Reciprocal consequences of mTOR activation in APCs and T cells may be host protective or disease promotive.

<p>Innate (e.g. TLRs) or adaptive signals (e.g. CD40) trigger the PI3 kinase-Akt-mTOR signaling cascade in the APCs. Activation of mTORc1 leads to the phosphorylation of 4E-BP1/2 and initiation of protein translation. Pathogenic virulence factors such as Gp63 and antibiotic rapamycin (RAPA) inhibit mTOR activation and hence downregulate translation of type I interferons and iNOS (inducible nitric oxide synthase). Inhibition of 4E-BP1/2 can selectively upregulate translation and hence may be an attractive drug target. mTOR activation can also upregulate anti-inflammatory molecule IL-10 and inhibits the proinflammatory molecules, such as IL-12. IL-10 may skew Th0 cells to the disease-promoting Th2/Treg cells, whereas IL-12 and other proinflammatory cytokines can enhance the Th1/Th17 axis. Activation of mTOR signaling by inhibition of TSC1/TSC2 (tuberous sclerosis complex) or inhibition of Rictor (rapamycin-insensitive companion of mTOR, an essential component of mTORc1 signaling), especially at the early stage of an infection, can boost the propensity of these cells to be skewed towards Th1 phenotype. mTOR inhibition of Treg cells by rapamycin can augment expansion of Treg cells with increased suppressive capacity. This can be prevented by the activation of mTOR by inhibiting TSC1/2 or PTEN (Phosphatase and TENsin homolog) and may be a lucrative drug target at the later stages of an infection. On the other hand, inhibition of mTOR signaling in memory cells can improve the memory cell differentiation. Blockade of mTOR by pharmacological and genetic ablation enhances the quality and quantity of surviving memory. Targeted inhibition of mTOR in memory cells can thus be an attractive drug target especially at the later stage of infection.</p

DNA viruses that target mTOR signaling pathways.

*<p>Virus activates the target. Unmarked, virus inhibits the target. <b>PKB</b>, Protein Kinase B; <b>AMPK</b>, AMP-activated kinase; <b>TSC</b>, tuberous sclerosis complex; <b>PP2A</b>, Protein Phosphatase 2A; <b>4E-BP</b>, eIF4E binding protein; <b>elF4F</b>, eukaryotic elongation factor complex consisting of elF4E, elF4G, elF4A, and MnK1; <b>PyV</b>, polyomavirus; <b>HPV</b>, human papillomavirus; <b>HCMV</b>, human cytomegalovirus; <b>HSV</b>, herpes simplex virus; <b>VV</b>, vaccinia virus.</p

The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

<div><p>The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.</p> </div

Motifs in the cytoplasmic tail of the M-4 isoform that regulate cell surface expression.

<p>(<b>A</b>) Potential amino acid motifs in the cytoplasmic tail of M-4 isoform (<a href="http://www.expasy.org" target="_blank">www.expasy.org</a>). (<b>B</b>) Quantitative analysis of the surface expression of the M-4 wild-type and mutant proteins expressed in H9 cell line using the anti-CD8β antibody (5F2) by flow cytometry. The amount of CD8β binding was normalized to GFP expression. Each value corresponds to an average of three experiments. The standard deviation and two-population Student’s paired t-test was used to determine statistical differences of the mutants relative to the wild type M-4 isoform, indicated as one star * for p<0.05 and ** p<0.01. (<b>C</b>) Surface expression levels of M-4 wild-type and mutant proteins normalized to GFP expression in primary CD4⁺ T cells. Peripheral blood CD4⁺ T cells were stimulated with antibodies against CD3 and CD28 and transduced with lentiviruses expressing GFP and wild type or mutant M-4 proteins. On day 8 cell surface staining with CD8β antibody was analyzed by flow cytometry. The data are the mean +/− S.D. from three independent experiments. Values that are statistically different from the wild type M-4 protein are indicated as * p<0.05 and ** for p<0.01. (<b>D</b>) CD8αβ expression on CD4⁺ T cells prepared as in (C) expressing M-4 wild-type or mutants S²³²A or LL^235–6AG/IL^240–1AA. One representative experiment of five experiments is shown.</p

The NPW motif in the M-4 cytoplasmic tail mediates binding to ubiquitinated proteins.

<p>(<b>A</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, and CD8β wild type M1, M4 or chimeric protein M1 with 15 amino acids of the C terminus of the M4 cytoplasmic tail. After 48 hrs cells were lysed with 1% BRIJ 97, precipitated with anti-CD8β mAb and run on a polyacrylamide gel. Western blotting was performed with the indicated antibody. The membrane was then stripped and re-probed with the anti-CD8β antibody. The experiment was repeated three times. (<b>B</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, and CD8β wild type or mutant proteins. The immunoprecipitation and Western blotting experiments were performed as in (A). A representative of four experiments is depicted. (<b>C</b>) The intensity of the 30 kDa band was analyzed and the amount of the 30 kDa band of the wild type relative to each mutant protein is represented. The levels of CD8β protein detected after reprobing with anti-CD8β antibody were used to correct for differences in protein expression between experiments. Student’s paired t-test was used to determine statistical differences of the mutants relative to the wild type M-4 isoform, indicated as one star * for p<0.05.</p

CD4⁺ T cells transduced with the M-4 CD8β isoform showed increased frequency of cells producing MIP-1β after stimulation.

<p>Peripheral blood CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours, and then co-transduced with a lentivirus expressing a NY-ESO-1 TCR and another lentivirus expressing CD8α and one of the CD8β isoforms. Cells were stimulated and analyzed for cytokine/chemokine production after day 10–12. (<b>A</b>) Schematic representation of lentiviral vectors used for co-transduction of primary CD4⁺ T cells is followed by histograms for surface expression of CD8αβ, CD8α and TCR and dot plots showing cell population co-expressing NY-ESO-1 TCR and CD8α. Data were collected by flow cytometry using antibodies against CD8 and an MHC tetramer specific to NY-ESO-1 (NY-ESO- tetramer). Live CD3⁺ lymphocytes were gated using side vs. forward scatter, anti-CD3 antibody and live/dead cell dye. (<b>B</b>) Frequency of transduced CD4⁺ T cells producing MIP-1β (top panel) after stimulation with K562 target cells expressing the NY-ESO-1 antigen. T cells without targets served as negative control and cells stimulated with PMA and Ionomycin (bottom panel) were used as positive control. One representative of three independent experiments is shown. Values that are statistically different from the wild type M-1 protein as determined by two-population Student’s paired t-test are indicated as one star (*) for p<0.05.</p

M-4 cytoplasmic tail binds to ubiquitinated proteins and is modified itself by ubiquitination.

<p>(<b>A</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, CD8α and each CD8β isoform or mutant proteins. After 48 hours cells were lysed with 1% BRIJ 97 and immunoprecipitated with anti-CD8β mAb, run on a polyacrylamide gel, transferred to a membrane and probed with the anti-HA antibody. Gels were reprobed with an anti-CD8β antibody (5F2). Small differences in size reflect different lengths of the isoforms. (<b>B</b>) Co-transfection of HEK-293T cells with either wild type HA-Ub or I⁴⁴A mutant of HA-Ub. The immunoprecipitation and Western blotting procedures were performed as in (A). (<b>C</b>) Determination of direct ubiquitination of CD8β isoforms. Cells expressing individual isoforms were immunoprecipitation with anti-HA mAb followed by blotting with anti-CD8β antibody. An aliquot of total cell lysate was similarly analyzed. (<b>D</b>) Effect of lysine mutants on M-4 ubiquitination. Cells expressing M-4 wild-type, single lysine (K²³⁴R, K²⁴²G) or double lysine (K²³⁴R/K²⁴²G labeled KRKG) mutants were lysed and protein analyzed as described in (C). (<b>E</b>) The JM T cells expressing CD8α and either CD8β M-4 or the double lysine mutant KRKG were incubated with the anti-CD8α (OKT8) and anti-CD3 (OKT3) mAbs for 0, 2.5 or 7 minutes at 37°C. Cells were lysed, immunoprecipitated with the anti-Ub mAb followed by Western blotting with the anti-CD8β antibody. A representative of three independent experiments is shown in each panel.</p

Identification of motifs signals in the cytoplasmic tail of M-4 isoform that modulate downregulation from the cell surface.

<p>(<b>A</b>) Receptor downregulation was measured by determining surface expression for CD8β M-4 wild-type and M-4 mutant levels by flow cytometry before and after stimulation of cells with PMA (100 ng/ml) for 60 minutes. One representative of 3 independent experiments is shown. (<b>B</b>) Receptor downregulation relative to CD8β M4 wild type was as in (A) quantitatively represented. Levels of CD3 protein after stimulation are indicated as well. Analyzing three experiments values that are statistically different from the wild type M-4 protein are indicated as * for p<0.05 and ** p<0.01.</p

Treg expansion performed in the absence of serum results in more durable Treg suppressive activity.

<p><b>A</b>. Enriched Tregs were expanded in the absence of human serum. Where indicated, 10 nM ATRA or 100 ng/ml RAPA were added to the medium and this concentration of drug was added to media used to feed the cultures. Cell counting was performed on the indicated days. The data reflects the average of 3 cultures using enriched Tregs from different healthy donors. Error bars represent the standard deviation. (<b>B</b>.) At the end of culture, FOXP3 expression was measured by flow cytometry, and (<b>C</b>.) an <i>in vitro</i> suppressive assay was performed. For parts B. and C., each symbol refers to data collected from a single donor.</p

ATRA and RAPA affect Treg subsets equally.

<p>CD4+ CD25high CD45RA+ (<b>A</b>.) or CD4+ CD25high CD45RA− (<b>B</b>.) populations were sort purified and expanded by anti-CD3 Ab-loaded K.64.86 aAPCs in the presence of 100 ng/ml of rapamycin, 10 nM ATRA or a combination of both. After 12–15 days of culture, FOXP3 expression was measured by flow cytometry. Data from 4 unique donors is displayed. <b>C</b>. Representative suppression data from a single donor using a 1∶4 Treg to Effector ratio. <b>D and E</b>. Compiled suppression data from 4 donors (the donor in C. is the closed triangle).</p