Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors

AbstractA major barrier to curing HIV is the long-lived latent reservoir that supports re-emergence of HIV upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV reservoir.Author SummaryActivation of CD4+ T cells facilitates HIV-1 infection; however, whether there are minimal signals required for the establishment of infection, replication, and latency has not been explored. To determine how T cell signaling influences HIV-1 infection and the generation of latently infected cells, we used chimeric antigen receptors to create a tunable model. Stronger signals result in robust HIV-1 expression and an inducible latent population. Minimal signals predispose cells towards latent infections that are refractory to reversal. We discovered that repression of HIV-1 transcription immediately after infection is due to RNA polymerase II pausing and inefficient transcription elongation. These studies demonstrate that signaling events influence the course of HIV-1 infection and have implications for cure strategies. They also provide a mechanistic explanation for why a significant portion of the HIV latent reservoir is not responsive to latency reversing agents which function by modifiying chromatin.

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Third Generation Chimeric Antigen Receptors Containing CD137 or CD134 Signaling Endodomains Augment CD19-Specific T-Cell Effector Function.

Blood ◽

10.1182/blood.v114.22.4097.4097 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4097-4097 ◽

Cited By ~ 1

Author(s):

Harjeet Singh ◽

Hillary N Gibbons ◽

Simon Olivares ◽

Matthew J. Figliola ◽

Margaret J. Dawson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Second Generation ◽

Third Generation ◽

Antigen Receptors ◽

T Cell Signaling ◽

Chimeric Antigen Receptors ◽

The Third

Abstract Abstract 4097 Poster Board III-1032 CD19-specific chimeric antigen receptors (CARs) based on first and second generation designs (Figure) expressed by genetically manipulated T cells are being evaluated in clinical trials and it is apparent that the long term in vivo survival of the infused T cells will be critical to achieving therapeutic successes. We have previously demonstrated that the first-generation CAR can be modified to include and provide an improved T-cell survival signal through addition of a chimeric CD28 endodomain (designated second generation CAR, CD19RCD28). However, it is recognized that optimal activation of T cells through CD28 for sustained proliferation may require co-stimulation through other signaling molecules. To address this issue we have altered the second generation CAR to include additional intracellular signal transduction domains from CD137 (4-1BB) or, CD134 (OX40) to generate (Figure) third generation CARs (CD19RCD28CD137 or, CD19RCD28CD134, respectively). The third generation CARs were electro-transferred into peripheral blood T cells using the Sleeping Beauty transposon/transposase system and propagated on K562-derived CD19+ artificial antigen presenting cells (aAPCs). We observed the selective outgrowth of CD19-specific T cells expressing similar percentages of expression and densities of CD19RCD28, CD19RCD28CD137, and CD19RCD28CD134 CARs with a subset of the propagated T cells displaying a central memory phenotype (CD62L+CD28+). The T cells expressing the third generation CARs exhibited redirected-killing and were able to produce IFN-g in response to CD19. Although the cytolytic ability and Tc1 cytokines released by both second and third generation CARs were similar, differences due to the presence of the CD137 and CD134 intracellular signaling endodomains were apparent using in vitro experiments designed to mimic in vivo T-cell persistence, by measuring long-term propagation in the absence of exogenous cytokines added to the T-cell culturing process. We observed an approximate 200% increase in the numeric expansion of CD19RCD28CD137+ and CD19RCD28CD134+ T cells as compared to the CD19RCD28+ T cells at the end of 14 days of co-culture on CD19+ aAPC. These data highlight the fact that CD19-dependent signaling through chimeric CD28 might be improved by the addition of signaling through chimeric CD137 or CD134 domains so as to fully maintain T-cell activation and sustain proliferation in vivo after adoptive transfer. Figure Schematic of first (CD19R), second (CD19RCD28), and two third (CD19RCD28CD137 and CD19RCD28CD134) generation CD19-specific CARs (shown as homodimers on the cell surface) expressing one, two, or three T-cell signaling chimeric endodomains. TM = transmembrane. Figure. Schematic of first (CD19R), second (CD19RCD28), and two third (CD19RCD28CD137 and CD19RCD28CD134) generation CD19-specific CARs (shown as homodimers on the cell surface) expressing one, two, or three T-cell signaling chimeric endodomains. TM = transmembrane. Disclosures: No relevant conflicts of interest to declare.

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