Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8+ T cell response during Mtb infection, CD8+ T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages. We derived CD8+ T cell lines that recognized the Mtb immunodominant epitope TB10.44−11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. While the CD4+ T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8+ T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8+ T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8+ T cells. CD8+ T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8+ T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner. As TB10.4 elicits a dominant CD8+ T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8+ T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8+ T cell response may explain why CD8+ T cells make a disproportionately small contribution to host defense compared to CD4+ T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.
CD4 T cell help prevents CD8 T cell exhaustion and promotes control of Mycobacterium tuberculosis infectionGraphical abstract Highlights d CD4 T cell help promotes CD8 T cell effector functions and prevents exhaustion d Synergy between CD4 and CD8 T cells promotes survival during murine tuberculosis d Helped, but not helpless, CD8 T cells restrict intracellular mycobacterial growth d Protection mediated by CD8 T cells
CD4 T cells are essential for immunity to tuberculosis because they produce cytokines including interferon-γ. Whether CD4 T cells act as "helper" cells to promote optimal CD8 T cell responses during Mycobacterium tuberculosis is unknown. Using two independent models, we show that CD4 T cell help enhances CD8 effector functions and prevents CD8 T cell exhaustion. We demonstrate synergy between CD4 and CD8 T cells in promoting the survival of infected mice. Purified helped, but not helpless, CD8 T cells efficiently restrict intracellular bacterial growth in vitro. Thus, CD4 T cell help plays an essential role in generating protective CD8 T cell responses against M. tuberculosis infection in vitro and in vivo. We infer vaccines that elicit both CD4 and CD8 T cells are more likely to be successful than vaccines that elicit only CD4 or CD8 T cells.
Human CD8 T cells express the antimicrobial peptide granulysin in their cytotoxic granules, and
in vitro
analysis suggest that it restricts growth of
Mycobacterium tuberculosis
and other intracellular pathogens. The murine model of tuberculosis cannot assess granulysin’s role
in vivo
, as rodents lack the granulysin gene.
Human cytotoxic lymphocytes kill infected cells through release of lytic proteins contained in cytotoxic granules. The cytotoxic granules of human NK and CD8 T cells contain perforin, granzyme and granulysin (GNLY), and GNLY has direct antimicrobial activity against Mycobacterium tuberculosis (Mtb). Murine studies suggest that CD8 T cells have only a modest role in immunity to tuberculosis (TB). We hypothesize that mouse CD8 T cells are ineffective at containing Mycobacterium tuberculosis (Mtb) as they don’t express GNLY. To test this hypothesis, GNLY-transgenic mice that express human granulysin protein under the control of human regulatory elements were infected with Mtb. GNLY-Tg and non-tg control mice control Mtb similarly. There was no difference in the ability of GNY-Tg and non-tg CD8 T cells to transfer protection to T cell deficient mice. Anti-CD3 stimulation led to expression of granulysin in human CD8 T cells, but not in murine CD8 T cells. However, after Mtb infection, GNLY was expressed in NK cells but not CD8 T cells. We conclude that GNLY-expressing NK cells do not alter resistance to Mtb. Importantly, GNLY-Tg mice are inadequate model to study CD8 function during TB. We are developing a new strategy to express granulysin in CD8 T cells. We expressed GNLY (isoform 2) in primary murine CD8 T cells by retroviral gene transfer and detected GNLY expression in 60% of CD8 T cells.
Future directions
We are assessing the cytolytic activity of GNLY-expressing CD8 T cells and their ability to kill intracellular pathogens in vitro and in vivo. We expect to develop a new GNLY-Tg model that can be used to study the function of CD8 T cells and granulysin in many different diseases. This work is supported by NIH AI159374.
This work is supported by NIH grant R21 AI159374.
Despite the central role of IFNγ in vitiligo pathogenesis, systemic IFNγ neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFNγ signaling during vitiligo, we hypothesized that tethering an IFNγ neutralizing antibody to keratinocytes would limit anti-IFNγ effects to the treated skin for the localized treatment. To that end, we developed a bispecific antibody (BsAb) capable of blocking IFNγ signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the BsAb in vitro, ex vivo, and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFNγ inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFNγ antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFNγ blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.
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