Effect of pembrolizumab on CD4+ CD25+ , CD4+ LAP+ and CD4+ TIM-3+ T cell subsets.

Tumor immune evasion involves the expansion of avidly proliferating immunosuppressive cells and inhibition of effector T cell proliferation. Immune checkpoints (IC) block the activation pathways of tumor-reactive T cells. IC pathways are often exploited by tumor cells to evade immune destruction, and blocking these pathways through IC inhibitors (ICI) has shown promising results in multiple malignancies. In this study, we investigated the effects of an ICI, pembrolizumab, on various T cell subsets in vitro. We compared the suppressive activity of CD4+ CD25+ regulatory T cells (conventional Treg ) with T cells expressing T cell immunoglobulin-3+ (TIM-3+ ) and latency-associated peptide (LAP)+ T cells. We found that LAP-expressing T cells were more suppressive than conventional Treg , but TIM-3-expressing T cells were not suppressive. Our results show that pembrolizumab does not modulate functions of Treg and mediates its immunostimulatory effects via the release of effector T cells from suppression. These findings may assist in the development of agents designed to intervene in IC pathways to overcome Treg resistance to ICI.

In-vitro effect of pembrolizumab on different T regulatory cell subsets.

Programmed death-1 (PD-1) and interactions with PD-ligand 1 (PD-L1) play critical roles in the tumour evasion of immune responses through different mechanisms, including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumour-infiltrating T cells and regulatory T cell (Treg ) expansion. Effective blockade of immune checkpoints can therefore potentially eliminate these detrimental effects. The aim of this study was to investigate the effect of anti-PD-1 antibody, pembrolizumab, on various Treg subpopulations. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and primary breast cancer patients (PBC) were treated in vitro with pembrolizumab, which effectively reduced PD-1 expression in both cohorts. We found that PD-1 was expressed mainly on CD4+ CD25+ T cells and pembrolizumab had a greater effect on PD-1 expression in CD4+ CD25- T cells, compared to CD4+ CD25+ cells. In addition, pembrolizumab did not affect the expression levels of Treg -related markers, including cytotoxic T lymphocyte antigen-4 (CTLA-4), CD15s, latency-associated peptide (LAP) and Ki-67. Moreover, we report that CD15s is expressed mainly on forkhead box P3 (FoxP3)- Helios+ Treg in HD, but it is expressed on FoxP3+ Helios- Treg subset in addition to FoxP3- Helios+ Treg in PBC. Pembrolizumab did not affect the levels of FoxP3+/- Helios+/- Treg subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect Treg or change their phenotype or function but rather blocks signalling via the PD-1/PD-L1 axis in activated T cells.

5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia and is associated with a more invasive and chemotactic phenotype.

Although the overall prognosis in childhood acute lymphoblastic leukemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterized by the occurrence of disease at extramedullary sites, such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofetal antigen transcript levels in cytogenetic high-risk subgroups of patients (P<0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4-positive leukemic blasts than healthy donors (P=0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4(+) (Sup5T4) and 5T4(-) (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared with Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4-positive Sup-B15 cells are susceptible to 5T4-specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high-risk pre-B-ALL.

Novel IFNγ ELISPOT assay for detection of functional carcinoembryonic antigen-specific chimeric antigen receptor-redirected T cells.

We here describe the development of a novel ELISPOT assay for the detection and enumeration of IFNγ-secreting functional chimeric antigen receptor (CAR)-redirected T cells against carcinoembryonic antigen (CEA). This method is valuable for clinical trials to monitor the presence of functional CEA-specific T cells transduced with a CAR. The same principle should be applicable for the detection of functional CAR-redirected T cells against any other tumour-associated antigens by immobilizing a particular biotinylated antigen to streptavidin-coated beads.

Phase II trial of imiquimod and HPV therapeutic vaccination in patients with vulval intraepithelial neoplasia.

BACKGROUND: Vulval intraepithelial neoplasia (VIN) is a premalignant condition, which is frequently associated with type HPV16 infection, and multifocal disease has high rates of surgical treatment failure. METHODS: We report a phase II clinical trial of the topical immunomodulator, imiquimod, for 8 weeks, followed by 3 doses (weeks 10, 14 and 18) of therapeutic human papillomavirus (HPV) vaccination (TA-CIN, fusion protein HPV16 E6E7L2) in 19 women with VIN grades 2 and 3. Histology and HPV testing of biopsies were performed at weeks 0, 10, 20 and 52. Intralesional infiltration of T-cell subsets and lymphocyte proliferation for HPV systemic immune responses were also assessed. RESULTS: Lesion response (complete regression of VIN on histology) was observed in 32% (6 out of 19) of women at week 10, increasing to 58% (11 out of 19) at week 20 and 63% (12 out of 19) at week 52. At this time, 36% (5 out of 14) of lesions showed HPV16 clearance and 79% (15 out of 19) of women were symptom free. At week 20, after treatment with imiquimod and vaccination, there was significantly increased local infiltration of CD8 and CD4 T cells in lesion responders; in contrast, non-responders (persistent VIN by histology) showed an increased density of T regulatory cells. After vaccination, only lesion responders had significantly increased lympho-proliferation to the HPV vaccine antigens. CONCLUSION: The therapeutic effect of treatment depends on the differential immune response of responders and non-responders with affect locally and systemically.

Novel therapeutic strategies by regulatory T cells in allergy.

Natural CD4+CD25+FoxP3+ regulatory T cells (Treg) actively suppress physiological and pathological responses, therefore playing a critical role in controlling peripheral tolerance to self antigens and maintaining immune homeostasis. In normal individuals, natural Treg and interleukin- 10-secreting Treg are able to suppress Th2 responses to allergens, whereas lower levels of Treg or defect in their functionality have been described as potential mechanisms for inducing allergic diseases. In animal models, adoptive transfer of CD4+CD25+Treg has been shown as a promising strategy for preventing or treating allergic disorders. Recent studies show that induction of Treg activity is associated with suppression of allergic responses in allergic patients treated with specific immunotherapy. Herein, I review the potential of Treg as exciting targets for developing new immunotherapeutic strategies for treating allergic diseases.

Immunology and immunotherapy approaches for prostate cancer.

Several mechanisms that impair the immune response to promote tumour progression are reported. These mechanisms aim to reduce the ability of antigen-presenting cells to present antigen and activate naïve T cells to support an active immune response or to create a suppressive environment that induce non-functional tumour-associated antigen-specific T cells. Prostate cancer (PC) alone accounts for 33% of incident cancer cases and about 9% of all cancer-related deaths among men in the USA during 2006. Whereas androgen deprivation has remained the first line of therapy for advanced PC, other therapies are still required due to progression to an androgen-resistant state and eventually loss of control in patients receiving hormonal therapy. Immunotherapy seems to be a promising approach to enhance tumour-specific T-cell responses in different cancers including prostate. More importantly, clinical trials in advanced PC patients have shown that immunotherapy may generate significant clinical responses. Immunology and immunotherapy aspects of PC with focus on prostate-specific antigen will be presented.