Kelly M. Beatty

Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function☆

We tested whether granulocytes, which contaminate PBMC isolates after prolonged blood storage at room temperature, are responsible for inhibited T cell function in aged blood. We extend previous observations by characterizing these contaminating granulocytes as CD11b+ CD15+ cells comparable to activated CD11b+ CD15+ granulocytes induced by incubation of blood with FMLP. Granulocyte contamination of PBMC was observed within 6-8 h after venipuncture and room temperature storage (2.3 fold increase), and increased 11.3-fold by 24-26 h in comparison to PBMC from fresh blood. Refrigerated 22-26 hour storage of blood exacerbated granulocyte contamination (84-fold increase). In contrast, granulocyte contamination was markedly reduced if blood was diluted in RPMI-1640 medium (3.9-fold increase) or PBS (1.8-fold increase) prior to 22-26 hour room temperature storage. Granulocyte contamination significantly correlated with reduced CD3zeta chain expression, a marker of T cell dysfunction. Correspondingly, T cell proliferation following PHA stimulation was significantly decreased in PBMC with contaminating granulocytes from aged blood (77% of control) or FMLP treated blood (44% of control). Minimizing granulocyte contamination in PBMC of aged blood by cell sorting, or by reducing granulocyte activation by diluting blood in PBS prior to storage, increased CD3zeta chain expression and increased T cell proliferation following stimulation. These data indicate that granulocytes inhibit T cell function in aged blood. Therefore, preventing granulocyte activation in blood specimens is critical to maintain optimal T cell function. This may be accomplished by limiting the time from venipuncture to PBMC isolation to <8 h and may be extended to 26 h by simply diluting blood in PBS prior to room temperature storage.

Activated CD11b+ CD15+ Granulocytes Increase in the Blood of Patients with Uveal Melanoma

PURPOSE To determine whether activated CD11b(+) CD15(+) granulocytes increase in the blood of patients with uveal melanoma. METHODS Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from the blood of patients with primary choroidal/ciliochoroidal uveal melanomas (six women, four men; age range, 46-91 years) and healthy control donors (14 women, 10 men; age range, 50-81 years). The expression of CD15 and CD68 on CD11b(+) myeloid cells within PBMCs and primary uveal melanomas was evaluated by flow cytometry. CD3zeta chain expression by CD3epsilon(+) T cells in PBMCs and within primary uveal melanomas was measured as an indirect indication of T-cell function. RESULTS The percentage of CD11b(+) cells in PBMCs of patients with uveal melanoma increased 1.8-fold in comparison to healthy donors and comprised three subsets: CD68 negative CD15(+) granulocytes, which increased 4.1-fold; CD68(-) CD15(-) cells, which increased threefold; and CD68(+) CD15(low) cells, which were unchanged. A significant (2.7-fold) reduction in CD3zeta chain expression on CD3epsilon(+) T cells, a marker of T-cell dysfunction, was observed in PBMCs of patients with uveal melanoma in comparison with healthy control subjects and correlated significantly with the percentage of CD11b(+) cells in PBMCs. CD3zeta chain expression on T cells within primary tumors was equivalent to CD3zeta expression in PBMCs of the same patient in four of five patients analyzed. CONCLUSIONS Activated CD11b(+) CD15(+) granulocytes expand in the blood of patients with uveal melanoma and may contribute to immune evasion by ocular tumors by inhibiting T-cell function via decreasing CD3zeta chain expression.

Ascorbate in Aqueous Humor Augments Nitric Oxide Production by Macrophages

Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting proinflammatory NO production by macrophages (Mϕs). Consistent with previous observations, we observed that although Mϕs stimulated in the presence of AqH expressed NO synthase 2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure of NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of l-arginine (l-Arg) into l-citrulline, was augmented in lysates of Mϕs stimulated in the presence of AqH. These data suggested that intracellular l-Arg may have been limited by AqH. However, we observed increased mRNA expression of the l-Arg transporter, cationic amino acid transporter 2B, and increased l-Arg uptake in Mϕs stimulated in the presence of AqH. Arginases were expressed by stimulated Mϕs, but competition for l-Arg with NOS2 was excluded. Expression of GTP cyclohydrolase, which produces tetrahydrobiopterin (H4B), an essential cofactor for NOS2 homodimerization, increased after Mϕ stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together, these data provided no evidence for inhibited NOS2 enzymatic activity by AqH, suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH, and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in Mϕs via ascorbate, which limited degradation of H4B. Therefore, AqH may augment NO production in macrophages by stabilizing H4B and increasing intracellular l-Arg.

Splenectomy Promotes Indirect Elimination of Intraocular Tumors by CD8+ T Cells That Is Associated with IFNγ- and Fas/FasL-Dependent Activation of Intratumoral Macrophages

Miller and colleagues demonstrate a mechanism by which splenectomy promotes rejection of intraocular tumors, which involves an IFNg-and-Fas/FasL-dependent interaction between CD8+ T cells and intratumoral macrophages eliciting severe ocular inflammation that indirectly eliminates intraocular tumors by inducing phthsis. Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. CD8+ T cells, IFNγ, and FasL, but not perforin, or TNFα were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNγ and FasL did not target tumor cells directly as the majority of SPLNX IFNγR1−/− mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNγR1. Bone marrow chimeras revealed that IFNγR1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mφ) within intraocular tumors in an IFNγ- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNγ and Fas. As depletion of Mφs limited CD8 T cell–mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNγ- and Fas/FasL-dependent activation of intratumoral Mφs by CD8+ T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis, and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interaction between CD8+ T cells and Mφs to limit the immunosurveillance of intraocular tumors. Cancer Immunol Res; 2(12); 1175–85. ©2014 AACR.

A caveat for T cell transfer studies: generation of cytotoxic anti-Thy1.2 antibodies in Thy1.1 congenic mice given Thy1.2+ tumors or T cells

Thy1.1 congenic B6.PL mice were used to simultaneously monitor Thy1.2+ E.G7‐OVA tumors transplanted in the a.c. of the eye and i.v.‐transferred tumor‐specific Thy1.2+ CTLs to determine mechanisms that inhibit the tumoricidal activity of CTL responses in mice with established ocular tumors. Transferred CTLs were systemically deleted in mice with established ocular tumors. However, this deletion was not a unique mechanism of immune evasion by ocular tumors. Rather, development of Thy1.2+ tumors in the eye or skin of B6.PL mice generated cytotoxic anti‐Thy1.2 antibodies that eliminated a subsequent Thy1.2+ T cell transfer. Anti‐Thy1.2 immune responses in B6.PL mice were influenced by the route of antigen administration, as the serum concentration of cytotoxic anti‐Thy1.2 antibodies was 92‐fold greater in mice with eye tumors in comparison with mice with skin tumors. In addition, anti‐Thy1.2 immune responses were detected in B6.PL mice given naïve Thy1.2+ T cells i.p. but not i.v. Anti‐Thy1.2 responses were augmented in B6.PL mice with ocular Thy1.2+ EL‐4 tumors that did not express OVA, suggesting immunodominance of OVA antigen over Thy1.2. Thy1.1+ T cells given i.p. was not immunogenic in Thy1.2 congenic mice. These data reaffirm that the introduction of antigens in the a.c. induces robust antibody responses. Experimentation using allotypic differences in Thy1 between donor cells and recipient mice must consider cytotoxic anti‐Thy1 antibody generation in the interpretation of results.