An age-dependent increase in IFN-γ expression by intestinal intraepithelial lymphocytes (IELs) contributes to the acquisition of resistance to infection by pathogens. However, how IELs acquire the ability to produce IFN-γ remains to be elucidated. Here, we report that IELs in the small intestine acquire the ability to rapidly produce IFN-γ at two distinct life stages. TCRαβ+ IELs (αβIELs) started producing IFN-γ at 4 weeks of age, within 1 week after weaning. In contrast, TCRγδ+ IELs (γδIELs) started producing IFN-γ at 7 weeks of age. In mice lacking Eγ4, an enhancer of the TCRγ locus (Eγ4-/- mice), Thy-1+ Vγ5+ γδIELs, a major subpopulation of γδIELs, were specifically reduced and their ability to produce IFN-γ was severely impaired, whereas Vγ2+ γδIELs normally produced IFN-γ. In Eγ4-/- mice, TCR expression levels were reduced in Vγ5+ γδIEL precursors in the thymus but unchanged in the Vγ5+ IELs. Nevertheless, TCR responsiveness in Vγ5+ γδIELs was impaired in Eγ4-/- mice, suggesting that the TCR signal received in the thymus may determine TCR responsiveness and the ability to produce IFN-γ in the gut. These results suggest that αβIELs and γδIELs start producing IFN-γ at different life stages and that the ability of Vγ5+ γδIELs to produce IFN-γ in the gut may be predetermined by TCR signaling in IEL precursors in the thymus.

BACKGROUND: Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown.
METHODS: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.
RESULTS: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.
CONCLUSIONS: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

γδ T cells play a crucial role in immune surveillance and serve as a bridge between innate and adaptive immunity. However, the metabolic requirements and regulation of γδ T-cell development and function remain poorly understood. In this study, we investigated the role of liver kinase B1 (Lkb1), a serine/threonine kinase that links cellular metabolism with cell growth and proliferation, in γδ T-cell biology. Our findings demonstrate that Lkb1 is not only involved in regulating γδ T lineage commitment but also plays a critical role in γδ T-cell effector function. Specifically, T-cell-specific deletion of Lkb1 resulted in impaired thymocyte development and distinct alterations in γδ T-cell subsets in both the thymus and peripheral lymphoid tissues. Notably, loss of Lkb1 inhibited the commitment of Vγ1 and Vγ4 γδ T cells, promoted the maturation of IL-17-producing Vγ6 γδ T cells, and led to the occurrence of fatal autoimmune hepatitis (AIH). Notably, clearance of γδ T cells or blockade of IL-17 significantly attenuated AIH. Mechanistically, Lkb1 deficiency disrupted metabolic homeostasis and AMPK activity, accompanied by increased mTORC1 activation, thereby causing overactivation of γδ T cells and enhanced apoptosis. Interestingly, activation of AMPK or suppression of mTORC1 signaling effectively inhibited IL-17 levels and attenuated AIH in Lkb1-deficient mice. Our findings highlight the pivotal role of Lkb1 in maintaining the homeostasis of γδ T cells and preventing IL-17-mediated autoimmune diseases, providing new insights into the metabolic programs governing the subset determination and functional differentiation of thymic γδ T cells.

Infection at barrier sites, e.g., skin, activates local immune defenses that limit pathogen spread, while preserving tissue integrity. Phenotypically distinct γδ T cell populations reside in skin, where they shape immunity to cutaneous infection prior to onset of an adaptive immune response by conventional αβ CD4+ (TCD4+) and CD8+ (TCD8+) T cells. To examine the mechanisms used by γδ T cells to control cutaneous virus replication and tissue pathology, we examined γδ T cells after infection with vaccinia virus (VACV). Resident γδ T cells expanded and combined with recruited γδ T cells to control pathology after VACV infection. However, γδ T cells did not play a role in control of local virus replication or blockade of systemic virus spread. We identified a unique wound healing signature that has features common to, but also features that antagonize, the sterile cutaneous wound healing response. Tissue repair generally occurs after clearance of a pathogen, but viral wound healing started prior to the peak of virus replication in the skin. γδ T cells contributed to wound healing through induction of multiple cytokines/growth factors required for efficient wound closure. Therefore, γδ T cells modulate the wound healing response following cutaneous virus infection, maintaining skin barrier function to prevent secondary bacterial infection.

Immuno-oncology has traditionally focused on conventional MHC-restricted αβ T cells. Yet, unconventional γδ T cells, which kill tumor cells in an MHC-unrestricted manner, display characteristics of effector activity and stemness without exhaustion and are nearly universally observed in human gynecologic malignancies, correlating with improved outcomes. These cells do not have a clear counterpart in mice but are also found in the healthy female reproductive tract. Interventions that modulate their in vivo activity, or cellular therapies utilizing γδ T cells as an allogeneic, \"off-the-shelf\" platform (e.g., for chimeric antigen receptor expression) hold significant potential against challenging tumors like ovarian cancer, which has been stubbornly resistant to the immune checkpoint inhibitors that change the landscape of other human tumors. Here, we discuss recent discoveries on the specific populations of γδ T cells that infiltrate human gynecologic cancers, their anti-tumor activity, and the prospect of redirecting their effector function against tumor cells to develop a new generation of immunotherapies that extends beyond the traditional αβ T cell-centric view of the field.

Invasive aspergillosis (IA) and mucormycosis are life-threatening diseases, especially among immunocompromised patients. Drug-resistant Aspergillus fumigatus strains have been isolated worldwide, which can pose a serious clinical problem. As IA mainly occurs in patients with compromised immune systems, the ideal therapeutic approach should aim to bolster the immune system. In this study, we focused on Vγ9Vδ2 T cells that exhibit immune effector functions and examined the possibility of harnessing this unconventional T cell subset as a novel therapeutic modality for IA. A potent antifungal effect was observed when A. fumigatus (Af293) hyphae were challenged by Vγ9Vδ2 T cells derived from peripheral blood. In addition, Vγ9Vδ2 T cells exhibited antifungal activity against hyphae of all Aspergillus spp., Cunninghamella bertholletiae, and Rhizopus microsporus but not against their conidia. Furthermore, Vγ9Vδ2 T cells also exhibited antifungal activity against azole-resistant A. fumigatus, indicating that Vγ9Vδ2 T cells could be used for treating drug-resistant A. fumigatus. The antifungal activity of Vγ9Vδ2 T cells depended on cell-to-cell contact with A. fumigatus hyphae, and degranulation characterized by CD107a mobilization seems essential for this activity against A. fumigatus. Vγ9Vδ2 T cells could be developed as a novel modality for treating IA or mucormycosis.
OBJECTIVE: Invasive aspergillosis (IA) and mucormycosis are often resistant to treatment with conventional antifungal agents and have a high mortality rate. Additionally, effective antifungal treatment is hindered by drug toxicity, given that both fungal and human cells are eukaryotic, and antifungal agents are also likely to act on human cells, resulting in adverse effects. Therefore, the development of novel therapeutic agents specifically targeting fungi is challenging. This study demonstrated the antifungal activity of Vγ9Vδ2 T cells against various Aspergillus spp. and several Mucorales in vitro and discussed the mechanism underlying their antifungal activity. We indicate that adoptive immunotherapy using Vγ9Vδ2 T cells may offer a new therapeutic approach to IA.

Toxin- and drug-induced tubulointerstitial nephritis (TIN), characterized by interstitial infiltration of immune cells, frequently necessitates dialysis for patients due to irreversible fibrosis. However, agents modulating interstitial immune cells are lacking. Here, we addressed whether the housekeeping enzyme glutamyl-prolyl-transfer RNA synthetase 1 (EPRS1), responsible for attaching glutamic acid and proline to transfer RNA, modulates immune cell activity during TIN and whether its pharmacological inhibition abrogates fibrotic transformation. The immunological feature following TIN induction by means of an adenine-mixed diet was infiltration of EPRS1high T cells, particularly proliferating T and γδ T cells. The proliferation capacity of both CD4+ and CD8+ T cells, along with interleukin-17 production of γδ T cells, was higher in the kidneys of TIN-induced Eprs1+/+ mice than in the kidneys of TIN-induced Eprs1+/- mice. This discrepancy contributed to the fibrotic amelioration observed in kidneys of Eprs1+/- mice. TIN-induced fibrosis was also reduced in Rag1-/- mice adoptively transferred with Eprs1+/- T cells compared to the Rag1-/- mice transferred with Eprs1+/+ T cells. The use of an EPRS1-targeting small molecule inhibitor (bersiporocin) under clinical trials to evaluate its therapeutic potential against idiopathic pulmonary fibrosis alleviated immunofibrotic aggravation in TIN. EPRS1 expression was also observed in human kidney tissues and blood-derived T cells, and high expression was associated with worse patient outcomes. Thus, EPRS1 may emerge as a therapeutic target in toxin- and drug-induced TIN, modulating the proliferation and activity of infiltrated T cells.

Adoptive cellular immunotherapy as a new paradigm to treat cancers is exemplified by the FDA approval of six chimeric antigen receptor-T cell therapies targeting hematological malignancies in recent years. Conventional αβ T cells applied in these therapies have proven efficacy but are confined almost exclusively to autologous use. When infused into patients with mismatched human leukocyte antigen, αβ T cells recognize tissues of such patients as foreign and elicit devastating graft-versus-host disease. Therefore, one way to overcome this challenge is to use naturally allogeneic immune cell types, such as γδ T cells. γδ T cells occupy the interface between innate and adaptive immunity and possess the capacity to detect a wide variety of ligands on transformed host cells. In this article, we review the fundamental biology of γδ T cells, including their subtypes, expression of ligands, contrasting roles in and association with cancer prognosis or survival, as well as discuss the gaps in knowledge pertaining to this cell type which we currently endeavor to elucidate. In addition, we propose how to harness the unique properties of γδ T cells for cellular immunotherapy based on lessons gleaned from past clinical trials and provide an update on ongoing trials involving these cells. Lastly, we elaborate strategies that have been tested or can be explored to improve the anti-tumor activity and durability of γδ T cells in vivo.

The migration of γδ T lymphocytes toward skin lesions and their concomitant pathogenic IL-17A production play a crucial role in the pathogenesis of psoriasis. However, the regulatory mechanisms of IL-17A production by γδ T cells and their migration remain to be fully explored. Intracellular GRP78 is a molecular chaperone that regulates endoplasmic reticulum stress, whereas secretory GRP78, as a member of the resolution-associated molecular patterns, exerts immunoregulatory effects. In this study, we reported that both the intracellular GRP78 in skin lesions and secretory GRP78 in the serum were significantly decreased in patients with psoriasis. A GRP78 knockdown exacerbated imiquimod-induced skin inflammation, whereas the application of recombinant GRP78 protein or BIP inducer X (a GRP78 inducer) attenuated the dermatitis. Mechanistically, the GRP78 knockdown in keratinocytes enhanced the production of chemokines, specifically CCL20, which regulates γδ T-cell migration. Moreover, recombinant GRP78 was found to directly bind to γδ T cells to suppress its migration ability and proinflammatory capacities by downregulating the CCR6 and IL-17A expression. Collectively, our results uncovered a pivotal role of GRP78 in the pathogenesis of psoriasis, which was mainly exerted by regulating the interaction between keratinocytes and γδ T cells, and might provide a promising target for psoriasis therapy.

γδ T cells and natural killer (NK) cells have attracted much attention as promising effector cell subsets for adoptive transfer for use in the treatment of malignant and infectious diseases, because they exhibit potent cytotoxic activity against a variety of malignant tumors, as well as virus-infected cells, in a major histocompatibility complex (MHC)-unrestricted manner. In addition, γδ T cells and NK cells express a high level of CD16, a receptor required for antibody-dependent cellular cytotoxicity. Adult T-cell leukemia-lymphoma (ATL) is caused by human T-lymphotropic virus type I (HTLV-1) and is characterized by the proliferation of malignant peripheral CD4+ T cells. Although several treatments, such as chemotherapy, monoclonal antibodies, and allogeneic hematopoietic stem cell transplantation, are currently available, their efficacy is limited. In order to develop alternative therapeutic modalities, we considered the possibility of infusion therapy harnessing γδ T cells and NK cells expanded using a novel nitrogen-containing bisphosphonate prodrug (PTA) and interleukin (IL)-2/IL-18, and we examined the efficacy of the cell-based therapy for ATL in vitro. Peripheral blood samples were collected from 55 patients with ATL and peripheral blood mononuclear cells (PBMCs) were stimulated with PTA and IL-2/IL-18 for 11 days to expand γδ T cells and NK cells. To expand NK cells alone, CD3+ T-cell-depleted PBMCs were cultured with IL-2/IL-18 for 10 days. Subsequently, the expanded cells were examined for cytotoxicity against ATL cell lines in vitro. The proportion of γδ T cells in PBMCs was markedly low in elderly ATL patients. The median expansion rate of the γδ T cells was 1998-fold, and it was 12-fold for the NK cells, indicating that γδ T cells derived from ATL patients were efficiently expanded ex vivo, irrespective of aging and HTLV-1 infection status. Anti-CCR4 antibodies enhanced the cytotoxic activity of the γδ T cells and NK cells against HTLV-1-infected CCR4-expressing CD4+ T cells in an antibody concentration-dependent manner. Taken together, the adoptive transfer of γδ T cells and NK cells expanded with PTA/IL-2/IL-18 is a promising alternative therapy for ATL.