also occur with various medications, including sulfonamides, phenytoin, and carbamazepine. These small chemicals are believed to act as haptens or activate T cells by directly binding to T cell Ag receptor (TCRs)/major histocompatibility complex (MHC).

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Originally, CH was thought to be skin delayed-type hypersensitivity reaction. However, recent studies indicate that CH can be caused by different immune mechanisms. That is, in CH, CD4⁺ T cells are major effector cells, with CD8⁺ cell playing a regulatory role at least in certain model systems. In rodents, chemically induced CH was reported by attenuated by recombinant IFN-γ.^[2]

In contrast, CH induced by chemicals was enhanced by IL-17A, which induces production of IL-6, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), and upregulated expression of intercellular adhesion molecule (ICAM)-1 in human keratinocytes.^{[1, 2]} In addtion, IL-4 KO mice revealed attenuated CH responses.^[4] Cytotoxic CD8⁺ T cells that produce IL-17A (Tc17 cells) are also implicated in induction of CH in rodent models.^[5] Thus, at leastinrodentmodels, induction of CH appears to be mediated by Th17 and Th2 cytokines; Th17 cells produce IL-17A, and Th2 cells are a major source of IL-4.

A delayed type hypersensitivity (DTH) reaction in the skin is initiated when certain Ags are presented by Ag-presenting cells (APCs) (ie, Langerhans cells to sensitized memory T cells). The Ag presentation and subsequent T-cell activation via CD3 and T-cell Ag receptor (TCR)(CD3/TCR) complex elicit an influx of macrophages, monocyte, and lymphocytes at the site of Ag exposure. These cells then produce inflammatory cytokines including tumor necrosis factor-α (TNF-α), IL-17A, and IFN-γ. At the onset of the reaction, vasopermeability is increased by serotonin and histamine, and adhesion molecules are up-regulated in the vascular endothelium, so that additional cellular components migrate into the local site of Ag presentation.

The APCs present Ags complex in the groove of major histocompatibility complex (MHC) molecules expressed on the cell surface of the APCs. For most proten antigens or haptens associated with delayed-type hypersensitivity skin reaction, CD4⁺ T cells are presented with Ags bound to MHC class II alleles, human leukocyte antigen (HLA)-DR, -DP, and –DQ. Specific MHC class II alleles are recognized to produce excessive immune activation to certain Ags.

T cells recognize Ags through TCRs, which are composed of heterodimers containing constant and variable regions analogous to the constant and variable regions of immunoglobulin (TCR α/ß and TCR γ/δ). Most delayed-type hypersensitivity responses are elicited through TCR α/β T cells. TCR γ/δ is commonly expressed on T cells in the epithelium boundaries with limited TCR diversities. The function of TCR γ/δ T cells are not well understood.

Ag elicited TCR activation is mediated by CD3 composed of γ, δ, and ε proteins and intracellular ζ chains. CD3. This CD3/TCR complex activation requires additional signaling mediated by CD4 or CD8 which are physically associated with the CD3/TCR complex. In addition, Ag-mediated TCR activation requires Ag-independent signaling via co-stimulatory molecules which are not physically associated with the CD3/TCR complex. Co-stimulatory molecules that mediate activation signals such as CD28 and ICOS are constitutively expressed on T cells while inhibitory co-stimulatory molecules such as CTLA-4 (CD152) and PD-1 are up-regulated following T cell activation to abrogate excessive inflammatory responses.

Delayed-type hypersensitivity responses to some organisms are predominantly mediated by CD8⁺ cytotoxic T cells and effector stage Th1 cells augment CD8⁺ T cell activation. CD8⁺ T cells activate macrophages via CD40-CD40L interactions and production of Th1 cytokines including IFN-γ. The differences in T cell immune responses to intracellular microbes determine disease outcomes in certain diseases. Namely, ineffective CMI is observed in patients with lepromatous leprosy with high Mycobacteria leprae load in macrophages and destructive skin lesions. While, in tuberculoid leprosy, strong CMI induces granulomas with M Leprae load but severe sensory nerve defects.

IFN-γ is the key cytokine that plays the dominant role in delayed-type hypersensitivity and is a major activator of macrophage-monocyte lineage cells by augmenting phagocytic function and production of reactive oxygen intermediates (ROIs). IFN-γ also up-regulate T cell activation markers (CD69, CD71, CD25, and HLA-DR) and MHC molecules on APCs, promotes differentiation of Th1 cells while suppressing Th2 cell differentiation, and promote isotype switching of antibody (Ab) to facilitate Ab-mediated phagocytic cell immune responses. Recent studies also revealed a role of Th17 cells that produce IL-17A, IL-17F, IL-21, and IL-22 (ref 1 and 2). IL-17A was reported to amplify human contact hypersensitivity (CH) by rendering keratinocytes to susceptible to ICAM-1 dependent, Ag-non-specific T cell killing.^[6]

IFN-γ also exerts regulatory actions on CD4⁺ Th cells.^[7] IFN-γ affects activation-induced cell death of Th cells, contributing to lymphocyte homeostasis. IFN-γ is also reported to covert CD4⁺ CD25^- Th cells into inducible CD4⁺ CD25⁺ regulatory T (Treg) cells by inducing Foxp3 expression. Such action of IFN-γ may be associated with excessive delayed-type hypersensitivity reactions in patients with defects of IL-12 – IFNγ signaling pathways.

TNF-α is also essential for an effective delayed-type hypersensitivity responses. Major cellular sources of TNF-α include macrophages and Th1 cells but this cytokine is also produced by many other lineage cells. TNF-α induces chemokine production from macrophages and endothelial cells and it also up-regulates expression of adhesion molecules on vascular endothelial cells, augmenting influx of inflammatory cells. In delayed-type hypersensitivity responses to poison ivy and nickel, mast cells are likely a major source of TNF-α, in addition to production of other inflammatory mediators including histamine. TNF-α also induces inflammatory cytokines including IL-1. In rodent models of delayed-type hypersensitivity, IL-1 but not IL-33 was found to be crucial for IFN-γ induced delayed-type hypersensitivity reactions.^[8]

Another key cytokine associated with delayed-type hypersensitivity responses is IL-12. IL-12 is mainly produced by APC and Th1 cells augments IFN-γ production and promotes Th1 cell differentiation. It also enhances cytolytic actions of NK and CD8⁺ T cells. IL-18 was also shown to augment IFN-γ production in the presence of IL-12. Osteopontin (OPN), a phosphoglycoprotein produced in the tissue, has also a role for promoting Th1 driven delayed-type hypersensitivity by supporting APC migration and IL-12 expression.^[9]

A defect in delayed-type hypersensitivity reaction is best illustrated in the gene mutations of IFN-γ and IL-12 pathway. Gene mutation of IFN-γ receptor 1 (IFNGR1) is characterized by ineffective granuloma formation and disseminated infection of atypical Mycobacterium species, BCG, and Salmonella species. IFNGR1 gene consists of 7 exons (50 kb) and highly polymorphic.

Mutations identified thus far include frameshift deletions, insertion, a splice mutation, and missense mutations at the up-stream end of the gene. These mutations generally lead to absence of IFN-γ protein expression or non-functional binding sites for IFN-γ, leading to impaired macrophage activation, hence decreased production of TNF-α and ROIs. The gene mutation of IFNGR2 also cause similar clinical pictures. In contrast, mutation in IFNGR1 at a downstream hotspot disrupt intracytoplasmic domain and results in dominantly expressed disorder with milder clinical features.^[10]

TH17 and Tc17 cells are also implicated with delayed-type hypersensitivity and CH responses in humans and rodents. For example, nickel-specific Th clones established from patients with nickel CH was shown to produce IL-17A.

Down regulation of CMI in the immunocompetent host is an active process and important for minimizing tissue injury caused by delayed-type hypersensitivity. Counter-regulatory cytokines such as IL-10 down-regulate production of Th1/Th17 cytokines or counteracts to Th1/Th17 cytokines. Naturally occurring and inducible T reg cells also play a crucial role in this process partly via production of IL-10 and TGF-ß. Drugs that block components of delayed-type hypersensitivity.

Drugs that block components of delayed-type hypersensitivity responses include anti-histamines, histamine 2 (H2) receptor antagonists, and prostaglandin antagonists such as indomethacin. As indicated before, IFN-γ can augment induction of inducible Treg cell that produce IL-10 and TGF-ß in the late stage of delayed-type hypersensitivity–induced inflammation.^[7]

Frequency

United States

Seventy-five percent of healthy children aged 12-36 months mount positive delayed-type hypersensitivity skin test reactivity to a Candida antigen. Evaluating CMI with in vitro lymphocyte proliferative responses to the specific antigen is necessary in the absence of a skin DTH response confirm anergic state.

Anergy is observed in patients who are malnourished, have severe atopic dermatitis, and those with severe infections caused by M tuberculosis, measles, mumps, HIV, influenza, mononucleosis, lepromatous leprosy, and certain fungi. Anergy is also observed in patients who have recently received the measles, mumps, and rubella (MMR) vaccine, patients with sarcoidosis, or patients with parasitic infestations. In addition, immunosuppressive drugs, such as cytotoxic medications and corticosteroids, lead to anergy. Malignancy, especially malignant lymphomas, also induces anergy.

Interestingly, delayed-type hypersensitivity reactions in patients with impaired IFN-γ and IL-12/IL-23 pathways tend to reveal excessive delayed-type hypersensitivity reactions. In contrast, in patients with autosomal dominant or autosomal recessive hyper immunoglobulin E (IgE) syndrome with STAT3 mutations or DOCK 8 deficiency, decreased delayed-type hypersensitivity responses against Candida may be observed due to Th17 cell deficiency.^{[11, 12]}

International

The presence of delayed-type hypersensitivity reactivity to tuberculin follows BCG vaccination. BCG is the most commonly administered vaccine throughout the world; however, it is not commonly used in the United States.

Mortality/Morbidity

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Delayed-type hypersensitivity skin testing is almost never associated with mortality or morbidity. The major error is associated with failure of differentiating anergy from negative delayed-type hypersensitivity reactivity. Anergy may result from overwhelming infection such as sepsis or immunodeficiency. Secondary immunodeficiency is commonly due to therapy with corticosteroids, chemotherapy, calcineurin inhibitors (eg, cyclosporine, tacrolimus), and various monoclonal antibodies directed against the immune system.

Patients with T-cell immunodeficiency diseases such as severe combined immunodeficiency (SCID) are anergic.

Patients with mutations in IL12P40 or in IL12RB1 may show a positive, often excessive, delayed-type hypersensitivity reactivity through activation of other components of delayed-type hypersensitivity responses such as IL-17 and perhaps due to lack of regulatory effects of IFN-γ.

Mutations in IFNGR2, STAT-1, IL12P40, and IL12RB1 are autosomal recessive. Mutations found in complete IFN-γR deficiency are also autosomal recessive. Mutations in IFNGR1 that affect the downstream intracytoplasmic domain are autosomal dominant and are manifested as partial IFN-γR deficiency with less severe clinical manifestations. Patients with complete IFN-γR deficiency often succumb to death from overwhelming infection caused by nontuberculosis mycobacteria (NTM) and BCG at young age. Severe cytomegalovirus (CMV) infection is also described in these patients.

Mutations partially impairing IFN-γ signaling pathway may be manifested with milder mycobacterial infection, nontyphus Salmonella infection, Legionella infection, and listeriosis.

Race

Some individuals from the Mediterranean area have mutations leading to insufficient IFN-γ function. Specifically, a family from Tunisia, several families from Malta, and 1 family from Italy have been reported. Genetic defects involving the IFN-γ/IL-12 axis are now increasingly reported in other races.

Sex

Anergy to BCG and of idiopathic disseminated BCG infection are equally distributed among males and females. As expected in autosomal recessive gene mutations, IFNGR1, IL12P40, and IL12RB1 mutations are found with equal frequency in males and females.

Age

Delayed-type hypersensitivity reactivity to Candida antigens can be detected in infants as young as 3-4 months, but reactivity depends on exposure to the candida antigen. Positive delayed-type hypersensitivity reactivity to tetanus toxoid requires completion of the primary immunization series of 3 injections administered 4-6 weeks apart; only one third of infants have a positive response to tetanus after the first dose of immunization. Patients with IFNGR1/IFNGR2 mutations that cause complete loss of IFN-receptor expression or IFN-γ–mediated signaling have presented as infants or in early childhood with disseminated BCG or NTM infections. This age-related infection seems to reflect the age of exposure to the causative organisms.