Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 162 (3), 516-27

Were Monocytes Responsible for Initiating the Cytokine Storm in the TGN1412 Clinical Trial Tragedy?

Affiliations

Were Monocytes Responsible for Initiating the Cytokine Storm in the TGN1412 Clinical Trial Tragedy?

G P Sandilands et al. Clin Exp Immunol.

Abstract

The precise biological mechanisms that caused the TGN1412 clinical trial tragedy (also known as 'The Elephant Man Clinical Trial') in March 2006 remain a mystery to this day. It is assumed widely that the drug used in this trial (TGN1412) bound to CD28 on T lymphocytes and following activation of these cells, a massive 'cytokine storm' ensued, leading ultimately to multi-organ failure in all recipients. The rapidity of this in vivo response (within 2 h), however, does not fit well with a classical T lymphocyte response, suggesting that other 'faster-acting' cell types may have been involved. In this study we have activated purified human peripheral blood leucocyte populations using various clones of mouse monoclonal anti-CD28 presented to cells in the form of a multimeric array. Cytokines were measured in cell-free supernatants at 2 h, and specific mRNA for tumour necrosis factor (TNF)-α, thought to be the initiator of the cytokine storm, was also measured in cell lysates by reverse transcription-polymerase chain reaction (RT-PCR). Monocytes were the only cell type found to show significant (P < 0·05) up-regulation of TNF-α at 2 h. Eleven other monocyte cytokines were also up-regulated by anti-CD28 within this time-frame. It therefore seems likely that monocytes and not T cells, as widely believed, were probably responsible, at least in part, for initiating the cytokine storm. Furthermore, we propose that a multimeric antibody array may have formed in vivo on the vascular endothelium via an interaction between TGN1412 and CD64 (FcγRI), and we provide some evidence in support of this hypothesis.

Figures

Fig. 1
Fig. 1
Demonstration of surface and cytoplasmic staining for CD28 by flow cytometry. Typical examples are shown as histogram overlays with an appropriate isotype control (shown as a grey infill) used to provide background values. (a) Dot-plot of forward-scatter (cell size) versus side-scatter (granularity) showing lymphocytes gated as region 1 (R1). The histogram overlay shows that CD28 was expressed on a distinct subpopulation of lymphocytes as indicated by the arrow i.e. T cells. (b) Dot-plot of forward- versus side-scatter showing monocytes gated as region 2 (R2). (c) Dot-plot of forward- versus side-scatter showing neutrophils gated as region 3 (R3). Cytoplasmic staining: cell surface staining (solid line) of viable cells was compared to staining following fixation and permeabilization of cells (dotted line). These experiments were conducted on leucocytes obtained from 10 healthy donors. Net mean fluorescence intensity (MFI) are shown below. A significant (Wilcoxon two-sample test) increase in net MFI following fixation and permeabilization, relative to surface values, indicates the presence of cytoplasmic ‘stores’ of CD28. Inspection of permeabilized granulocytes, showing distinctive polymorphonuclear morphology, by fluorescence microscopy confirmed that cytoplasmic CD28 (green) was confined to granules. Nuclear staining (blue) was demonstrated using 4′,6-diamidino-2-phenylindole (DAPI). No significant ‘stores’ were detected within T cells or monocytes. Data obtained from 10 healthy donors are shown below. Average MFI ± standard error of the mean shown
Fig. 2
Fig. 2
Effect of cross-linking (X–L) CD28 on cell surface expression of CD28. (a) Flow cytometry dot-plots of forward-scatter (cell size) versus log fluorescence [fluorescein isothiocyanate (FITC)-anti CD28, clone15E8] shows baseline values for lymphocytes and neutrophils. CD28 was detected on a distinct subpopulation of cells (T cells) as indicated by the population appearing above the dotted line with a mean fluorescence intensity (MFI) of 45. In contrast, all neutrophils in this example showed very weak surface staining for CD28 (MFI = 22). (b) Flow cytometry dot-plots of forward-scatter (cell size) versus log fluorescence (FITC-anti CD28, clone 15E8) shows the effect of cross-linking CD28 at 37°C for 1 h. No change was observed in the MFI for CD28 on the surface of T cells. There was a striking increase, however, in surface staining for CD28 on all neutrophils (MFI = 75). (c) Cells visualized by fluorescence microscopy using FITC-anti-CD28 (green) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Lymphocytes show surface staining for CD28 in a continuous ring pattern. This was not altered by cross-linking CD28. In contrast, CD28 was not visible by fluorescence microscopy on the surface of control neutrophils but following cross-linking of CD28 at 37°C for 1 h, CD28 was clearly visible in the form of large heterogeneous clusters on the cell surface.
Fig. 3
Fig. 3
The effect of cross-linking (X–L) granulocyte CD28 on surface expression of CD66 and CD28: kinetic studies. Purified granulocytes were incubated on a multimeric array of anti-CD28 (clone 204.12) or mouse IgG1 antibody as a control, at 37°C for various time-intervals prior to measurement of cell surface CD66 (clone Kat4c) and CD28 (clone 15E8) by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated antibodies. Results are shown as the mean fluorescence intensity (MFI). Mean ± standard error of the mean of three experiments.
Fig. 4
Fig. 4
The effect of cross-linking (X–L) CD28 using a multimeric array (clone 204.12) on neutrophil granulocyte nuclear morphology. Cells were incubated at 37°C for 1 h on a multimeric array of anti-CD28 (or isotype control immunoglobulin G) as described in Methods prior to flow cytometry or morphological inspection using cytospin preparations of cells. (a) Dot-plot of forward- versus side-scatter (cell size versus granularity) showing control neutrophils and a haematoxylin and eosin (H&E)-stained cytospin preparation of neutrophils showing the characteristic multi-lobed structure of the nucleus. The bar chart shows counts performed on 300 cells showing that the vast majority of control neutrophils have three or four lobes. (b) Cross-linking of neutrophil CD28 produces a reduction in side-scatter (granularity) and a reduction in nuclear lobes with the vast majority of cells (76%) now showing one or two lobes. This is known as the pseudo-Pelger–Huët anomaly that was observed in all six recipients of TGN1412 [4].
Fig. 5
Fig. 5
Human umbilical vein endothelial cells (HUVECs) express FcγRI (CD64). (a,b) HUVECs before and after incubation with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD64 (FcγRI), clone 10.1 [immunoglobulin (Ig) G1]. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Cell surface CD64 appears as punctate staining. (c) Human peripheral blood mononuclear cells (PBMCs), i.e. lymphocytes (L) plus monocytes (M) adhering spontaneously to HUVECs following incubation for 18 h at 37°C. Cells were visualized using an inverted microscope at a magnification of ×400. Examples of adherent cells are indicated by arrows. (d) Increased binding of PBMCs to HUVECS (18 h at 37°C) as a result of preincubating the same number of cells (opsonization) with mouse anti-CD28 (clone 204.12). No increase in binding was observed when cells were preincubated at 4°C for 1 h with mouse IgG2a at the same concentration (4 µg per 106 cells). Based on microscopic inspection of 100 HUVECs the ratio of PBMC : HUVEC was 1:1 for non-opsonized cells and 2:1 for anti-CD28 opsonized cells.
Fig. 6
Fig. 6
Hypothesis: (1) TGN1412 binds to CD28+ cells, including monocytes, to form a complex. (2) Complex binds to CD64 (FcγRI) on vascular endothelial cells displacing any passively bound plasma immunoglobulin (Ig)G. (3) Cross-linking of monocyte CD28 occurs leading to cell activation and rapid up-regulation of multiple cytokines, including tumour necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-1α. (4) MIP-1α recruits natural killer (NK) cells. (5) Cross-linking of CD28 on NK cells (known to express a variant form of CD28: vCD28) causes release of other ‘storm’ cytokines including interferon (IFN)-γ, that in turn activate monocytes. (6) Opsonized NK cells could also interact with CD64 on adjacent monocytes to provide a further amplification signal.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

MeSH terms

LinkOut - more resources

Feedback