The aged tumor microenvironment limits T cell control of cancer.

The etiology and effect of age-related immune dysfunction in cancer is not completely understood. Here we show that limited priming of CD8+ T cells in the aged tumor microenvironment (TME) outweighs cell-intrinsic defects in limiting tumor control. Increased tumor growth in aging is associated with reduced CD8+ T cell infiltration and function. Transfer of T cells from young mice does not restore tumor control in aged mice owing to rapid induction of T cell dysfunction. Cell-extrinsic signals in the aged TME drive a tumor-infiltrating age-associated dysfunctional (TTAD) cell state that is functionally, transcriptionally and epigenetically distinct from canonical T cell exhaustion. Altered natural killer cell-dendritic cell-CD8+ T cell cross-talk in aged tumors impairs T cell priming by conventional type 1 dendritic cells and promotes TTAD cell formation. Aged mice are thereby unable to benefit from therapeutic tumor vaccination. Critically, myeloid-targeted therapy to reinvigorate conventional type 1 dendritic cells can improve tumor control and restore CD8+ T cell immunity in aging.

Surface immobilized α-1 acid glycoprotein and collagen VI modulate mouse macrophage polarization and reduce the foreign body capsule.

Macrophages are widely recognized in modulating the foreign body response, and the manner in which they do so largely depends on their activation state, often referred to as their polarization. This preliminary study demonstrates that surface immobilized α-1 acid glycoprotein (AGP), as well as collagen VI (Col6) in conjunction with AGP, can direct macrophages towards the M2 polarization state in vitro and modify the foreign body response in vivo. AGP and Col6 are immobilized onto poly(2-hydroxyethyl methacrylate) (pHEMA) surfaces using carbonyl diimidazole chemistry. Mouse bone marrow derived macrophages are cultured on modified surfaces with or without lipopolysaccharide stimulation. Surface modified pHEMA discs are implanted subcutaneously into mice to observe differences in the foreign body response. After stimulation with lipopolysaccharide, macrophages cultured on AGP or Col6 modified surfaces showed a reduction in TNF-α expression compared to controls. Arg1 expression was also increased in macrophages cultured on modified surfaces. Explanted tissues showed that the foreign body capsule around implants with AGP or AGP and Col6 modification had reduced thickness, while also being more highly vascularized. These data demonstrate that α-1 acid glycoprotein and collagen VI could potentially be used for the surface modification of medical devices to influence macrophage polarization leading to a reduced and modulated foreign body response.

CD4+ T cells engineered with FVIII-CAR and murine Foxp3 suppress anti-factor VIII immune responses in hemophilia a mice.

Although protein replacement therapy provides effective treatment for hemophilia A patients, about a third of severe patients develop neutralizing inhibitor antibodies to factor VIII. Adoptive transfer of regulatory T cells (Tregs) has shown promise in treating unwanted immune responses. In previous studies, transferred polyclonal Tregs ameliorated the anti-factor VIII immune responses in hemophilia A mice. In addition, factor VIII-primed Tregs demonstrated increased suppressive function. However, antigen-specific Tregs are a small fraction of the total lymphocyte population. To generate large numbers of factor VIII-specific Tregs, the more abundant murine primary CD4+ T cells were lentivirally transduced ex vivo to express Foxp3 and a chimeric antigen receptor specific to factor VIII (F8CAR). Transduced cells significantly inhibited the proliferation of factor VIII-specific effector T cells in suppression assays. To monitor the suppressive function of the transduced chimeric antigen receptor expressing T cells in vivo, engineered CD4+CD25+Foxp3+F8CAR-Tregs were sorted and adoptively transferred into hemophilia A mice that are treated with hydrodynamically injected factor VIII plasmid. Mice receiving engineered F8CAR-Tregs showed maintenance of factor VIII clotting activity and did not develop anti-factor VIII inhibitors, while control CD4+T cell or PBS recipient mice developed inhibitors and had a sharp decrease in factor VIII activity. These results show that CD4+ cells lentivirally transduced to express Foxp3 and F8CAR can promote factor VIII tolerance in a murine model. With further development and testing, this approach could potentially be applied to human hemophilia patients.

A Treg-Selective IL-2 Mutein Prevents the Formation of Factor VIII Inhibitors in Hemophilia Mice Treated With Factor VIII Gene Therapy.

Hemophilia A is a genetic disorder that results in the deficiency of functional factor VIII protein, which plays a key role in blood coagulation. Currently, the majority of hemophilia A patients are treated with repeated infusions of factor VIII protein. Approximately 30% of severe hemophilia A patients develop neutralizing antibodies to factor VIII (known as factor VIII inhibitors) due to treatment, rendering factor VIII protein infusions ineffective. Previously, mice receiving murine IL-2 complexed with α-murine IL-2 mAbs (JES6-1A12) showed a lack of factor VIII inhibitor formation after factor VIII treatment, which was associated with the proliferation and the activation of factor VIII-specific regulatory T cells (Tregs). In this paper, we evaluated if an Fc-fused mutated protein analog of mouse IL-2, named Fc.Mut24, engineered to selectively promote the expansion of Tregs in vivo can modulate factor VIII-specific immune responses. The mice received one intraperitoneal injection of Fc.Mut24. When the regulatory T cell population reached its highest frequency and peak activation, the mice received a hydrodynamic injection of factor VIII plasmid (day 4) followed by a second Fc.Mut24 dose (day 7). Peripheral blood was collected weekly. Flow cytometry was used to characterize the peripheral blood cell populations, while ELISA and Bethesda assays were used to assess the inhibitor concentrations and the functional titers in plasma. The activated partial thromboplastin time assay was used to assess the functional activities of factor VIII in blood. The mice receiving Fc.Mut24 showed a dramatic and transient increase in the population of activated Tregs after Fc.Mut24 injection. Factor VIII gene therapy via hydrodynamic injection resulted in high anti-factor VIII inhibitor concentrations in control PBS-injected mice, whereas the mice treated with Fc.Mut24 produced no inhibitors. Most significantly, there were no inhibitors generated after a second hydrodynamic injection of factor VIII plasmid administered at 19 weeks after the first injection in Fc.Mut24-treated mice. The mice receiving Fc.Mut24 maintained high levels of factor VIII activity throughout the experiment, while the control mice had the factor VIII activity dropped to undetectable levels a few weeks after the first factor VIII plasmid injection. Our data show that human therapies analogous to Fc.Mut24 could potentially provide a method to prevent inhibitor formation and induce long-term immune tolerance to factor VIII in hemophilia patients.

Antigen-specific in vitro expansion of factor VIII-specific regulatory T cells induces tolerance in hemophilia A mice.

BACKGROUND: Following protein replacement therapy, one-third of severe hemophilia A patients develop antibodies to factor VIII (FVIII), which also hinders the efficacy of gene therapy. Regulatory T cells (Tregs) have a naturally suppressive function that potentially reduces the immune response to FVIII therapy. Furthermore, antigen-specific Tregs are functionally much more potent than polyclonal cells. Adoptive transfer of antigen-specific Tregs can effectively suppress anti-FVIII antibody responses. OBJECTIVE: Develop a clinically feasible protocol to enrich and expand Tregs specific to FVIII for suppressing anti-FVIII immune responses. METHODS: Regulatory T cells are isolated from FVIII-sensitized mice, sorted on CD25high markers, and expanded specifically with FVIII, antigen-presenting cells, and interleukin 2 (IL 2). Subsequently, Tregs are further cultured with anti-CD3/anti-CD28 beads, anti-Crry antibodies, and IL 2 to achieve 10-fold to 20-fold expansion. Expanded Tregs are characterized and tested for their suppressive activity in vitro and in vivo. RESULTS: In vitro FVIII-specific suppressive assays indicate that FVIII specifically expanded Tregs are more suppressive than non-specifically expanded and naive Tregs. Adoptive transfer of expanded Tregs into HemA mice showed that FVIII-specifically expanded Tregs are significantly more potent in suppressing anti-FVIII immune responses in FVIII plasmid-treated HemA mice. Moreover, the FVIII-specific immune tolerance is maintained after a secondary challenge with FVIII plasmid. CONCLUSIONS: Our results demonstrate that the FVIII-specific sensitization and expansion protocol yields more potent Tregs to suppress anti-FVIII antibody responses and induce long-term tolerance to FVIII, increasing the potential for adoptive Treg cell therapy to modulate anti-FVIII immune responses.

Pyrolytic graphite foam: a passive magnetic susceptibility matching material.

PURPOSE: To evaluate a novel soft, lightweight cushion that can match the magnetic susceptibility of human tissue. The magnetic susceptibility difference between air and tissue produces field inhomogeneities in the B(0) field, which leads to susceptibility artifacts in magnetic resonance imaging (MRI) studies. MATERIALS AND METHODS: Pyrolytic graphite (PG) microparticles were uniformly embedded into a foam cushion to reduce or eliminate field inhomogeneities at accessible air and tissue interfaces. 3T MR images and field maps of an air/water/PG foam phantom were acquired. Q measurements on a 4T tuned head coil and pulse sequence heating tests at 3T were also performed. RESULTS: The PG foam improved susceptibility matching, reduced the field perturbations in phantoms, does not heat, and is nonconductive. CONCLUSION: The susceptibility matched PG foam is lightweight, safe for patient use, adds no noise or MRI artifacts, is compatible with radiofrequency coil arrays, and improves B(0) homogeneity, which enables more robust MR studies.