Predictors of responses to immune checkpoint blockade in advanced melanoma.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

Lupus anti-DNA autoantibodies cross-react with a glomerular structural protein: a case for tissue injury by molecular mimicry.

Anti-DNA autoantibodies are the hallmark of human and murine systemic lupus erythematosus (SLE), an autoimmune rheumatic disease of unknown etiology. Some of these antibodies are believed to be pathogenic for kidney tissue and to initiate immune glomerulonephritis. However, the mechanisms by which anti-DNA antibodies participate in tissue injury remain controversial. We have studied the in vivo pathogenicity of anti-DNA monoclonal antibodies in immune deficient mice, using a panel of murine B cell hybridomas. No consistent genetic or immunochemical differences were found between pathogenic and non-pathogenic anti-DNA antibodies. However, the two antibody populations differed in their cross-reaction with the acidic actin-binding protein, alpha-actinin, that is known to play a major role in the structural integrity of glomerular filtration components. These results suggest that kidney dysfunction in SLE may be facilitated by protein-nucleic acid antigenic mimicry.

The cytoskeleton-associated TCR zeta chain is constitutively phosphorylated in the absence of an active p56(lck) form.

The TCR recognizes peptide-MHC complexes and transmits activation signals leading to cellular responses. We have previously characterized two TCR populations expressed on the T cell surface; one is linked to the cytoskeleton via a detergent-insoluble cytoskeleton-associated zeta (cska-zeta) chain, while the other is detergent soluble and not linked to the cytoskeleton. The cska-zeta form displays unique properties: it is constitutively phosphorylated, does not undergo hyperphosphorylation upon TCR stimulation as opposed to its non-cytoskeleton-associated counterpart (non-cska-zeta) and it maintains a molecular mass of 16 kDa. It is well established that p56(lck) and possibly p59(fyn) are responsible for the generation of the 21/23-kDa phosphorylated detergent-soluble zeta form. We now demonstrate that the phosphorylation of cska-zeta does not require the activity of p56(lck). We also show that although Lck does not phosphorylate cska-zeta in vivo, it retains the capacity to phosphorylate cska-zeta in vitro. Moreover, differences in zeta-associated kinase activity were detected for non-cska-zeta and cska-zeta. Our results indicating that different kinases phosphorylate the two zeta forms are consistent with a growing consensus that each TCR form may regulate distinct cellular functions.

V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.

Searching for significance in TCR-cytoskeleton interactions.

Two T-cell receptor (TCR) populations are expressed on T cells; one is linked to the cytoskeleton via its zeta chain. These cytoskeleton-linked receptors (30-40% of the total number of TCRs) might be important in TCR-mediated signaling and/or concurrent events. Here, differences between the two populations are summarized, and new data are examined to speculate on the functional significance of cytoskeleton-linked TCRs.

Expression of the T cell antigen receptor zeta chain following activation is controlled at distinct checkpoints. Implications for cell surface receptor down-modulation and re-expression.

The multisubunit T cell antigen receptor (TCR) is involved in antigen recognition and signal transduction, leading to T cell activation and rapid down-modulation of the cell surface expressed TCRs. Although the levels of TCR cell surface expression are pivotal to the efficiency and duration of the immune response, the molecular mechanisms controlling TCR down-modulation and re-expression upon activation, remain obscure. Here, we provide a biochemical characterization of the regulatory mechanisms governing TCR expression following long-term T cell activation. We focused primarily on the TCR zeta chain, as this is considered the limiting factor in TCR complex formation and transport to the cell surface. We found that following TCR-mediated activation zeta mRNA is up-regulated by a transcription-dependent mechanism. Concomitantly, zeta protein levels are modified according to a biphasic pattern: rapid degradation coinciding with TCR cell surface down-regulation, followed by a rebound to normal levels 24 h subsequent to T cell activation. Even though there are adequate levels of all the TCR subunits within the cell following 24 h of activation, TCR cell surface expression remained very low, provided the activating antibody is continuously present. Correlative with the latter, we detected a previously undescribed monomeric form of the zeta chain. This form could be indicative of adverse endoplasmic reticulum conditions affecting correct protein folding, dimerization, and TCR assembly, all critical for optimal receptor surface re-expression. Cumulatively, our results indicate that the levels of TCR expression following activation, are tightly controlled at several checkpoints.

Enhanced lymphoid cell recovery after bone marrow transplantation: correlation with the presence of costimulatory antibodies in serum.

We describe a patient with T cell deficiency who underwent bone marrow transplantation (BMT) from an HLA-identical brother. The patient's white blood cell count recovered with exceptional rapidity post-BMT: after 7 to 9 days it rose sharply to 98x10(9) cells/L, 76% of which were mononuclear leukocytes. It then decreased, and a second peak was observed 250 days post-BMT. Lymphocytes from both peaks displayed a phenotype of mature T cells together with characteristics of a constitutively activated state; that is, they 1) exhibited high levels of tyrosine-phosphorylated T cell receptor (TCR) zeta chain, 2) spontaneously secreted IL-2, 3) expressed activation specific cell surface markers, and 4) were unresponsive to in vitro stimuli. The increased cell counts in both peaks correlated with the presence of anti-lymphocytic antibodies in the patient's serum, which reacted with peripheral blood lymphocytes (PBLs) both from the donor and from unrelated individuals. These antibodies were present before BMT and reappeared post-BMT. Variable number tandem repeats analysis revealed that the patient's PBLs were chimeras for up to 2 years post-BMT. This finding could explain the newly synthesized post-BMT anti-lymphocytic antibodies and the appearance of the second WBC peak during that period. The patient's anti-lymphocytic antibodies displayed costimulatory activity, enhancing the in vitro proliferation of normal T cells suboptimally activated via the TCR. The unique characteristics of these antibodies could explain the enhanced T cell recovery observed post-BMT as well as the constitutive activation state of these cells. Furthermore, such antibodies may eventually facilitate development of a therapeutic method for inducing enhanced post-BMT recovery.

Complement receptor 3 of macrophages is associated with galectin-1-like protein.

We have previously identified a 16-kDa protein with a pI of 5.1 (P16/5.1) that is associated with macrophage CR3. Microsequencing of P16/5.1 indicated exclusive homology to the beta-galactoside-binding lectin, galectin-1. Abs specific to a galectin-1 unique peptide reacted with P16/5.1. The association of P16/5.1 with CR3 was specifically inhibited by lactose, which binds with high affinity to galectin-1. These data together with similarities in molecular mass and pI suggest that P16/5.1 is galectin-1. Two-color immunofluorescence staining revealed the expression of galectin-1 on the macrophage surface and its colocalization with CR3. However, a surplus of CR3 was free of galectin-1, and some galectin-1 molecules were associated with cell surface receptors other than CR3. Based on these results we propose two models depicting the functional significance of CR3-galectin-1 association: 1) homodimeric galectin-1 possessing a divalent sugar binding site may act as an extracellular adapter molecule that cross-links CR3 with other receptors; and 2) association of galectin-1 with beta-galactosides on the extracellular domain of CR3 may modify the binding affinity of the receptor to its ligand. These possibilities are not mutually exclusive and can clarify the mode by which CR3 transmits signals in macrophages.

The tryphostin AG17 induces apoptosis and inhibition of cdk2 activity in a lymphoma cell line that overexpresses bcl-2.

Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases. We studied the effects of tyrphostins on OCI-Ly8, a cell line derived from a patient with immunoblastic lymphoma that carries the t(14;18) translocation and overexpresses the B-cell lymphoma/leukemia-2 gene (bcl-2). To test the possibility that tyrphostins induce apoptosis in these cells, overcoming the protection rendered by bcl-2, we screened 16 tyrphostins representing different families at a concentration of 0.5-50 microM. We found that AG17 was the most potent in this regard. Cell cycle analysis demonstrated that AG17 induces arrest at the G1 phase followed by apoptosis with general reduction of the intracellular level of tyrosine-phosphorylated proteins. To further elucidate the mechanism of action of AG17, we investigated its effect on some of the key proteins that regulate the cell cycle. Bcl-2 and cdk2 protein levels were not altered with AG17, whereas cdk2 kinase activity, as well as p21 and p16 protein levels, were reduced markedly. These results suggest that the target of AG17 is inactivation of cdk2. Because lymphoma cells with the t(14;18) translocation and bcl-2 overexpression are resistant to chemotherapy, novel drugs selectively able to induce apoptosis in these cells could offer a new approach to the treatment of lymphoma patients.

Normal T cells express two T cell antigen receptor populations, one of which is linked to the cytoskeleton via zeta chain and displays a unique activation-dependent phosphorylation pattern.

The TCR couples antigen recognition and the transmission of activation signals. We report the expression of two TCR populations on the surface of T lymphocytes, one of which is linked to the cytoskeleton via the zeta chain. We also demonstrate that assembly of the CD3 subunits with cytoskeleton-associated zeta is necessary for their maximal localization to the cytoskeleton. The potential significance of these two receptor forms is underscored by differences observed in non-activated T cells; while detergent-soluble phosphorylated zeta appears as a 21-kDa protein, phosphorylated cytoskeleton-associated zeta appears as a 16-kDa form. This dichotomous phosphorylation pattern is rigidly maintained following activation, although each of the receptor populations undergoes different activation-dependent modifications: 1) levels of soluble phosphorylated 21-kDa zeta are enhanced, while phosphorylated 16-kDa cytoskeleton-associated zeta exhibits little change; 2) soluble non-phosphorylated 16-kDa zeta translocates to the cytoskeleton; 3) activation-dependent ubiquitinated zeta forms localize to both fractions, albeit with different kinetics. We also show that the protein tyrosine kinase Lck undergoes activation-dependent modifications and translocates to the cytoskeleton. The phosphorylation profiles of the dichotomous TCR populations in both non-activated and activated lymphocytes suggest that each population could regulate distinct cellular functions, possibly by select intermolecular associations.

Transcriptional regulation of the murine TCR zeta gene.

The TCR zeta chain plays a significant role in the assembly of the receptor complex and in coupling antigen recognition to the intracellular signal transduction apparatus. Since the zeta protein level is considered the limiting factor for receptor assembly in mature T cells, aberrant expression of the zeta chain affects both receptor structure and function. To understand the regulatory mechanisms controlling zeta gene expression, we characterized the 5' flanking region of the gene. Our analysis reveals the existence of at least three regions within the -784 to +121 fragment involved in the transcription of the zeta gene in T cells: (i) the fragment from -216 to +121 contains the basal promoter; (ii) the sequence between -561 and -216 includes positive elements which confer strong transcriptional activity; and (iii) the region between -784 and -561 which contains negative element(s) that down-regulate zeta gene transcription. The entire 5' flanking region of the zeta gene is functional in both T and fibroblast cell lines, although the transcriptional levels and the specific regions required for maximal activity differ between the two cell types. Maximal transcriptional activity is achieved when T cells are stimulated simultaneously via the TCR and with PMA. The transcriptional activity of the zeta gene can be induced by PMA alone in T cells but not in fibroblasts, suggesting that this effect is mediated by T cell-specific factors. We also demonstrate that upregulation of the transcriptional activity induced by the different stimuli is consistent with increased expression of zeta mRNAm, pointing to the possibility that signal transduction events initiated during T cell activation may be involved in controlling zeta gene expression.

Identification and characterization of a novel protein associated with macrophage complement receptor 3.

CR3 is a member of the beta 2 integrin family which functions as a bidirectional signaling receptor. The CR3 is composed of the alpha (CD11b) and beta (CD18) subunits, which contain a short intracytoplasmic domain devoid of catalytic activity. It was therefore postulated that CR3 is associated with intracellular molecules that link it to the cytoplasmic signal transduction apparatus. However, no direct association between such molecules and CR3 have been identified so far. We searched for CR3 co-associated molecules that might regulate the function of this receptor. For this purpose CR3 was immunoprecipitated from radiolabeled bone marrow macrophages using a combination of anti-CD11b and anti-CD18 mAbs. Two-dimensional isoelectric focusing analysis of the immunoprecipitates revealed the two CR3 subunits and an additional 16-kDa protein with an apparent isoelectric point of 5.1. This protein, designated p16/5.1, was intracellular, monomeric, nonglycosylated and noncovalently associated with CR3 but not with CR4. CR3-associated p16/5.1 was also detected in four of six macrophage lines as well as in thymic large macrophages, all of which express cell-surface CR3. We suggest that p16/5.1 may be involved in CR3-mediated function.

Cell-surface-expressed T-cell antigen-receptor zeta chain is associated with the cytoskeleton.

The T-cell antigen receptor zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. zeta chain can associate with certain protein tyrosine kinases and retains the capacity to transduce signals independently of the other receptor subunits. Thus, zeta chain could couple cell-surface-expressed T-cell antigen receptors to the intracellular signal-transduction apparatus by its association with various intracellular molecules in addition to tyrosine kinases. In the process of searching for zeta chain-associated molecules we observed that after lysis of resting T cells with Triton X-100, zeta chain is localized in the detergent-insoluble fraction, in addition to its presence in the detergent-soluble fraction. Treatment of T cells with cytochalasin B, an actin-depolymerizing agent, leads to the complete dissociation of zeta chain from the Triton-insoluble fraction, suggesting a linkage between zeta chain and the cytoskeletal matrix. We have also determined that cytoskeletal-associated zeta chain is expressed on the cell surface. Furthermore, a tyrosine-phosphorylated 16-kDa zeta chain was detected only in the Triton-insoluble cytoskeletal fraction of resting T cells. zeta chain also maintains its association with the cytoskeleton when expressed in COS cells, inferring that the cytoskeletal elements involved in this linkage may be ubiquitous. Finally, we have localized a 42-amino acid region in the intracytoplasmic domain of zeta chain, which is crucial for maximal interaction between zeta chain and the cytoskeleton. Anchorage of cell-surface-expressed zeta chain to the cytoskeleton in resting T cells may facilitate recycling of receptor complexes and/or allow the transduction of external stimuli into the cell.

Multisubunit receptors in the immune system and their association with the cytoskeleton: in search of functional significance.

Various multisubunit receptors of the immune system share similarities in structure and induce closely related signal transduction pathways upon ligand binding. Examples include the T cell antigen receptor (TCR), the B cell antigen receptor (BCR), and the high-affinity receptor for immunoglobulin E (Fc epsilon RI). Although these receptors are devoid of intrinsic kinase activity, they can associate with a similar array of intracellular kinases, phosphatases and other signaling molecules. Furthermore, these receptor complexes all form an association with the cytoskeletal matrix. In this review, we compare the structural and functional characteristics of the TCR, BCR and Fc epsilon RI. We examine the role of the cytoskeleton in regulating receptor-mediated signal transduction, as analyzed in other well-characterized receptors, including the epidermal growth factor receptor and integrin receptors. On the basis of this evidence, we review the current data depicting a cytoskeletal association for multisubunit immune system receptors and explore the potential bearing of this interaction on signaling function.

Changes in the methylation pattern of the TCR zeta-chain gene correlate with its expression in T cells and developing thymocytes.

The tight regulation of T-cell gene expression during thymic ontogeny is essential to the development of a normal immune system. One set of developmentally regulated genes encodes the multicomponent T-cell antigen receptor (TCR). The zeta chain, a component of the TCR, has been shown to play important roles in signal transduction from antigen binding to T-cell activation and in transport of the TCR complex to the cell surface. In this study, we examine the regulation of zeta gene expression in murine T-cell hybridomas. In these cells, zeta expression is correlated with complex, but predictable, changes in the pattern of cytosine methylation of its gene. Some of these structural changes are identical to those observed in murine fetal thymocytes and correlate with the rapid alteration of zeta message seen in the thymus between days 15 and 18 of gestation.

The isolation and characterization of the murine T cell antigen receptor zeta chain gene.

The T cell antigen receptor (TCR) is a multisubunit complex which has a dual function of antigen recognition and signal transduction. One of its invariant subunits, the zeta chain, has been shown to have a significant role in the expression and function of the TCR on the cell surface. The mouse and human zeta cDNAs share significant homologies to each other but are distinct from all of the previously characterized TCR components. We now report the isolation and structural analysis of the complete murine zeta gene. This gene spans at least 31 kilobases and divides into eight exons. The first exon, which is located at least 20 kilobases upstream from the second exon, codes for the 5'-untranslated region and most of the signal peptide. The second exon codes for the remainder of the signal peptide, the extracellular domain, the transmembrane domain, and the first three amino acids of the intracytoplasmic domain. Exons 3-7 encode the majority of the intracytoplasmic domain. The eight exon encodes the carboxyl-terminal 21 amino acids and the 3'-untranslated region. Four groups of mRNA initiation sites have been identified at approximately 140 base pairs upstream to the AUG codon. No TATA-like box has been detected. The gene is localized to the distal part of chromosome 1 in a linkage group highly conserved between man and mouse.

The T cell antigen receptor zeta chain is tyrosine phosphorylated upon activation.

The T cell antigen receptor is composed of at least seven chains derived from six different gene products. Upon stimulation, several chains can be phosphorylated. Two of these, CD3-gamma and CD3-epsilon are phosphorylated on serine residues. In addition, a 21-kDa nonglycosylated receptor component is phosphorylated, upon activation, on tyrosine residues. We have referred to this phosphoprotein as p21 because we have previously not been able to assign the tyrosine phosphorylation to any of the described receptor subunits (Samelson, L. E., Patel, M. D., Weissman, A. M., Harford, J. B., and Klausner, R. D. (1986) Cell 46, 1083-1090). In this paper, we demonstrate that it is the 16-kDa zeta chain which is the tyrosine phosphorylated subunit, and thus the p21 nomenclature can be replaced. This phosphorylation results in a shift of the apparent Mr of zeta to 21 kDa. Proof that p21 is tyrosine phosphorylated zeta was afforded by a number of approaches. Specific anti-zeta antibodies directly precipitated phospho-p21. Metabolically labeled protein corresponding to p21 could only be observed after activation. When this 21-kDa band was isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with alkaline phosphatase, its migration was identical with that of zeta. Furthermore, peptide mapping of metabolically labeled p21 (after gel isolation and dephosphorylation) showed it to be indistinguishable from p21. Thus, one of the early events of T cell activation is the tyrosine phosphorylation of the zeta chain of the T cell antigen receptor.

Possible association between IL-2 receptors and class I HLA molecules on T cells.

In the process of evaluating murine hybridomas for an antibody to the beta-subunit of the IL-2R (p70) we identified an antibody that immunoprecipitated a 55- to 57-kDa complex from cross-linked lysates. We demonstrate that this complex is composed of IL-2 (15.5 kDa) cross-linked to the H chain of HLA class I (40 to 42 kDa), suggesting a molecular interaction between HLA class I molecules and IL-2R. Although the exact role of this association remains to be determined, the specific cross-linking of IL-2 to HLA class I Ag is intriguing in view of published claims for a role of HLA class I in OKT3-induced lymphocyte proliferation and in NK cell lytic activity.

Anti-IgE monoclonal antibodies directed at the Fc epsilon receptor binding site.

In a search for the region in the IgE molecule, which is recognized by the Fc epsilon receptor (Fc epsilon R) on mast cells and basophils, we have generated and characterized anti-IgE monoclonal antibodies (MAbs). The novel rat anti-mouse IgE MAb described herein (denoted 84-1c) interacts with an antigenic determinant which is associated with the Fc epsilon R recognition site on the IgE molecule. The MAb can bind to the Fc epsilon of IgE and block its binding to rat basophil leukemia (RBL) cells. The epitope recognized by 84-1c MAb was completely masked by the Fc epsilon R either in its cellular or soluble form. This epitope was dependent on the native conformation of the IgE molecule and differed from the ones that were recognized by the anti-IgE MAbs we described before.