Aging Impairs Recipient T Cell Intrinsic and Extrinsic Factors in Response to Transplantation

As increasing numbers of older people are listed for solid organ transplantation, there is an urgent need to better understand how aging modifies alloimmune responses. Here, we investigated whether aging impairs the ability of donor dendritic cells or recipient immunity to prime alloimmune responses to organ transplantation.Using murine experimental models, we found that aging impaired the host environment to expand and activate antigen specific CD8(+) T cells. Additionally, aging impaired the ability of polyclonal T cells to induce acute allograft rejection. However, the alloimmune priming capability of donor dendritic cells was preserved with aging.Aging impairs recipient responses, both T cell intrinsic and extrinsic, in response to organ transplantation

Targeting transcription regulation in cancer with a covalent CDK7 inhibitor

Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state1, but direct pharmacological inhibition of transcription factors has thus far proven difficult2. However, the transcriptional machinery contains various enzymatic co-factors that can be targeted for development of new therapeutic candidates3, including cyclin-dependent kinases (CDKs)4. Here we present the discovery and characterization of the first covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-ALL, exhibit exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and this transcription factor’s key role in the core transcriptional regulatory circuitry of these tumor cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state

Targeting transcription regulation in cancer with a covalent CDK7 inhibitor

Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.National Institutes of Health (U.S.) (Grant HG002668)National Institutes of Health (U.S.) (Grant CA109901

Aging impairs T cell alloimmune responses.

<p>A: Young C57BL/6 T cell deficient mice reconstituted with 5×10⁶ aged (20 mths) syngeneic, polyclonal T cells rejected BALB/c skin allografts at a slower tempo than counterparts reconstituted with young (2–4 mths) T cells. (*p = 0.007; Log rank). B: Aged or young CBA recipients were transplanted with young C57BL/6 skin transplants. At two weeks post transplantation, recipient spleen cells were harvested and CD4⁺ T cells purified. The results demonstrate that aged CD4⁺ T cells that were stimulated ex vivo with irradiated donor spleen cells manifest impaired IFNγ (*p<0.0001; T test) and IL-2 (**p<0.0001; T test) responses (ELISPOT) compared to young CD4⁺ T cells. N = 3/group, representative data from two independent experiments with consistent results. C: Aged and young purified, polyclonal T cells were stimulated with C57BL/6 BMDCs and the upregulation of indicated activation markers shown after 96 h of culture. N = 3/group, representative data three independent experiments with consistent results.</p

Aged BMDCs exhibit a similar alloimmune priming ability as compared to young BMDCs.

<p>A: Young C57BL/6 mice were immunized (via i.p. injection) with 1×10⁷ young CBA spleen cells. After two weeks, recipient spleen cells were harvested and cultured <i>ex vivo</i> overnight with increasing numbers of either aged or young irradiated CBA BMDCs and IFN-γ responses measured (ELISPOT). B: Aged or young CBA BMDCs were i.p. injected into young C57BL/6 mice. Two weeks later, recipient spleen cells were harvested and cultured <i>ex vivo</i> overnight with young irradiated CBA spleen cells. IFN-γ responses measured via ELISPOT. Similar results were obtained when the re-stimulating irradiated donor spleen cells were aged (data not shown).</p

Recipient age impairs the expansion of transplant reactive CD8⁺ T cells.

<p>C57BL/6 aged and young CD45.2⁺, Thy1.2⁺ mice were adoptively transferred with 5×10⁵ CD45.1⁺, OTI T cells 2 days prior to receiving a B6.Act-mOVA skin transplant. A: At various points after transplantation, recipient spleen cells were harvested and the number of OTI T cells or OTI T cells that produced IFN-γ calculated. Expansion of transplant/antigen specific T cells was reduced in an aged environment as compared to a young environment. (*P<0.02; T test at day +7 post transplantation). Representative data of three independent experiments with N = 2–3/experiment. B: Representative flow cytometric dot plots are shown at day +7 post transplantation in young and aged mice. A representative, non-transplanted recipient that received OTI T cells is shown. The transferred cells (CD45.1⁺, CD8⁺) are shown in the rectangle. Representative data of three independent experiments with N = 2–3/experiment.</p

Transplant specific CD8⁺ T cells produced similar cytokine profiles but impaired activation in aged transplant recipients as compared to young recipients.

<p>A: Representative flow cytometric plots on day +7 post transplantation are shown along with isotype controls (IgG2b for IFNγ, IgG1 for IL-2 and TNFα). Young antigen specific CD8⁺ T cells produced similar amounts of TNFα, IL-2 and IFN-γ during <i>ex vivo</i> culture with OVA peptide regardless of the age of the recipient environment (cytokines were not produced if peptide was not added, data not shown). Proportions are shown in right upper quadrant. Note, that cytokines were not detected in either aged or young mice that were adoptively transferred but did not receive an OVA expressing skin transplant (data not shown). Similar results were noted at day +14 post transplantation (data not shown). Flow cytometric plots are gated on CD45.1⁺ cells. N = 3/group, representative data from three independent experiments with consistent results. B: Representative flow cytometric plots of adoptively transferred CD45.1⁺, OTI young T cells on day +7 post transplantation. Young antigen specific CD8⁺ T cells upregulated CD27 and downregulated CD62L to a greater degree in young transplant recipients compared to antigen specific CD8⁺ T cells in aged transplant recipients.</p

Aged splenic DCs loaded with cognate peptide exhibit a similar ability to prime antigen specific CD8⁺ T cells as compared to young splenic DCs.

<p>A: Aged and young splenic DCs were cultured with OTI T cells in the presence of OVA peptide and proliferation measured. Representative from one experiment, which was repeated with similar results. B: Young hosts (N = 3/group) were adoptively transferred with young CD45.1⁺ OTI T cells and either young or aged splenic DCs that had been pulsed with OVA peptide (or irrelevant peptide, GP33, as a control). After 5 days, expansion of transferred young OTI T cells was assessed. Proportion of transferred T cells is shown in the box in each flow plot. Representative data from one experiment, which was repeated with similar results. C: Same experiment as B except that cytokine production by the transferred OTI T cells (gated on CD45.1⁺ population) is measured by intracellular cytokine staining. Proportion % is shown in each flow plot.</p

Aging does not impair the ability of graft originating APCs to prime alloimmunity.

<p>C: Young CBA recipients received either aged or young C57BL/6 skin allografts. Two weeks after transplantation, recipient spleens were removed and then restimulated overnight in the presence of irradiated donor spleen cells. (No differences were noted if aged or young donor irradiated stimulators were used, data not shown). D: Young CBA recipients were transplanted with either an aged or young C57BL/6 skin allograft and the time to rejection was monitored. Young recipients of aged allografts manifest similar rejection kinetics compared to young counterparts that received young allografts (p = 0.5; Log rank).</p