Nadia Caccamo

Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer

The increasing evidence that gammadelta T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormone-refractory prostate cancer to examine the feasibility and consequences of using the gammadelta T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood gammadelta cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant long-term shift of peripheral gammadelta cells toward an activated effector-memory-like state (T(EM)), producing IFN-gamma and perforin. These patients also maintained serum levels of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by gammadelta cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T(EM) gammadelta cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either gammadelta cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, gammadelta cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma.

Differentiation of Effector/Memory Vδ2 T Cells and Migratory Routes in Lymph Nodes or Inflammatory Sites

Vδ2 T lymphocytes recognize nonpeptidic antigens without presentation by MHC molecules and mount both immediate effector functions and memory responses after microbial infection. However, how Vδ2 T cells mediate different facets of a memory response remains unknown. Here, we show that the expression of CD45RA and CD27 antigens defines four subsets of human Vδ2 T cells with distinctive compartmentalization routes. Naive CD45RA+CD27+ and memory CD45RA−CD27+ cells express lymph node homing receptors, abound in lymph nodes, and lack immediate effector functions. Conversely, memory CD45RA−CD27− and terminally differentiated CD45RA+CD27− cells, which express receptors for homing to inflamed tissues, are poorly represented in the lymph nodes while abounding at sites of inflammation, and display immediate effector functions. These observations and additional in vitro experiments indicate a lineage differentiation pattern for human Vδ2 T cells that generates naive cells circulating in lymph nodes, effector/memory cells patrolling the blood, and terminally differentiated effector cells residing in inflamed tissues.

Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4+ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4+ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4+ T‐cell subsets. Proportions of the other double or single CD4+ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4+ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4+ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.

Efficient Killing of Human Colon Cancer Stem Cells by γδ T Lymphocytes

Colon cancer comprises a small population of cancer stem cells (CSC) that is responsible for tumor maintenance and resistant to cancer therapies, possibly allowing for tumor recapitulation once treatment stops. We previously demonstrated that such chemoresistance is mediated by autocrine production of IL-4 through the up-regulation of antiapoptotic proteins. Several innate and adaptive immune effector cells allow for the recognition and destruction of cancer precursors before they constitute the tumor mass. However, cellular immune-based therapies have not been experimented yet in the population of CSCs. Here, we show that the bisphosphonate zoledronate sensitizes colon CSCs to Vγ9Vδ2 T cell cytotoxicity. Proliferation and production of cytokines (TNF-α and IFN-γ) and cytotoxic and apoptotic molecules (TRAIL and granzymes) were also induced after exposure of Vγ9Vδ2 T cells to sensitized targets. Vγ9Vδ2 T cell cytotoxicity was mediated by the granule exocytosis pathway and was highly dependent on isoprenoid production by of tumor cells. Moreover, CSCs recognition and killing was mainly TCR mediated, whereas NKG2D played a role only when tumor targets expressed several NKG2D ligands. We conclude that intentional activation of Vγ9Vδ2 T cells by zoledronate may substantially increase antitumor activities and represent a novel strategy for colon cancer immunotherapy.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti‐tumour activities of γδ T cells have prompted the development of protocols in which γδ‐agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vγ9Vδ2 T cell agonist, plus low‐dose interleukin (IL)‐2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vγ9Vδ2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vγ9Vδ2 T cell numbers emerged, as seven patients who failed to sustain Vγ9Vδ2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vγ9Vδ2 cell populations showed declining CA15‐3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vγ9Vδ2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low‐dose IL‐2 offers a novel, safe and feasible approach to enhance this in a subset of treatment‐refractory patients with advanced breast cancer.

Differentiation, phenotype, and function of interleukin-17–producing human Vγ9Vδ2 T cells

In healthy adults, the major peripheral blood γδ T-cell subset expresses the Vγ9Vδ2 TCR and displays pleiotropic features. Here we report that coculture of naive Vγ9Vδ2 T cells with phosphoantigens and a cocktail of cytokines (IL-1-β, TGF-β, IL-6, and IL-23), leads to selective expression of the transcription factor RORγt and polarization toward IL-17 production. IL-17(+) Vγ9Vδ2 T cells express the chemokine receptor CCR6 and produce IL-17 but neither IL-22 nor IFN-γ; they have a predominant terminally differentiated (CD27(-)CD45RA(+)) phenotype and express granzyme B, TRAIL, FasL, and CD161. On antigen activation, IL-17(+) Vγ9Vδ2 T cells rapidly induce CXCL8-mediated migration and phagocytosis of neutrophils and IL-17-dependent production of β-defensin by epithelial cells, indicating that they may be involved in host immune responses against infectious microorganisms. Accordingly, an increased percentage of IL-17(+) Vγ9Vδ2 lymphocytes is detected in the peripheral blood and at the site of disease in children with bacterial meningitis, and this pattern was reversed after successful antibacterial therapy. Most notably, the phenotype of IL-17(+) Vγ9Vδ2 T cells in children with meningitis matches that of in vitro differentiated IL-17(+) Vγ9Vδ2 T cells. Our findings delineate a previously unknown subset of human IL-17(+) Vγ9Vδ2 T lymphocytes implicated in the pathophysiology of inflammatory responses during bacterial infections.

Functional Signatures of Human CD4 and CD8 T Cell Responses to Mycobacterium tuberculosis

With 1.4 million deaths and 8.7 million new cases in 2011, tuberculosis (TB) remains a global health care problem and together with HIV and Malaria represents one of the three infectious diseases world-wide. Control of the global TB epidemic has been impaired by the lack of an effective vaccine, by the emergence of drug-resistant forms of Mycobacterium tuberculosis (Mtb) and by the lack of sensitive and rapid diagnostics. It is estimated, by epidemiological reports, that one third of the world’s population is latently infected with Mtb, but the majority of infected individuals develop long-lived protective immunity, which controls and contains Mtb in a T cell-dependent manner. Development of TB disease results from interactions among the environment, the host, and the pathogen, and known risk factors include HIV co-infection, immunodeficiency, diabetes mellitus, overcrowding, malnutrition, and general poverty; therefore, an effective T cell response determines whether the infection resolves or develops into clinically evident disease. Consequently, there is great interest in determining which T cells subsets mediate anti-mycobacterial immunity, delineating their effector functions. On the other hand, many aspects remain unsolved in understanding why some individuals are protected from Mtb infection while others go on to develop disease. Several studies have demonstrated that CD4+ T cells are involved in protection against Mtb, as supported by the evidence that CD4+ T cell depletion is responsible for Mtb reactivation in HIV-infected individuals. There are many subsets of CD4+ T cells, such as T-helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs), and all these subsets co-operate or interfere with each other to control infection; the dominant subset may differ between active and latent Mtb infection cases. Mtb-specific-CD4+ Th1 cell response is considered to have a protective role for the ability to produce cytokines such as IFN-γ or TNF-α that contribute to the recruitment and activation of innate immune cells, like monocytes and granulocytes. Thus, while other antigen (Ag)-specific T cells such as CD8+ T cells, natural killer (NK) cells, γδ T cells, and CD1-restricted T cells can also produce IFN-γ during Mtb infection, they cannot compensate for the lack of CD4+ T cells. The detection of Ag-specific cytokine production by intracellular cytokine staining (ICS) and the use of flow cytometry techniques are a common routine that supports the studies aimed at focusing the role of the immune system in infectious diseases. Flow cytometry permits to evaluate simultaneously the presence of different cytokines that can delineate different subsets of cells as having “multifunctional/polyfunctional” profile. It has been proposed that polyfunctional T cells, are associated with protective immunity toward Mtb, in particular it has been highlighted that the number of Mtb-specific T cells producing a combination of IFN-γ, IL-2, and/or TNF-α may be correlated with the mycobacterial load, while other studies have associated the presence of this particular functional profile as marker of TB disease activity. Although the role of CD8 T cells in TB is less clear than CD4 T cells, they are generally considered to contribute to optimal immunity and protection. CD8 T cells possess a number of anti-microbial effector mechanisms that are less prominent or absent in CD4 Th1 and Th17 T cells. The interest in studying CD8 T cells that are either MHC-class Ia or MHC-class Ib-restricted, has gained more attention. These studies include the role of HLA-E-restricted cells, lung mucosal-associated invariant T-cells (MAIT), and CD1-restricted cells. Nevertheless, the knowledge about the role of CD8+ T cells in Mtb infection is relatively new and recent studies have delineated that CD8 T cells, which display a functional profile termed “multifunctional,” can be a better marker of protection in TB than CD4+ T cells. Their effector mechanisms could contribute to control Mtb infection, as upon activation, CD8 T cells release cytokines or cytotoxic molecules, which cause apoptosis of target cells. Taken together, the balance of the immune response in the control of infection and possibly bacterial eradication is important in understanding whether the host immune response will be appropriate in contrasting the infection or not, and, consequently, the inability of the immune response, will determine the dissemination and the transmission of bacilli to new subjects. In conclusion, the recent highlights on the role of different functional signatures of T cell subsets in the immune response toward Mtb infection will be discerned in this review, in order to summarize what is known about the immune response in human TB. In particular, we will discuss the role of CD4 and CD8 T cells in contrasting the advance of the intracellular pathogen in already infected people or the progression to active disease in subjects with latent infection. All the information will be aimed at increasing the knowledge of this complex disease in order to improve diagnosis, prognosis, drug treatment, and vaccination.

V gamma 9V delta 2 T lymphocytes efficiently recognize and kill zoledronate-sensitized, imatinib-sensitive, and imatinib-resistant chronic myelogenous leukemia cells.

Imatinib mesylate (imatinib), a competitive inhibitor of the BCR-ABL tyrosine kinase, is highly effective against chronic myelogenous leukemia (CML) cells. However, because 20–30% of patients affected by CML display either primary or secondary resistance to imatinib, intentional activation of Vγ9Vδ2 T cells by phosphoantigens or by agents that cause their accumulation within cells, such as zoledronate, may represent a promising strategy for the design of a novel and highly innovative immunotherapy capable to overcome imatinib resistance. In this study, we show that Vγ9Vδ2 T lymphocytes recognize, trogocytose, and efficiently kill imatinib-sensitive and -resistant CML cell lines pretreated with zoledronate. Vγ9Vδ2 T cell cytotoxicity was largely dependent on the granule exocytosis- and partly on TRAIL-mediated pathways, was TCR-mediated, and required isoprenoid biosynthesis by zoledronate-treated CML cells. Importantly, Vγ9Vδ2 T cells from patients with CML can be induced by zoledronate to develop antitumor activity against autologous and allogeneic zoledronate-treated leukemia cells, both in vitro and when transferred into immunodeficient mice in vivo. We conclude that intentional activation of Vγ9Vδ2 T cells by zoledronate may substantially increase their antileukemia activities and represent a novel strategy for CML immunotherapy.

Damping Excessive Inflammation and Tissue Damage in Mycobacterium tuberculosis Infection by Toll IL-1 Receptor 8/Single Ig IL-1-Related Receptor, a Negative Regulator of IL-1/TLR Signaling

Toll IL-1R 8/single Ig IL-1-related receptor (TIR8/SIGIRR) is a member of the IL-1R family, expressed by epithelial tissues and immature dendritic cells, and is regarded as a negative regulator of TLR/IL-1R signaling. Tir8-deficient mice were rapidly killed by intranasal administration of low doses of Mycobacterium tuberculosis, despite controlling efficiently the number of viable bacilli in different organs. Tir8−/−-infected mice showed an increased number of neutrophils and macrophages in the lungs; however, mycobacteria-specific CD4 and CD8 T cells were similar in Tir8−/− and Tir8+/+ mice. Exaggerated mortality of Tir8−/− mice was due to massive liver necrosis and was accompanied by increased levels of IL-1β and TNF-α in lung mononuclear cells and serum, as well as by increased production of IL-1β and TNF-α by M. tuberculosis-infected dendritic cells in vitro. Accordingly, blocking IL-1β and TNF-α with a mix of anti-cytokine Abs, significantly prolonged survival of Tir8−/− mice. Thus, TIR8/SIGIRR plays a key role in damping inflammation and tissue damage in M. tuberculosis infection.

Characterization of Lung γδ T Cells Following Intranasal Infection with Mycobacterium bovis Bacillus Calmette-Guérin

The lungs are considered to have an impaired capacity to contain infection by pathogenic mycobacteria, even in the presence of effective systemic immunity. In an attempt to understand the underlying cellular mechanisms, we characterized the γδ T cell population following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). The peak of γδ T cell expansion at 7 days postinfection preceded the 30 day peak of αβ T cell expansion and bacterial count. The expanded population of γδ T cells in the lungs of BCG-infected mice represents an expansion of the resident Vγ2 T cell subset as well as an influx of Vγ1 and of four different Vδ gene-bearing T cell subsets. The γδ T cells in the lungs of BCG-infected mice secreted IFN-γ following in vitro stimulation with ionomycin and PMA and were cytotoxic against BCG-infected peritoneal macrophages as well as against the uninfected J774 macrophage cell line. The cytotoxicity was selectively blocked by anti-γδ TCR mAb and strontium ions, suggesting a granule-exocytosis killing pathway. Depletion of γδ T cells by injection of specific mAb had no effect on the subsequent developing CD4 T cell response in the lungs of BCG-infected mice, but significantly reduced cytotoxic activity and IFN-γ production by lung CD8 T cells. Thus, γδ T cells in the lungs might help to control mycobacterial infection in the period between innate and classical adaptive immunity and may also play an important regulatory role in the subsequent onset of αβ T lymphocytes.