T-cell cloning for HPRT mutations is arguably the most frequently used assay for assessing somatic gene mutations arising in vivo in humans. Although its major use is in population monitoring for environmental mutagens, there are other applications of the assay. One is in characterizing the target cells in which the mutations arise, i.e., the T-cells. That use is illustrated by the results presented here. The goal of HPRT T-cell selection in melanoma patients is to identify a probe for the in vivo T cell response to human melanoma that provides mechanistic insights and helps direct subsequent monitoring and/or treatment strategies for patients with melanoma.
Malignant melanoma, a malignancy present in all five patients reported here, is expected to elicit a T-cell immunological response against tumor antigens, with resultant T-cell proliferation. To provide the rationale for studying surrogate selection in the context of melanoma, additional background information about T-cell responses to melanoma will be provided. Definitive evidence that melanoma-associated antigens (MAA) can stimulate cytotoxic and regulatory T-cell responses against human melanoma as well as serve as tumor-rejection antigens for T-cells in model systems has been present for many years [Mukherji et al., 1989
; Van der Bruggen et al., 1991
]. The characterized MAA can effectively stimulate cellular immune responses in vitro
, and immunodominant epitopes have been identified and characterized [Kawakami et al., 1995
; Kawakami et al., 1994
; Kierstead et al., 2001
; Topalian et al., 1994
, Zarour et al., 2002
]. Ongoing investigations are attempting to translate this understanding of MAA into effective in vivo
therapeutic strategies [Pardoll, 2002
]. However, clinical responses remain infrequent. While tumor-rejection antigens on human melanomas would provide an opportunity for effective targeted immune-based therapy, the MAA associated with effective in vivo
anti-melanoma T-cell activity still need to be identified. It is possible that study of in vivo
expanding T-cells in melanoma patients will provide insights into the in vivo
immune response to this disease.
Several methods have been utilized to investigate in vivo
clonally amplified T-cells including TCR-β analysis by Southern blot [Albertini MR et al., 2001
], MaGiK method [Killian et al., 2002
], RT in situ
PCR [Nuovo et al., 2001
], padlock probes and microarrays [Baner et al., 2005
] and quantitative RT-PCR with spectratyping [Degauque et al., 2004
]. These methods can provide a global look at the TCR-β repertoire in melanoma patients and have identified CDR3 length alterations [Degauque et al., 2004
; Speiser et al., 2006
] over-expression of TCR-β V-region subfamilies [Degauque et al., 2004
; Sensi et al., 1997
; Speiser et al., 2006
; Willhauck et al., 2003
] and clonal expansion of T-cells after melanoma vaccination or cytokine immunotherapy [Degauque et al., 2004
; Meidenbauer et al., 2004
; Sensi et al., 1997
; Speiser et al., 2006
; Willhauck et al, 1996
]. Additional insights into the detailed antigen specificity of in vivo
T-cell responses to melanoma could be provided by identifying T cells undergoing ongoing or repetitive in vivo
cell division and using a multiplex PCR-based method and subsequent analysis of TCR-β V-D-J gene sequences.
Elsewhere, we have suggested that a preferential occurrence of clonality among MT T-cells compared to WT T-cells is a result of an increased likelihood of spontaneous mutations in rapidly proliferating cells as opposed to quiescent G0
cells — the condition of the vast majority of mature T-cells in vivo
[O’Neill et al., 1994
; Falta et al., 1999
; Albertini RJ, 2001
]. It may be that the rapidly proliferating cells may have more opportunity to make replication errors or less time to repair DNA damage. In part, preferential clonality in the MT fractions of T-cells may be a reflection of the need for amplification of HPRT
mutations in order to be recovered in cloning assays. In any case, we have suggested that this phenomenon could be exploited for isolation of T-cells of immunological relevance — a form of surrogate selection. This strategy has been used to recognize such cells in autoimmune disorders and in organ transplantation [Albertini RJ, 2001
and references therein]. The study reported here suggests its application in cancer patients.
It may be asked —as was done in this study — the degree to which T-cell clonality identified by cloning assays represents the broader in vivo situation. Does this clonality reflect what is present in peripheral blood? More importantly for cancer patients, does it reflect what might be occurring within the tumors themselves?
clonality, as defined here, is a characteristic of the cells used to detect HPRT
mutations, and not necessarily of the HPRT
mutations themselves. Cellular clonality for the post-thymic, mature T-cells originates when the TCR genes undergo rearrangement, usually during thymic differentiation. HPRT
mutational clonality begins with the occurrence of that mutation, which may be before, during, or after thymic differentiation. The timing of HPRT
mutations relative to T-cell maturation has implications as to the anatomical sites of mutation [Albertini RJ, 2001
]. Post-thymic mutation implies origination in the peripheral lymphoid tissue, i.e., LNs, spleen, peripheral blood, or other tissues where T-cells circulate; pre-thymic mutation implies origination in bone marrow or in the thymus itself.
In this regard, the finding of an identical TCR-β V-D-J sequence in a MT SCD-T clone and in the WT fraction of TIL derived mass culture cells from Patient 5 () is of note. We have only rarely observed MT–WT sharing among clonal SCD-T derived from HPRT
cloning assays, i.e., as SCD-T clones [O’Neill et al., 1994
]. However, similar analyses of MT and WT fractions obtained from mass cultures of PBMCs or tissue-derived lymphocytes (TILN and TIL in the current study) have not previously been undertaken. The studies reported here are being extended to examine potential preferential usage of V-regions as well as potential clonal relationships between patients, with the addition of more patients and analyses of restricted TCR-β gene usage among and within patients. Indeed, we have observed MT–WT sharing of TCR-β gene patterns in mass cultures derived from blood and TILN/TIL (data not shown). The MT-WT sharing data at the mass culture level suggests that the preferential recovery of expanding MT T-cell clones is not simply due to dilution of clonality of WT T-cells because clonality, in this fraction, can be observed if it is extensive enough. However, when it is this extensive, an increased frequency of somatic mutations occur, as reflected in the presence of HPRT
mutations, with the finding that these expanding in vivo
clones usually have MT representatives. Importantly, the presence of identical TCR-β gene patterns in both the MT and WT fractions of expanding T-cell clones indicates that the HPRT
mutation has occurred in mature T-cells in the periphery, i.e., after the TCR gene rearrangement has occurred.
As shown in , and , preferential clusterings of TCR-β gene patterns were clearly reflected in the cDNAs derived from mass cultures of T-cells in peripheral blood and in tumors (in LN or other tissue). There were more clusters, and clusters of larger sizes, in the cDNAs derived from MT cultures than in those derived from WT cultures. These differences were statistically significant for all patients. Although some preferential clustering may have been expected because of size differences in starter population between the selected and non-selected in vitro cultures (the MT populations were much smaller based on calculations involving starting numbers of cells and MF) and/or differences in in vitro outgrowth of selected cells, allowances in the statistical analyses were made for that consideration.
The most compelling demonstration that MT T-cell clonality as reflected in cloning assays represents the broader in vivo situation is the identity of the TCR-β gene rearrangements in MT SCD-T to the rearrangement clusters of TCR-β cDNAs derived from both PBMC and TILN/TIL MT mass cultures in Patients 1 and 9 (). The finding of identical MT T-cells both in the peripheral blood and at sites of tumor suggests trafficking of expanding MT in vivo T-cells clones between the peripheral blood and sites of tumor. These findings validate analysis of MT isolates from blood when the target organ is not accessible, and these analyses can be used to investigate T-cells potentially involved in the in vivo T-cell response to human melanoma. Members of these expanding MT T-cell clones are expected to be present at frequencies difficult to detect within the WT fraction of T-cells from the same patient.
In conclusion, in vivo clonal amplifications of MT T-cells are present in the peripheral blood and at sites of tumors of melanoma patients. These expanding in vivo T-cell clones can be identified by analysis of TCR-β gene usage of TG-selected clonal SCD-T and of TG-selected mass cultures. In vivo MT T-cells in melanoma patients provide candidate probes to investigate the in vivo T-cell response to human melanoma.