Deciphering human γδ T cell response in cancer: Lessons from tumor-infiltrating γδ T cells.

The finding that γδ T cells are present among tumor-infiltrating lymphocytes in humans suggests they participate in tumor immune surveillance, but their relevance is unclear because the relative abundance of tumor-infiltrating γδ T cells correlates with positive or negative, or even do not correlate with prognosis. This likely depends on the fact that tumor-infiltrating γδ T cells may play substantially different effector or regulatory functions, and correlation with patient's prognosis relies on distinct γδ T cell subsets in the context of the tumor. There is interest to exploit γδ T cells in tumor immunotherapy, but to make this approach successful there is urgent need to fully understand the biological functions of γδ T cells and of how they can be manipulated in vivo and ex vivo to safely provide benefit to the host. This review focuses on our previous and ongoing studies of tumor-infiltrating γδ T lymphocytes in different types of human cancer. Moreover, we discuss the interaction of tumor-infiltrating γδ T cells with other cells and molecules present in the tumor microenvironment, and their clinical relevance on the ground, that deep knowledge in this field can be used further for better immunotherapeutic intervention in cancer.

Profile of gamma-delta (γδ) T lymphocytes in the peripheral blood of crossbreed dogs during stages of life and implication in aging.

BACKGROUND: Data on gamma-delta (γδ) T lymphocytes in the peripheral blood of dogs are scant, related only to healthy pure breed dogs and limited to a restricted age range. The aim of the study was to investigate the modulation of the γδ T lymphocyte (TCRγδ+) subpopulation in peripheral blood of crossbreed healthy dogs according to five identified stages of life: Puppy, Junior, Adult, Mature, Senior and to determine its implication in aging. A rigorous method of recruitment was used to minimize the influence of internal or external pressure on the immune response. Twenty-three intact female and twenty-four intact male dogs were enrolled. Blood samples were collected and immunophenotyping of peripheral blood T lymphocytes and γδ T cell subpopulations was performed. RESULTS: The percentage of γδ T cells in peripheral blood lymphocytes was comparable with the value of 2.5% published by Faldyna and co-workers (2001), despite the percentage reported was investigated in less arranged age range groups and coming from four different dog pure breeds, whereas our data were recorded on wider age range groups and coming from crossbreed dogs. Therefore, the γδ T cell percentage (2.5%) is consistent and points out that such value is breed-independent. Statistical analysis highlighted differences in both percentage and absolute γδ T cells according to the stage of life. γδ T cells decreased significantly in the peripheral blood of elder dogs (Senior group) in comparison with previous stages of life (Puppy, Junior, and Adult groups). Differences in γδ T cells are significant and they are reported, for the first time, related to dog aging. CONCLUSIONS: The study confirms dogs to be among the animals with a low TCRγδ+ cell profile. A decrease of the TCRγδ+ subpopulation percentage was observed in elder dogs. TCRγδ+ cells of group S were different from those of groups P, J, and A. The differences are reported for the first time in dog aging. Identifying the stage of life when the decrease of γδ T lymphocytes starts can be useful for providing a rationale for drafting a wellness plan trial to support thymus immune functions and mitigate its functional exhaustion.

Increased proportions of γδ T lymphocytes in atypical SCID associate with disease manifestations.

Severe combined immunodeficiencies (SCID) comprise a group of genetic diseases characterized by abrogated development of T lymphocytes. In some case reports of atypical SCID patients elevated proportions of γδ T lymphocytes have been reported. However, it is unknown whether these γδ T cells modulate or reflect the patient's clinical phenotype. We investigated the frequency of elevated γδ T cell proportions and associations with clinical disease manifestations in a cohort of 76 atypical SCID patients. Increased proportions of γδ T lymphocytes were present in approximately 60% of these patients. Furthermore, we identified positive correlations between elevated proportions of γδ T cells and the occurrence of CMV infections and autoimmune cytopenias. We discuss that CMV infections might trigger an expansion of γδ T lymphocytes, which could drive the development of autoimmune cytopenias. We advocate that atypical SCID patients should be screened for elevated proportions of γδ T lymphocytes, CMV infection and autoimmune cytopenias.

Antigen-Presenting Cell Characteristics of Human γδ T Lymphocytes in Chronic Myeloid Leukemia.

Human γδ T lymphocytes play a role in the immune system defense against cancer. Their broad anti-cancer activity against different types of cancers makes them outstanding candidates for cancer immunotherapy. An issue of recent interest is whether their antigen presentation features are similar to mature dendritic cells. The antigen-presenting cell (APC)-like phenotype and function of γδ T lymphocytes have been confirmed in many clinical trials. In this study, to support the strong role played by Vγ9Vδ2 T cells against cancer, we provide evidence that Vγ9Vδ2 T cells activated with chronic myeloid leukemia (CML) cell lysate antigens can efficiently express an APC phenotype and function. Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with tumor cell lysate, and the tumor-activated Vγ9Vδ2 T cells could recognize and kill CML through their cytotoxic activity. In conclusion, the Vγ9Vδ2 T cells activated by cancer cell lysate showed APC characteristics, and this may greatly increase interest in investigating their therapeutic potential in hematologic malignancies. Abbreviations: CML: chronic myeloid leukemia; APC: antigen-presenting cell; TCR: T cell receptor; MHC: major histocompatibility complex; N-BPs: nitrogen-containing bisphosphonates; IPP: isopentenyl pyrophosphate; PBMC: peripheral blood mononuclear cells; NKG2D: natural killer receptor group 2, member D; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand.

The atypical chemokine receptor ACKR2 drives pulmonary fibrosis by tuning influx of CCR2+ and CCR5+ IFNγ-producing γδT cells in mice.

Chemokines coordinate lung inflammation and fibrosis by acting on chemokine receptors expressed on leukocytes and other cell types. Atypical chemokine receptors (ACKRs) bind, internalize, and degrade chemokines, tuning homeostasis and immune responses. ACKR2 recognizes and decreases the levels of inflammatory CC chemokines. The role of ACKR2 in fibrogenesis is unknown. The purpose of the study was to investigate the role of ACKR2 in the context of pulmonary fibrosis. The effects of ACKR2 expression and deficiency during inflammation and fibrosis were analyzed using a bleomycin-model of fibrosis, ACKR2-deficient mice, bone marrow chimeras, and antibody-mediated leukocyte depletion. ACKR2 was upregulated acutely in response to bleomycin and normalized over time. ACKR2-/- mice showed reduced lethality and lung fibrosis. Bone marrow chimeras showed that lethality and fibrosis depended on ACKR2 expression in pulmonary resident (nonhematopoietic) cells but not on leukocytes. ACKR2-/- mice exhibited decreased expression of tissue-remodeling genes, reduced leukocyte influx, pulmonary injury, and dysfunction. ACKR2-/- mice had early increased levels of CCL5, CCL12, CCL17, and IFNγ and an increased number of CCR2+ and CCR5+ IFNγ-producing γδT cells in the airways counterbalanced by low Th17-lymphocyte influx. There was reduced accumulation of IFNγ-producing γδT cells in CCR2-/- and CCR5-/- mice. Moreover, depletion of γδT cells worsened the clinical symptoms induced by bleomycin and reversed the phenotype of ACKR2-/- mice exposed to bleomycin. ACKR2 controls the CC chemokine expression that drives the influx of CCR2+ and CCR5+ IFNγ-producing γδT cells, tuning the Th17 response that mediated pulmonary fibrosis triggered by bleomycin instillation.

Subpopulation Structure of IFNγ-Producing T Lymphocytes in Patients with Pulmonary Tuberculosis.

The study of subpopulation structure of IFNγ-producing T cells in patients with pulmonary tuberculosis revealed a decrease in the number of CD3+ IFNγ+ cells against the background of significantly increased IFNγ secretion in vitro irrespective of the clinical form of the disease and drug sensitivity of M. tuberculosis, most strongly expressed in case of the disseminated tuberculosis. In patients with infiltrative drug-sensitive and drug-resistant pulmonary tuberculosis, increased number of Th1/Th17 lymphocytes (CD4+ IFNγ+IL-17A+) and, conversely, decreased number of blood γδT cells was detected.

Bovine peripheral blood WC1+ and WC1neg γδ T lymphocytes modulate monocyte-derived macrophage effector functions during in vitro Mycobacterium avium subspecies paratuberculosis infection.

The importance of bovine γδ T lymphocytes during anti-mycobacterial immunity is recognized; however, the role of major subsets of γδ T lymphocytes (WC1+ and WC1neg) in this process remains unclear. We investigated how WC1+ and WC1neg γδ T lymphocyte subsets of calves modulate monocyte-derived macrophage (MDM) functions during Map infection in vitro. To achieve this, Map-infected or uninfected MDMs from young calves were co-cultured with autologous WC1+ or WC1neg γδ T lymphocytes. Our data indicate that WC1+ and WC1neg γδ T lymphocytes of young calves modulate effector functions of MDMs with respect to Map killing, CD11b and MHC-II expression. We observed differences in IFN-γ production and CD25 expression on γδ T lymphocyte subsets, as well as MDM expression of CD1b when in contact with WC1neg γδ T lymphocytes.

Erratum: γδ T cells as immunotherapy for malaria: balancing challenges and opportunities.

[This corrects the article DOI: 10.3389/fimmu.2023.1242306.].

Suppression of pro-inflammatory T-cell responses by human mesothelial cells.

BACKGROUND: Human γδ T cells reactive to the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) contribute to acute inflammatory responses. We have previously shown that peritoneal dialysis (PD)-associated infections with HMB-PP producing bacteria are characterized by locally elevated γδ T-cell frequencies and poorer clinical outcome compared with HMB-PP negative infections, implying that γδ T cells may be of diagnostic, prognostic and therapeutic value in acute disease. The regulation by local tissue cells of these potentially detrimental γδ T-cell responses remains to be investigated. METHODS: Freshly isolated γδ or αß T cells were cultured with primary mesothelial cells derived from omental tissue, or with mesothelial cell-conditioned medium. Stimulation of cytokine production and proliferation by peripheral T cells in response to HMB-PP or CD3/CD28 beads was assessed by flow cytometry. RESULTS: Resting mesothelial cells were potent suppressors of pro-inflammatory γδ T cells as well as CD4+ and CD8+ αß T cells. The suppression of γδ T-cell responses was mediated through soluble factors released by primary mesothelial cells and could be counteracted by SB-431542, a selective inhibitor of TGF-ß and activin signalling. Recombinant TGF-ß1 but not activin-A mimicked the mesothelial cell-mediated suppression of γδ T-cell responses to HMB-PP. CONCLUSIONS: The present findings indicate an important regulatory function of mesothelial cells in the peritoneal cavity by dampening pro-inflammatory T-cell responses, which may help preserve the tissue integrity of the peritoneal membrane in the steady state and possibly during the resolution of acute inflammation.

Changes in γδT Cells in Peripheral Blood of Patients with Ulcerative Colitis Exacerbations.

The role of γδT cells in ulcerative colitis (UC) is well confirmed in experimental animals and demonstrated in many clinical observations. Recent investigations have indicated that UC is associated with several forms of immune imbalance, such as an imbalance between effector T cells and regulatory T cells. However, little is known about the cellular aspect of clinical colitis exacerbations. We observed 140 patients with histologically confirmed UC over the course of 8 years. We investigated the percentage of γδT and αßT cells in peripheral blood of patients and also the expression of various surface markers (CD25, CD54, CD62L). Patients were assembled into stable colitis and exacerbated colitis groups. The percentage of γδT and αßT cells was evaluated by Ortho Cytorone Absolute flow cytometer. In patients with exacerbated colitis we observed a decrease of γδT cells in peripheral blood and an increased ratio of αßT/γδT. Additionally, we found that exacerbation results in a significant increase of percentage of γδTCD25, γδTCD54 and γδTCD62L lymphocytes in peripheral blood when compared to patients with stable colitis. Exacerbation of ulcerative colitis results in a decreased percentage of γδT cells in peripheral blood with increase of CD25, CD54 and CD62L expressing γδT cells. This may represent the effect of cell activation and migration, similar to that observed after the surgical trauma. We hope that this observation may help to predict exacerbations in colitis patients.

γδ T Lymphocytes in Asthma: a Complicated Picture.

A minor subset (approximately 5%) of peripheral T cells has their TCR build up from γ and δ chains instead of α and ß-those are the γδ T lymphocytes. They can be functionally divided into subsets, e.g., Th1-, Th2-, Th9-, Th17-, Tfh-, and Treg-like γδ T cells. They share some specifics of both innate and adaptive immunity, and are capable of rapid response to a range of stimuli, including some viral and bacterial infections. Atopic diseases, including asthma, are one of major health-related problems of modern western societies. Asthma is one of the most common airway diseases, affecting people of all ages and having potential life-threatening consequences. In this paper, we review the current knowledge about the involvement of γδ T cells in the pathogenesis of asthma and its exacerbations. We summarize both the studies performed on human subjects as well as on the murine model of asthma. γδ T cells seem to be involved in the pathogenesis of asthma, different subsets probably perform opposite functions, e.g., symptom-exacerbating Vγ1 and symptom-suppressing Vγ4 in mice model of asthma.

Phenotypic and functional changes in gamma delta T lymphocytes from HTLV-1 carriers.

Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic inflammatory disease that leads to gradual loss of motor movement as a result of the death of spinal cord cells through immune mediated mechanisms. The risk to develop HAM/TSP disease positively correlates with the magnitude of HTLV-1 proviral load. Gamma-delta T lymphocytes have been recognized as important players in a variety of infectious diseases. Therefore, we have investigated interactions between HTLV-1 infection and γδ T lymphocytes during HAM/TSP. Similar frequencies of total γδ T lymphocytes and their Vγ9δ2+ and Vγ9δ2neg subpopulations were observed in HAM/TSP patients. However, T lymphocytes obtained from HTLV-1 carriers displayed significantly higher rates of spontaneous proliferation and NKp30 expression when compared to cells from uninfected donors. In addition, an important decrease in the frequency of granzyme B+ γδ T lymphocytes (approximately 50%) was observed in HAM/TSP patients. Higher proportion of IFN-γ+ γδ T lymphocytes was found in HTLV-1-infected patients, which positively correlated with the HTLV-1 proviral load in peripheral blood mononuclear cells. Collectively, our data indicates that HTLV-1 infection leads to phenotypic and functional changes in the population of γδ T lymphocyte population, suggesting that HTLV-1 infection modulates functions associated to these cells, which might be involved in controlling the infection or in the development of HTLV-1-associated diseases.

Human Vδ1+ T Cells in the Immune Response to Plasmodium falciparum Infection.

Naturally acquired protective immunity to Plasmodium falciparum malaria is mainly antibody-mediated. However, other cells of the innate and adaptive immune system also play important roles. These include so-called unconventional T cells, which express a γδ T-cell receptor (TCR) rather than the αß TCR expressed by the majority of T cells-the conventional T cells. The γδ T-cell compartment can be divided into distinct subsets. One expresses a TCR involving Vγ9 and Vδ2, while another major subset uses instead a TCR composed of Vδ1 paired with one of several types of γ chains. The former of these subsets uses a largely semi-invariant TCR repertoire and responds in an innate-like fashion to pyrophosphate antigens generated by various stressed host cells and infectious pathogens, including P. falciparum. In this short review, we focus instead on the Vδ1 subset, which appears to have a more adaptive immunobiology, but which has been much less studied in general and in malaria in particular. We discuss the evidence that Vδ1+ cells do indeed play a role in malaria and speculate on the function and specificity of this cell type, which is increasingly attracting the attention of immunologists.

γδ T Lymphocytes: An Effector Cell in Autoimmunity and Infection.

γδ T cells are non-conventional lymphocytes which show several properties of innate immune cells. They present a limited TCR repertoire and circulate as cells with a pre-activated phenotype thus being able to generate rapid immune responses. γδ T cells do not recognize classical peptide antigens, their TCRs are non-MHC restricted and they can respond to pathogen-associated molecular patterns and to cytokines in absence of TCR ligands. They also recognize self-molecules induced by stress, which indicate infection and cellular transformation. All these features let γδ T cells act as a first line of defense in sterile and non-sterile inflammation. γδ T cells represent 1-10% of circulating lymphocytes in the adult human peripheral blood, they are widely localized in non-lymphoid tissues and constitute the majority of immune cells in some epithelial surfaces, where they participate in the maintenance of the epithelial barriers. γδ T cells produce a wide range of cytokines that orchestrate the course of immune responses and also exert high cytotoxic activity against infected and transformed cells. In contrast to their beneficial role during infection, γδ T cells are also implicated in the development and progression of autoimmune diseases. Interestingly, several functions of γδ T cells are susceptible to modulation by interaction with other cells. In this review, we give an overview of the γδ T cell participation in infection and autoimmunity. We also revise the underlying mechanisms that modulate γδ T cell function that might provide tools to control pathological immune responses.

A highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) strongly modulates cellular innate and adaptive immune subsets upon experimental infection.

Highly pathogenic (HP) PRRSV isolates have been discovered within both PRRSV-1 and PRRSV-2 genotypes and investigated in recent years especially for their ability to cause extremely severe disease in conventional pig herds. The exacerbation of general and respiratory clinical signs has been attributed not only to an efficient replication (virulence) but also to the ability to dysregulate viral recognition and induce mechanisms of immune evasion or immune enhancement of humoral and cellular anti-viral responses differently from non-HP PRRSV isolates in terms of intensity and temporal onset. Thus, the understanding of the immunopathogenesis of HP PRRSV is a major concern for the study of virus biology and development of efficacious vaccines. The present study aims at addressing the modulation of relevant immune cell subsets by flow cytometry in the blood of 4-week-old pigs experimentally infected with the recently discovered PR40/2014 HP PRRSV-1.1 strain phenotypically characterized in Canelli et al. (2017) compared to pigs infected with a non-HP PRRSV isolate (PR11/2014) and uninfected controls. PR40 infected animals showed an early and marked reduction of pro-inflammatory CD172α+ CD14+CD16+ and CD14+CD163+ monocytes and TCRγδ+CD8α+/CD8α- lymphocytes when pigs were most infected, possibly due to a recruitment sustaining an acute inflammatory response in target tissues. The prolonged increased CD3+CD16+ NKT cell levels may sustain peripheral inflammation and/or the anti-viral response. The late reduction (potential depletion) of γ/δ T lymphocytes and CD3+CD4+CD8α- naïve Th lymphocytes paralleled with the delayed increase of CD3+CD4+CD8α+ memory and CD3+CD4-CD8α/ß + cytotoxic T lymphocytes. In addition, PR40 infection showed an early depletion of activated CD4+CD25+ T lymphocytes and Tregs together with an intense and lasting depletion of CD21+ B lymphocytes. Overall, these features demonstrate that the more severe clinical signs observed upon infection with the HP PR40 strain are sustained by remarkable changes in the peripheral blood distribution of immune cells and provide further insights into the immune regulation/immunopathogenesis induced by PRRSV-1 subtype 1 European isolates.

Differential effect of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) on leukocyte infiltration during contact hypersensitivity responses.

BACKGROUND: 2'-4' Dinitrofluorobenzene (DNFB) induced contact hypersensitivity is an established model of contact sensitivity and leukocyte migration. Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) deficient mice were used to examine the role of PECAM-1 in the migration capacity of several different leukocyte populations after primary and secondary application. RESULTS: γδ T lymphocytes, granulocytes, and Natural Killer cells were most affected by PECAM-1 deficiency at the primary site of application. γδ T lymphocytes, granulocytes, DX5+ Natural Killer cells, and, interestingly, effector CD4+ T lymphocytes were most affected by the loss of PECAM-1 at the secondary site of application. CONCLUSIONS: PECAM-1 is used by many leukocyte populations for migration, but there are clearly differential effects on the usage by each subset. Further, the overall kinetics of each population varied between primary and secondary application, with large relative increases in γδ T lymphocytes during the secondary response.

Bovine WC1+ and WC1neg γδ T Lymphocytes Influence Monocyte Differentiation and Monocyte-Derived Dendritic Cell Maturation during In Vitro Mycobacterium avium Subspecies paratuberculosis Infection.

During early Mycobacterium avium subspecies paratuberculosis (Map) infection, complex interactions occur between the bacteria, cells from the mononuclear phagocyte system (MPS) including both resident (macrophages and dendritic cells) and recruited (monocytes) cells, and other mucosal sentinel cells such as γδ T lymphocytes. Though the details of early host-pathogen interactions in cattle remain largely underexplored, our hypothesis is that these significantly influence development of host immunity and ultimate success or failure of the host to respond to Map infection. The aims of the present study were to first characterize monocyte-derived MPS cells from young calves with respect to their immunophenotype and function. Then, we set out to investigate the effects of WC1+ and WC1neg γδ T lymphocytes on (1) the differentiation of autologous monocytes and (2) the maturation of autologous monocyte-derived dendritic cells (MDDCs). To achieve this, peripheral blood WC1+ or WC1neg γδ T lymphocytes were cocultured with either autologous freshly isolated peripheral blood-derived monocytes or autologous immature MDDCs (iMDDCs). We began by measuring several markers of interest on MPS cells. Useful markers to distinguish monocyte-derived macrophages (MDMs) from MDDCs include CD11b, CD163, and CD172a, which are expressed significantly higher on MDMs compared with MDDCs. Function, but not phenotype, was influenced by WC1neg γδ T lymphocytes: viability of Map harvested from monocytes differentiated in the presence of WC1neg γδ T lymphocytes (dMonWC1neg) was significantly lower compared to MDMs and MDDCs. With respect to DC maturation, we first showed that mature MDDCs (mMDDCs) have significantly higher expression of CD11c, CD80, and CD86 compared with iMDDCs, and the phagocytic capacity of mMDDCs is significantly reduced compared to iMDDCs. We then showed that γδ T lymphocyte subsets induce functional (reduced phagocytosis) but not phenotypic (surface marker expression) iMDDC maturation. These data collectively show that γδ T lymphocytes influence differentiation, maturation, and ultimately the function of monocytes during Map infection, which has significant implications on survival of Map and success of host defense during early Map infection.

Bovine WC1(+) γδ T lymphocytes modify monocyte-derived macrophage responses during early Mycobacterium avium subspecies paratuberculosis infection.

Following Mycobacterium avium subspecies paratuberculosis (Map) infection, some calves are apparently able to successfully clear the pathogen whereas others become persistently infected; however the reasons for this remain unknown. The importance of innate immunity, and in particular the role of γδ T lymphocytes, during early anti-mycobacterial immune response is recognized but specific mechanisms remain incompletely characterized. The objective of this study was to investigate how bovine WC1(+) γδ T lymphocytes mediate macrophage function during early Map infection. To achieve this objective, Map-infected monocyte-derived macrophages (MDMs) were co-cultured either in direct contact with, or separated by a semi-permeable membrane from, autologous WC1(+) γδ T lymphocytes. Nitrites, IL-17A, IFN-γ, IL-4 and IL-10 from cell culture supernatants were measured. Expression of CD25 on WC1(+) γδ T lymphocytes, expression of MHC-I and MHC-II on MDMs and the viability of Map recovered from MDM cultures 72h after Map infection were also assessed. Map viability was significantly reduced when WC1(+) γδ T lymphocytes were co-cultured in direct contact with Map-infected MDMs. Both MDMs and WC1(+) γδ T lymphocytes generated increased concentrations of IFN-γ and IL-4 in our system, and MDM/WC1(+) γδ T lymphocyte synergism was identified for IFN-γ production. MDMs but not WC1(+) γδ T lymphocytes were a significant source of IL-17A. The presence of WC1(+) γδ T lymphocytes was associated with higher expression of MHC-I on MDMs and increased concentration of nitrites in supernatants 72h after Map infection. In conclusion, this study showed that WC1(+) γδ lymphocytes had differential effects on Map-infected macrophages in vitro.