CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia.

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

Engagement of the T lymphocyte antigen receptor regulates association of son-of-sevenless homologues with the SH3 domain of phospholipase Cgamma1.

One mechanism for transducing signals downstream of lymphocyte receptor activation involves the stable association between signaling proteins. To identify protein ligands of the signal activator phospholipase Cgamma1 (PLCgamma1), we screened T cell cDNA libraries with the PLCgamma1-SH3 domain by the yeast two-hybrid assay. We observed association between the PLCgamma1-SH3 domain and the human Ras guanine nucleotide exchange factor son-of-sevenless-2 (hSos2) through a proline-rich domain interaction. Stable and abundant hSos2 / PLCgamma1 and hSos1 / PLCgamma1 complexes were observed in unstimulated T cells. The interaction between these enzymes was augmented following engagement of the T cell antigen receptor (TCR / CD3). The kinetics of protein complex enhancement correlated with TCR / CD3-induced tyrosine phosphorylation of PLCgamma1; however, those PLCgamma1 molecules in complex with hSos2 were non-phosphorylated after TCR / CD3 stimulation, in contrast to the phosphorylated PLCgamma1 associated with the linker for activation of T cells, LAT. The Grb2 adapter protein was detected in complex with hSos / PLCgamma1, suggesting a regulatory role for Grb2. SH3 domains from both Grb2 and PLCgamma1, but not RasGAP, bound directly to hSos homologues. The SH2 domain from Grb2 formed an association with the hSos / PLCgamma1 complex, which was enhanced following TCR / CD3 ligation. Together, the data suggest a mechanism for the son-of-sevenless and PLCgamma1 signal transducing enzymes in recruitment to protein complexes with potentially differential signaling consequences in T lymphocytes.

LeIF: a recombinant Leishmania protein that induces an IL-12-mediated Th1 cytokine profile.

We have evaluated the ability of the Leishmania protein LeIF to influence the Th1/Th2 cytokine responses and the generation of LeIF-specific T cell clones in the absence of adjuvant. We characterized LeIF-specific T cell responses in Leishmania major-infected and uninfected BALB/c mice. These mice develop a strong Th2 response during infection with L. major. When lymph node cells from infected BALB/c mice were stimulated in vitro with LeIF, only IFN-gamma (and no detectable IL-4) was found in the culture supernatant. In addition, LeIF down-regulated Leishmania Ag-specific IL-4 production by lymph node cells from infected BALB/c mice. Subsequently, Th responses were evaluated in naive BALB/c mice following immunization with LeIF. T cell clones derived from mice immunized with LeIF preferentially secreted IFN-gamma. Finally, to understand the basis for the preferential Th1 cytokine bias observed with LeIF, the ability of LeIF to influence the early cytokine profile was evaluated in splenocytes of SCID mice. We found that LeIF stimulated fresh spleen cells from naive SCID mice to secrete IFN-gamma by IL-12/IL-18-dependent mechanisms. The N-terminal half of the molecule (amino acid residues 1-226) maintained the ability to stimulate IFN-gamma from splenocytes of SCID mice. Finally, we also demonstrated that LeIF was able to provide partial protection of BALB/c mice against L. major. Thus, our results suggest the potential of LeIF as a Th1-type adjuvant and as a therapeutic and prophylactic vaccine Ag for leishmaniasis when used with other leishmanial Ags.

Detection of fetal congenital heart disease in a low-risk population.

Our purpose was to evaluate the efficacy of level two ultrasound screening for the detection of congenital heart defects (CHD) in a low-risk population by using three standardized cuts. Within a period of four years a total of 6727 pregnant women of a low-risk population undertook several ultrasound examinations on the basis of screening for fetal malformations. All ultrasound examinations were performed by three experienced doctors. At every single scan three standardized cuts (apical and lateral four-chamber view, crossing over of the great arteries) were obtained in order to detect congenital heart defects. Of 87 CHDs (1.33 per cent of the examined women) 39 (43.8 per cent) were diagnosed prenatally. The detection rate was 10/48 (20.8 per cent) in the presence of VSD, ASD2 or combined ASD2 + VSD, the detection rate was 29/39 (74.3 per cent) in the presence of other forms of congenital heart disease. None of the 38 missed cases in the first group but three of the ten missed CHDs in the second group had emergency neonatological problems. Aneuploidy and/or other malformations existed in 22/87 cases of CHD. The obstetrical management was changed in nearly all cases after the diagnosis of a CHD. Twenty-two women opted for termination of pregnancy because of additional fetal malformations or chromosomal defects. Five women were transferred prenatally to a tertiary centre for neonatal cardiac surgery. Ten deliveries were performed in the presence of a neonatologist. Good detection rates for CHD can be achieved in a low-risk population on the basis of level two ultrasound screening by using the above mentioned three cuts and thus, the perinatal mortality and morbidity can be improved.

[Relation between common allelic variation in a cross-sectional longitudinal population study. Vitamin D receptor locus, bone mineral density and postmenopausal bone loss]. / Relation mellem simpel allelisk variation i et tvaersnits- og longitudinelt befolkningsstudie. Vitamin D-receptorlocus, knoglemineraldensitet og postmenopausalt knogletab.

The aim of this study was to determine whether common allelic variation at the vitamin D receptor locus is related to bone mineral density and postmenopausal bone loss. Five hundred and ninety-nine healthy women aged 27 to 72 and 125 women with low bone mass aged 55-77 were measured once in a cross-sectional design. Furthermore, 136 women aged 45-54 were followed longitudinally for 18 years. The vitamin D receptor genotypes were determined using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism analysis for three different polymorphic restriction sites (BsmI, ApaI and TaqI). Bone mineral density was measured by photon and X-ray absorptiometry. The cross-sectional study showed no significant relationship between vitamin D receptor genotype and bone mineral density. The maximum difference between homozygotes was 1.3%, P = 0.33, N = 723. The low bone mineral density group had virtually the same genotype frequencies as the normal bone mineral density group. The results of the longitudinal study showed that vitamin D receptor genotype was neither related to early postmenopausal bone loss: age 51-53 (total bone loss at the lower forearm: -3.6% [3.6%], mean [standard deviation]), late postmenopausal bone loss: age 63-69 (total bone loss at the hip: -6.2% [8.7%]) nor to long term postmenopausal bone loss: age 51-69 (total bone loss at the lower forearm: -24.5% [11.4%]). Using analysis of variance to test for differences in the rates of bone loss between the vitamin D receptor genotypes, P values ranged from 0.07 to 0.79. We conclude that common allelic variation at the vitamin D receptor locus as defined by the endonucleases ApaI, BsmI and TaqI is neither related to bone mineral density nor to the rate of bone loss in healthy postmenopausal Danish women.

Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes.

The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvIg exhibited binding activity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than the mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvIg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation.

The human p167 gene encodes a unique structural protein that contains centrosomin A homology and associates with a multicomponent complex.

The characterization of novel cytoplasmic, structural, and enzymatic proteins has been enhanced by a panel of monoclonal antibodies specific for protein substrates of transforming and nontransforming c-Src mutants. These protein substrates have included the focal adhesion kinase (FAK), cortactin, AFAP-110, p120CAS, and p130CAS. The monoclonal antibody 4G8 was generated as part of this panel of antibodies and was used to isolate the human gene for a 167-kD polypeptide. The cDNA sequence is 5,238 nucleotides in length with a predicted open reading frame consisting of 1,382 amino acids. The polypeptide is largely hydrophilic and highly charged. The central region of p167 has 88% identity with the entire 278-amino-acid encoded sequence of the murine centrosomin A gene. The carboxyl third of p167 contains a unique cluster of 10 amino acid repeats with the consensus sequence (A/M)DDDRGPRRG. The p167 protein was found primarily in the cytoplasm of lymphocytes and is part of a multicomponent protein complex with prominent members of 167, 120, 64, 45, 40, 38, and 25 kD. Finally, we illustrate the conservation of p167 and its associated complex, and demonstrate its expression in different human tissues and cell types. The data suggest that p167 is novel and has an important cellular function as a cytoplasmic structural protein.

Prenatal diagnosis of facial malformations.

In a prospective study, 5407 pregnant women were screened by ultrasound to detect malformations of the fetal face. Of a total of 11 facial anomalies, eight were detected by prenatal ultrasound (72 per cent). Three pregnancies were terminated because of associated developmental abnormalities or aneuploidy. In all of them, the facial malformations were correctly diagnosed. When associated with other developmental abnormalities, facial malformations were picked up at a rate of 100 per cent. Isolated facial malformations, by contrast, were detected in no more than 50 per cent of cases. Eight cases with suspected facial dysmorphism ended with the delivery of normal babies (specificity 99.8 per cent). None of them prompted karyotyping or any other invasive testing. Only two correctly detected facial malformations (bilateral cleft lips/palate) had a minor influence on obstetrical management. There would not have been disadvantages for the newborns in any of the cases if the malformations had been missed.

[Effects of progesterone on proliferation and differentiation of fetal rat calvarial osteoblasts in vitro].

OBJECTIVES: To investigate the influence of progesterone on proliferation and differentiation of osteoblast at the levels of gene expression and cell functions. METHODS: Fetal rat calvarial osteoblasts were cultured in vitro in the presence of (10(-9) mol/L-10(-6) mol/L) progesterone. Cell proliferation, alkaline phosphalase (ALP) activity, osteocalcin mRNA expression and osteocalcin secretion in the medium and bone nodule formation were analyzed. RESULTS: Progesterone did not influence cell proliferation; Progesterone enhanced the ALP activity in rat osteoblasts; Progesterone stimulated osteocalcin mRNA expression in a dose-dependent manner and increased the amount of osteocalcin in the culture medium; Progesterone increased both number and area of bone nodule formation. CONCLUSION: Progesterone has a multi-stimulating effect on the differentiation of fetal rat calvarial osteoblast, hut no effect on cell proliferation.

Functional characterization of the human biglycan 5'-flanking DNA and binding of the transcription factor c-Krox.

The transcriptional regulation of human biglycan expression under normal and pathological conditions was studied. The 5'-flanking regions of the human and mouse genes were isolated and analyzed; the two promoter regions share 81% identity. Both promoters are without a TATA and CAT box and contain multiple Sp1 sites. Human dermal fibroblasts were transiently transfected with progressive deletional human biglycan 5'-flanking DNA-CAT constructs, and a significant variation in activity among the individual constructs was found. A small deletion in several cases caused a more than 2-fold increase or decrease in promoter activity, thereby mapping the target sites for repressors or activators. Human biglycan expression is reduced in females with Ullrich-Turner syndrome (45,X) and increased in individuals with supernumerary sex chromosomes, and it has been speculated that biglycan plays a role in the short stature phenotype of Turner syndrome. Analysis of the transcriptional regulation of biglycan in individuals with sex chromosome anomalies showed that a -262 to -218 region of the biglycan promoter was differentially regulated. This region was extensively analyzed by DNAse footprinting and electrophoretic mobility shift assays, and a putative binding site for the transcription factor c-Krox was discovered. The binding of c-Krox to a site located at approximately -248 to -230 in the human biglycan promoter was confirmed by using extracts from COS cells expressing recombinant human c-Krox. The expression of c-Krox in bone was then examined by reverse-transcribed polymerase chain reaction and Northern blotting analysis; an approximately 3.4 kb transcript was detected in primary osteoblastic cells, in MG-63 cells, and in human bone marrow stromal cells. This is the first detection of c-Krox in bone cells, and it suggests that c-Krox, like another member of the Krox family, Krox-20, might play a regulatory role in bone.

Interaction between effects of parathyroid hormone and bisphosphonate on regulation of osteoclast activity by the osteoblast-like cell line UMR-106.

Treatment of osteoblasts by either parathyroid hormone (PTH) or bisphosphonate can affect their regulation of the bone-resorbing activity of osteoclasts in vitro, leading to increased and decreased resorption, respectively. To address this issue, we have examined the interaction between the effects of PTH and bisphosphonate on the regulation of osteoclast activity by the PTH-responsive osteoblast-like cell line UMR-106. When rat osteoclasts were cocultured with UMR-106 cells on bovine bone slices in the presence of 10(-8) mol/L PTH, the number of resorption pits was increased 4.2-fold, whereas the addition of UMR-106 cells or PTH alone had no effect. Pretreatment of the UMR-106 cells for 5 min with increasing concentrations of either of the bisphosphonates, clodronate, and ibandronate before coculture with osteoclasts in 10(-8) mol/L PTH, caused a dose-dependent reduction in the formation of resorption pits, reaching the maximal inhibition level (60%-75% below the control) at approximately 10(-9) mol/L clodronate and 10(-11) mol/L ibandronate. Addition of conditioned medium (CM) from untreated UMR-106 cells to rat osteoclasts had no effect on pit formation, whereas CM from UMR-106 cells pretreated with ibandronate reduced the osteoclastic bone resorption by approximately 40%. However, this effect was abolished by the subsequent culture of the ibandronate-pretreated UMR-106 cells in 10(-8) mol/L PTH before harvesting the CM, because both this CM and CM from non-pretreated UMR-106 cells cultured in 10(-8) mol/L PTH caused an approximately 75% increase in pit formation when added to rat osteoclasts. In conclusion, osteoclastic bone resorption can be directly affected independently as well as at the same time by the ibandronate-induced osteoclast-inhibiting factor and the PTH-induced osteoclast-stimulating factor. The final level of bone resorption depends on the relative concentration of these two factors.

Relation of common allelic variation at vitamin D receptor locus to bone mineral density and postmenopausal bone loss: cross sectional and longitudinal population study.

OBJECTIVE: To determine whether common allelic variation at the vitamin D receptor locus is related to bone mineral density and postmenopausal bone loss. DESIGN: Cross sectional and longitudinal population study. SETTING: Outpatient clinic in research centre. SUBJECTS: 599 healthy women aged 27 to 72 and 125 women with low bone mass aged 55-77 had bone mineral density measured once in the cross sectional study. 136 women aged 45-54 were followed up for 18 years in the longitudinal study. MAIN OUTCOME MEASURES: Bone mineral density measured at the lumbar spine, hip, and forearm and rate of bone loss at different times over 18 years in relation to vitamin D receptor genotype as defined by the endonucleases ApaI, EsmI, and TaqI. RESULTS: Vitamin D receptor genotype was not related to bone mineral density at any site. The maximum difference between homozygotes was 1.3% (P = 0.33, n = 723). Women with low bone mineral density had almost the same genotype frequencies as the women with normal bone mineral densities. Vitamin D receptor genotype was not related to early postmenopausal bone loss from age 51 to 53 (mean (SD) total loss at the lower forearm -3.6% (3.6%)), late postmenopausal bone loss from age 63 to 69 (at the hip-6.2% (8.7%)), or to long term postmenopausal loss from age 51 to 69 (at the lower forearm-24.5% (11.4%)). CONCLUSION: Common allelic variation at the vitamin D receptor locus as defined by the endonucleases ApaI, EsmI, and TaqI is related neither to bone mineral density nor to the rate of bone loss in healthy postmenopausal Danish women.

ALP induction by beta-glycerophosphate during the non-mineralization phase in vitro.

beta-GP influences on rat osteoblast development at the early period of culture i.e., the non-mineralization phase, and changes with the different cell passages were investigated. Alkaline phosphatase (ALP) was chosen as a main object. Northern blot analysis revealed up to two-fold increase in the steady state level of ALP mRNA after stimulation of rat osteoblast with 10 mM beta-GP. Likewise, 10 mM beta-GP induced a 10-30% increase in ALP activity (P < 0.01) of early passages (1 to 4), but not of later passages (5 to 6). The beta-GP induced increase in ALP activity was totally inhibited by the protein synthesis inhibitor, cycloheximide (50 microM). beta-GP stimulation was found to be without effect on cell proliferation measured as 3H-thymidine incorporation. It is concluded that beta-GP has no effect on proliferation but induces an increase in both mRNA level and activity of ALP in the non-mineralization phase of cultures of fetal rat calvarial cells, which lasts for several passages but will disappear in older cultures.

Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

Analysis of T cell receptor alpha beta variability in lymphocytes infiltrating melanoma primary tumours and metastatic lesions.

The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumour-infiltrating lymphocytes (TIL) in four different primary human malignant melanomas and their corresponding metastatic lesions was characterized using a recently developed method based on the reverse-transcription-coupled polymerase chain reaction (RT-PCR). All patients were typed for HLA-A1 and -A2, either serologically or by a newly developed RT-PCR method. Two of these patients expressed HLA-A2, one the HLA-A1 haplotype and one further patient was heterozygous HLA-A1/-A2. The prognostic parameters for all four patients indicated that rapid progression of the disease was to be expected. However, only two of the patients showed rapid progression, while the remaining two patients are still alive after more than 3 years. In TIL in primary melanomas, a possible correlation was suggested between HLA-A2 and the preferential usage of the TCR V gene families V alpha 4, V alpha 5, V alpha 22 and V beta 8, whereas the V beta 3 gene family appeared to be expressed together with HLA-A1. Other highly expressed V gene families, apparently not restricted to either HLA-A1 or -A2, were V alpha 1 (expressed in three of four primary tumours) and V alpha 21 (expressed in two of four tumours). We found no evidence suggesting any correlations between the haplotypes HLA-A1 and -A2 and preferential V gene family expression in the metastatic lesions, and the only common feature was V alpha 8, which was found to be highly expressed in two out of three subcutaneous metastases. The V gene families, which were highly expressed in the primary tumour were generally not, or only very weakly, expressed in metastases and vice versa, possibly reflecting a change in the phenotype of the metastatic melanoma target cells. With regards to patient 0368, it was possible to obtain and study material from two subcutaneous metastases. The first metastasis was excised more than a year after the primary tumour, showing a completely different V region repertoire. The second metastasis was excised at surgery 2 years after primary surgery and likewise showed a dramatic shift in comparison to the first subcutaneous metastasis. Although the present study only included a small number of patients, it suggests that the estimation of V gene expression, if applied to a larger amount of patient material, might make it possible to substantiate further the suggested correlations between the T cell response against the tumour, HLA and antigen expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Preferential usage of T-cell receptor alpha beta variable regions among tumor-infiltrating lymphocytes in primary human malignant melanomas.

The usage of T-cell-receptor (TCR) alpha beta variable (V) regions among tumor infiltrating lymphocytes (TILs) in primary human malignant melanomas was characterized using a method based on the polymerase chain reaction (PCR). A panel of 57 different variable-region primers specific for the TCR V alpha I-29 and V beta I-28 was designed, and a semi-quantitative PCR method applicable to formalin-fixed, paraffin-embedded tissues was developed. This semi-quantitative method was demonstrated to be reproducible and to be useful for the assessment of V alpha- and V beta-gene-family usage in formalin-fixed, paraffin-embedded tissue samples. A total of 9 different histopathologically characterized primary tumors were analyzed in this study. The TILs in these tumors were found to preferentially express certain TCR V alpha and V beta regions. The differential usage of certain V alpha regions was very pronounced as illustrated by V alpha 4, which was highly expressed in 3/8 tumors, and V alpha 22, which was highly expressed in 4/8 tumors. For comparison, specific highly expressed V alpha regions in control samples of peripheral-blood lymphocytes rarely exceeded 10%. The most highly expressed V beta region was V beta 8, which was highly expressed in 2/8 tumors. For the highly expressed V alpha 4 and V alpha 22 and V beta 8 regions, the high levels may be explained by the in situ clonal or oligoclonal expansion of TIL. In one specific case, the high expression of V beta 8 was due to expansion of a single clone of TILs, as evidenced by a fully readable sequence of the CDR3 (V-D-J) region, determined by direct sequencing of the PCR product corresponding to V beta 8. In contrast, sequence analysis of V alpha 22, which was expressed in the same tumor sample at similar levels, demonstrated the simultaneous presence of 3 different CDR3 (V-J) sequences derived from V alpha 22 transcripts of exactly the same length. The observed preferential use of TCR V alpha and V beta regions suggests the in situ clonal expansion of specific populations of T-cells, possibly reactive with melanoma-associated peptides presented by HLA molecules. The preferential use of TCR V alpha and V beta regions may imply the involvement of a limited number of shared melanoma-associated peptides.

T-cell receptor v-alpha and v-Beta gene usage in interleukin-2-cultured tumor-infiltrating lymphocytes from patients with breast-cancer.

Tumor-infiltrating lymphocytes (TIL) are often found in malignant breast tumors, and have been claimed to be of prognostic value. It has been proposed that TIL may represent an enriched population of tumor-specific cytotoxic lymphocytes, reacting with antigenic determinants on the tumor cell surface through the T cell receptor (TCR) complex. We have studied the phenotype, cytotoxicity, and expression of TCR variable (V) alpha and beta chain on in vitro IL-2-cultured TIL isolated from primary malignant breast tumors from 11 patients. 10/11 cultures were dominated by CD4(+) (T-helper) cells. The different TIL cultures exhibited varying levels of cytotoxicity against the natural killer (NK)-sensitive cell line K562 and breast cancer cell line T47D. The level of clonality, as measured by PCR-based analyses of usage of the different V segments was low, as only a few tumors showed patterns of restricted V gene expression. The mean number of V alpha segments per TIL culture was higher than the number of V beta segments per culture. A significant negative correlation was observed between the number of CD4+ cells and the number of V beta segments per culture, and no other correlations between phenotypes and expression of any particular V segments were found. Neither was there any correlation between the expression of specific V alpha/V beta segments and cytotoxicity against allogeneic tumor cells.