Microbiome-based interventions: therapeutic strategies in cancer immunotherapy.

The composition of the commensal microbiota has recently emerged as a key element influencing the efficacy of cancer treatments. It has become apparent that the interplay between the microbiome and immune system within the host influences the response to immunotherapy, particularly immune checkpoint inhibitor therapy. Identifying the key components of the gut microbiota that influence this response is paramount for designing therapeutic interventions to enhance the response to cancer therapy. This review will discuss strategies being considered to modulate the gut microbiota, including fecal microbiota transplantation, administration of defined bacterial isolates as well as bacterial consortia, supplementation with probiotics, and lifestyle modifications such as dietary changes. Understanding the influence of the complex variables of the human microbiota on the effectiveness of cancer therapy will help drive the clinical design of microbial-based interventions in the field of oncology.

Major pathologic response on biopsy (MPRbx) in patients with advanced melanoma treated with anti-PD-1: evidence for an early, on-therapy biomarker of response.

BACKGROUND: With increasing anti-PD-1 therapy use in patients with melanoma and other tumor types, there is interest in developing early on-treatment biomarkers that correlate with long-term patient outcome. An understanding of the pathologic features of immune-mediated tumor regression is key in this endeavor. MATERIALS AND METHODS: Histologic features of immune-related pathologic response (irPR) following anti-PD-1 therapy were identified on hematoxylin and eosin (H&E)-stained slides in a discovery cohort of pre- and on-treatment specimens from n = 16 patients with advanced melanoma. These features were used to generate an irPR score [from 0 = no irPR features to 3 = major pathologic response on biopsy (MPRbx, ≤10% residual viable tumor)]. This scoring system was then tested for an association with objective response by RECIST1.1 and overall survival in a prospectively collected validation cohort of pre- and on-treatment biopsies (n = 51 on-treatment at 4-week timepoint) from melanoma patients enrolled on the nivolumab monotherapy arm of CA209-038 (NCT01621490). RESULTS: Specimens from responders in the discovery cohort had features of immune-activation (moderate-high TIL densities, plasma cells) and wound-healing/tissue repair (neovascularization, proliferative fibrosis) compared to nonresponders, (P ≤ 0.021, for each feature). In the validation cohort, increasing irPR score associated with objective response (P = 0.009) and MPRbx associated with increased overall survival (n = 51; HR 0.13; 95%CI, 0.054-0.31, P = 0.015). Neither tumoral necrosis nor pretreatment histologic features were associated with response. Eight of 16 (50%) of patients with stable disease showed irPR features, two of which were MPRbx, indicating a disconnect between pathologic and radiographic features at the 4-week on-therapy timepoint for some patients. CONCLUSIONS: Features of immune-mediated tumor regression on routine H&E-stained biopsy slides from patients with advanced melanoma correlate with objective response to anti-PD-1 and overall survival. An on-therapy biopsy may be particularly clinically useful for informing treatment decisions in patients with radiographic stable disease. This approach is inexpensive, straightforward, and widely available.

Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints.

T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8(+) T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for Gαi-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8(+) effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8(+) effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy.

Efficacy and safety of ipilimumab monotherapy in patients with pretreated advanced melanoma: a multicenter single-arm phase II study.

BACKGROUND: This phase II study evaluated the safety and activity of ipilimumab, a fully human mAb that blocks cytotoxic T-lymphocyte antigen-4, in patients with advanced melanoma. PATIENTS AND METHODS: Patients with previously treated, unresectable stage III/stage IV melanoma received 10 mg/kg ipilimumab every 3 weeks for four cycles (induction) followed by maintenance therapy every 3 months. The primary end point was best overall response rate (BORR) using modified World Health Organization (WHO) criteria. We also carried out an exploratory analysis of proposed immune-related response criteria (irRC). RESULTS: BORR was 5.8% with a disease control rate (DCR) of 27% (N = 155). One- and 2-year survival rates (95% confidence interval) were 47.2% (39.5% to 55.1%) and 32.8% (25.4% to 40.5%), respectively, with a median overall survival of 10.2 months (7.6-16.3). Of 43 patients with disease progression by modified WHO criteria, 12 had disease control by irRC (8% of all treated patients), resulting in a total DCR of 35%. Adverse events (AEs) were largely immune related, occurring mainly in the skin and gastrointestinal tract, with 19% grade 3 and 3.2% grade 4. Immune-related AEs were manageable and generally reversible with corticosteroids. CONCLUSION: Ipilimumab demonstrated clinical activity with encouraging long-term survival in a previously treated advanced melanoma population.

Peripheral survival of naïve CD8+ T cells.

Maintenance of a sufficient population of naive CD8+ T cells in the peripheral lymphoid compartment is critical for immunocompetence. Peripheral T cell number is a function of T cell generation, survival, and death. Homeostasis, a critical balance between survival and death, must exist to prevent either lymphopenia or lymphocytosis. In the current review, we discuss known requirements for the survival of naive peripheral CD8+ T cells as well as mechanisms of death when survival signals are lost. We also discuss associations between survival and homeostasis-driven proliferation, and highlight the gaps in our knowledge of these critical processes.

Long-term follow-up of nonmyeloablative allogeneic stem cell transplantation for renal cell carcinoma: The University of Chicago Experience.

Nonmyeloablative allogeneic stem cell transplantation (NST) has considerable activity in patients with metastatic renal cell carcinoma (RCC), although there are limited long-term follow-up data. Between February 1999 and May 2003, 18 patients with metastatic RCC underwent 19 matched-sibling NSTs after conditioning with fludarabine and cyclophosphamide with tacrolimus and mycophenolate mofetil as post-transplant immunosuppression. Among the four objective responses, all were partial and have relapsed with a median response duration of 609 days (range, 107-926). All responders are alive at a median of 41 months. Median overall survival for the entire cohort was 14 months. There were four early treatment-related deaths and one late treatment-related death. Eight patients died from progressive disease and five (28%) from treatment-related mortality. Stratifying transplant outcome as early death, intermediate (no response, no early death), or response, the combination of pre-treatment anemia and decreased performance status, was associated with adverse outcome (P = 0.015) and reduced survival (HR 5.4, 95% confidence interval of 1.4 to 21, P = 0.007). Responders demonstrated prolonged survival compared to nonresponders (P = 0.002). NST leads to durable responses in a minority of metastatic RCC patients. Appropriate patient selection is paramount. Anemia and decreased performance status may enable risk stratification.

Depletion of normal B cells with rituximab as an adjunct to IL-2 therapy for renal cell carcinoma and melanoma.

BACKGROUND: We postulated that in patients with metastatic renal cell carcinoma (RCC) or melanoma, depletion of normal B cells using the anti-CD20 mAb rituximab before treatment with low-dose interleukin (IL)-2 would improve clinical outcome. PATIENTS AND METHODS: Rituximab (375 mg/m(2)) weekly for 4 weeks. IL-2 [11 (million units) daily] s.c., 4 days a week for weeks 5-8, followed by a 2-week rest (weeks 9 and 10). Patients without disease progression continued on IL-2. Disease re-evaluation was performed after rituximab and after every course of IL-2. RESULTS: Fifteen patients with RCC and six with melanoma were enrolled. One patient had a partial response and seven patients had stable disease. Toxicities were similar to those expected with IL-2 alone, and there were no grade 4 events. Circulating B cells were depleted in all patients. The subsequent low-dose IL-2 increased absolute numbers of natural killer cells, activated CD4(+) and activated CD8(+) T cells. Expanded T cells produced interferon-gamma, but not IL-4. Proliferation of peripheral blood lymphocytes to phytohemagglutinin was diminished following rituximab treatment, suggesting that B cells participate in this response in vitro. CONCLUSIONS: Our results suggest that depletion of circulating B cells with rituximab does not increase the response rate, alter the toxicity profile or change the biological activity in response to low-dose IL-2 in patients with RCC or melanoma.

Clinical responses following nonmyeloablative allogeneic stem cell transplantation for renal cell carcinoma are associated with expansion of CD8+ IFN-gamma-producing T cells.

Nonmyeloablative allogeneic stem cell transplantation (NST) is thought to be an immunologic therapy in which donor T cells mediate a graft-versus-tumor effect. We recently reported the clinical outcome of a phase II trial of NST in metastatic renal cell carcinoma (RCC). However, the immune response correlates of clinical activity remain unknown. We now describe the analysis of T-cell subsets and T-cell cytokine-producing potential for those patients evaluable for immune monitoring. The incidence of graft-versus-host disease (GVHD) correlated with clinical outcome, with all responders exhibiting chronic GVHD. Following initial tapering of immunosuppression, an increase in the total numbers of CD8+ T cells but not CD4+ T cells was observed among responders compared to nonresponders. In addition, a greater ratio of CD8+ to CD4+ T cells producing IFN-gamma and IL-2 was seen in clinical responders at the time when clinical responses were first detected (day 180 after transplantation). Our results support the hypothesis that the antitumor effects of NST may be mediated by IFN-gamma-producing CD8+ T cells, and indicate that isolation of putative tumor antigen-specific T cells, ideally, should be pursued around day +180.

CD28 is not required for c-Jun N-terminal kinase activation in T cells.

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28(+/+) or CD28(-/-) TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28(+/+) and CD28(-/-) 2C/recombinase-activating gene 2(RAG2)(-/-) T cells stimulated with anti-CD3 mAb alone, and with L(d)/peptide dimers, a direct alphabeta TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2(-/-) T cells stimulated with P815 cells that express the relevant alloantigen L(d) whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28(+/+) 2C/RAG2(-/-) T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.

Antigen-specific blockade of T cells in vivo using dimeric MHC peptide.

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR zeta-chain and recruitment and phosphorylation of zeta-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.

Epitope spreading upon P815 tumor rejection triggered by vaccination with the single class I MHC-restricted peptide P1A.

Epitope spreading has been best characterized as an exacerbating factor in CD4(+) T cell-dependent autoimmune disease models and is believed to occur via presentation of antigens liberated by tissue destruction initiated by CD4(+) T cells specific for a primary epitope. The growing evidence that exogenous antigens can also be processed and presented by class I MHC molecules has suggested that epitope spreading could occur for CD8(+) cytotoxic T lymphocyte (CTL) responses as well. In the context of anti-tumor immunity, expansion of a CTL response to include secondary epitopes could improve the efficacy of therapeutic vaccines. To determine directly whether epitope spreading can occur during an anti-tumor immune response, two defined class I MHC-binding peptides in the P815 tumor model were utilized. We observed that immunization against the single tumor peptide, P1A, followed by rejection of a P1A(+) tumor, subsequently yielded CTL activity and tumor protection against a P1A(-) tumor variant. P1A immunized mice that subsequently rejected tumor challenge developed CTL against a second defined epitope, P1E. These results indicate that, as for class II-restricted peptides in autoimmune disease, epitope spreading can occur for class I-restricted peptides during tumor rejection. A broadened CTL response may help eliminate outgrowth of antigen-negative tumor variants.

Immunization of HLA-A2+ melanoma patients with MAGE-3 or MelanA peptide-pulsed autologous peripheral blood mononuclear cells plus recombinant human interleukin 12.

Vaccination with dendritic cells (DCs) pulsed with tumor antigen peptides has shown promise in the treatment of melanoma. Interleukin (IL)-12 production by DCs is a key component for their efficacy. Murine studies have shown that IL-12 promotes potent antitumor immunization when coadministered with peptides loaded onto other class I MHC+ cells, thus bypassing the need to use DCs. The easiest cell source to obtain in large quantity from human patients is peripheral blood mononuclear cells (PBMCs). A Phase I clinical trial was thus performed in patients with metastatic melanoma using immunization with autologous PBMCs pulsed with a MAGE-3 or a MelanA peptide, coadministered with various doses of recombinant human (rh)IL-12. Patients receiving low-to-moderate doses of rhIL-12 developed increased specific CD8+ T-cell responses. Of the eight patients showing increased immunity, six had evidence of clinical activity, with one complete, one partial, one minor, and three mixed responses observed. In two patients with mixed responses, growing tumors were found to lack expression of the antigen used to immunize. Thus, vaccination with peptide-pulsed PBMCs plus rhIL-12 induces specific immunity and has clinical activity, without the need to generate DCs. Outgrowth of antigen-negative tumors argues for the future development of polyepitope vaccines.

Absence of CTLA-4 lowers the activation threshold of primed CD8+ TCR-transgenic T cells: lack of correlation with Src homology domain 2-containing protein tyrosine phosphatase.

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.

Diagnosis and treatment of mycoplasma-contaminated cell cultures.

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.

The p815 mastocytoma tumor model.

This unit presents an experimental tumor model which has led to pivotal advances in tumor immunology culminating in the preclinical development of human cancer vaccines for melanoma. The model employs the use of the P815 mastocytoma cell line. Although the P815 cell line belongs to the mast cell lineage, it offers several advantages for in vivo experimentation of the tumor-host relationship. It grows progressively in the majority of syngeneic DBA/2 mice and can be implanted either intraperitoneally or subcutaneously. Moreover, immunogeneic variants have been created yielding tumors that are spontaneously rejected BB a behavior that has provided a context in which to study the immunologically relevant molecules and cells that dictate a successful anti-tumor response.

Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in naive T cells.

The inability to easily and efficiently introduce genes into primary T cells has hampered the investigation of the pathways controlling T cell fate. To enable adenoviral-mediated gene transfer into normal naive T cells, transgenic (Tg) mice expressing the coxsackie/adenovirus receptor (CAR) in their T cell compartment were constructed. Whereas naive T cells are resistant to adenoviral infection, Tg expression of CAR on T cells greatly facilitates adenoviral-mediated gene expression ex vivo, in vivo, and in differentiated T helper cells. Thus we have developed a technology for efficient gene delivery to naive T cells. By using adenoviral vectors encoding specific inhibitors, we show that G1 cyclin-dependent kinase, NF-kappaB, and caspase activities are required for the proliferation of primary T cells. In addition, by expressing Bcl-x(L) protein at a level that closely approximates mitogen-induced levels, we demonstrate that Bcl-x(L) expression is sufficient to account for mitogen-mediated survival of primary T cells. Thus, adenoviral-mediated gene delivery to CAR Tg T cells should be useful for the analysis of many genes controlling T cell fate.

Cutting edge: spontaneous rejection of poorly immunogenic P1.HTR tumors by Stat6-deficient mice.

Experimental evidence suggests that a type 1 T cell response may result in optimal tumor rejection in vivo. This phenotype is determined in part by cytokines that influence T cell differentiation. In transplantable tumor models such as P1.HTR, tumors grow progressively despite expression of defined tumor Ags. We hypothesized that this failure to reject may be due to poor generation of a type 1 phenotype, through a dominant influence of the type 2-promoting cytokines IL-4 and/or IL-13. This hypothesis was tested by implanting P1.HTR tumors into mice deficient in Stat6. In contrast to progressive growth of P1.HTR tumors in wild-type mice, and aggressive growth even of IL-12-transfected P1.HTR in Stat1(-/-) mice, P1.HTR was spontaneously rejected by Stat6(-/-) mice. Rejection was accompanied by augmented tumor-specific IFN-gamma production and CTL activity. These results suggest that pharmacologic inhibition of Stat6 signaling could potentiate anti-tumor immunity in vivo.

Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152).

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3epsilon and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.