3.1 Emergence of TCR clonality and preferential usage of TCR chains
In general, healthy, 'antigen-experienced' individuals may show clonally altered, or even monoclonal TCRs. These 'private' alterations in the TCR repertoire have been reported in a number of apparently healthy individuals and identified in the CD8+ T-cell population, preferentially in the CD57+ T-
cell subset (18). Monoclonal or oligoclonal TCR VB families may occur in EBV or CMV-specific T-cell responses and reflect the immunological memory of previous encounters with antigens (19, 20). Some alterations can also be identified in CD4+ T-cells, presumably reacting to common infectious agents, e.g. CMV (21). Due to structural constraints of the TCR interaction with its nominal MHC/peptide ligand, some T-cell responses are characterized by a common usage of a TCR VB family, but not with a common CDR3 motif. For instance, the HLA-A2 restricted CD8+ T-cell response targeting the influenza matrix peptide Ml (aa 58-66) shows a preferential usage of the TCR VB17 family (22) which is polyclonal in nature. Other, preferential usage of certain TCR variable families may also be present in the human population. This is difficult to assess, since these studies depend either on the generation of bona fide T-cell clones directed against a single MHC/peptide complex, or alternatively on tetramer-sorted T-cells. For instance, the TCR VA2.1 chain appears to be frequently utilized in CD 8+ T-cells recognizing the human HLA-A2 restricted melanoma -associated antigen Melan-A/MART-1 as determined by tetramer-sorting and T-cell cloning analysis (23, 24).
Monoclonal expansion of T-cells may be indicative for antigen-driven process either in blood or in tissue. Since each monoclonal TCR can be molecularly defined (see above), a molecular probe specific for the respective CDR3 region can be designed and the individual T-cell clone can either be traced in different compartments or longitudinally over time in a patient. This methodology has been used to tag clonal T-cell populations in autoimmune myositis (25), or in patients with multiple sclerosis (MS) suggesting that expansion of certain TCR VB families may be associated with disease onset or progression (26, 27). Similarly, if a monoclonal T-cell response can be linked to MHC peptide recognition, it allows to visualize monoclonal TCRs with defined specificity in situ, e.g. in a patient with melanoma responding to peptide vaccination: An TCR VB16+ Melan-A/MART-1 reactive T-cell clone could be demonstrated in a vitiligo lesion associated with destruction of melanin-positive cells (i.e. melanoma cells and normal melanocytes) (28). Similar studies have been performed to track HPV-specific T-cells in patients with cervical cancer (29). Specific T-cells can be determined in tissue by using fluorochrome-labeled probes and realtime PCR (30) using the unique composition of the CDR3 region of a T-cell clone.
Antigen specific T-cells undergo a selection process driving T-cell proliferation. In patients with cancer, clonal expansions may be associated with tumor-surveillance. This has been demonstrated in patients with melanoma undergoing peptide vaccination: molecularly defined TCRs, present in PBL and in blood, were able to recognize autologous tumor cells (9). Indeed, some T-cell responses involved in tumor containment may be mediated by a few effective T-cell clones or even a single clonotypic TCR (31).
TCR CDR3 analysis has been determined in patients with severe combined immunodeficiency syndrome (SCID). Of interest, the appearance of CD45RO + T-cells has been associated with a skewed TCR repertoire, and low TREC levels (T-cell receptor excision circles). In contrast, the appearance of CD45RA+ T-cells was associated with high TREC levels, a marker for thymic output and a broadened TCR repertoire (32). Particularly in patients with HIV infection, the advent of highly active retroviral therapy (HAART) has changed the prognosis and outcome of HIV infection. Progression to AIDS, the clinical presentation with opportunistic infections is associated with perturbation of the TCR repertoire. HAART is able to restore the TCR repertoire. Thus, CDR3 analysis may represent a valuable marker to compare efficacy of novel treatment protocols, together with other markers, e.g. CD4 counts and viral load determination (16, 33).
HAART drastically reduces viral load and also anti-HIV directed CD8+ T-cell responses. In order to boost anti-HIV directed T-cell responses, HAART therapy may be interrupted to stimulate CD8+ anti-HIV specific T-cells. The monitoring of this critical state is crucial for clinical management and to determine the time point to restart HAART therapy. TCR CDR3 analysis may present an objective marker to study the TCR 'perturbation' in the structured stop (and start) of HAART therapy.
Allograft rejection is mediated by T-lymphocytes, the appreciation of the T-cell infiltrate into the transplant may represent an interesting prognostic marker, or alternatively, represent a surrogate marker for testing novel drugs preventing graft rejection. TCR CDR3 analysis has been studied in the context of allograft rejection and tolerance induction, e.g. in the setting of heart transplants (15) or in the context of allogeneic hematopoietic stem cell transplantation (34), the detailed analysis of the TCR usage may also be helpful in the molecular definition of the effector cell population mediating either graft-versus-leukemia and graft-versus-host reactions (35). More recent studies suggest that TCR CDR3 analysis may represent a valuable tool to monitor GVHD after allogeneic stem cell transplantation (36).
Dramatic clonal expansion of T-cells in human healthy subjects has been reported, most of them are possibly linked to common viral pathogens, e.g. EBV or CMV. Similarly to infection with influenza A, leading to expansion of the TCR VB17 family in HLA-A2 positive individuals, the anti-EBV response is apparently driving the TCR VB6 family if the infected individual carries the HLA-B8 allele. Recognition can be linked to the HLA-B8 restricted EBV-derived peptide FLRGRAYGL (37). Tetramer complexes and TCR CDR3 analysis may also be combined to determine it the TCR repertoire changes over time as a result of therapy or vaccination. For instance, if T-cells responding to a defined antigen exist prior to (therapeutic) vaccination, a tetramer - based analysis is not able to answer the question if antigen-specific T-cells induced by the vaccine are similar or different as compared to the pre-existing T-cells. Tetramer-guided sorting, followed by TCR CDR3 analysis is able to identify molecular differences in the TCR repertoire reacting to the nominal antigens used in the vaccine (38).
The pathogenesis of a number of diseases is still not very well understood. The exact mechanisms of containment of either transformed or virally infected cells have not been determined. In general, effective adaptive T-cell responses are desirable in these diseases. The flip side of the coin - in the context of cellular immune responses - is a strong T-cell response which mediates auto-immune disorders. Many parameters exist to measure disease activity in autoimmune diseases, but the magnitude of a cellular immune response is hard to assess, particularly if no molecular targets have been identified. Since TCR CDR3 analysis visualizes objectively every alteration in the TCR composition, it may be helpful to define new markers of disease activity in autoimmune diseases; it may also present a potential matrix to gauge immuno-suppressive effects of novel drugs. For instance, TCR diversity has been suggested as a readout in PBL from patient suffering from SLE (39), or in the synovial fluid from patients with RA (40) and TCR CDR3 analysis suggested that the T-cell infiltrates in patients with Crohn's disease are indicative of an antigen-driven process (41). Recently, TCR
CDR3 analysis supported the notion that T-cells play a major role in mediating inflammation in patients with MS (42, 43) or in patients with Guillain-Barre and Fisher syndrome (44) and shed light on the association of psoriatic lesions and joint inflammation (45) as well as a certain form of polymyositis and HTLV-infection (46). In addition, TCR CDR3 analysis may be helpful to appreciate the TCR repertoire of individuals with exposure to defined pathogens. For instance, TCR CDR3 analysis in HIV+ and HIV negative men, but with 'high-risk- exposure to the pathogen showed that - in general - high antigenic exposure is associated with a restricted T-cell repertoire (47). This underlines the need to select appropriate control populations if the TCR repertoire is compared to 'healthy controls'.
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