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With expanding indications for programmed death 1 (PD-1) axis inhibitors in non–small cell lung cancer (NSCLC), acquired resistance (AR) to these therapies is increasingly being encountered. We sought to characterize clinical patterns of AR to PD-1 axis inhibitors in patients with advanced NSCLC, and evaluate subsequent outcome and management strategies for such patients.

The intracellular survival of the bacterial pathogen Chlamydia trachomatis depends on protein synthesis by the microbe soon after internalization. Pharmacologic inhibition of bacterial translation inhibits early trafficking of the parasitophorous vacuole (inclusion) to the microtubule-organizing center (MTOC) and promotes its fusion with lysosomes, which is normally blocked by CHLAMYDIA: Depletion of cellular tryptophan pools by gamma interferon-inducible indoleamine-2,3-dioxygenase (IDO) is believed to be the major innate immune mechanism controlling C. trachomatis infection in human cells, an action to which the bacteria can respond by converting into a nonreplicating but highly reactivatable persistent state. However, whether severe IDO-mediated tryptophan starvation can be sufficient to fully arrest the chlamydial life cycle and thereby counteract the onset of persistence is unknown. Here we demonstrate that at low exogenous tryptophan concentrations a substantial fraction of C. trachomatis bacteria fail to traffic to the MTOC or to switch into the conventional persistent state in gamma interferon-induced human cells. The organisms stay scattered in the cell periphery, do not retain infectivity, and display only low transcriptional activity. Importantly, the rate at which these aberrant Chlamydia bacteria become reactivated upon replenishment of cellular tryptophan pools is substantially lower. Thus, severe tryptophan depletion in cells with high IDO activity affects chlamydial development more rigorously than previously described.

Class I molecules, encoded by diverse alleles at several loci of the major histocompatibility complex (MHC) are assembled in the endoplasmic reticulum (ER) from heavy chain, β2 microglobulin and peptide in association with accessory proteins of the peptide loading complex. We show here, that mutations in the α2 domain (Q115A; D122A) of the human class I allele HLA-A2 cause a lack of apparent association with the loading complex and a faster assembly. Despite the drastically reduced association with the TAP loading complex, i.  e. less than 20 % of HLA-A2 expressed in the cells can be co-precipitated with either TAP, calreticulin or tapasin, the mutant proteins are expressed on the cell surface in a stable conformation, and bind a complex set of peptides almost identical to that of wild-type HLA-A2. Furthermore, the mutant class I molecules are more rapidly exported from the ER than wild-type HLA-A2 and undergo faster maturation. The mutation Q115A does not destroy a binding site for the loading complex as this HLA-A2 mutant associates with the loading complex when peptide supply is limited. The association of class I molecules with the TAP-associated loading complex appears to be a reflection of how quickly the stable conformation is gained.

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.

In our previous work, DNase hypersensitivity mapping was used to identify an enhancer within the human CD8 alpha (hCD8 alpha) gene which allowed T cell-specific expression of a reporter construct in transiently transfected cell lines. To study the role of this intronic enhancer in vivo, transgenic mice were made using human CD8 genomic constructs. We found that while a 14 kb wild-type human CD8 alpha (WThCD8) genomic construct did not lead to expression in mature peripheral CD8+ T cells, this transgene was consistently expressed in small populations of T cells and B cells, and in a subset of mouse NK cells. While murine CD8 is not normally expressed on resting NK cells, expression of the human CD8 transgene on mouse NK cells is appropriate since CD8 is expressed on a subset of human NK cells. Deletion of the intronic enhancer resulted in a complete loss of transgene expression in most lines and a loss of expression only in NK cells in one line. Our results indicate, firstly, that cis-acting sequences within the 14 kb genomic fragment are sufficient for NK cell-specific expression. In addition, our results suggest that the enhancer may have dual roles in regulation of transgene expression. It may enhance general expression of the transgene and may also be required for NK cell-specific expression.

Three new kinds of recombinant DNA constructs were used to transfer cloned human class I HLA genes (A2 and B8) into unique HLA mutant lymphoblastoid cells: pHeBo(x): a class I gene, "x," in plasmid vector pHeBo, which contains a hygromycin resistance gene and Epstein-Barr virus oriP element that sustains extrachromosomal replication; pHPT(x): gene x in a vector with a hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene; pHPTe(x): gene x in a vector with the HPRT gene and oriP element. Cell surface class I antigen expression was strong in transferents made with class I-deficient lymphoblastoid cell line mutants .144 (A-null), .53 (B-null), and .184 (A-null, B-null). Transferents expressing HLA-A2 were recognized specifically by HLA-A2-specific cytotoxic T lymphocytes. When introduced on either of the vectors with the Epstein-Barr virus oriP element, the class I gene replicated extrachromosomally and was lost at rates of 0.2 to 0.3 per cell division. When introduced with vector pHPT (lacking Epstein-Barr virus oriP), the B8 gene was inserted at different chromosomal locations. Introduction of the HLA-B8 gene failed to restore antigen expression by HLA-B-null mutant .174, providing evidence that, unlike mutants exemplified by .53, .144, and .184, some HLA antigen loss mutants are deficient in a trans-acting function needed for class I antigen expression. Of more general interest, the results obtained with HLA class I genes in vectors that replicate extrachromosomally suggest ways of relating genic expression to chromatin structure and function and of attempting to clone functional human centromeres.

We previously identified a T cell-specific enhancer in the last intron of the human CD8α gene that is adjacent to a sequence element that significantly represses enhancer function. This negative regulatory region consists of a half-Alusequence that has potential to base-pair with a downstreamAluelement, which is part of the fully active enhancer, to form a cruciform structure. The actvity of this half-Alusilencer sequences is position and orientation-dependent, suggesting that DNA structure plays an important role in its function. Using site-directed mutational analysis and P1nuclease mapping, we directly demonstrate that formation of a cruciform structure is required for repression of enhancer function in transient transfection assays. Finally, a P1nuclease-sensitive site is present in the endogenous CD8α gene in T cell lines providing indirect evidence that the stem-loop may formin vivo. Taken together, these results suggest thatAluelements may contribute to the regulation of the CD8α gene enhancer through the formation of secondary structure that disrupts enhancer function.

We report the isolation of genomic and cDNA clones encoding the human T-lymphocyte cell-surface differentiation antigen, Leu-2 (T8), by use of a combination of transfection, fluorescence-activated cell-sorting, and subtractive cDNA hybridization. We constructed a cDNA library with mRNA from a mouse L-cell transfectant in which the human Leu-2 gene is expressed and amplified. We identified Leu-2 cDNA clones by screening with a selected cDNA probe from a second amplified Leu-2 transfectant. This probe contained cDNA species not removed by hybridization with L-cell mRNA. A Leu-2 cDNA clone was used to isolate a genomic clone. Transfection with DNA from this clone resulted in a high number of Leu-2 transfectants. This approach can be used to clone genes coding for other cell-surface molecules.

The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. We demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8+ T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern we observe differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

Gamma rays from a cesium source were used to generate human lymphoblastoid cell line variants that had lost expression of all major histocompatibility complex antigens coded for by a single haplotype. The cell line was heterozygous at the glyoxalase I locus and had the HLA haplotypes HLA-A1, B8, DRw3, and HLA-A2, B5, DRw1. We selected with anti-HLA-B8 antiserum in a population of cells that had been irradiated with 300 R. The incidence of B8-loss variants was 4.1 X 10(-5) on day 5 after irradiation. Analysis of variants showed that expressions of HLA and GLO alleles trans to B8 were retained. However, expression of additional cis-linked HLA and GLO gene products was lost in 12 of 17 variants. Variants that had lost expression of (i) HLA-B8, (ii) HLA-B8, A1, (iii) HLA-B8, A1, DRw3, or (iv) HLA-B8, A1, DRw3 and the cis-linked glyoxalase I allele were obtained. Karyotype analysis was performed on eight variants that had lost expression of two or more cis-linked alleles. Three variants had two normal appearing no. 6 chromosomes, four variants had a deletion that included the region coding for HLA genes on the short arm of one no. 6 chromosome, and one variant had an inversion or translocation involving the short arm of one no. 6.

Leu 7 (HNK-1) is a membrane antigen expressed on human natural killer cells and some other lymphoid cells. Starting with two clones of mouse-human hybrid lymphoid cells that had 1.6% and 35% Leu 7-positive cells, respectively, we viably sorted Leu 7-positive and -negative cells using a fluorescence-activated cell sorter (FACS). Short-term progeny of the sorted cells were then karyotyped. Chromosome 11 was the only human chromosome that was absent from the Leu 7-negative population and present in nearly all of the progeny of the Leu 7-positive selected cells. Thus, we assigned the Leu 7 gene to chromosome 11.

The CD8 dimer interacts with the alpha 3 domain of major histocompatibility complex class I molecules through two immunoglobulin variable-like domains. In this study a crystal structure-informed mutational analysis has been performed to identify amino acids in the CD8 alpha/alpha homodimer that are likely to be involved in binding to class I. Several key residues are situated on the top face of the dimer within loops analogous to the complementarity-determining regions (CDRs) of immunoglobulin. In addition, other important amino acids are located in the A and B beta-strands on the sides of the dimer. The potential involvement of amino acids on both the top and the side faces of the molecule is consistent with a bivalent model for the interaction between a single CD8 alpha/alpha homodimer and two class I molecules and may have important implications for signal transduction in class I-expressing cells. This study also demonstrates a role for the positive surface potential of CD8 in class I binding and complements previous work demonstrating the importance of a negatively charged loop on the alpha 3 domain of class I for CD8 alpha/alpha-class I interaction. We propose a model whereby residues located on the CDR-like loops of the CD8 homodimer interact with the alpha 3 domain of MHC class I while amino acids on the side of the molecule containing the A and B beta-strands contact the alpha 2 domain of class I.

We isolated stable transformants of mouse L cells expressing human cell surface differentiation antigens by using immunofluorescence with monoclonal antibodies and selection with a fluorescence-activated cell sorter (FACS). Mouse L cells (TK-) were cotransformed with human cellular DNA and the herpes simplex virus thymidine kinase (TK) gene. TK+ transformants were first selected. The TK+ populations were stained with various fluorescent antibodies to membrane antigens, and positive cells were sorted and cloned by using a FACS. Transformants for HLA class I antigens, for beta 2-microglobulin, and for the T-cell differentiation antigens Leu-1 and Leu-2 were isolated. The frequency of antigen transformants among the TK+ transformants was about 0.5 X 10(-3). The sizes of the HLA, Leu-1, and Leu-2 molecules expressed by the transformants were the same as those of the proteins present on DNA-donor cells.

Abstract:  Lack of expression of a cell surface protein can occur by means of transcriptional and/or post-transcriptional mechanisms. Expression of the CD8A gene has been shown to be regulated by post-transcriptional mechanisms when 1) CD4–CD8lo thymocytes are blocked from differentiating into CD4+CD8+ cells by TCR crosslinking and 2) upon activation of mature CD8+ T cells. We demonstrate in this paper that there is also post-transcriptional regulation of CD8A expression in a CD4+CD8– T-cell line Jurkat. On the basis of northern blotting, mRNA for CD8A was not seen to be present in the Jurkat cells, but the gene was observed to be transcriptionally active in nuclear run-on analysis. In addition, we provide evidence that post-transcriptional mechanisms also contribute to the regulation of CD8A expression in mature CD4+CD8– T cells, challenging the assumption that the regulation is due solely to transcriptional mechanisms.

Class I loci of the human major histocompatibility complex (MHC) encode 44,000-molecular weight polypeptides that associate nonconvalently with β-microglobulin1. Included in this category are the HLA-A, -B and -C loci (Fig. 1a), which are extensively polymorphic2. HLA-specific cDNA clones3,4now allow this polymorphism to be studied at the DNA level. However, the lack of sufficient amino acid sequence data for all but a few of the class 1 antigens presents a major difficulty in the allelic assignment of those DNA clones obtained in man3–5and mouse6–8. HLA loss variants produced with deletion-inducing ionizing radiation offer a means of partially circumventing the time-consuming and difficult task of sequencing HLA class I gene products for identifying restriction enzyme digest fragments of DNA and recombinant DNA clones. P.K. et al.9used γ-ray irradiation followed by immunoselection to obtain many mutants that no longer expressed one or more HLA specificities from the human lymphoblastoid cell line LCL 721. Only the expression of HLA-B8 was lost in one class of mutant, while expressions of HLA-B8 and at least one additional cis- linked HLA allele were lost in mutants of the second class. Some mutants lost expression of the entire haplotype, that is, HLA-A1, -B8 and -DR3 (ref. 9) as well as SB (refs 4, 10 and unpublished results). Recently, anti-HLA-B5 and anti-HLA-A2 sera have been used to isolate mutants that have lost expressions of one or more loci of the haplotype on the other chromosome 6 of LCL 721 (Table 1)10. Data presented here illustrate how one cDNA clone of an HLA class I gene, pHLA-1, in combination with γ-ray-induced HLA loss variants, will be used to identify recombinant DNA HLA clones and to correlate individual DNA restriction fragments with expression of specific HLA alleles.

Although the genes of the major histocompatibility complex (MHC) are tightly linked, four loci (HLA-A, B, C and DR) have been defined using rare families in which antigenic specificities segregated from each other. As an alternative approach for defining loci, Kavathas et al.1selected HLA-mutant lymphoblastoid cell lines (LCLs) that had lost expression of specific genetic markers. An HLA-GLO heterozygous LCL, LCL-721, was mutagenized with ionizing radiation which is known to induce multigenic deletions2; variants no longer expressing a specific HLA antigen were selected. While some mutants lost expression of only the antigen selected against, most simultaneously lost expression of several identifiable cis-linked gene products. Many of the multiple-loss mutants resulted from chromosome rearrangements, especially deletions1. Five new secondary B-cell (SB) antigens have recently been defined3,4, which evoke strong secondary allogeneic proliferative and cytotoxic responses. Because they resemble the HLA-DR antigens both functionally and genetically, it was not known whether they were coded for by a gene distinct from the gene(s) encoding HLA-DR. We have now characterized LCL-721 mutants for expression of both HLA-DR and SB. Of 20 lines which had lost expression of the HLA-A, -B and -DR antigens from one haplotype, four retained expression of the cis-linked SB antigen. One of these mutants had a visible deletion that included the region coding for HLA on the short arm of one chromosome 6. Because the cis-linked SB antigen continued to be expressed, the gene coding lor SB antigens must be different from that coding for HLA-DR antigens.

Gene products of the human HLA region are the principal histocompatibility barrier to human allogeneic tissue transplantation1. In addition, HLA genes regulate immune responses to environmental antigens2and affect susceptibility to various diseases3. Although the HLA region has been studied intensively, only four histocompatibility loci (HLA-A, -B, -C and -DR) have been identified within this ‘supergene’, but the existence of additional loci has been suggested by immunochemical4–9and genetic recombination10–15studies. Recently, a new segregant series of five histocompatibility antigens, named ‘Secondary B cell’ (SB) antigens, was defined16–18. We now report studies of 21 families which indicate that the gene encoding the SB antigens is closely linked to HLA-DR, but is distinguished from it by recombination in two families and can be mapped between HLA-DR and GLO in additional recombinant families.

Using previously isolated mouse L-cell transferees for the human T-cell differentiation antigen Leu-2, we now report the first example of spontaneous gene amplification for membrane antigens. The Leu-2 (or T8)1,2antigen is normally expressed on T lymphocytes that have cytotoxic or suppressor functions. Cells of a Leu-2 transfected clone were stained with fluorescein-tagged monoclonal anti-Leu-2, and the brightest 0.1–0.3% of cells were viably separated using a fluorescence activated cell sorter (FACS). After growth of these selected cells, sorting and regrowth was repeated six times, resulting in a population of cells that, compared with the starting population, stains 40 times brighter for Leu-2 and whose DNA transforms 20 times more efficiently for Leu-2. In addition, these cells have 10- to 50-fold amplified human DNA sequences and numerous double minute chromosome fragments, a common indicator of gene amplication in mouse cells.

We have shown previously1that transfection of mouse L-cells with DNA from JM, a human T-cell line expressing certain T-cell differentiation antigens, yields stable transfectants expressing one or another of these antigens. The identities of the antigens were confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. We now report that our procedure—co-transfection2with the chicken thymidine kinase gene (tk) and whole cellular DNA, selection with hypoxanthine–aminopterin–thymidine (HAT)3, and staining of the cells with fluorochrome-conjugated monoclonal antibodies and fluorescence-activated cell-sorter (FACS) selection—yields transfectants expressing a variety of cell-surface molecules (19 of 21 investigated), most at a frequency of about one per 103Tk+transformants. Of these, 9 of 12 were transferred and expressed as readily using DNA from cells which did not express the cell-surface antigens as from tissues or cells that did express them.