•Neoadjuvant ipilimumab and IFNα were evaluated in regionally advanced melanoma.•Immune cellular profiling investigated tumor immune susceptibility and resistance.•Higher levels of peripheral Th1 ...cell subsets predicted favorable clinical outcomes.•Higher levels of peripheral Th2 cells was associated with poor prognosis.•Significant reductions in peripheral T-reg and MDSC were seen in responders.
Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial NCT01608594. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.
A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months ...in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks) was also ineffective, while proinsulin DNA (weekly for up to 12 weeks) showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04). In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.
Human Vγ2Vδ2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent ...anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vγ2Vδ2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vγ2Vδ2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vγ2Vδ2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vγ2Vδ2 T cells in vivo upon engraftment into severe combined immunodeficiency–beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vγ2Vδ2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vγ2Vδ2 T cells to respond to a broad array of pathogens using this pathway.
BackgroundNatural Killer (NK) cells have garnered increasing interest as potential cellular therapies or as targets of biotherapeutic agents due to their ability to kill tumor cells in a non-antigen ...dependent manner. Hence, measurement of NK cell proliferation and/or activation following treatment can serve as a useful biomarker for assessing the efficacy of immunomodulatory therapies.MethodsWe developed a novel 13-parameter flow cytometry panel incorporating cell differentiation (CD) markers important for identification of NK cell subsets (CD56, CD16), their proliferation (Ki-67), activation (CD25, CD335, NKG2D) and inhibition (CD159a) status. Additionally, CD markers that identify other cellular subsets known to be amenable to cytokine modulation (e.g., CD3 and CD14) were included for concurrent monitoring of T cell proliferation and monocyte activation. Method validation focused on analytical sensitivity, specificity and precision as key criteria of assay performance using peripheral blood mononuclear cells (PBMCs) stimulated with NK cell-activating cytokines and resting PBMCs from healthy donors.ResultsThe assay design allowed for robust quantitation of NK cell, T cell and monocyte functionalities. Lower limit of quantification (LLOQ) of target biomarker population was determined to be 1.0% of the parent population, based upon an analysis of 110 key target populations that displayed a co-efficient of variation (CV) of ≤25% and their frequencies ranged from 0.1% to 97.8% of the parent population. Additionally, ≤25% CV was observed in precision assessments, confirming the repeatability and reproducibility of the assay. Clinical trial utility of the assay was verified on cryopreserved PBMCs from patients with a variety of solid tumor malignancies. In these patients, the assay could clearly identify proliferating and activated NK cells, as well as proliferating T cells and activated monocytes, thus demonstrating its suitability for clinical trial applications.ConclusionsWe developed and validated a novel multiparameter flow cytometry assay that allows for simultaneous measurement of proliferation, activation and inhibitory status of key immune cell subsets. Thus, this assay can help shed light on the mode of efficacy of novel therapeutic agents that modulate the immune system, aimed at treatment of cancer and autoimmune diseases.
The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, ...such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4(+) T cells of the BDC12-4.1 clone to convert into Foxp3(+) iTreg cells. We found that in vitro polarization toward Foxp3(+) iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3(+) BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4(+) T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3(+) cell phenotype and its associated in vivo regulatory potential.
Multiple immune parameters such as frequencies of autoreactive CD4(+), CD8(+) T-cells and CD4(+)CD25(+)Foxp3(+) T-cells have been explored as biomarkers in human T1D. However, intra-individual ...temporal variation of these parameters has not been assessed systematically over time. We determined the variation in each of these parameters in a cohort of T1D and healthy donors (HDs), at monthly intervals for one year. Despite low intra- and inter-assay co-efficient of variation (CV), mean CVs for each of the immune parameters were 119.1% for CD4(+) T-cell-derived IFN-γ, 50.44% for autoreactive CD8(+) T-cells, and 31.24% for CD4(+)CD25(+)Foxp3(+) T-cells. Further, both HDs and T1D donors had similar CVs. The variation neither correlated with BMI, age, disease duration or insulin usage, nor were there detectable cyclical patterns of variation. However, averaging results from multiple visits for an individual provided a better estimate of the CV between visits. Based on our data we predict that by averaging values from three visits a treatment effect on these parameters with a 50% effect size could be detected with the same power using 1.8-4-fold fewer patients within a trial compared to using values from a single visit. Thus, our present data contribute to a more robust, accurate endpoint design for future clinical trials in T1D and aid in the identification of truly efficacious therapies.
Human Vγ2Vδ2 T cells play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. Vγ2Vδ2 T cells recognize the pyrophosphorylated isoprenoid ...intermediates (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the foreign 2-C-methyl- d-erythritol 4-phosphate (MEP) pathway, and isopentenyl pyrophosphate (IPP), an intermediate in the self-mevalonate pathway. Infection with bacteria and protozoa using the MEP pathway leads to the rapid expansion of Vγ2Vδ2 T cells to very high numbers through preferential recognition of HMBPP. Activated Vγ2Vδ2 T cells produce proinflammatory cytokines and chemokines, kill infected cells, secrete growth factors for epithelial cells, and present antigens to αβ T cells. Vγ2Vδ2 T cells can also recognize high levels of IPP in certain tumors and in cells treated with pharmacological agents, such as bisphosphonates and alkylamines, that block farnesyl pyrophosphate synthase. Activated Vγ2Vδ2 T cells are able to kill most tumor cells because of recognition by T-cell receptor and natural killer receptors. The ubiquitous nature of the antigens converts essentially all Vγ2Vδ2 T cells to memory cells at an early age. Thus, primary infections with HMBPP-producing bacteria are perceived by Vγ2Vδ2 T cells as a repeat infection. Extensive efforts are underway to harness these cells to treat a variety of cancers and to provide microbial immunity.
Human V gamma 2V delta 2 T cells play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. V gamma 2V delta 2 T cells recognize the ...pyrophosphorylated isoprenoid intermediates (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the foreign 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, and isopentenyl pyrophosphate (IPP), an intermediate in the self-mevalonate pathway. Infection with bacteria and protozoa using the MEP pathway leads to the rapid expansion of V gamma 2V delta 2 T cells to very high numbers through preferential recognition of HMBPP. Activated V gamma 2V delta 2 T cells produce proinflammatory cytokines and chemokines, kill infected cells, secrete growth factors for epithelial cells, and present antigens to alpha beta T cells. V gamma 2V delta 2 T cells can also recognize high levels of IPP in certain tumors and in cells treated with pharmacological agents, such as bisphosphonates and alkylamines, that block farnesyl pyrophosphate synthase. Activated V gamma 2V delta 2 T cells are able to kill most tumor cells because of recognition by T-cell receptor and natural killer receptors. The ubiquitous nature of the antigens converts essentially all V gamma 2V delta 2 T cells to memory cells at an early age. Thus, primary infections with HMBPP-producing bacteria are perceived by V gamma 2V delta 2 T cells as a repeat infection. Extensive efforts are underway to harness these cells to treat a variety of cancers and to provide microbial immunity.
Exceptional clinical responses produced by the first chimeric antigen receptor T CAR‐T cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next ...generation CAR‐T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these “living” drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR‐T therapies. Herein, three main areas for monitoring CAR‐T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR‐T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit‐for‐purpose tools and help to more rapidly advance the development of next generation CAR‐T therapies.
Human V gamma 2V delta 2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-D-erythritol-4-phosphate ...pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate V gamma 2V delta 2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of V gamma 2V delta 2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for V gamma 2V delta 2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of V gamma 2V delta 2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of V gamma 2V delta 2 T cells for adoptive cancer immunotherapy.
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