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Immunotherapy is now experiencing unprecedented successes in treating various cancers based on new understandings of cancer immunopathogenesis. Nonetheless, although ageing is the biggest risk factor for cancer, the majority of cancer immunotherapy preclinical studies are conducted in young hosts. This review will explore age-related changes in immunity as they relate to cancer immune surveillance, immunopathogenesis and responses to immunotherapy. Although it is recognized that declining T cell function with age poses a great challenge to developing effective age-related cancer immunotherapies, examples of successful approaches to overcome this hurdle have been developed. Further, it is now recognized that immune functions do not simply decline with age, but rather change in ways than can be detrimental. For example, with age, specific immune cell populations with detrimental functions can become predominant (such as cells producing proinflammatory cytokines), suppressive cells can become more numerous or more suppressive (such as myeloid-derived suppressor cells), drugs can affect aged immune cells distinctly and the aged microenvironment is becoming recognized as a significant barrier to address. Key developments in these and other areas will be surveyed as they relate to cancer immunotherapy in aged hosts, and areas in need of more study will be assessed with some speculations for the future. We propose the term 'age-related immune dysfunction' (ARID) as best representative of age-associated changes in immunity. [ABSTRACT FROM AUTHOR]

The authors investigated the effect of IVIg on T-cell proliferation induced by the superantigen, staphylococcal enterotoxin B (SEB). The addition of IVIg to normal PBMC stimulated with SEB resulted in a threefold increase in the proportion of CD3+ blast cells expressing Vβ3 and Vβ17, two subsets of T cells that selectively expand in the presence of SEB. There was no increase in the proportion of T-cell blasts expressing Vβ2, Vβ8 and Vβ13.6 antigens that do not respond to SEB in the absence of IVIg. As described previously, IVIg inhibited T-cell proliferation independently of Vβ specificity. The effects of IVIg were mediated by variable regions of immunoglobulins since they were reproduced with F(ab′)2 fragments of IVIg but not with purified Fc fragments of IgG. The observation that SEB-activated T cells are rescued from inhibition of proliferation by IVIg indicates that IVIg modulates the effects of superantigen on T cells. These results may be of relevance for understanding the mechanisms underlying the effects of IVIg in patients with diseases in which T-cell superantigens are of pathophysiological significance. [ABSTRACT FROM AUTHOR]

Abstract Background Gene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor. Results To render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCARΔcyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCARΔcyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCARΔcyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCARΔcyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCARΔcyt with high efficiency. Expression of hCARΔcyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCARΔcyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization. Conclusion The DO11.hCARΔcyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation.

Several PI3K inhibitors are in clinical development for the treatment of various forms of cancers, including pan-PI3K inhibitors targeting all four PI3K isoforms (α, β, γ, and δ), and isoform-selective inhibitors. Diarrhea and immune-mediated colitis are among the adverse events observed with PI3K inhibition which limits the maximal tolerated dose. A quantitative systems pharmacology model was developed to investigate PI3K-inhibitor-induced colitis. The effects of individual PI3K isoforms on relevant cellular pathways were incorporated into a mechanistic representation of mucosal inflammation. A virtual clinical population captures the observed clinical variability in the onset timing and rates of diarrhea and colitis for seven clinically tested PI3K inhibitors. Model-based analysis suggests that colitis development is governed by both the inhibition of PI3Kδ, which drives T cell differentiation and proliferation, and PI3Kα, which regulates epithelial barrier integrity. Specifically, when PI3Kα is inhibited below a given threshold, epithelial barrier dysfunction precipitates an exaggerated T effector response due to PI3Kδ-inhibition, leading to risk of diarrhea and colitis. This synergy explains why the lowest diarrhea and colitis rates are seen with the weakest PI3Kδ inhibition (alpelisib), and higher rates are seen with strong PI3Kδ inhibition if PI3Kα is even mildly inhibited (e.g., idelalisib), whereas strong PI3Kδ inhibition in the absence of PI3Kα inhibition does not result in high colitis rates (umbralisib). Thus, the model-based analysis suggests that PI3Kα and δ inhibition play unique but synergistic roles in driving colitis. Finally, we explore if and how dose-regimen might influence colitis rates for molecules that inhibit both PI3Kα and PI3Kδ., (© 2021 Genentech, Inc. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Purpose: Immunotherapy treats some cancers, but not ovarian cancer. Regulatory T cells (Tregs) impede anti-ovarian cancer immunity but effective human Treg-directed treatments are lacking. We tested Treg depletion with denileukin diftitox (DD) ± IFNα as ovarian cancer immunotherapy., Patients and Methods: Mice with syngeneic ID8 ovarian cancer challenge were treated with DD, IFNα, or both. The phase 0/I trial tested one dose-escalated DD infusion for functional Treg reduction, safety, and tolerability. The phase II trial added IFNα2a to DD if DD alone failed clinically., Results: DD depleted Tregs, and improved antitumor immunity and survival in mice. IFNα significantly improved antitumor immunity and survival with DD. IFNα did not alter Treg numbers or function but boosted tumor-specific immunity and reduced tumor Treg function with DD by inducing dendritic cell IL6. DD alone was well tolerated, depleted functional blood Tregs and improved immunity in patients with various malignancies in phase 0/I. A patient with ovarian cancer in phase 0/I experienced partial clinical response prompting a phase II ovarian cancer trial, but DD alone failed phase II. Another phase II trial added pegylated IFNα2a to failed DD, producing immunologic and clinical benefit in two of two patients before a DD shortage halt. DD alone was well tolerated. Adding IFNα increased toxicities but was tolerable, and reduced human Treg numbers in blood, and function through dendritic cell-induced IL6 in vitro ., Conclusions: Treg depletion is clinically useful but unlikely alone to cure ovarian cancer. Rational treatment agent combinations can salvage clinical failure of Treg depletion alone, even when neither single agent provides meaningful clinical benefit., (©2021 American Association for Cancer Research.)

The IL2 receptor (IL2R) is an attractive cancer immunotherapy target that controls immunosuppressive T regulatory cells (Treg) and antitumor T cells. Here we used IL2Rβ-selective IL2/anti-IL2 complexes (IL2c) to stimulate effector T cells preferentially in the orthotopic mouse ID8agg ovarian cancer model. Despite strong tumor rejection, IL2c unexpectedly lowered the tumor microenvironmental CD8 + /Treg ratio. IL2c reduced tumor microenvironmental Treg suppression and induced a fragile Treg phenotype, helping explain improved efficacy despite numerically increased Tregs without affecting Treg in draining lymph nodes. IL2c also reduced Treg-mediated, high-affinity IL2R signaling needed for optimal Treg functions, a likely mechanism for reduced Treg suppression. Effector T-cell IL2R signaling was simultaneously improved, suggesting that IL2c inhibits Treg functions without hindering effector T cells, a limitation of most Treg depletion agents. Anti-PD-L1 antibody did not treat ID8agg, but adding IL2c generated complete tumor regressions and protective immune memory not achieved by either monotherapy. Similar anti-PD-L1 augmentation of IL2c and degradation of Treg functions were seen in subcutaneous B16 melanoma. Thus, IL2c is a multifunctional immunotherapy agent that stimulates immunity, reduces immunosuppression in a site-specific manner, and combines with other immunotherapies to treat distinct tumors in distinct anatomic compartments. SIGNIFICANCE: These findings present CD122-targeted IL2 complexes as an advancement in cancer immunotherapy, as they reduce Treg immunosuppression, improve anticancer immunity, and boost PD-L1 immune checkpoint blockade efficacy in distinct tumors and anatomic locations., (©2020 American Association for Cancer Research.)

Background : Intravesical bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for treating patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, relapse rates remain high and BCG unresponsive NMIBC often requires bladder removal. Preclinical data suggest that priming with percutaneous BCG vaccine could improve response to intravesical BCG. Methods : A single-arm trial (NCT02326168) was performed to study the safety, immunogenicity, and preliminary efficacy of priming. Percutaneous BCG was given 21 days prior to intravesical BCG instillation in patients ( n = 13) with high-risk NMIBC. Immune responses were monitored and compared to a sequentially enrolled cohort of nine control patients receiving only intravesical BCG. The effect of BCG on natural killer (NK) and γδ T cell in vitro cytotoxicity was tested. γδ T cell subsets were determined by T cell receptor gene expression with NanoString. Results : Priming was well tolerated and caused no grade ≥3 adverse events. The 3-month disease-free rate for prime patients was 85% (target goal ≥ 75%). Priming boosted BCG-specific immunity at 3 months and increased the activation status of in vitro expanded circulating NK and γδ T cells and their cytotoxicity against bladder cancer cells through receptor NKG2D. BCG enhanced the cytotoxicity of NK and γδ T cells against K562, RT4, and UM-UC6 but not against T24, UM-UC-3, or UM-UC-14 cells. Infiltrating γδ T cell subsets identified in the bladder includes γ9δ2 and γ8δ2. Conclusions : BCG priming is safe and tolerable. Poor sensitivity to NK and γδ T cell cytotoxicity by some bladder tumors represents a potential BCG-resistance mechanism.

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients ( P = 0.008). Rapamycin reduced surgery-induced T-cell exhaustion in patients, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1)-expressing T cells. Grade 3 to 4 adverse event rates were similar between groups, but rapamycin-treated patients had a higher rate of wound complications versus controls. In conclusion, surgery promoted bladder cancer metastasis and decreased the efficacy of postoperative bladder cancer immunotherapy. Low-dose (3 mg daily) oral rapamycin has favorable pharmacodynamic and immune modulating activity in surgical patients and has the potential to decrease surgery-induced immune dysfunction., (©2018 American Association for Cancer Research.)

Background: Natural killer (NK) cells are effective at killing tumors in a non-MHC restricted manner and are emerging targets for cancer therapy but their importance in bladder cancer (BC) is poorly defined. NK cells are commonly subdivided into populations based on relative surface expression of CD56. Two major subsets are CD56 bright and CD56 dim NK cells., Methods: The prevalence of intratumoral lymphocytes was examined via flow cytometric analysis of bladder tissue from a local cohort of patients with non-invasive and invasive BC (n=28). The association of NK cell subsets with cancer-specific survival (CSS) and overall survival (OS) was examined in 50 patients with BC using Cox regression. Fluorescence-activated cell sorting (FACS) of intratumoral lymphocytes isolated CD56 NK cell subsets were used for examination of function, including cytokine production and in vitro cytotoxicity., Results: NK cells predominated among bladder intratumoral lymphocytes. Intratumoral CD56 bright NK cells showed increased cytokine production and cytotoxicity compared to their CD56 dim counterparts and were associated with improved CSS and OS independent of pathologic tumor stage. On the other hand, CD56 dim NK cells were not associated with improved outcomes but were associated with higher pathologic stage., Conclusions: NK cells are frequent among intratumoral lymphocytes in BC. Bladder intratumoral CD56 bright NK cells are functional and prognostically relevant whereas CD56 dim NK cells are dysfunctional and prevalent in higher stage tumors. Thus, CD56 bright NK cells are promising targets in BC., Competing Interests: CONFLICTS OF INTEREST The authors declare that no conflicts of interest exist.

Improvements in understanding cancer immunopathogenesis have now led to unprecedented successes in immunotherapy to treat numerous cancers. Although aging is the most important risk factor for cancer, most pre-clinical cancer immunotherapy studies are undertaken in young hosts. This review covers age-related immune changes as they affect cancer immune surveillance, immunopathogenesis and immune therapy responses. Declining T cell function with age can impede efficacy of age-related cancer immunotherapies, but examples of successful approaches to breach this barrier have been reported. It is further recognized now that immune functions with age do not simply decline, but that they change in potentially detrimental ways. For example, detrimental immune cell populations can become predominant during aging (notably pro-inflammatory cells), the prevalence or function of suppressive cells can increase (notably myeloid derived suppressor cells), drugs can have age-specific effects on immune cells, and attributes of the aged microenvironment can impede or subvert immunity. Key advances in these and related areas will be reviewed as they pertain to cancer immunotherapy in the aged, and areas requiring additional study and some speculations on future research directions will be addressed. We prefer the term Age Related Immune Dysfunction (ARID) as most encompassing the totality of age-associated immune changes., (Copyright © 2017. Published by Elsevier Inc.)

Cancer immunotherapy has shown remarkable recent progress. Immune checkpoint blocking antibodies have become the most successful anti-cancer agent class ever developed, with six distinct agents approved since 2011 for a wide variety of cancers. Although age is the biggest risk factor for cancer (aside from selected early-onset pediatric cancers), these agents were tested pre-clinically in young hosts, and there is remarkably little published on the effects of host age on treatment outcomes in pre-clinical studies or human clinical trials. The three principal immune checkpoints against which blocking antibodies have been FDA-approved for human use are CTLA-4, PD-1 and PD-L1. We used a mouse model of transplantable, orthotopic B16 melanoma to test age effects of treatments with anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies. All three agents were highly effective in treating young tumor-bearing hosts as expected. Anti-PD-L1 as a single agent had no effect on tumor growth in aged hosts, anti-CTLA-4 had detectable, modest effects and anti-PD-1 was essentially as effective in aged as in young hosts, the first single agent we have identified not to lose efficacy with age in this model. Other important differences in young versus aged hosts included lack of anti-CTLA-4-mediated depletion of intratumor regulatory T cells in aged hosts and poorer ability of all three agents to activate T cells in aged versus young hosts. Anti-CTLA-4 efficacy appeared to improve when combined with anti-PD-L1. Regulatory T cell depletion with FDA-approved denileukin diftitox did not improve treatment by any single agent. Aged mice tolerated treatments as well as young mice without obvious toxicities at equivalent doses., (Copyright © 2017. Published by Elsevier Inc.)

mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1-8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)-mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell-dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L + CD8 + central memory T cells in EL4 challenge, but without clinical benefit. LD rapamycin significantly enhanced DD treatment efficacy, but DD plus LD rapamycin treatment effects were independent of antitumor immunity. Instead, rapamycin upregulated EL4 IL2 receptor in vitro and in vivo, facilitating direct DD tumor cell killing. LD rapamycin augmented DD efficacy against B16 melanoma and a human B-cell lymphoma, but not against human Jurkat T-cell lymphoma or ID8agg ovarian cancer cells. Treatment effects correlated with IL2R expression, but mechanisms in some tumors were not fully defined. Overall, our data define a distinct, biphasic mechanisms of action of mTOR inhibition at doses that are clinically exploitable, including in T-cell lymphomas. Cancer Res; 77(2); 520-31. ©2016 AACR., Competing Interests: The authors have no relevant financial conflicts of interest to declare., (©2016 American Association for Cancer Research.)

PD-L1 antibodies produce efficacious clinical responses in diverse human cancers, but the basis for their effects remains unclear, leaving a gap in the understanding of how to rationally leverage therapeutic activity. PD-L1 is widely expressed in tumor cells, but its contributions to tumor pathogenicity are incompletely understood. In this study, we evaluated the hypothesis that PD-L1 exerts tumor cell-intrinsic signals that are critical for pathogenesis. Using RNAi methodology, we attenuated PD-L1 in the murine ovarian cell line ID8agg and the melanoma cell line B16 (termed PD-L1 lo cells), which express basal PD-L1. We observed that PD-L1 lo cells proliferated more weakly than control cells in vitro As expected, PD-L1 lo cells formed tumors in immunocompetent mice relatively more slowly, but unexpectedly, they also formed tumors more slowly in immunodeficient NSG mice. RNA sequencing analysis identified a number of genes involved in autophagy and mTOR signaling that were affected by PD-L1 expression. In support of a functional role, PD-L1 attenuation augmented autophagy and blunted the ability of autophagy inhibitors to limit proliferation in vitro and in vivo in NSG mice. PD-L1 attenuation also reduced mTORC1 activity and augmented the antiproliferative effects of the mTORC1 inhibitor rapamycin. PD-L1 lo cells were also relatively deficient in metastasis to the lung, and we found that anti-PD-L1 administration could block tumor cell growth and metastasis in NSG mice. This therapeutic effect was observed with B16 cells but not ID8agg cells, illustrating tumor- or compartmental-specific effects in the therapeutic setting. Overall, our findings extend understanding of PD-L1 functions, illustrate nonimmune effects of anti-PD-L1 immunotherapy, and suggest broader uses for PD-L1 as a biomarker for assessing cancer therapeutic responses. Cancer Res; 76(23); 6964-74. ©2016 AACR., Competing Interests: The authors have no conflicting financial interests to declare., (©2016 American Association for Cancer Research.)

The FDA-approved mTOR inhibitor rapamycin mediates important immune effects, but its contributions to the anticancer effects of the drug are unclear. Here we report evidence that rapamycin-mediated cancer protection relies upon stimulation of γδ T cells. In a well-established mouse model of carcinogen and inflammation-driven skin carcinogenesis, IFNγ recruited γδ TCR mid T cells to the epidermis where rapamycin boosted their perforin-dependent antitumor properties. These antitumor cells were mostly Vγ5 - Vγ4 - Vγ1 - in phenotype. IFNγ signals were required in both hematopoietic and nonhematopoietic cells for rapamycin to optimally promote epidermal infiltration of γδ TCR mid T cells, as mediated by CXCR3-CXCL10 interactions, along with the antitumor effects of these cells. In mouse xenograft models of human squamous cell carcinoma, rapamycin improved human γδ T-cell-mediated cancer cell killing. Our results identify immune mechanisms for the cancer prevention and treatment properties of rapamycin, challenging the paradigm that mTOR inhibition acts primarily by direct action on tumor cells. Cancer Res; 76(20); 5970-82. ©2016 AACR., (©2016 American Association for Cancer Research.)

Hydroxychloroquine (HCQ) enhances the anti-cancer activity of the histone deacetylase inhibitor, vorinostat (VOR), in pre-clinical models and early phase clinical studies of metastatic colorectal cancer (mCRC). Mechanisms could include autophagy inhibition, accumulation of ubiquitinated proteins, and subsequent tumor cell apoptosis. There is growing evidence that autophagy inhibition could lead to improved anti-cancer immunity. To date, effects of autophagy on immunity have not been reported in cancer patients. To address this, we expanded an ongoing clinical study to include patients with advanced, refractory mCRC to evaluate further the clinical efficacy and immune effects of VOR plus HCQ. Refractory mCRC patients received VOR 400 milligrams orally with HCQ 600 milligrams orally daily, in a 3-week cycle. The primary endpoint was median progression-free survival (mPFS). Secondary endpoints include median overall survival (mOS), adverse events (AE), pharmacodynamic of inhibition of autophagy in primary tumors, immune cell analyses, and cytokine levels. Twenty patients were enrolled (19 evaluable for survival) with a mPFS of 2.8 months and mOS of 6.7 months. Treatment-related grade 3-4 AEs occurred in 8 patients (40%), with fatigue, nausea/vomiting, and anemia being the most common. Treatment significantly reduced CD4+CD25hiFoxp3+ regulatory and PD-1+ (exhausted) CD4+ and CD8+ T cells and decreased CD45RO-CD62L+ (naive) T cells, consistent with improved anti-tumor immunity. On-study tumor biopsies showed increases in lysosomal protease cathepsin D and p62 accumulation, consistent with autophagy inhibition. Taken together, VOR plus HCQ is active, safe and well tolerated in refractory CRC patients, resulting in potentially improved anti-tumor immunity and inhibition of autophagy.

As tumor PD-L1 provides signals to anti-tumor PD-1 + T cells that blunt their functions, αPD-1 and αPD-L1 antibodies have been developed as anti-cancer immunotherapies based on interrupting this signaling axis. However, tumor cell-intrinsic PD-L1 signals also regulate immune-independent tumor cell proliferation and mTOR signals, among other important effects. Tumor initiating cells (TIC) generate carcinomas, resist treatments and promote relapse. We show here that in murine B16 melanoma and ID8agg ovarian carcinoma cells, TIC express more PD-L1 versus non-TIC. Silencing PD-L1 in B16 and ID8agg cells by shRNA ("PD-L1 lo ") reduced TIC numbers, the canonical TIC genes nanog and pou5f1 (oct4), and functions as assessed by tumorosphere development, immune-dependent and immune-independent tumorigenesis, and serial transplantability in vivo . Strikingly, tumor PD-L1 sensitized TIC to interferon-γ and rapamycin in vitro. Cell-intrinsic PD-L1 similarly drove functional TIC generation, canonical TIC gene expression, and sensitivity to interferon-γ and rapamycin in human ES2 ovarian cancer cells. Thus, tumor-intrinsic PD-L1 signals promote TIC generation and virulence, possibly by promoting canonical TIC gene expression, suggesting that PD-L1 has novel signaling effects on cancer pathogenesis and treatment responses., Competing Interests: The authors disclose no potential conflicts of interest

The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age-related debilities including increasing antigen-specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T- and B-lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer- and infection-prone mice supporting disease mitigation as a mechanism for mTOR suppression-mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Cancer prevention is a cost-effective alternative to treatment. In mice, the mTOR inhibitor rapamycin prevents distinct spontaneous, noninflammatory cancers, making it a candidate broad-spectrum cancer prevention agent. We now show that oral microencapsulated rapamycin (eRapa) prevents skin cancer in dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) carcinogen-induced, inflammation-driven carcinogenesis. eRapa given before DMBA/TPA exposure significantly increased tumor latency, reduced papilloma prevalence and numbers, and completely inhibited malignant degeneration into squamous cell carcinoma. Rapamycin is primarily an mTORC1-specific inhibitor, but eRapa did not reduce mTORC1 signaling in skin or papillomas, and did not reduce important proinflammatory factors in this model, including p-Stat3, IL17A, IL23, IL12, IL1β, IL6, or TNFα. In support of lack of mTORC1 inhibition, eRapa did not reduce numbers or proliferation of CD45(-)CD34(+)CD49f(mid) skin cancer initiating stem cells in vivo and marginally reduced epidermal hyperplasia. Interestingly, eRapa reduced DMBA/TPA-induced skin DNA damage and the hras codon 61 mutation that specifically drives carcinogenesis in this model, suggesting reduction of DNA damage as a cancer prevention mechanism. In support, cancer prevention and DNA damage reduction effects were lost when eRapa was given after DMBA-induced DNA damage in vivo. eRapa afforded picomolar concentrations of rapamycin in skin of DMBA/TPA-exposed mice, concentrations that also reduced DMBA-induced DNA damage in mouse and human fibroblasts in vitro. Thus, we have identified DNA damage reduction as a novel mechanism by which rapamycin can prevent cancer, which could lay the foundation for its use as a cancer prevention agent in selected human populations., (©2015 American Association for Cancer Research.)

Purpose: To determine the safety and toxicities of sequential MMC (mitomycin C) + BCG (bacillus Calmette-Guérin) in patients with non-muscle-invasive bladder cancer (NMIBC) and explore evidence for potentiation of BCG activity by MMC., Experimental Design: A 3 + 3 phase I dose-escalation trial of six weekly treatments was conducted in patients with NMIBC. MMC (10, 20, or 40 mg) was instilled intravesically for 30 minutes, followed by a 10-minute washout with gentle saline irrigation and then instillation of BCG (half or full strength) for 2 hours. Urine cytokines were monitored and compared with levels in a control cohort receiving BCG only. Murine experiments were carried out as described previously., Results: Twelve patients completed therapy, including 3 patients receiving full doses. The regimen was well tolerated with no treatment-related dose-limiting toxicities. Urinary frequency and urgency, and fatigue were common. Eleven (91.7%) patients were free of disease at a mean (range) follow-up of 21.4 (8.4-27.0) months. Median posttreatment urine concentrations of IL2, IL8, IL10, and TNFα increased over the 6-week treatment period. A greater increase in posttreatment urinary IL8 during the 6-week period was observed in patients receiving MMC + BCG compared with patients receiving BCG monotherapy. In mice, intravesical MMC + BCG skewed tumor-associated macrophages (TAM) toward a beneficial M1 phenotype., Conclusions: Instillation of sequential MMC + BCG is safe tolerable up to 40-mg MMC plus full-strength BCG. This approach could provide improved antitumor activity over BCG monotherapy by augmenting beneficial M1 TAMs., (©2014 American Association for Cancer Research.)

Opinion Statement: All work referenced herein relates to treatment of epithelial ovarian carcinomas, as their treatment differs from ovarian germ cell cancers and other rare ovarian cancers, the treatments of which are addressed elsewhere. Fallopian tube cancers and primary peritoneal adenocarcinomatosis are also generally treated as epithelial ovarian cancers. The standard of care initial treatment of advanced stage epithelial ovarian cancer is optimal debulking surgery as feasible plus chemotherapy with a platinum plus a taxane agent. If this front-line approach fails, as it too often the case, several FDA-approved agents are available for salvage therapy. However, because no second-line therapy for advanced-stage epithelial ovarian cancer is typically curative, we prefer referral to clinical trials as logistically feasible, even if it means referring patients outside our system. Immune therapy has a sound theoretical basis for treating carcinomas generally, and for treating ovarian cancer in particular. Advances in understanding the immunopathogenic basis of ovarian cancer, and the immunopathologic basis for prior failures of immunotherapy for it and other carcinomas promises to afford novel treatment approaches with potential for significant efficacy, and reduced toxicities compared with cytotoxic agents. Thus, referral to early phase immunotherapy trials for ovarian cancer patients that fail conventional treatment merits consideration.

Mutation of a single copy of the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer. Apc(Min/+) mice exhibit multiple intestinal neoplasia (MIN) that causes anemia and death from bleeding by 6 months. Mechanistic target of rapamycin complex 1 (mTORC1) inhibitors were shown to improve Apc(Min/+) mouse survival when administered by oral gavage or added directly to the chow, but these mice still died from neoplasia well short of a natural life span. The National Institute of Aging Intervention Testing Program showed that enterically targeted rapamycin (eRapa) extended life span for wild-type genetically heterogeneous mice in part by inhibiting age-associated cancer. We hypothesized that eRapa would be effective in preventing neoplasia and extend survival of Apc(Min/+) mice. We show that eRapa improved survival of Apc(Min/+) mice in a dose-dependent manner. Remarkably, and in contrast to previous reports, most of the Apc(Min/+) mice fed 42 parts per million eRapa lived beyond the median life span reported for wild-type syngeneic mice. Furthermore, chronic eRapa did not cause detrimental immune effects in mouse models of cancer, infection, or autoimmunity, thus assuaging concerns that chronic rapamycin treatment suppresses immunity. Our studies suggest that a novel formulation (enteric targeting) of a well-known and widely used drug (rapamycin) can dramatically improve its efficacy in targeted settings. eRapa or other mTORC1 inhibitors could serve as effective cancer preventatives for people with FAP without suppressing the immune system, thus reducing the dependency on surgery as standard therapy., (©2013 AACR.)

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

Although studies have shown that the Notch2 family member is critical for embryonic development, little is known concerning its role in hematopoiesis. In this study, we show that the effects of an activated form of Notch2 (N2IC) on the T-cell lineage are dosage related. High-level expression of N2IC results in the development of T-cell leukemias. In contrast, lower-level expression of N2IC does not lead to transformation but skews thymocyte development to the CD8 lineage. Underlying this skew is a dramatic enhancement in positive selection and CD8SP maturation. N2IC permits early B-cell development but blocks the maturation of conventional B2 cells at the pre-B stage, which is the limit of endogenous Notch2 protein expression in developing B cells. Most strikingly, while B2 B cell development is blocked at the pre-B-cell stage, N2IC promotes the selective development of LPS-responsive B1 B cells. This study implicates a role for Notch2 in the maturation of the CD8 lineage and suggests a novel function for Notch2 in the development of the B1 B-cell subset.

Recent studies have implicated a role for Notch in the generation of marginal zone (MZ) B cells. To further investigate the role of Notch in the B cell lineage, we have analyzed the effects of reduced Notch2 signaling in mice expressing one functional allele of Notch2 (Notch2(+/-)). Notch2(+/-) mice have reduced B1 B cells of the peritoneal cavity and show a severe reduction in MZ B cells of the spleen. The reduction in MZ B cells was not due to the disruption of splenic architecture, disregulated terminal differentiation, nor to increased apoptosis within the MZ B cell compartment. Rather, our data suggest that Notch2 haploinsufficiency leads to impaired development of MZ B cells, possibly by impacting the formation of immediate MZ B precursors. These results provide evidence that Notch2 plays a determining role in the development and/or the maintenance of B1 B and MZ B cells.

Limited frequencies of T cells express IL-2 in primary antigenic responses, despite activation marker expression and proliferation by most clonal members. To define the basis for restricted IL-2 expression, a videomicroscopic system and IL-2 reporter transgenic model were used to characterize dendritic cell (DC)-T cell interactions. T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2. Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression. Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

Background: Gene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor., Results: To render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCAR(Delta)cyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCAR(Delta)cyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCAR(Delta)cyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCAR(Delta)cyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCAR(Delta)cyt with high efficiency. Expression of hCAR(Delta)cyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCAR(Delta)cyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization., Conclusion: The DO11.hCAR(Delta)cyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation.

Technologies for transfer of exogenous genes into primary T cells have been limited until recently. The introduction of new approaches for gene transfer via different viral vectors has expanded the options for genetic manipulation of primary T cells and has provided powerful tools for studies of T cell activation and differentiation. We provide a brief overview of the systems currently available and contrast the advantages and disadvantages of each. We also describe a new transgenic model that enables highly efficient gene delivery into primary T cells by nonreplicating adenoviral vectors.

A diverse pattern of polymorphism is defined for the paired Ig-like receptors (PIRs) that serve as activating (PIR-A) and inhibitory (PIR-B) receptors on B lymphocytes, dendritic cells, and myeloid-lineage cells in mice. The monoclonal anti-PIR antibody 10.4 is shown to recognize an allelic PIR-A/PIR-B determinant on cells from BALB/c but not C57BL/6 mice. Other strains of inbred mice also can be typed on the basis of their expression of this PIR allelic determinant. Analysis of (BALB/c x C57BL/6) F1 hybrid offspring indicates that PIR molecules bearing the paternal PIR allotype are expressed whereas PIR-A and PIR-B molecules bearing the maternal allotype are not. The monoallelic expression of the polymorphic PIR-A and PIR-B molecules, and possibly of their human Ig-like transcript/leukocyte Ig-like receptor/monocyte/macrophage Ig-like receptor and killer cell inhibitory receptor relatives, may influence innate and specific immune responses in outbred populations.

Normal human serum contains IgM antibodies that regulate the natural autoantibody activity of IgG in autologous serum. In the present study, we show that pooled normal human IgM (IVIgM) purified from plasma of more than 2,500 healthy donors and processed in a similar fashion to that of therapeutic preparations of pooled normal human IgG (IVIg) suppresses activity of IgG autoantibodies purified from the serum of patients with autoimmune diseases in vitro. The inhibitory effect of IVIgM was greater or equivalent to that of IVIg on a molar basis. We show that IVIgM contains anti-idiotypic antibodies directed against idiotypic determinants of autoantibodies, in particular by showing that Sepharose-bound IVIgM selectively retained F(ab')2 fragments of IgG autoantibodies. The infusion of (Lewis x Brown-Norway) F1 rats with IVIgM protected the animals against experimental autoimmune uveitis induced by immunization with the soluble retinal S antigen, as evidenced by clinical scoring and histopathological analysis. The present findings provide a rationale for considering pooled IgM for immunomodulation of autoimmune disease.

Significant progress has been made in understanding the mechanisms by which intravenous immunoglobulins (IVIg) exert immunomodulatory effects in the treatment of autoimmune diseases. A unique property of immunoglobulins is the diversity of variable (V) regions. The evidence discussed in this communication supports our notion that the diversity of V regions in IVIg preparations is a determining factor for the anti-inflammatory substitutive and immunomodulatory functions of IVIg therapy. We have demonstrated the presence in IVIg, of anti-idiotypic antibodies directed against various autoantibodies. The ability of IVIg to interact through V regions with complementary V regions of antibodies and antigen receptors as well as with relevant soluble and surface molecules provides the basis for inducing the selection of immune repertoires. The study of the mechanisms by which IVIg mediates selection of autoreactive repertoires is essential for our understanding of the mechanisms underlying the emergence of pathological autoimmunity and of the physiological role of natural antibodies in the establishment and maintenance of tolerance to self and homeostasis of autoreactivity in healthy individuals.

The authors investigated the effect of IVIg on T-cell proliferation induced by the superantigen, staphylococcal enterotoxin B (SEB). The addition of IVIg to normal PBMC stimulated with SEB resulted in a threefold increase in the proportion of CD3+ blast cells expressing V beta 3 and V beta 17, two subsets of T cells that selectively expand in the presence of SEB. There was no increase in the proportion of T-cell blasts expressing V beta 2, V beta 8 and V beta 13.6 antigens that do not respond to SEB in the absence of IVIg. As described previously, IVIg inhibited T-cell proliferation independently of V beta specificity. The effects of IVIg were mediated by variable regions of immunoglobulins since they were reproduced with F(ab')2 fragments of IVIg but not with purified Fc fragments of IgG. The observation that SEB-activated T cells are rescued from inhibition of proliferation by IVIg indicates that Mg modulates the effects of superantigen on T cells. These results may be of relevance for understanding the mechanisms underlying the effects of IVIg in patients with diseases in which T-cell superantigens are of pathophysiological significance.

The effects of intravenously administered normal immunoglobulin G (IVIg) in autoimmune diseases are dependent on the ability of IVIg to interact with surface molecules of lymphocytes. In the present study, we demonstrate the presence of anti-CD4 activity in IVIg by showing the ability of IVIg to bind to CD4 and to inhibit CD4-dependent cellular functions. Binding of IVIg to recombinant soluble human CD4 was assessed by ELISA, immunoblotting and real time analysis of complex formation. Anti-CD4 antibodies isolated from IVIg by affinity-chromatography bound to human CD4+ T cells. These anti-CD4 antibodies inhibited proliferative responses in MLR and infection of CD4+ human T cells with HIV. These results indicate that IVIg contains antibodies reactive with human CD4 and that these anti-CD4 antibodies exhibit biological functions. The presence of anti-CD4 antibodies in IVIg may be relevant to the immunoregulatory effects of normal polyspecific immunoglobulin G.

We have evaluated the effect of human Igs for intravenous use (IVIg) on the onset and development of experimental autoimmune uveoretinitis (EAU), a T cell-dependent autoimmune disease induced in rats by a single immunization with retinal S-antigen (S-Ag). Five consecutive daily infusions of IVIg, starting on the same day as S-Ag immunization, protected (Lewis x Brown-Norway) F1 rats against EAU. The prevention of EAU was IVIg-specific, i.e. mediated by pooled human IgG from multiple donors, since neither infusions of BSA nor infusions of pooled Ig from only two healthy individuals were effective. Treatment with IVIg decreased lymphocyte proliferative and antibody responses to S-Ag and the proliferative response to concanavalin A. Lack of proliferation was not dependent upon generation of suppressor cells. Lymph node (LN) cells from IVIg-treated and S-Ag-immunized animals neither proliferated nor secreted IL-2 in response to S-Ag but proliferated when co-cultured with LN cells from rats immunized with S-Ag. Our findings are compatible with an induction of a state of functional inactivation/anergy of T lymphocytes by infusions of IVIg. This functional inactivation may be due to the presence in IVIg of antibodies that bind both in vivo and in vitro to rat lymphocytes. Results from the present study suggest a novel mechanism by which IVIg may be beneficial in human autoimmune diseases.

Infusion of intravenous immunoglobulin (IVIg) has resulted in clinical improvement and/or a fall in autoantibody in a number of autoimmune and inflammatory diseases. Several lines of evidence demonstrate that IVIg may react with disease-associated autoantibodies through idiotypic interactions. In addition, infusion of IVIg results in changes in the expressed autoantibody repertoire that are dependent on interactions between variable regions of infused IgG and autoantibodies and in the subsequent alterations in the regulatory function of immune networks. Thus, pooled normal immunoglobulin restored in a patient with autoimmune thyroiditis the dynamics of spontaneous fluctuations of serum autoantibodies characteristic of physiological conditions. The ability of IVIg to interact with V regions of autoantibodies and with surface molecules of lymphocytes in vitro provides a basis for the selective modulation of B-cell clones observed in patients treated with immunoglobulins. The natural intrinsic complexity of IVIg provides a physiological rationale for immunoregulatory therapy of autoimmune disease.

In the present study we have analyzed the changes in the expressed antibody repertoire and in temporal fluctuations of antibody levels in serum that followed infusion of normal IgG (IVIg) in a patient with autoimmune thyroiditis. Administration of IVIg resulted in the stimulation of IgM production, in alterations of expressed antibody activity in serum that could not merely be accounted for by the passive transfer of antibody specificities contained in IVIg, in transient down-regulation of B cells clones expressing a specific disease-related idiotype and in the increase in serum in recipient's autoantibodies specifically reactive with F(ab')2 fragments of IVIg. In addition, infusion of IVIg shifted the pattern of spontaneous fluctuations of autoantibody activities in the patient's serum from a pattern indicative of disconnected events in the immune network to a pattern similar to that which is consistently observed in healthy controls. These results suggest that normal IgG may modulate autoreactivity by selecting expressed antibody repertoire through V region-dependent interactions with antibodies.