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Combining transcriptomic and TCRαβ repertoire analysis of circulating and tumor-infiltrating CD8 T cells from patients with metastatic melanoma, Lucca et al. identify a blood-based population with effector properties that reflect those of clonally related tumor-resident T cells., Understanding the relationship between tumor and peripheral immune environments could allow longitudinal immune monitoring in cancer. Here, we examined whether T cells that share the same TCRαβ and are found in both tumor and blood can be interrogated to gain insight into the ongoing tumor T cell response. Paired transcriptome and TCRαβ repertoire of circulating and tumor-infiltrating T cells were analyzed at the single-cell level from matched tumor and blood from patients with metastatic melanoma. We found that in circulating T cells matching clonally expanded tumor-infiltrating T cells (circulating TILs), gene signatures of effector functions, but not terminal exhaustion, reflect those observed in the tumor. In contrast, features of exhaustion are displayed predominantly by tumor-exclusive T cells. Finally, genes associated with a high degree of blood–tumor TCR sharing were overexpressed in tumor tissue after immunotherapy. These data demonstrate that circulating TILs have unique transcriptional patterns that may have utility for the interrogation of T cell function in cancer immunotherapy.

Resting autoreactive T cells are present in the circulation of normal individuals without pathologic consequences. In autoimmune animal models, stimulation of these self-reactive T cells in the presence of costimulatory molecules B7-1 results in T cell-mediated autoimmune disease, whereas B7-2 stimulation generates regulatory autoreactive T cells that abrogate disease severity. Thus, reactivation in the brain of myelin-autoreactive T cells by antigen with costimulatory molecules may be a critical event in the pathophysiology of multiple sclerosis (MS), a putative autoimmune disease of central nervous system (CNS) myelin. We investigated the expression of cytokines and costimulatory molecules in a panel of 41 histologically characterized CNS specimens from 15 MS and 10 control cases using semiquantitative reverse transcriptase-polymerase chain reaction and immunocytochemistry. In four cases, vascular CNS infarcts with inflammation were compared with MS plaques from the same brain. We observed increased expression of B7-1 and interleukin (IL) 12p40 in acute MS plaques, particularly from early disease cases but not in inflammatory infarcts. B7-1 staining was localized predominantly to the lymphocytes in perivenular inflammatory cuffs but not the parenchyma. In contrast, B7-2 was expressed predominantly on macrophages both in MS lesions of varied time duration and in inflammatory infarcts. These findings indicate that an early event in the initiation of MS involves upregulation of B7-1 and IL-12, resulting in conditions that maximally stimulate T cell activation and induction of T helper 1-type immune responses.

Equal numbers of CD4+ T cells recognizing myelin basic protein (MBP) and proteolipid protein (PLP) are found in the circulation of normal individuals and multiple sclerosis (MS) patients. We hypothesized that if myelin-reactive T cells are critical for the pathogenesis of MS, they would exist in a different state of activation as compared with myelin-reactive T cells cloned from the blood of normal individuals. This was investigated in a total of 62 subjects with definitive MS. While there were no differences in the frequencies of MBP- and PLP-reactive T cells after primary antigen stimulation, the frequency of MBP or PLP but not tetanus toxoid-reactive T cells generated after primary recombinant interleukin (rIL-2) stimulation was significantly higher in MS patients as compared with control individuals. Primary rIL-2-stimulated MBP-reactive T cell lines were CD4+ and recognized MBP epitopes 84-102 and 143-168 similar to MBP-reactive T cell lines generated with primary MBP stimulation. In the cerebrospinal fluid (CSF) of MS patients, MBP-reactive T cells generated with primary rIL-2 stimulation accounted for 7% of the IL-2-responsive cells, greater than 10-fold higher than paired blood samples, and these T cells also selectively recognized MBP peptides 84-102 and 143-168. In striking contrast, MBP-reactive T cells were not detected in CSF obtained from patients with other neurologic diseases. These results provide definitive in vitro evidence of an absolute difference in the activation state of myelin-reactive T cells in the central nervous system of patients with MS and provide evidence of a pathogenic role of autoreactive T cells in the disease.

Major histocompatibility complex class II-positive human T cell clones are nontraditional antigen-presenting cells (APCs) that are able to simultaneously present and respond to peptide or degraded antigen, but are unable to process intact protein. Although T cell presentation of peptide antigen resulted in a primary proliferative response, T cells that had been previously stimulated by T cells presenting antigen were completely unresponsive to antigen but not to interleukin 2 (IL-2). In contrast, peptide antigen presented by B cells or DR2+ L cell transfectants resulted in T cell activation and responsiveness to restimulation. The anergy induced by T cell presentation of peptide could not be prevented by the addition of either autologous or allogeneic B cells or B7+ DR2+ L cell transfectants, suggesting that the induction of anergy could occur in the presence of costimulation. T cell anergy was induced within 24 h of T cell presentation of antigen and was long lasting. Anergized T cells expressed normal levels of T cell receptor/CD3 but were defective in their ability to release [Ca2+]i to both alpha CD3 and APCs. Moreover, anergized T cells did not proliferate to alpha CD2 monoclonal antibodies or alpha CD3 plus phorbol myristate acetate (PMA), nor did they synthesize IL-2, IL-4, or interferon gamma mRNA in response to either peptide or peptide plus PMA. In contrast, ionomycin plus PMA induced both normal proliferative responses and synthesis of cytokine mRNA, suggesting that the signaling defect in anergized cells occurs before protein kinase C activation and [Ca2+]i release.

T cell immunoglobulin- and mucin domain–containing molecule (TIM)3 is a T helper cell (Th)1–associated cell surface molecule that regulates Th1 responses and promotes tolerance in mice, but its expression and function in human T cells is unknown. We generated 104 T cell clones from the cerebrospinal fluid (CSF) of six patients with multiple sclerosis (MS) (n = 72) and four control subjects (n = 32) and assessed their cytokine profiles and expression levels of TIM3 and related molecules. MS CSF clones secreted higher amounts of interferon (IFN)-γ than did those from control subjects, but paradoxically expressed lower levels of TIM3 and T-bet. Interleukin 12–mediated polarization of CSF clones induced substantially higher amounts of IFN-γ secretion but lower levels of TIM3 in MS clones relative to control clones, demonstrating that TIM3 expression is dysregulated in MS CSF clones. Reduced levels of TIM3 on MS CSF clones correlated with resistance to tolerance induced by costimulatory blockade. Finally, reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-γ secretion, directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-γ secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel, intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases.

Regulatory T cells have been clearly implicated in the control of disease in murine models of autoimmunity. The paucity of data regarding the role of these lymphocytes in human autoimmune disease has prompted us to examine their function in patients with rheumatoid arthritis (RA). Regulatory (CD4+CD25+) T cells isolated from patients with active RA displayed an anergic phenotype upon stimulation with anti-CD3 and anti-CD28 antibodies, and suppressed the proliferation of effector T cells in vitro. However, they were unable to suppress proinflammatory cytokine secretion from activated T cells and monocytes, or to convey a suppressive phenotype to effector CD4+CD25− T cells. Treatment with antitumor necrosis factor α (TNFα; Infliximab) restored the capacity of regulatory T cells to inhibit cytokine production and to convey a suppressive phenotype to “conventional” T cells. Furthermore, anti-TNFα treatment led to a significant rise in the number of peripheral blood regulatory T cells in RA patients responding to this treatment, which correlated with a reduction in C reactive protein. These data are the first to demonstrate that regulatory T cells are functionally compromised in RA, and indicate that modulation of regulatory T cells by anti-TNFα therapy may be a further mechanism by which this disease is ameliorated.

Substantial numbers of both alpha/beta and gamma/delta T cells are present in human fetal liver, which suggests a role of the fetal liver in T cell development. The diversity of fetal liver T cell receptor (TCR) gamma and delta chain rearrangements was examined among both CD4+CD8- and CD4-CD8- gamma/delta T cell clones. In addition, TCR delta chain transcripts from three fetal livers were sequenced after polymerase chain reaction amplification of TCR delta chains with V delta 1 or V delta 2 rearrangements. Five of six fetal liver gamma/delta T cell clones had a V delta 2-D delta 3-J delta 3 gene rearrangement with limited junctional diversity; three of these clones had an unusual CD4+CD8- phenotype. V delta 2-D delta 3-J delta 3 gene rearrangements were also common among both in-frame and out-of-frame transcripts from three fetal livers, indicating that they are the result of an ordered rearrangement process. TCR gamma chain sequences of the fetal liver gamma/delta T cell clones revealed V gamma 1-J gamma 2.3, V gamma 2-J gamma 1.2, and V gamma 3-J gamma 1.1 rearrangements with minimal incorporation of template-independent N region nucleotides. TCR gamma chain rearrangements found in these fetal liver T cell clones were different from those that have been observed among early thymic gamma/delta T cell populations, while similar TCR delta chain rearrangements are found among gamma/delta T cells from both sites. These data demonstrate that the fetal liver harbors gamma/delta T cell populations distinct from those found in the fetal thymus, suggesting that the fetal liver is a site of gamma/delta T cell development in humans. These unusual T cell populations may serve a specific function in the fetal immune system.

We have derived 33 independent T cell clones from the cerebrospinal fluid (CSF) of a patient with subacute sclerosing panencephalitis using a single T cell cloning method. 6% (2 of 33) of these clones express the T cell receptor gamma (TCR-gamma) protein and are called CSF TCR-gamma clones. Phenotypic analyses of the CSF TCR-gamma clones indicate that they are WT-31-, CD3+, CD4-, and CD8-. The TCR-gamma protein exists on the cell surface as part of an 85-kD disulphide-linked dimer noncovalently associated with the CD3 polypeptides. The CSF TCR-gamma clones have NK-like activity that can be inhibited by anti-CD3 mAbs. Both CSF TCR-gamma clones proliferated in response to anti-CD3 mAbs coupled to Sepharose beads and/or IL-2. Furthermore, stimulation of one of these clones with anti-CD3 mAbs results in a rapid rise in intracellular calcium. These data suggest that T cells bearing the CD3-TCR-gamma protein complex are functional and play a role in the human immune response.

We have investigated the T cell populations in the cerebrospinal fluid (CSF) of chronic progressive multiple sclerosis (MS) patients. Individual T cells from the CSF and blood were cloned before expansion and their clonotypes were defined by analysis of rearranged T cell receptor beta chain and gamma chain genes. 87 T cell clones from blood and CSF of two patients with chronic progressive MS were examined for common TCR gene rearrangement patterns. In one patient, 18 of 28 CSF-derived T cell clones demonstrated common TCR gene rearrangements indicating oligoclonal T cell populations; in the blood, two patterns were found twice among 26 T cell clones. In another patient, 5 of 27 CSF-derived clones had common TCR gene rearrangement patterns. In contrast, no common beta chain rearrangement pattern was found among 67 T cell clones derived from the blood or CSF of a patient with subacute sclerosing panencephalitis, among 20 clones from the CSF of a patient with herpes zoster meningoencephalitis, or among 66 clones from a normal subject. A subject with atypical, fatal MS of 8-mo duration was also studied and did not have oligoclonal T cells in the CSF or blood. These results demonstrate that distinct oligoclonal T cell populations can be found in the CSF immune compartment of subjects with nonmalignant inflammatory disease and they can create a new avenue for the investigation of the specificity of the T cell response within the central nervous system.

A second type of TCR molecule has been identified on human and murine T lymphocytes, which involves the protein products of the gamma and delta genes. T lymphocytes bearing this receptor may constitute a separate cell lineage with a distinct immune function. We have produced an mAb, which specifically detects human TCR-gamma/delta in native as well as denatured states, this in contrast to previously used anti-gamma chain peptide sera, which only reacted with denatured protein. The receptor occurs in different molecular forms, with or without interchain disulphide bonds, in which a delta chain may or may not be detected by cell surface iodination. The mAb is reactive with all these receptor forms. Therefore, this antibody could be used to determine the expression of TCR-gamma/delta on viable human T lymphocytes. In normal individuals, TCR-gamma/delta was found on a subset composing 2-7% of CD3+ lymphocytes in peripheral blood and 0.1-1.0% in thymus. The majority of these cells do not express the CD4 or CD8 antigens, although a significant percentage of CD8+ cells was found. TCR-gamma/delta+ cells in peripheral blood are resting lymphocytes, as judged by ultrastructural analysis. T cell clones with different receptor types can display MHC-nonrestricted cytolytic activity, which is shown to be induced by the culture conditions, most likely by growth factors such as IL-2. This strongly suggests that TCR-gamma/delta does not play a role in target cell recognition in MHC-nonrestricted cytotoxicity. The anti-TCR-gamma/delta antibody can specifically induce cytotoxic activity in clones expressing the receptor, but in addition inhibit growth factor induced cytotoxicity, which indicates a regulatory role of the TCR-gamma/delta/CD3 complex in MHC-nonrestricted cytotoxicity.

Whether the expression of higher molecular weight isoforms of the T-200 complex represents different lineages of T cells and/or a sequential stage of the differential pathway of T cells has been unclear. Understanding T cell expression of higher molecular weight isoforms of the T-200 complex (CD45R) may be important because of their association with regulation of immune responses. By direct single cell cloning, we observed a number of long-term T cell clones that expressed CD45RA (2H4). CD45RA expression could be further regulated by ionomycin or the cytokines IL-1 and IL-6, but not IL-2, IL-4, or IFN-gamma. These results indicate that CD45RA expression may define T cell lineages of activated T cells partially controlled by the cytokines IL-1 and IL-6. Further, these results may associate regulatory actions of IL-1 and IL-6 with their ability to increase CD45RA expression in subpopulations of human T cells.