K P Lee

CTLA-4 is an adhesion receptor expressed on activated T cells. The amino acid sequence of CTLA-4 is related to CD28, and although the function of CTLA-4 remains unknown, it shares several features with CD28, including a common counter-receptor, B7, that is present on Ag-presenting cells. In a recent study we found that CD28 and CTLA-4 were coexpressed at the mRNA level on activated T cells but that only CD28 was expressed on resting T cells. Here we show that within the T cell population, CTLA-4 expression is restricted to the subset of T cells that also express cell surface CD28. CTLA-4 mRNA expression can be induced on quiescent T cells via phorbol ester-mediated activation of protein kinase C but not with calcium ionophore treatment alone. Phorbol ester-induced expression of CTLA-4 mRNA could be enhanced with calcium ionophore treatment, and treatment of cells in this manner resulted in a reciprocal decrease in expression of CD28 mRNA. Ligation of CD28 with monoclonal antibody also resulted in the specific and rapid induction of CTLA-4 mRNA. To study the expression of CTLA-4 at the protein level, a rabbit antiserum against a recombinant protein derived from CTLA-4 cDNA was generated. When activated T cells were labeled with [35S]methionine, the rabbit antiserum precipitated a 41- to 43-kDa protein from whole cell lysates. Similar results were found when detergent-soluble lysates from 125I surface-labeled resting and activated T cells were analyzed by SDS-PAGE. Surprisingly, under the conditions tested, CTLA-4 migrated primarily as a monomer at the cell surface, and could not be shown to exist as a disulfide-bonded homodimer or as a heterodimer consisting of CTLA-4 and CD28. These results suggest that B7 can bind to T cells via distinct receptor complexes consisting of either CD28 or CTLA-4, and that these complexes may potentially mediate distinct biologic functions. Further, the present results suggest that noncovalent interactions might mediate association of CTLA-4 and/or CD28 at the cell surface.

CD28 is a 90-kDa homodimeric glycoprotein present on the surface of a large subset of T cells that appears to play an important role in the modulation of T cell activation. Although a number of physiologic effects associated with CD28 stimulation have been defined, relatively less is known about the structure and expression of the CD28 gene itself. We now show that CD28 is expressed in both Th cells and plasma cells as a series of four distinct CD28 mRNA species: 1.3-, 1.5-, 3.5-, and 3.7-kb transcripts. The steady state expression of all four transcripts in CD28+ T cells was stimulated by PMA, suggesting that they might share a common phorbol-sensitive promoter. Consistent with this hypothesis, CD28 was found to be encoded by a single copy gene organized into four exons, each exon defining a functional domain of the predicted protein. All CD28 transcripts appear to initiate within a 61-bp palindrome. Generation of the four CD28 mRNA species from the CD28 gene involves two distinct posttranscriptional events. The longer pair of transcripts (3.5/3.7 kb) is generated by the use of an alternate nonconsensus polyadenylation signal. This results in the addition of 2167 bp beyond the first polyadenylation site utilized by the shorter (1.3/1.5 kb) pair of transcripts. The size difference between the 3.7- and 3.5-kb messages and between the 1.5- and 1.3-kb messages is generated by an internal splicing event that deletes 252 bp within exon 2, which encodes the extracellular domain. This deletion would result in the loss of 84 amino acids, including 4 of 5 extracellular cysteine residues. Although this deletion would result in significant disruption of CD28 secondary structure, it would not be expected to interfere with the ability of the resultant protein to be expressed on the cell surface. These findings suggest that variant isotypes of CD28 may be expressed on the cell surface with potentially different physiologic roles.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal microenvironment and profound hypoxia, which contribute to therapeutic resistance. Using an in vitro system incorporating pancreatic cancer cells and cancer-associated fibroblasts (CAFs), we show that hypoxia suppresses CD8+ T cell accumulation and, in combination with cancer cell- and CAF-derived factors, further impairs T cell fitness by increasing cell death and reducing proliferation. Although the combination of hypoxia and cancer cell/CAF-derived factors enhances IFNγ and granzyme B expression in CD8+ T cells on a per-cell basis, the overall number of functional effector T cells is markedly reduced. Analysis of human PDAC single-cell RNA sequencing data corroborates these findings, revealing that CD8+ T cells enriched for hypoxia signatures exhibit elevated apoptosis and stress-response pathways. Furthermore, hypoxia is associated with downregulation of stemness-related genes and upregulation of terminal differentiation markers. Together, these data suggest that the integration of intrinsic hypoxic responses and extrinsic cues from tumor cells and CAFs impairs CD8+ T cell fitness and correlates with a terminally differentiated, dysfunctional T cell state.