CD8⁺ T cell dialyzable extract activity is dependent on TCR and MHC‐I

Short abstract

Author response.

Abbreviations

^−/−

knockout/deficient

β2m

β2 microglobulin

DC

dendritic cell

DLE

dialyzable leukocyte extract

IP

immunoprecipitation

MHC‐I/II

MHC types I/II

MS

mass spectrometric

TF

transfer factor

WT

wild‐type

The authors thank Dr. Levis’ group for their interest in our research [1]. We agree that full molecular identification will be key to unraveling the biology of TF, also referred to as DLE. Our central finding from the previous work indicated that cell lysates derived from antigen‐specific CD8⁺ T cells induce IL‐6 from antigen‐pulsed DCs [2]. Furthermore, we demonstrated that the TCR was implicated in the mechanism of DLE. We reported that cell lysates from unimmunized OT‐I mice, but not OT‐II sourced lysates, could impart anti‐OVA immune reactivity in OVA‐naive mice.

The only difference between WT and OT‐I mice is the presence of a transgene encoding for the TCR that recognizes the CD8 OVA epitope [3]. Thus, we hypothesized that the anti‐OVA TCR from CD8⁺ cells may interact with the OVA‐presenting DC on its MHC‐I. We have since confirmed TCR dependence with IP experiments using dialyzed OT‐I cell lysates ( Fig. 1A ). Incubation of OT‐I lysates with anti‐TCR‐β antibody, but not isotype control, depleted the IL‐6 induction activity; the elution of the IP product from the anti‐TCR‐β antibody restored this activity (Fig. 1A).

Figure 1.

DLE activity is dependent on TCR and CD8 T cells but not MHC‐II.

(A) DLE from OT‐I mice was incubated overnight with anti‐TCR‐β antibody or isotope control. The antibody‐bound product was eluted, as described previously [2]. DCs were pulsed with OVA and incubated, as described previously, with nonprecipitated OT‐I lysate (–), depleted lysate (Dep), or the dialyzed elution product (Elute). Change in IL‐6 (ΔIL‐6) from media control at 24 h is shown. (B) WT, MHC‐II^−/−, CD8^−/−, β2m^−/−, TCR‐α^−/−, or TCR‐β^−/− mice were immunized with OVA in CFA, as described previously. DLE was extracted from the mice 3 wk later and used to stimulate OVA‐pulsed DCs from WT mice. Change in IL‐6 from diluent control is shown. (C) DCs were harvested from β2m^−/−, C3H, MHC‐II^−/−, or WT mice as before and stimulated for 24 h with DLE from OT‐I mice. Data shown are representative of 3 independent experiments and displayed as means + sem. Significance shown vs. nonprecipitated control (B) or WT control (C). ****P < 0.0001, as determined by ANOVA.

Furthermore, we extracted DLE from various knockout mice as an additional approach to assess the importance of CD8 T cells and their antigen‐specific TCR. The ability to induce IL‐6 from DC was preserved in cell lysates from MHC‐II^−/− mice immunized with OVA (Fig. 1B). However, IL‐6 induction was significantly reduced when the lysates were extracted from mice deficient in CD8 T cells as a result of direct genetic knockout (CD8^−/−) or β2m^−/− (Fig. 1B). IL‐6 induction by DLE was completely abrogated by the absence of TCR‐α or TCR‐β in the immunized mice from which the lysates were harvested (Fig. 1B). To assess DLE signaling further, we harvested DCs from MHC‐I^−/− mice, as a result of β2m genetic depletion, TLR4 signaling (C3H mice), or MHC‐II. As seen previously [2], cell lysate activity was dependent on both MHC‐I and TLR4 on the DC, but this signaling was independent of MHC‐II ( Fig. 1C ). Taken together, these data substantiate our prior reports that DLE activity is dependent on the antigen specificity of the TCR in the CD8 T cell lysate source, as well as MHC‐I and TLR4 signaling in the target DC.

We are currently pursuing various approaches to molecular identification. However, we have been limited by the paucity of TCR sequences deposited in standard MS protein identification databases. This has made it difficult to assess or confirm TCR content in protein bands that stain positive in anti‐TCR immunoblots. Furthermore, the MS signal from the relatively high concentration of antibody fragments used during IP has proven difficult to separate from the remaining peptide signatures. This inability to assess TCR content has precluded our ability to identify other molecules that may be linked to the presumed TCR fragment, such as S100a9 or as Dr. Levis points out, noncoding RNA [1].

Whereas molecular identification of the TF/DLE complex is the ultimate goal, we also agree that a stable cell line made from antigen‐specific CD8 T cells would be a markedly improved source of DLE compared with our current reliance on mixed cellular populations. To this end, we are investigating immortalization techniques for pathogen‐specific CD8⁺ T cell lines from humans, which we hope will function similarly to our OVA tetramer‐sorted cells or those from the OT‐I spleen lysates. Like Dr. Levis [1], we hope continued studies to clarify the physiologic function and molecular nature of DLE will move us closer to harnessing its full therapeutic potential. This work was supported by the Intramural Research Program of The National Institute of Allergy and Infectious Diseases and The National Institutes of Health.