OBJECTIVE--To assess the nature of T cell receptor (TCR) utilisation by CD4 T cells in the rheumatoid joint. METHODS--Sequencing of the joining (NDJ) region of TCR beta chain mRNA isolated from synovial fluid CD4 T cells was performed in three patients in order to determine if oligoclonal expansion of particular sequences was present. Two patients were studied longitudinally to determine if these sequences changed over time. RESULTS--A number of dominant clonotypes were found within the TCR transcripts sequenced in each patient. In the two patients who were studied longitudinally, different dominant clonotypes were detected over time. No single clonotype was persistently dominant during the period of study. CONCLUSIONS--The pattern of TCR usage showed multiple oligoclonally expanded CD4 T cells within the rheumatoid joint. The change in clonotypes within the joint over time suggests that different antigens may be able to elicit synovial inflammation during the course of rheumatoid disease.
Clonal expansion of T cell specificities in the synovial fluid of patients has been taken as evidence for a local stimulation of T cells. By studying the T cell receptor (TCR) repertoire of CD4+ T cells in the synovial and peripheral blood compartments of patients with early rheumatoid arthritis (RA), we have identified clonally expanded CD4+ populations. Expanded clonotypes were present in the peripheral blood and the synovial fluid but were not preferentially accumulated in the joint. Dominant single clonotypes could not be isolated from CD4+ cells of HLA-DRB1*04+ normal individuals. Clonal expansion involved several distinct clonotypes with a preference for V beta 3+, V beta 14+, and V beta 17+CD4+ T cells. A fraction of clonally related T cells expressed IL-2 receptors, indicating recent activation. The frequencies of clonally expanded V beta 17+CD4+ T cells fluctuated widely over a period of one year. Independent variations in the frequencies of two distinct clonotypes in the same patient indicated that different mechanisms, and not stimulation by a single arthritogenic antigen, were involved in clonal proliferation. These data support the concept that RA patients have a grossly imbalanced TCR repertoire. Clonal expansion may result from intrinsic defects in T cell generation and regulation. The dominance of expanded clonotypes in the periphery emphasizes the systemic nature of RA and suggests that T cell proliferation occurs outside of the joint.
OBJECTIVE—To characterise the type and kinetics of T cell clones in synovial lesions of patients with rheumatoid arthritis (RA).
METHODS—Mononuclear cells from serial samples of synovial fluid (SF) and peripheral blood from nine RA patients were separated phenotypically using antibody coated magnetic beads. After mRNA preparation, reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify V-D(N)-J (that is, the third complementarity determining, CDR3) regions of their T cell receptor beta chain genes. This was followed by single strand conformation polymorphism (SSCP) analysis to detect the clonotypes of accumulating T cells. Amino acid sequences of the dominant clones were also determined.
RESULTS—Although peripheral T cells were heterogeneous, accumulation of oligoclonal T cells was detected in SF. The predominant accumulating clone was the CD8 subset, which was persistently present in serial samples obtained over almost one year of follow up. A proportion of these cells expressed CD25 or CD45RO, or both, suggesting they are `memory' clones.
CONCLUSION—The persistent presence of CD8+ T cell clones in RA joints indicates that they may be involved in the perpetuation of the chronic inflammatory process in RA joints.
OBJECTIVE—Recent studies have suggested that interleukin (IL) 15 induces T cell accumulation in synovial lesions of rheumatoid arthritis (RA). This study aimed at determining whether this cytokine could explain in vivo T cell clonality in RA.
METHODS—Peripheral blood mononuclear cells (PBMC) from patients with RA were stimulated in vitro with IL15 or IL2. After isolation of mRNA from stimulated cells and synovial T cells, genes coding the V-D(N)-J (CDR3) region of T cell receptor β chains were amplified by a reverse transcriptase polymerase chain reaction. A single strand conformation polymorphism analysis was used to detect the clonotype(s) of accumulating T cells. Nucleotide and amino acid sequencing was also performed.
RESULTS—Stimulation of PBMC with IL15 resulted in oligoclonal expansion of T cells. However, IL15 induced clones from PBMC were mostly different from the dominantly expanding T cell clones in synovial fluid. Furthermore, IL15 and IL2 responding clones were only partially identical.
CONCLUSIONS—Although IL15 results in clonal accumulation of T cells, T cell clonality in rheumatoid joints could not be explained by the effect of IL15 alone. The results indicated the requirement of other factor(s), in addition to IL15, in the pathological process affecting RA joints. The results also suggested different responses by each T cell clone to IL15 or IL2.
Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single-strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS.
Clonal expansion of CD4+CD28- T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4+ clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4+CD28- T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4+CD28- T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4+CD28- T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4+CD28- T cells may be a marker correlating with extra-articular manifestations and joint involvement.
arthritis; CD4+CD28-; lymphocytes
Clonal expansion of CD4+ T cells is a characteristic finding in patients with RA and is only infrequently found in patients with psoriatic arthritis and healthy controls. Expanded CD4+ clonotypes are present in the blood, infiltrate into the joint, and persist over years. We have not addressed the question of whether the expanded clonotypes have unique functional and phenotypic properties which may explain the preferential in vivo expansion in RA. In contrast to most CD4+ T cells, expanded clonotypes lacked the expression of the CD28 and CD7 cell surface molecules. Accordingly, the subsets of CD4+ CD28- (9.7 vs 1.7, P = 0.00002) and CD4+ CD7- T cells (21.5 vs 12.26, P = 0.018) were increased in RA patients compared with age-matched normal individuals. Despite the lack of CD28 expression, clonally expanded CD4+ T cells were not anergic but proliferated in response to immobilized anti-CD3 and could be maintained in tissue culture. In vivo expanded CD4+ T cells were autoreactive to ubiquitously distributed autoantigens. They responded in an autologous mixed lymphocyte reaction, and T cell clones isolated from selected patients proliferated to autologous peripheral blood adherent cells. These data suggest that in RA patients selected CD4+ T cells which share the CD7- CD28- phenotype escape from peripheral tolerance.
BACKGROUND: The synovial T cell infiltrate in rheumatoid arthritis (RA) is diverse but contains clonally expanded CD4+ populations. Recent reports have emphasized that RA patients have a tendency to develop CD4+ T cell oligoclonality which also manifests in the peripheral blood. Clonal dominance in the tissue may thus result from antigen specific stimulation in the synovial membrane or may reflect the infiltration of expanded clonotypes present throughout the lymphoid system. We have explored to what extent clonal populations amongst tissue CD4+ T cells display joint specificity as defined by their restriction to the joint, their persistence over time, and their expression of markers indicative for local activation. MATERIALS AND METHODS: Matched samples of peripheral blood and synovial fluid or synovial tissue were collected from 14 patients with active RA and CD4+ IL-2R+ and CD4+ IL-2R- T cells from both compartments were purified. Clonal populations of CD4+ T cells were detected by RT-PCR amplification of T cell receptor (TCR) transcripts with BV and BJ specific primers followed by size fractionation and direct sequencing of dominant size classes of TCR transcripts. RESULTS: Clonal CD4+ T cells were detected in the synovial fluid and synovial tissue of all patients. All patients carried synovial clonotypes that were undetectable in the blood but were present in independent joints or at several non-adjacent areas of the same joint. These joint restricted CD4+ clonotypes were generally small in size, were preferentially found in the IL-2R+ subpopulation, and persisted over time. A second type of clonogenic T cells in the synovial infiltrate had an unrestricted tissue distribution and was present at similar frequencies amongst activated and nonactivated T cells in the blood and affected joints. Ubiquitous clonotypes isolated from two different patients expressed sequence homologies of the TCR beta chain. CONCLUSIONS: Two types of expanded CD4+ clonotypes contribute to the T cell infiltrate in rheumatoid synovitis. Differences in the distribution pattern and in molecular features suggest that distinct mechanisms are supporting the clonal outgrowth of these two groups of clonotypes. Clonally expanded T cells restricted to the joint but present in several independent joints appear to respond to locally residing antigens. Clonogenic cells with an unrestricted distribution pattern and widespread activation in the blood and tissue may react to a different class of antigens which appear to be shared by multiple patients. T cell recognition in RA may be involved at several different levels and may be related to more than one pathomechanism.
OBJECTIVE—To investigate whether type II collagen (CII) is recognised by oligoclonally expanded synovial T cells of patients with rheumatoid arthritis (RA).
METHODS—Peripheral blood mononuclear cells (PBMC) from 15 RA patients were stimulated with CII in vitro. T cell clones expanded by such stimulation were compared with the clonally expanded synovial T cells by using T cell receptor (TCR) B chain gene specific reverse transcription-polymerase chain reaction and subsequent single strand conformation polymorphism analyses.
RESULTS—Stimulation of the heterogeneous peripheral T cells with CII induced clonal expansion of T cells. In three of 15 patients, a proportion of these clones (approximately 17% to 25%) was found to be identical to expanded T cell clones in the synovium in vivo.
CONCLUSION—T cell clones that had TCR CDR3 sequences identical to those induced by purified CII were found in a proportion of RA patients. This finding suggests that CII is recognised by T cells that accumulate clonally in RA joints. Oligoclonal T cell expansion in RA joints is probably driven, at least in part, by intra-articular components such as CII.
In a phase I clinical trial on the effects of preoperative adjuvant IL-2 therapy given to patients undergoing hepatic resection of colorectal adenocarcinoma metastases, we monitored the putative induction of T cell clonal expansion in both tissues and blood. The presence of T cell clonotypes was analyzed with a PCR-based method that determines V-D-J junction size patterns in T cell receptor (TCR) V beta subfamilies in samples before and after a 5-d IL-2 infusion. This high resolution method analyzing CDR3 sizes of TCR transcripts was used in conjunction with FACS analysis of the corresponding T cell subpopulations with TCR V beta-specific mAb. At time of surgery (day 8 after starting IL-2), we found in the three patients analyzed with V beta-C beta primers multiple dominant T cell clonotypes in the tumor and peritumoral tissues which had probably expanded as a result of therapy. In three control patients not treated with IL-2, multiple oligoclonal patterns were not observed with this set of primers. In the fourth control patient a unique V beta 21-C beta CDR3 pattern which corresponds to two dominant clonotypes was found in the tumor. The same dominant clonotypes identified in the tumor after IL-2 were also detectable in the blood and comparison of the profiles obtained before and after IL-2 therapy indicates that they were induced by IL-2. The relative expansion of the corresponding T cell subpopulations was maintained for varying periods of time after surgery (4-7 d and almost 2 yr in one case). Together, these results indicate that IL-2 induces marked expansion of several T cell clones. Systemic IL-2 administration may represent, either alone or as a vaccine adjuvant, an appropriate way of boosting antigen-specific immune responses.
Clonal responses of Mycobacterium tuberculosis-specific CD4+ or CD8+ T effector cells producing antituberculosis cytokine IFN-γ in the context of immune protection against tuberculosis remain poorly characterized in humans. Utilizing decade-long TCR expertise, we previously developed a useful method to isolate clonotypic TCR sequences from Ag-specific IFN-γ–producing T cells and to specifically measure clonotypic TCR frequencies in the T cell pool. In this study, we investigated TCR Vβ repertoires/CDR3 usage, clonal expansion or dominance, and pulmonary trafficking or accumulation for purified protein deritative (PPD)-specific T effector cells producing IFN-γ during bacillus Calmette-Guérin (BCG) vaccination and subsequent M. tuberculosis challenge of macaques. We found that while PPD-specific CD4+ and CD8+ T effector clones employed diverse TCR Vβ repertoires, 30–33% of IFN-γ+CD4+ T cell clones from three M. tuberculosis-infected macaques expressed TCR bearing a conserved residue leucine in CDR3. Many Ag-specific IFN-γ+ CD4+ and few CD8+ T effector cells emerged as dominant clones during mycobacterial infections and underwent major recall expansion after pulmonary M. tuberculosis infection of BCG-vaccinated macaques. PPD-specific T cell clones readily trafficked to the airway or lung after BCG vaccination or M. tuberculosis infection, and some of them continuously accumulated in lungs during M. tuberculosis infection even after they became undetectable in the circulation. Importantly, remarkable recall expansion and pulmonary accumulation of T effector cells coincided with BCG-induced protection against tuberculosis. Thus, rapid clonal expansion and pulmonary accumulation of Ag-specific T effector cells appear to be one of the immune mechanisms underlying immunity against tuberculosis.
In this study, we combined a novel sequencing method, which can identify individual clonotypes based on their unique T cell receptor (TCR) rearrangement, with existing immune assays to characterize antigen-specific T cell responses. We validated this approach using three types of assays routinely used to measure antigen-specific responses: pentamers which enable identification of T cells bearing specific TCRs, activation marker expression following antigen stimulation and antigen-induced proliferation to identify cytomegalovirus (CMV) specific clonotypes. In one individual, 8 clonotypes were identified using a pentamer reagent derived from the CMV pp65 protein. The same 8 clonotypes were also identified following sequencing of cells that upregulated an activation marker following incubation with an identical peptide derived from pp65. These 8 and an additional 8 clonotypes were identified using a more sensitive CFSE-based proliferation assay. We found clear sequence homology among some of the clonotypes identified, and the CDR3 region in one clonotype was identical to a previously published pp65-specific clonotype sequence. Many of these CMV-specific clonotypes were present at frequencies below 10−5 which are undetectable using standard flow-cytometric methods. These studies suggest that an immune response is comprised of a diverse set of clones, many of which are present at very low frequencies. Thus, the combination of immune assays and sequencing depicts the richness and diversity of an immune response at a level that is not possible using standard immune assays alone. The methods articulated in this work provide an enhanced understanding of T cell-mediated immune responses at the clonal level.
Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8+ T cell populations in Mamu-A*01+ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8+ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8+ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection.
In multiple myeloma, circulating “clonotypic” B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential “feeder” cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10−3, this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology.
Transfer of autologous tumor-specific tumor infiltrating lymphocytes (TILs) in adoptive immunotherapy can mediate the regression of tumor in patients with metastatic melanoma. In this procedure, TILs from resected tumors are expanded in vitro, then administered to patients and further stimulated to proliferate in vivo by the administration of high dose IL-2. After in vitro expansion, TILs are often dominated by a few specific clonotypes, and recently it was reported that the persistence in vivo of one or more of these clonotypes correlated with positive therapeutic response. We and others have previously shown that repeated in vitro stimulation and clonal expansion of normal human T lymphocytes results in progressive decrease in telomerase activity and shortening of telomeres, ultimately resulting in replicative senescence. In the studies reported here, we therefore compared telomerase activity and telomere length in persistent and nonpersistent TIL clonotypes before transfer in vivo, and found a correlation between telomere length and clonal persistence. We also observed that TILs proliferate extensively in vivo in the days after transfer, but fail to induce substantial telomerase activity, and undergo rapid decreases in telomere length within days after transfer. Thus, in vivo loss of telomeres by clonotypes that have the shortest telomeres at the time of administration may drive these clones to replicative senescence, whereas cells with longer telomeres are able to persist and mediate antitumor effects. These findings are relevant both to predicting effectiveness of adoptive immunotherapy and in deriving strategies for improving effectiveness by sustaining telomere length.
cancer; immunotherapy; telomere; telomerase
Objective clinical responses have been observed in approximately 50% of patients who received non-myeloablative chemotherapy prior to the adoptive transfer of autologous melanoma-reactive tumor-infiltrating lymphocytes (TILs). Recent studies carried out through the use of antibodies directed against T-cell-receptor beta chain variable region (TRBV) products, as well as by direct sequencing of the expressed TRBV gene products, indicated that clinical responses in this trial were associated with the level of persistence of adoptively transferred T cells. In an attempt to further characterize T cells that persist in vivo following adoptive transfer, five dominant T-cell clonotypes were identified in TIL 2035, an adoptively transferred TIL that was associated with the complete regression of multiple metastases. The most highly persistent clonotype, which expressed the BV1 TR gene product, recognized the MAGE-6 cancer/testis antigen in the context of HLA-A23. This clonotype was detected in peripheral blood for over 16 months following adoptive transfer, expressed relatively higher levels of the co-stimulatory markers CD28 and CD27, and possessed telomeres that were long relative to other clonotypes present in TIL 2035 that showed only short-term persistence. The long-term persistent BV1 clonotype appeared to differentiate more slowly toward an end-stage effector in vivo than short-term persistent clonotypes, as manifested by the downregulation of CD28, CD27, and CD45RO and upregulation of CD57 and CD45RA expression on these T cells. These results indicated that the differentiation stage and replicative history of individual TIL clonotypes might be associated with their ability to survive and to persist in vivo, and progressive differentiation of the persistent clonotypes occurred following adoptive transfer.
cell differentiation; human; T-cell receptors; T cells; tumor immunity
We sought to delineate further the immunological significance of T lymphocytes infiltrating the valve leaflets in calcific aortic stenosis (CAS) and determine whether there were associated alterations in circulating T cells. Using clonotypic TCR β-chain length and sequence analysis we confirmed the repertoire of tricuspid CAS valves contains numerous expanded T cell clones with varying degrees of additional polyclonality, which was greatest in cases with severe calcification. We now report a similar proportion of clonal expansions in the much younger bicuspid valve CAS cases. Peripheral blood flow cytometry revealed elevations in HLA-DR+ activated CD8 cells and in the CD8+CD28nullCD57+ memory-effector subset that were significantly greater in both bicuspid and tricuspid CAS cases with more severe valve calcification. Lesser increases of CD4+CD28null T cells were identified, principally in cases with concurrent atherosclerotic disease. Upon immunostaining the CD8 T cells in all valves were mainly CD28null, and CD8 T cell percentages were greatest in valves with oligoclonal repertoires. T cell clones identified by their clonotypic sequence as expanded in the valve were also found expanded in the circulating blood CD28nullCD8+ T cells and to a lesser degree in the CD8+CD28+ subset, directly supporting the relationship between immunologic events in the blood and the valve. The results suggest that an ongoing systemic adaptive immune response is occurring in cases with bicuspid and tricuspid CAS, involving circulating CD8 T cell activation, clonal expansion and differentiation to a memory-effector phenotype, with trafficking of T cells in expanded clones between blood and the valve.
Human; calcific aortic stenosis; bicuspid aortic valve; T cell receptors; activated T cells; memory-effector T cells; clonal expansions; cell trafficking
We characterized the T-cell receptor (TCR) repertoire in freshly harvested tumor lesions, in short-term-expanded CD4+ tumor infiltrating lymphocytes (TIL) as well as in CD4+ and CD8+ peripheral blood lymphocytes (PBL) from three patients with cervical cancer. Skewing of the T-cell repertoire as defined by measuring the length of the complementarity-determining region 3 (CDR3) of the TCR VA and VB chains was observed in CD8+ PBL, in freshly harvested tumor tissue, as well as in CD4+ TIL. Comparative analysis of the TCR repertoire revealed unique monoclonal TCR transcripts within the tumor lesion which were not present in PBL, suggesting selection of TCR clonotypes due to antigenic stimulation. TCR repertoire analysis of the short-term (7-day) CD4+ TIL lines revealed that the TCR composition is markedly different from that in CD4+ PBL or in the freshly harvested tumor tissue. Only one-third of CD4+ TIL lines showed HLA-DR-restricted recognition of autologous tumor cells as defined by cytolysis. These data provide support for the antigen-driven selection of T cells within cervical cancer lesions and suggest that analysis of the TCR repertoire may aid in obtaining an objective description of the immune response in patients with cervical cancer who are undergoing epitope-based immunotherapy.
T cell receptors (TCRs) mediate cytotoxic T lymphocyte (CTL) specificity, but TCRs recognizing the same epitope often differ between persons due to their stochastic derivation. The role of this variability in the pathogenesis of virus infections and malignancies has been technically difficult to study. We apply a novel adaptation of TCR spectratyping to study HIV-specific CTLs, defining the clonal breadth and sequences of epitope-specific TCRs from PBMC without cellular sorting or molecular cloning. Examining 48 CTL responses in 12 persons reveals a mean of 4.5 ± 2.7 clones per response, of both public and private clonotypes. The number of identified epitope-specific TCRs correlates with CTL frequency across epitopes, suggesting that clonal breadth limits the magnitude of the CTL response against HIV-1 in vivo. HLA A- and B-restricted CTLs are similar in their TCR breadth in this small cohort, preliminarily suggesting that qualitative differences may account for their disparate impacts on pathogenesis. Overall, these findings demonstrate that the magnitude of the CTL response in chronic HIV-1 infection is constrained by TCR clonal breadth, suggesting maximal expansion of CTLs in response to chronic antigenic stimulation.
To better define the immunologic character of the T cell infiltrate in lupus nephritis.
We performed double immunohistochemical staining and clonotypic T cell receptor (TCR) beta-chain sequencing in multiple anatomic regions isolated by laser-capture microdissection from renal biopsies.
SLE kidneys have a variably patterned and often extensive infiltrate of predominantly clonally expanded T cells of CD4 and CD8 lineages. CD4 T cells were prominent in nearly two-thirds of SLE biopsies, and distributed as broad periglomerular aggregates or intermixed with CD8 T cells forming periglomerular caps. Sequencing of the T cell TCR from periglomerular regions showed a predominance of clonally expanded T cells. The CD8 T cells, which were present in all biopsies, often adhered to Bowman's capsule and infiltrated the tubular epithelium. They exhibited features that suggest participation in an adaptive immune response: differentiation into CD28null memory-effector phenotype, trafficking of the same expanded clonotype to different regions of the kidney and to the peripheral blood, and clonal persistence for years in repeat biopsies. CD8 T cell tubulitis was especially associated with progressive changes.
The immunological characteristics of the infiltrating CD4 and CD8 T cells in the lupus kidney indicate they have the potential to mediate injury, which may be relevant to development of progressive renal failure. Whereas the oligoclonality of the CD4 T cell infiltrate is consistent with the paradigm of SLE as a class II-associated autoimmune disease, the finding of CD8 T cell clonality and trafficking implies participation in a distinct systemic adaptive immune response.
Metastatic melanoma patients treated with an autologous DNP-modified tumor cell vaccine develop inflammatory responses in metastatic tumors characterized by infiltration of CD8+ T cells. To further define this immune response, we analyzed T cell receptor beta-chain variable (TCRBV) region repertoire in biopsy specimens and peripheral blood lymphocytes of six patients. After administration of DNP vaccine, a restricted set of TCRBV gene families was found to be expanded compared with prevaccine metastases. In several postvaccine lesions of one patient, obtained over a 2-yr period, TCRBV14+ T cells were clonally expanded and identical T cell clonotypes could be detected. Two major recurring clones were biased toward the use of TCRBJ1S5. Furthermore, T cell lines derived from two such infiltrated skin lesions and, enriched in TCRBV14+ T cells, displayed HLA-class I-restricted lysis of the autologous melanoma cells. Clonal expansion of T cells was demonstrated in the T cell-infiltrated, postvaccine metastasis of a second patient as well. These results indicate that vaccination with autologous, DNP-modified melanoma cells can expand selected clones of T cells at the tumor site and that such clones are potentially destructive to the tumor.
Assessment of T-cell diversity, besides giving insights about the molecular basis of tumor antigen recognition, has clinical implications since it provides criteria for evaluating antigen-specific T cells clinically relevant for spontaneous and vaccine-induced anti-tumor activity. Melan-A is one of the melanoma antigens most frequently recognized by peripheral and tumor-infiltrating lymphocytes in HLA-A2+ melanoma patients. Many clinical trials involving anti-tumor vaccination have been conducted using modified versions of this peptide.
We conducted an in-depth characterization of 210 T-cell receptor beta chain (TRB) clonotypes derived from T cells of HLA-A2+ melanoma patients displaying cytotoxic activity against natural and A27L-modified Melan-A peptides. One hundred and thirteen Melan-A-specific clonotypes from melanoma-free subjects, 199 clonotypes from T-cell clones from melanoma patients specific for melanoma antigens other than Melan-A, and 305 clonotypes derived from T cells of HLA-A2+ individuals showing unrelated specificities, were used as control. After sequence analysis, performed according to the IMGT definitions, TRBV and TRBJ usage, CDR3 length and amino acid composition were compared in the four groups of clonotypes.
TRB sequences of Melan-A-specific clonotypes obtained from melanoma patients were highly heterogeneous, but displayed a preferential usage of few TRBV and TRBJ segments. Furthermore, they included a recurrent "public" amino acid motif (Glycine-Leucine-Glycine at positions 110-112-113 of the CDR3) rearranged with dominant TRBV and TRBJ segments and, in one case, associated with a full conservation of the entire TRB sequence.
Contrary to what observed for public anti-Melan-A T-cell receptor alpha motifs, which had been identified in several clonotypes of both melanoma patients and healthy controls, the unexpectedly high contribution of a public TRB motif in the recognition of a dominant melanoma epitope in melanoma patients may provide important information about the biology of anti-tumor T-cell responses and improve monitoring strategies of anti-tumor vaccines.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) are required to restrain the immune system from mounting an autoaggressive systemic inflammatory response, but why their activity can prevent (or allow) organ-specific autoimmunity remains poorly understood. We have examined how TCR specificity contributes to Treg activity using a mouse model of spontaneous autoimmune arthritis, in which CD4+ T cells expressing a clonotypic TCR induce disease by an IL-17-dependent mechanism. Administration of polyclonal Tregs suppressed Th17 cell formation and prevented arthritis development; notably, Tregs expressing the clonotypic TCR did not. These clonotypic Tregs exerted antigen-specific suppression of effector CD4+ T cells using the clonotypic TCR in vivo, but failed to mediate bystander suppression and did not prevent Th17 cells using nonclonotypic TCRs from accumulating in joint-draining lymph nodes of arthritic mice. These studies indicate that the availability of Tregs with diverse TCR specificities can be crucial to their activity in autoimmune arthritis.
T cell receptor (TCR) gene therapy is an approach being considered for HIV-1, but epitope mutation is a significant barrier. We assessed whether HIV-specific TCR can be modified to have broader coverage of epitope variants by recombining polymorphisms between public clonotype TCR sequences.
Public clonotype TCRs recognizing the same epitope often differ by polymorphisms in their third complementarity determining (CDR3) regions. We assessed whether novel combinations of such polymorphisms could improve TCR recognition of epitope variation.
A TCR recognizing the HLA A*0201-restricted epitope SLYNTVATL (Gag 77-85, SL9) was engineered to have combinations of four polymorphisms in the CDR3 regions compared to another SL9-specific TCR. These novel TCRs were screened for functional avidities against SL9 epitope variants and abilities to mediate cytotoxic T lymphocyte suppression of HIV-1 containing the same epitope variants.
The TCRs varied modestly in functional avidities (FAs) for SL9 variants, due to alterations in affinity. This translated to differences in antiviral activities against HIV-1 when FA changes crossed the previously defined threshold required for efficient recognition of HIV-1 infected cells. Higher avidity TCR mutants had generally broader recognition of SL9 variants.
These results indicate that rationally targeted increases in FA can be utilized to maximize the antiviral breadth of transgenic TCRs. In contrast to previously reported random mutagenesis to markedly increase FA, tuning through recombining naturally occurring polymorphisms may offer a more physiologic approach that minimizes the risk of deleterious TCR reactivities.
T-cell receptor; HIV; immune evasion; cytotoxic T-lymphocytes; gene therapy
In multiple myeloma (MM), the immunoglobulin heavy chain VDJ gene rearrangement is a unique clonotypic signature that identifies all members of the myeloma clone independent of morphology or phenotype. Each clonotypic MM cell has only one genomic copy of the rearranged IgH VDJ.
Pre-treatment bone marrow aspirates from myeloma patients at diagnosis or in relapse were evaluated for the number of clonotypic cells using real time quantitative PCR (RPCR). RPCR measured the level of clonal cells, termed VDJ%, in 139 diagnosis and relapse BM aspirates from MM patients.
Patients with a VDJ% below the median had a significantly longer event free survival (EFS) then those with a VDJ% higher than the median (p=0.0077, HR=0.57). Further, although the VDJ% from non-transplant patients predicted EFS (p=0.0093), VDJ% failed to predict outcome after autologous stem cell transplant (p=0.53).
Our results suggest that for non-transplant patients, the tumor burden before treatment, perhaps reflecting cancer stem cell progeny/output, is an indirect measure that may indicate the number of MM cancer stem cells and hence event free survival.
Multiple myeloma; Clonotypic signature; Quantitative analysis; Clinical correlates; Cancer stem cell; Autologous transplant; Tumor burden