Imbalanced Randomization in Vaccine Clinical Safety Trials

Clinical safety and immunogenicity data in vaccines are commonly dichotomized into responder and nonresponder binary data. This article expands upon Fagerland, Lyndersen, and Laake to further study Newcombe’s hybrid score and Fisher’s conditional exact test using imbalanced randomization in vaccine clinical studies. The intent of this article is to study a potential statistical issue in clinical study design and analysis; hence, no reference to any particular product is made.</p

SHIP+/+ and −/− GM-DCs suppress T cell activation.

<p>2×10^{5<b> </b>}WT splenocytes were stimulated with soluble αCD3+ αCD28 antibodies and incubated with the indicated number of SHIP+/+ or −/− <b>A)</b> CD11c⁺ splenic DCs, FL- or GM-DCs. Proliferation was determined after 72 hrs by incorporation of ³H-thymidine for the last 18 hrs. Data shown are the mean ± SEM of triplicate cultures and are representative of more than 3 independent experiments. Supernatants were collected after 72 hrs from <b>B)</b> Splenic (25×10³) <b>C)</b> Flt3L-derived (25×10³) and <b>D)</b> GM-derived DCs (50×10³) co-cultures and subjected to cytokine ELISAs or Griess assays for NO determination. Data shown are the mean ± SEM of triplicate cultures and are representative of 2–3 independent experiments. *p<0.05 relative to stimulated splenocytes in the absence of DCs (Ctrl).</p

Secreted cytokines are not responsible for T cell suppression.

<p><b>A)</b> WT splenocytes (2×10⁵) were stimulated with soluble αCD3+ αCD28 Abs and co-incubated with SHIP+/+ or −/− GM-DCs (50×10³) containing isotype control Ab (iso) or the indicated neutralizing cytokine Ab (10 µg/ml) or LAP (250 ng/ml). <b>B)</b> WT splenocytes were stimulated with soluble αCD3+ αCD28 Abs and incubated with the indicated number of SHIP+/+ and −/− GM-DCs and IL-2 (100 U) was added as indicated and proliferation determined after 72 hrs. Data shown are the mean ± SEM of triplicate cultures and is representative of at least 2 independent experiments. <b>C)</b> CD4⁺ T cells from SHIP+/+ and −/− GM-DC (50×10³) co-cultures were analyzed for expression of CD25 by flow cytometry. Splenocyte control  =  grey fill, WT GM-DCs  =  black line and SHIP−/− GM-DCs  =  grey line. Data shown are representative of 2 independent experiments.</p

Model of SHIP+/+ and −/− GM-DC-induced T cell suppression.

<p>SHIP+/+ and −/− GM-DCs both suppress T cell proliferation in a contact-dependent manner. αCD3+ αCD28-stimulated T cells secrete IFNγ, which acts on WT GM-DCs to upregulate iNOS and secrete NO. This NO then suppresses T cell proliferation. SHIP−/− GM-DCs express Arg 1 and do not produce NO, but may use an alternate direct mechanism of suppression or induce the expansion or differentiation of a regulatory cell, likely not Tregs, to suppress T cell proliferation. If a second cell type is involved in SHIP−/− GM-DC-induced suppression, its induction or activation is contact-dependent.</p

SHIP−/− GM-DCs express Arg 1.

<p><b>A)F</b> Day 8 SHIP+/+ and −/− GM- and FL-DCs were subjected to Western analysis using Abs to SHIP, Arg1 and Grb2 as a loading control. Data shown are representative of at least 3 independent experiments. <b>B)</b> mRNA expression of <i>Arg 1</i>, <i>Arg 2</i>, <i>Nos2</i>, and <i>Indo</i> in SHIP+/+ and SHIP−/− GM- and FL-DCs. Data shown is mean ± SEM of duplicate determinations from 2–3 independent experiments. *p<0.05 relative to SHIP+/+. <b>C)</b> WT splenocytes were stimulated with soluble αCD3+ αCD28 Abs and incubated with SHIP+/+ or −/− GM-DCs (50×10³) ±100 µM of the arginase inhibitor, Bec, 2 mM L-arginine (L-Arg), 1 µM of the IDO inhibitor, exiguamine A (Exi) or 200 µM L-tryptophan (L-Tryp). Data shown are the mean ± SEM of triplicate determinations and are representative of 2 independent experiments.</p

SHIP+/+ and −/− GM-CSF-derived DCs suppress via a contact-dependent mechanism.

<p><b>A)</b> The indicated number of SHIP<b>+/+</b> and −/− GM-DCs were plated in the bottom chamber of a 0.4 µm 96 well transwell plate and WT splenocytes (2×10⁵ ) were stimulated with soluble αCD3+ αCD28 Abs and plated in the top chamber. Proliferation was determined after 72 hrs by incorporation of ³H-thymidine for the last 18 hrs. Data shown are the mean ± SEM of triplicate cultures and are representative of 3 independent experiments. <b>B)</b> Left panel, relative percent suppression with the addition of agents that reduce the presence of ROS (2 mM NAC, 100 U/ml catalase, 200 U/ml SOD). Right panel, relative percent suppression with the addition of blocking antibodies to CTLA4 (10 µg/ml), LFA1+ mac1 (5 µg/ml each) and PD-L2 (10 µg/ml). Data shown are the mean ± SEM of triplicate cultures and are representative of at least 2 independent experiments with the exception of PD-L2 which was only performed once. <b>C)</b> SHIP+/+ and −/− GM-DCs were cultured for 4 days with WT sorted conventional T cells at a ratio of 1∶2 DCs to T cells and analyzed for Treg induction by flow cytometry. Data shown are the mean ± SEM of two independent experiments.</p

SHIP+/+ but not −/− GM- DC-induced T cell suppression is mediated by IFNγ-dependent NO production.

<p>WT splenocytes were stimulated with soluble αCD3+ αCD28 Abs and incubated with SHIP+/+ or −/− GM-DCs (50×10³) for 72 hrs. <b>A)</b> Left panel, relative percent suppression of proliferation in the absence (ctrl) or presence of 25 µg/ml PTIO, 0.5 mM–2.5 mM L-NMMA or 25 µg/ml PTIO +0.5 mM L-NMMA). Right panel, NO production using the same concentrations of PTIO and/or L-NMMA. <b>B)</b> WT splenocytes were stimulated with soluble αCD3+ αCD28 Abs and incubated with SHIP+/+ or −/− GM-DCs (50×10³) ±10 µg/ml neutralizing αIFNγ. Left panel, percent suppression of T cell proliferation. Right panel, NO production. Data shown are mean ± SEM of triplicate cultures and are representative of 3 independent experiments. *p<0.05 relative to genotype control, ns  =  not significantly different. ▾ indicates level is below detection.</p