Protocadherin-18 Is a Novel Differentiation Marker and an Inhibitory Signaling Receptor for CD8+ Effector Memory T Cells

CD8+ tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling previously shown to result from inactivation of p56lck kinase. We identify a novel interacting partner for p56lck in nonlytic TIL, Protocadherin-18 (‘pcdh18’), and show that pcdh18 is transcribed upon in vitro or in vivo activation of all CD8+ central memory T cells (CD44+CD62LhiCD127+) coincident with conversion into effector memory cells (CD44+CD62LloCD127+). Expression of pcdh18 in primary CD8+ effector cells induces the phenotype of nonlytic TIL: defective proximal TCR signaling, cytokine secretion, and cytolysis, and enhanced AICD. pcdh18 contains a motif (centered at Y842) shared with src kinases (QGQYQP) that is required for the inhibitory phenotype. Thus, pcdh18 is a novel activation marker of CD8+ memory T cells that can function as an inhibitory signaling receptor and restrict the effector phase

Biochemical and functional analyses of lytic T cells transfected with pcdh18.

<p>(6a) Flow cytometry analysis of primary lytic effector cells generated from spleens of 7 week old mice (prepared as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’). PI^lo cells are >80% CD8⁺, PI^hi cells are ∼40% CD8⁺ (top panel). >70% of CD8⁺CD44^hi cells are CD62L^hiCD127^hi (bottom panel). (6b) (top) Reciprocal immunoblot of p56^lck isolated from nonlytic TIL. Analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#pone-0036101-g001" target="_blank">Figure 1a</a> (after conjugation with cognate MCA38 tumor cells for 0, 5, or 15 min as indicted and immune precipitated with anti-p56^lck Ab 2102- left panels- or Ab 3A5- right panels) and blots were probed with anti-pY or anti-Pcdh18 as indicated. (bottom) Expression of pcdh18 protein in transfected effector cells by flow cytometry was as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’. Cells were stained with control or anti-pcdh18 Ab as indicated (top). (6c) Phase contrast microscopy of transfected cells. Effector cells were transfected as indicated and cultured <i>in vitro</i> in the presence or absence of IL-2 for ∼24 h before microscopy. Arrows indicate cell clusters. (6d) RNA was extracted from transfected effector cells (‘control’ or ‘pcdh18’), nonlytic TIL (‘TIL 0 h’), or lytic TIL that were activated with anti-CD3 for 4 hours (‘TIL 24 h’) and used for cytokine qRT-PCR, or cells were assessed for viability (PI and Annexin V staining), as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’. In the viability assay two different plasmid constructs were used in separate experiments (bottom left panel) or the Y842F mutant (bottom right panel). (6e) Transfected effector cells were assayed for lytic function by re-directed cytolysis assay as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’. (6f) Transfected cells were assayed for binding of anti-Zap70 pY493 after permeabilization following activation with anti-TCR for 2 min as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Material and Methods</a>’.</p

RNA analyses of spleen cells after induction of memory <i>in vivo</i>.

<p>(a) qRT-PCR analysis of spleen CD8⁺ T cells isolated from BL/6 mice previously infected with either 5,000 recombinant <i>Listeria monocytogenes</i>, buffer controls, or EL4 cells at a subtumorigenic dose (‘regressor tumor’). 28 days after exposure spleen CD8⁺ T cells were purified, activated <i>in vitro</i> with Con A for the indicated times, RNA prepared and qRT-PCR performed as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’. (This analysis has been performed using mice previously infected for times up to 1 year with similar results). Insert shows gel analysis of pcdh18 RT-PCR expression from <i>L. monocytogenes</i>-immune mice. (3b) qRT-PCR analysis of purified CD8⁺ T cells from mice injected with <i>L. monocytogenes</i> or allogeneic H-2D spleen cells. (3c) qRT-PCR analysis of purified CD8⁺ T cells from mice originally infected with <i>L. monocytogenes</i> which were challenged by <i>in vivo</i> infection by <i>L. monocytogenes</i>. Spleens were isolated at the indicated times following challenge. Age-matched naive mice received only primary exposure given at the time of secondary challenge. Nonlytic TIL are shown for comparison. (3d) qRT-PCR analysis of purified control CD8⁺ spleen T cells and activated <i>in vitro</i> with anti-CD3e for the indicated times before RNA isolation and analysis by qRT-PCR. The data shown are representative of multiple repetitions.</p

RT-PCR analysis of protocadherin 18 in tissues and immune cells.

<p>(2a) Amino acid sequence comparison of the p56^lck Y505 motif in protocadherins. (2b) RT-PCR analysis of mouse tissues. The indicated tissues and organs were isolated from a control mouse, RNA was extracted and used to prepare cDNA, and PCR performed using control (pcdh8 and β-actin) or pch18 primers as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’. (2c) RT-PCR analysis of spleen cells. Spleens were isolated from a 7 week old mouse and the indicated cells were purified by FACS (top panel). TIL were isolated from an MCA38 tumor. CTLL-2 is a CD8⁺ lytic cell line. RNA was isolated and used to program RT-PCR as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’. Similarly spleen CD8⁺ T cells were purified and activated <i>in vitro</i> with anti-CD3 for the indicated times before analysis (bottom panel). (2d) TIL qRT-PCR analyses. Single cell suspensions of MCA38 tumors were prepared and CD4⁺ or CD8⁺ TIL were isolated by magnetic immunobeading. (Liver tissue was isolated and used for RNA isolation in certain control analyses. TIL were labeled with anti-CD4 or CD8 Ab and further purified by FACS (example of flow cytometry analysis shown in left panels) before RNA isolation and qRT-PCR analysis. TIL used to prepare RNA immediately after isolation are indicated in as ‘nonlytic’ or ‘TIL 0 hr’. As indicated some TIL samples were cultured <i>in vitro</i> for 8 or 24 h before RNA isolation during which time TIL recover both proximal TCR-mediated signaling and lytic function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#pone.0036101-Monu1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#pone.0036101-Koneru1" target="_blank">[7]</a>. PCR analyses of various target RNAs are shown and include several control reactions that demonstrate specificity of the expression patterns observed (e.g. pcdh18 and pcdh12 in CD8⁺ TIL, Dab1 and Dab2a in CD8⁺ TIL, granzyme B in CD8⁺ TIL and liver, as well as TNF, IFN, IL-2, PD-1, and PD-1L). Data show SD from three independent experiments. (2e) qRT-PCR analysis of purified spleen cells. Spleen immune cells were isolated by FACS from young (4 week) or old (>48 week) control mice and RNA was isolated and used to program pcdh18 qRT-PCR as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#s4" target="_blank">Materials and Methods</a>’. The data shown are representative of multiple repetitions.</p

Conversion of Cm into Em cells upon activation <i>in vitro</i>.

<p>(4a) FACS purification of spleen CD8⁺ CD44^hiCD62L^loCD127^hi (Effector-memory) and CD44^hiCD62L^hiCD127^hi (Central-memory) cells obtained from young (<5 week) and >50 week old naïve, and 50 week old mice that were previously injected with allogeneic spleen cells as indicated. Following sorting, an aliquot of cells was immediately taken for RNA isolation without activation. Analyses in the left panels are gated on CD8⁺ cells and show the distribution of CD44⁺ cells. Analyses in the right panels show Em and Cm cells within the CD44^lo and CD44^hi populations as indicated. (4b) Cm and Em cells were isolated by FACS from young, old, and memory (<i>L. monocytogenes</i>-infected) mice and activated <i>in vitro</i> with ConA for the indicated times before qRT-PCR. The % of CD8⁺CD44^hi cells as CD62L^loCD127^hi (Em) and CD62L^hiCD127^hi (Cm from the ‘young’ cohort are shown in tabular form in this figure. Flow cytometry analyses of some samples from the kinetic experiment are shown to illustrate gating. (4c) Enriched CD8⁺ spleen cells prior to sorting are shown in the left panels. After labeling Cm were isolated and activated <i>in vitro</i>. The recovery of Em and Cm cells was determined by flow cytometry after re-staining and flow cytometric analysis. Shown are the FACS analyses after the indicated times of Cm activation. Also shown is the number of Cm and Em cells derived from <i>in vitro</i> activation of Cm cells represented as a ratio of Cm to Em cells at the various times of analyses.</p

Primers for qPCR.

<p>(Ta = annealing temperature).</p

Reciprocal immunoblot analysis of p56^lck isolated from nonlytic and lytic MCA38 TIL.

<p>(1a) TIL were purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036101#pone.0036101-Radoja1" target="_blank">[6]</a> and either immediately used to form conjugates with MCA38 cells for the indicated times (upper panel) or plated in complete RPMI medium (lower panel) until performance of conjugation and reciprocal immunoblotting. Following incubation, detergent lysates were prepared and immuneprecipitated with Ab reactive with an epitope in the amino terminal portion of the protein (clone 3A5). Immuneprecipitated p56^lck was subjected to immunoblotting using anti-p56^lck (pY505) and detected by chemiluminescence following reaction with peroxidase-conjugated anti-rabbit. (1b) Schematic diagram of p56^lck. The two primary sites of p56^lck regulation- the kinase activation motif (centered at Y394) and the inhibitory motif (centered at Y505) are indicated by boxes and key regulators of Y phosphorylation at each site are indicated. ‘+’ and ‘−’ indicate whether a given enzyme causes activation or inhibition of p56^lck activity. Activation of kinase function is mediated by phosphorylation of Y394 which is autophosphorylated upon dephosphorylation of Y505 (by CD45). Once phosphorylated, control of kinase function is mediated by Shp-1 dephosphorylation of Y394. Phosphorylation of Y505 (by Csk) prevents autophosphorylation of Y394 and Csk activity is controlled by cAMP regulation of PKA activity. The amino acid sequence of the Y505 motif is also indicated. (1c) Reciprocal immunoblot analysis of p56^lck using anti-pY Ab. TIL:MCA38 conjugates were formed for the indicated times as described above, extracts were prepared and immuneprecipitated with anti-p56^lck (Ab 3A5), and blotted with anti-pY (4G10). The position of mouse IgG (the species used for p56^lck immuneprecipitation, indicated as ‘mIgG’, and which obscures p56^lck) is validated by analysis of purified mouse IgG in the last gel lane and the arrowhead indicates the interacting protein detected by anti-pY blotting. (1d) High mw band is not p56^lck dimer. Substitution of anti-pY505 with an anti-p56^lck reactive to a different p56^lck epitope than 3A5 (Ab 2102) was performed and detected only p56^lck migrating at the appropriate mw, and not a higher molecular weight species, eliminating the possibility that the higher mw band represented dimeric p56^lck.</p