Clinical variability of target volume description in conformal radiotherapy planning.

PURPOSE: The pivotal step in radiation planning is delineation of the target volume and production of a treatment plan to encompass this. This study assesses the variation of physicians in creation of these volumes. METHODS AND MATERIALS: Three radiologists and eight radiation oncologists outlined the gross tumour volume (GTV) on the planning CT scans of four cases with T3 bladder cancer. In addition, the radiation oncologists (RO) created a planning target volume according to a set protocol for all cases. Volumes were produced and comparison of these volumes and the position of the isocenters were analysed. In addition, the margins allowed were measured and compared. RESULTS: There was a maximum variation ratio (largest to smallest volume outlined) of the GTV in the four cases of 1.74 among radiologists and 3.74 among oncologists. There was a significant difference (p = 0.01) in mean GTV between RO and the radiologists. The mean GTV of the RO exceeded the radiologists by a factor of 1.29 with a mean difference of 13.4 cm3. The variation ratio in PTV among oncologists ranged from 1.25 to 3.33. There was no significant difference in mean PTV values between the two groups of ROs divided by specialization in uro-oncology. The mean variation in location of the isocenter from the centroid of the radiologists' volume in the four cases was from 2.6 to 5.7 mm. There was, however, a wide range of values from 1.4 mm to 24.1 mm. Median margin per case ranged from 14.7 to 18.7 mm. Minimum margins allowed in each case varied from minus 7 mm to 9 mm. CONCLUSION: This study demonstrates significant interphysician variability in producing target volumes and radiation plans for conformal radiotherapy. The scale of this difference is clearly of significance, with up to 3-fold variation in volumes delineated by clinicians. The factors leading to these differences will be further addressed. The existence of such variability, however, clearly needs to be accepted as a factor in the overall uncertainty analysis in conformal radiotherapy planning.

Limiting dilution analysis of the allo-MHC anti-paternal cytotoxic T cell response. I: Normal primigravid and multiparous pregnancies.

Anti-paternal cytotoxic T lymphocyte precursor frequencies (CTLpF) were determined by limiting dilution analysis (LDA) in the peripheral blood of eight primigravid and seven multiparous women during the three trimesters of pregnancy. In five of these women the responses to cord blood lymphocytes (CBL) and paternal lymphocytes were also determined at the time of delivery and at 6 weeks post delivery. As controls, CTLpF against unrelated third party donors were determined. A wide range of CTLpF against all three groups of targets was found in both the primigravid and multiparous women, reflecting the wide range of frequencies found in random populations. These frequencies remained fairly constant during and 6 weeks after the pregnancy. Splitwell analysis demonstrated that the responses generated in our culture system were specific to the stimulator. The LDA data conform to single-hit kinetics, indicating that only cytotoxic T cells were limiting in the assay. Proliferative responses of maternal lymphocytes to paternal, cord blood and third party MHC antigens also remained unchanged as determined by time-course mixed lymphocyte reactions (MLR). Our data suggest that there is no significant allo-stimulation or suppression of the maternal immune system during normal pregnancy. The mother remains immunocompetent and is capable of both cytotoxic and proliferative responses to paternally-derived fetal MHC antigens. Our findings confirm that in normal pregnancy the trophoblast, which is devoid of classical MHC antigens, forms an effective immune barrier which prevents interaction of the maternal and fetal immune systems.

Limiting dilution analysis of the allo-MHC anti-paternal cytotoxic T cell response. II: Recurrent spontaneous abortion and the effect of immunotherapy.

Using limiting dilution analysis (LDA) we determined anti-paternal cytotoxic T lymphocyte precursor (CTLp) frequencies in the peripheral blood of 10 women with unexplained recurrent spontaneous abortion (RSA) before and after immunization with paternal lymphocytes. The women and their partners were HLA tissue-typed and none of the women had anti-paternal cytotoxic antibodies (APCA) before immunization. All other known causes of RSA were excluded. All 10 women were found to have high frequencies of specific anti-paternal cytotoxic T cells before immunization (range 1/1030 to 1/9574). Splitwell analysis showed that these cytotoxic cells were specific to paternal MHC antigens. These frequencies rose significantly following immunization (range 1/683 to 1/4652). The cytotoxic T lymphocyte frequencies against an HLA-mismatched third party varied from woman to woman, but were not affected by the immunization. The LDA data conformed to single-hit kinetics, indicating that only cytotoxic T cells were limiting in the assay. Our data are in sharp contrast to the previously held view that women with RSA may be hyporesponsive to paternal MHC antigens. Immunizing such women with paternal leucocytes further sensitizes them. These findings cannot be reconciled with a favourable outcome in the treatment of RSA with immunotherapy. We would argue that this treatment is at best of unproven value, and may even be harmful. That these women may sometimes have successful pregnancies following immunotherapy testifies to the effectiveness of the classical MHC antigen-deficient trophoblast as an immunological barrier between mother and fetus.

Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease.

The T cell proliferative responses of peripheral blood lymphocytes from 20 patients with autoimmune thyroid disease (AITD) and 20 healthy controls were analysed to immunoaffinity-purified thyroid peroxidase (TPO) and recombinant antigen preparations generated in Escherichia coli as glutathione-s-transferase fusion proteins. The epitope specificity of the T cell response was investigated using a selection of eight discrete recombinant fragments encompassing the whole of the extracellular region of the TPO molecule. Significant differences in the proliferative responses between patients and controls were observed to the full length, affinity-purified TPO molecule (P less than 0.002) as well as to the recombinant fragments R1c (residues 145-250) (P less than 0.001) and R2b (residues 457-589) (P less than 0.001) suggesting the presence of at least two distinct T cell determinants on this autoantigen. One of these T cell epitopes, localized within the region R1c, has not previously been identified by studies with synthetic peptides.

High frequency of cord blood lymphocytes against mycobacterial 65-kDa heat-shock protein.

A high frequency of nonadherent mononuclear cells in human cord blood proliferates in response to mycobacterial 65-kDa heat-shock protein. The frequency range in cord blood is not different from that in peripheral blood of Bacillus Calmette-Guérin vaccinated adults. In comparison we found 10 to 100 times lower frequencies to purified protein derivative in nonadherent cord blood mononuclear cells than in adult peripheral blood mononuclear cells. These findings may provide experimental support for Cohen's theory of the immunological homonculus.

Limiting dilution analysis of proliferative T cell responses to mycobacterial 65-kDa heat-shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis.

Recent evidence has pointed to the mycobacterial 65-kDa heat-shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65-responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti-PPD or anti-hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross-reacting antigen, in RA.

A monoclonal antibody with broad anti-HLA-DR activity fails to bind to DRw11/Dw5: possible effect of a unique polymorphism on the beta 1 domain alpha-helix.

We describe the generation and characterization of a murine monoclonal antibody with broad anti-HLA-DR beta activity, but which does not recognize DRw11/Dw5. A BALB/c mouse was immunized with a human alloreactive T-cell clone, with the intention of generating monoclonal anti-T-cell-receptor antibodies. In the course of screening the resulting hybridomas, a clone was detected which secreted an antibody (MP2) with anti-HLA-DR activity. This was shown by flow cytometry as well as immunoprecipitation followed by gel electrophoresis. Flow cytometry experiments using transfectants bearing hybrid human/murine class II molecules demonstrated that MP2 binds to the DR beta chain. MP2 bound to a wide range of Epstein-Barr-Virus-transformed cell lines and transfectants expressing different DR beta 1 and DR beta 3 subtypes: the only exceptions were three transfectants expressing DRw11/Dw5. One of these (RGT1) was shown to be functionally normal in DRw11-restricted alloreactive and antigen-specific systems. The specificity of MP2 was confirmed in functional assays: it was able to inhibit the recognition of DR1 and DRw15 but not DRw11 by alloreactive T-cell clones. Previously reported sequence data show that the beta chain of DRw11 differs from all other DR and DQ beta chains at position 58 by a glutamic acid for alanine substitution. This may account for the inability of DRw11/Dw5 to be recognized by MP2. The data emphasize the value of transfectants in defining precisely the allelic and chain specificity of anti-major-histocompatibility-complex (MHC) antibodies, and illustrate the influence that inaccessible residues can have on the conformation of MHC molecules.

Limiting dilution analysis of human T cells: a useful clinical tool.

Limiting dilution analysis (LDA) provides a practical and simple method for determining the frequency of defined clones of lymphocytes responding to a specific antigen or with a particular effector function. The importance of the technique stems from the fact that it is the only way to assess the immune response in humans, at the level of the cell, in a quantitative manner. In this article Claire Sharrock, Edward Kaminski and Stephen Man review the current status of the technique and its applications in human immunology.

Bone-marrow transplant from father to son and subsequent graft from son to father.

An 11-year-old boy with acute myeloblastic leukaemia in first remission received an allogeneic mismatched bone-marrow transplant (BMT) from his father in 1979; subsequent HLA typing showed that his haemopoietic system had been repopulated by the donor cells. In 1986 hypereosinophilic syndrome, secondary to a T-cell lymphocytic lymphoma, developed in the father, then aged 45 years. A full haematological remission was obtained by means of standard acute lymphoblastic leukaemia treatment. He then received melphalan, total body irradiation, and a BMT from his son. Graft-versus-host disease was transient in both patients, and father and son remain well and disease-free 20 months and 10 years, respectively, after BMT.

Individual variation in the frequency of HLA class II-specific cytotoxic T lymphocyte precursors.

The frequencies of HLA class II-specific cytotoxic T lymphocyte precursors (CTLp) were studied in number of unrelated individuals using a limiting dilution analysis system optimized for the detection of CD4+ CTLp. Peripheral blood mononuclear cells (PBMC) were enriched for CD4+ T cells by immunomagnetic depletion of CD8+ T cells. In some allogeneic combinations high CTLp frequencies were obtained with no significant difference between PBMC and CD4-enriched PBMC populations. In other combinations CTLp frequencies in CD4-enriched PBMC were found to be at least twentyfold lower than in the starting, unfractionated PBMC, suggesting a predominance in these pairs of CD8+ CTLp. In addition there was variation in CTLp frequencies against the same set of HLA class II gene products between individuals, and variation in CTLp frequencies against different HLA class II gene products within individuals. The HLA class II specificity of the assay system was demonstrated unequivocally with detection of CTLp against HLA-DR1 expressed on a murine L cell transfectant.

Frequency analysis of cytotoxic T lymphocyte precursors--possible relevance to HLA-matched unrelated donor bone marrow transplantation.

HLA-matched unrelated donor (MUD) bone marrow transplants and transplants between HLA mismatched family members are associated with an increased incidence and severity of graft-versus-host disease (GVHD) in comparison with HLA-identical sibling transplants. A limiting dilution analysis system was set up to measure the frequency of alloreactive cytotoxic T lymphocyte precursors (CTL-p) in normal individuals and in potential donor/patient pairs selected for bone marrow transplantation. The donor/recipient pairs were divided into four groups depending on their degree of HLA disparity. A distinct range of CTL-p frequencies was obtained for each group and these showed a hierarchy of response related to the degree of HLA disparity between donors and recipients in that particular group. This assay system may be of value in selecting potential matched unrelated and mismatched family donor/patient pairs for those at lower risk of GVHD.

Analysis of the alloreactive T cell repertoire in man. I. Differences in precursor frequency for cytotoxic T cell responses against allogeneic MHC molecules in unrelated individuals.

We demonstrate here that in man the frequency of cytotoxic T cells specific for a given set of allo-major histocompatibility complex (MHC) antigens varies among unrelated individuals. This has been established by limiting dilution analysis of human peripheral blood lymphocytes (HPBL) in the presence of irradiated autologous filler cells, T cell growth factors, and irradiated HPBL carrying allo-MHC antigens. Two unrelated individuals were tested against the same panel of allo-MHC antigens. We have been able to identify frequencies of T cells ranging from 1:5,000 to 1:20,000(high), 1:20,000 to 1:60,000(intermediate) and 1:60,000 to 1:100,000(low). In some cases, the precursor frequency of cytotoxic T cells was so low that it was considered to represent nonresponsiveness. The clear differences in precursor frequency suggest that this system is suitable for further analysis of the allo-MHC-specific repertoire in man.

T-cell responses to minor histocompatibility antigens.

We have investigated the helper and cytotoxic T-cell response to minor histocompatibility antigens and generated long term antigen-specific cell lines to them. Antigen-specific activity was selected for by regular restimulation with irradiated cells bearing the antigens in the presence of interleukin 2, so that alloreactivity to other cell surface antigens was gradually lost. Helper T cells cultured over several months were active in vivo and in vitro, but the culturing method eventually selected for cytotoxic T cells at the expense of helper T cells, with concomitant changes in the proportions of cells expressing the Lyt phenotypes. Individual long term cultures of cytotoxic T cells specific for minor histocompatibility antigens were restricted by either H2K or D but not both. Helper T cells to minor histocompatibility antigens derived directly from primed F1 mice did not show restriction to the priming parental haplotype. This is consistent with antigen reprocessing by the F1 antigen presenting cells such that populations of helper T cells restricted by both parental H-2 haplotypes were primed. F1 cytotoxic T cells were restricted to the parental H-2 haplotype used for in vitro boosting, irrespective of which H-2 was used for in vivo priming.