Identification of melanoma cells and lymphocyte subpopulations in lymph node metastases by FTIR imaging histopathology.

While early stages of melanoma are usually cured by surgery, metastatic melanomas are difficult to treat because the widely available options have low response rates. Careful and precise diagnosis and staging are essential to determine patient's risk and to select appropriate treatments. Fortunately, the recent progress in immunotherapy is very encouraging. In this context, it is important to characterize the intratumoral infiltration of immune cells in each patient, which is however not done routinely due to the lack of standardized methods. In this study, we used Fourier transform infrared (FTIR) imaging combined with multivariate statistical analyses to investigate non-metastatic and metastatic lymph nodes from melanoma patients. Our results show that the different cell types have different infrared spectral features allowing automated identification of these cell types. High recognition rates were obtained using a supervised partial least square discriminant analysis (PLS-DA) model. Melanoma cells were recognized with 87.1% sensitivity and 85.7% specificity, showing that FTIR spectroscopy has similar detection power as immunohistochemistry. Besides, FTIR imaging could also distinguish lymphocyte subpopulations (B and T cells). Finally, we investigated the changes in lymphocytes due to the presence of metastases. Interestingly, specific features of spectra of lymphocytes present in metastatic or tumor-free lymph nodes could be evidenced by PCA. A PLS-DA model was capable of predicting whether lymphocytes originated from invaded or non-invaded lymph nodes. These data demonstrate that FTIR imaging is capable to distinguish known and also novel biological features in human tissues, with potential practical relevance for histopathological diagnosis and biomarker assessment.

An infrared spectral signature of human lymphocyte subpopulations from peripheral blood.

Metastatic melanomas are frequently refractory to most adjuvant therapies such as chemotherapies and radiotherapies. Recently, immunotherapies have shown good results in the treatment of some metastatic melanomas. Immune cell infiltration in the tumor has been associated with successful immunotherapy. More generally, tumor infiltrating lymphocytes (TILs) in the primary tumor and in metastases of melanoma patients have been demonstrated to correlate positively with favorable clinical outcomes. Altogether, these findings suggest the importance of being able to identify, quantify and characterize immune infiltration at the tumor site for a better diagnostic and treatment choice. In this paper, we used Fourier Transform Infrared (FTIR) imaging to identify and quantify different subpopulations of T cells: the cytotoxic T cells (CD8+), the helper T cells (CD4+) and the regulatory T cells (T reg). As a proof of concept, we investigated pure populations isolated from human peripheral blood from 6 healthy donors. These subpopulations were isolated from blood samples by magnetic labeling and purities were assessed by Fluorescence Activated Cell Sorting (FACS). The results presented here show that Fourier Transform Infrared (FTIR) imaging followed by supervised Partial Least Square Discriminant Analysis (PLS-DA) allows an accurate identification of CD4+ T cells and CD8+ T cells (>86%). We then developed a PLS regression allowing the quantification of T reg in a different mix of immune cells (e.g. Peripheral Blood Mononuclear Cells (PBMCs)). Altogether, these results demonstrate the sensitivity of infrared imaging to detect the low biological variability observed in T cell subpopulations.

Breast cancer and melanoma cell line identification by FTIR imaging after formalin-fixation and paraffin-embedding.

Over the past few decades, Fourier transform infrared (FTIR) spectroscopy coupled to microscopy has been recognized as an emerging and potentially powerful tool in cancer research and diagnosis. For this purpose, histological analyses performed by pathologists are mostly carried out on biopsied tissue that undergoes the formalin-fixation and paraffin-embedding (FFPE) procedure. This processing method ensures an optimal and permanent preservation of the samples, making FFPE-archived tissue an extremely valuable source for retrospective studies. Nevertheless, as highlighted by previous studies, this fixation procedure significantly changes the principal constituents of cells, resulting in important effects on their infrared (IR) spectrum. Despite the chemical and spectral influence of FFPE processing, some studies demonstrate that FTIR imaging allows precise identification of the different cell types present in biopsied tissue, indicating that the FFPE process preserves spectral differences between distinct cell types. In this study, we investigated whether this is also the case for closely related cell lines. We analyzed spectra from 8 cancerous epithelial cell lines: 4 breast cancer cell lines and 4 melanoma cell lines. For each cell line, we harvested cells at subconfluence and divided them into two sets. We first tested the "original" capability of FTIR imaging to identify these closely related cell lines on cells just dried on BaF2 slides. We then repeated the test after submitting the cells to the FFPE procedure. Our results show that the IR spectra of FFPE processed cancerous cell lines undergo small but significant changes due to the treatment. The spectral modifications were interpreted as a potential decrease in the phospholipid content and protein denaturation, in line with the scientific literature on the topic. Nevertheless, unsupervised analyses showed that spectral proximities and distances between closely related cell lines were mostly, but not entirely, conserved after FFPE processing. Finally, PLS-DA statistical analyses highlighted that closely related cell lines are still successfully identified and efficiently distinguished by FTIR spectroscopy after FFPE treatment. This last result paves the way towards identification and characterization of cellular subtypes on FFPE tissue sections by FTIR imaging, indicating that this analysis technique could become a potential useful tool in cancer research.

Distinct fates of self-specific T cells developing in irradiation bone marrow chimeras: clonal deletion, clonal anergy, or in vitro responsiveness to self-Mls-1a controlled by hemopoietic cells in the thymus.

Elimination of potentially self-reactive T lymphocytes during their maturation in the thymus has been shown to be a major mechanism in accomplishing self-tolerance. Previous reports demonstrated that clonal deletion of self-Mls-1a-specific V beta 6+ T lymphocyte is controlled by a radiosensitive I-E+ thymic component. Irradiation chimeras reconstituted with I-E- bone marrow showed substantial numbers of mature V beta 6+ T cells despite host Mls-1a expression. Analysis of the functional properties of such chimeric T cells revealed a surprising variability in their in vitro reactivity to host Mls-1a, depending on the H-2 haplotype of stem cells used for reconstitution. In chimeras reconstituted with B10.S (H-2s) stem cells, mature V beta 6+ lymphocytes were present but functionally anergic to host-type Mls-1a in vitro. In contrast, in chimeras reconstituted with B10.G (H-2q) bone marrow, nondeleted V beta 6+ cells were highly responsive to Mls-1a in vitro. These findings suggest that clonal anergy of V beta 6+ cells to self-Mls-1a may be controlled by the affinity/avidity of T cell receptor interactions with bone marrow-derived cells in the thymus depending on the major histocompatibility complex class II molecules involved. Furthermore, chimeras bearing host (Mls-1a)-reactive V beta 6+ cells did not differ clinically from those with anergic or deleted V beta 6+ cells and survived more than one year without signs of autoimmune disease. Interestingly, their spleen cells had no Mls-1a stimulatory capacity in vitro. Therefore, regulation at the level of antigen presentation may be an alternative mechanism for maintenance of tolerance to certain self-antigens such as Mls-1a.

Positive and negative selection of T cell receptor V beta domains controlled by distinct cell populations in the thymus.

The role of thymic MHC class II-bearing cells in the selection of the TCR repertoire has been investigated in allogeneic radiation bone marrow chimeras. Positive selection of mature CD4+ T lymphocytes expressing the V beta 6+ TCR domain was found to depend upon radioresistant (presumably epithelial) I-E+ thymic cells. On the other hand, negative selection of CD4+ V beta 6+ cells (which was additionally dependent upon expression of the Mls-1a gene product) was controlled by a radiosensitive I-E+ thymic component (most likely dendritic cells). These data argue in favor of a compartmentalization of positive and negative selection events during T cell development.

Clonal deletion induced by either radioresistant thymic host cells or lymphohemopoietic donor cells at different stages of class I-restricted T cell ontogeny.

Major histocompatibility complex (MHC) products and self-antigens expressed in the thymus determine the repertoire of mature alpha/beta T cells. While positive selection of self-MHC-restricted T cells is directed by MHC molecules expressed by thymic epithelial cells, negative selection depends to a large extent on self-antigens presented by lymphohemopoietic cells. However, radioresistant components of the thymus also influence negative selection, but it remains controversial whether this is accomplished by clonal deletion, clonal anergy, or other mechanisms. In this study, T cell development in mice expressing a transgenic T cell receptor (TCR) specific for lymphocytic choriomeningitis virus (LCMV) plus H-2Db was analyzed in the presence or absence of the viral antigen. A novel approach to analyze the thymic tissue requirements for negative selection was possible by comparing thymocyte selection in H-2Db versus H-2Dbm13 mice, since the latter allowed positive selection but not LCMV-specific deletion of transgenic TCR-expressing thymocytes. In irradiation bone marrow chimeras expressing the restriction element for negative selection (H-2Db) on host tissue, we show that radioresistant recipient cells in the thymus deleted developing T cells at an early stage of differentiation. In contrast, chimeras expressing H-2Db on lymphohemopoietic donor cells showed clonal deletion at a later stage during ontogeny.

Clonal deletion of self-reactive T cells in irradiation bone marrow chimeras and neonatally tolerant mice. Evidence for intercellular transfer of Mlsa.

Tolerance to Mlsa has been shown to be associated with clonal deletion of cells carrying TCR beta chain variable regions V beta 6 or V beta 8.1 in mice possessing I-E antigens. To evaluate the rules of tolerance induction to Mlsa we prepared irradiation bone marrow chimeras expressing Mlsa or Mlsb and I-E by different cell types. Deletion of V beta 6+, Mlsa-reactive T cells required the presence of Mlsa and I-E products either on bone marrow-derived cells or on irradiated recipient cells. Tolerance was induced when Mlsa and I-E were expressed by distinct cells of the chimera. Also neonatally tolerized mice exhibited depletion of V beta 6+ cells after injection of I-E- Mlsa spleen cells (DBA/1) into newborn I-E+ Mlsb mice (BALB/c x B10.G)F1. These results suggest that the product of the Mlsa locus is soluble and/or may be transferred from cell to cell and bound to I-E antigens. The chimera experiments also showed that tolerance to Mlsa is H-2 allele independent, i.e., is apparently unrestricted. Differentiation of chimeric (H-2d/Mlsa x H-2q/Mlsb)F1 stem cells in either an H-2d or an H-2q thymus revealed that tolerance assessed by absence of V beta 6+ T cells is not dependent on the thymically determined restriction specificity of T cells.

A regulatory role for TRAF1 in antigen-induced apoptosis of T cells.

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF1 were found as components of the TNFR2 signaling complex, which exerts multiple biological effects on cells such as cell proliferation, cytokine production, and cell death. In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-kappaB, but the role of TRAF1 has not been previously determined. Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8(+) T lymphocytes. Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.

Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy.

Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic beta-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP x Tag2) mice (137 +/- 18 d) as opposed to control RIP-Tag2 mice (88 +/- 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP-specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP x Tag2) mice further prolonged survival (168 +/- 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.

In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.

High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I.

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.

Magnetic nanoparticle-induced hyperthermia with appropriate payloads: Paul Ehrlich's "magic (nano)bullet" for cancer theranostics?

Effective multimodal cancer management requires the optimal integration of diagnostic and therapeutic modalities. Radiation therapy, chemotherapy and immunotherapy, alone or in combination, are integral parts of various cancer treatment protocols. Hyperthermia at 39-45°C is a potent radiosensitiser and has been shown to improve therapeutic outcomes in various tumours through its synergy with chemotherapy. Gene silencing approaches, using small interfering RNAs and microRNAs, are also being explored in clinical trials in oncology. The rapid developments in multifunctional nanoparticles provide ample opportunities to integrate both diagnostic and therapeutic modalities into a single effective cancer "theranostic" vector. Nanoparticles could extravasate passively into the tumour tissues in preference to the adjacent normal tissues by capitalizing on the enhanced permeability and retention effect. Tumour targeting might be further augmented by conjugating tumour-specific peptides and antibodies onto the surface of these nanoparticles or by activation through electromagnetic radiations, laser or ultrasound. Magnetic nanoparticles can induce hyperthermia in the presence of an alternating magnetic field, thereby multifunctionally with tumour-specific payloads empowering tumour specific radiotheranostics (for both imaging and radiotherapy), chemotherapy drug delivery, immunotherapy and gene silencing therapy. Such a (nano)bullet could realise the "magic bullet" conceived by Paul Ehrlich more than a century ago. This article discusses the various aspects of this "magic (nano)bullet" and the challenges that need to be addressed to usher in this new paradigm in modern cancer diagnostics and therapeutics.

Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8+ and CD4+ T-cell responses with multiple specificities including a novel DR7-restricted epitope.

Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8+ and CD4+ T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8+ and NY-ESO-183-99 for CD4+ T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7+ patients generated strong CD4+ T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8+ and CD4+ T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach.

Expansion and functional maturation of human tumor antigen-specific CD8+ T cells after vaccination with antigenic peptide.

Peptide-based vaccines are currently being tested for their ability to induce or augment tumor antigen (Ag)-specific CD8+ T-cell responses in cancer patients. Here we report that the frequency of circulating CD8+ T cells directed against the Melan-A/MART-1 Ag increased >20-fold in an HLA-A2 melanoma patient immunized repeatedly with the corresponding antigenic peptide, as assessed by staining with HLA-A2/peptide tetramers. Multiparameter flow cytometric analysis demonstrated that the increase in total Melan-A-specific cell number was accompanied by a marked increase in the proportion of the cells that expressed an activated/memory surface phenotype. As assessed by ELISPOT assays and intracellular staining, the absolute number of Melan-A-specific cells able to secrete IFN-gamma increased >50-fold upon vaccination. When tested directly after cell sorting on the basis of tetramer staining, Melan-A-specific cells were weakly cytolytic but became highly active after in vitro restimulation. Altogether, these results indicate that large numbers of functionally active tumor Ag-specific CD8+ T cells can be obtained and maintained at high levels after in vivo activation by repeated peptide-based vaccination.

Acute graft-versus-host disease without costimulation via CD28.

T lymphocyte activity is enhanced by costimulatory signals mediated through CD28 binding to B7-1/B7-2 on antigen-presenting cells. Several recent studies have shown that graft-versus-host disease (GVHD) can be inhibited by in vivo treatment with CTLA4Ig, which blocks CD28-B7 interactions. These findings prompted us to investigate the role of CD28 in acute GVHD, using gene-targeted mice. We performed the experiments in the context of strong allogeneic MHC stimulation (H2(b) anti-H2(d)) and weak stimulation (H2(d) anti-H2(b)). In both directions, efficient in vitro T-cell cytotoxicity and acute lethal GVHD were induced by CD28-deficient lymphocytes, which was only partially delayed when compared with wild-type mice. We conclude that lethal GVHD can develop without costimulation via CD28.

Alloreactive cytotoxic T cells recognize minor transplantation antigens presented by major histocompatibility complex class Ib molecules.

BACKGROUND: Cytotoxic T lymphocytes (CTLs) contribute to the rejection of transplanted tissues through two pathways: first, by direct recognition of foreign graft major histocompatibility complex (MHC) class I molecules; and second, by recognition of foreign graft-derived peptides presented by classical MHC class Ia molecules that are matched between graft and donor. However, a number of observations suggest that additional categories of CTL recognition patterns may exist, but they remain to be defined molecularly. METHODS: Previous studies showed that the murine nonclassical MHC molecule H2 M3 may be involved in allorecognition. We investigated whether other members of nonclassical MHC class Ib, namely Qa1 and Qa2, may be recognized. Alloreactive CTLs were generated from mice mismatched for non-MHC and/or MHC genetic backgrounds and tested using various target cells, including cells transfected with Qa1 or Qa2. Furthermore, candidate peptides were synthesized and used to generate CTLs specific for peptide presented by Qa1 or Qa2. RESULTS: The experiments demonstrate that allogeneic and xenogeneic peptides were recognized by CTLs when presented on shared nonclassical MHC class Ib Qa1 and Qa2 molecules. CONCLUSIONS: The results confirm that MHC class Ib molecules present peptides to CTLs. This potentially important alloreactivity pathway may be functional between most individuals because sharing of MHC class Ib alleles is frequent.

Oligotyping of HLA-A2, -A3, and -B44 subtypes. Detection of subtype incompatibilities between patients and their serologically matched unrelated bone marrow donors.

We have set up a simple PCR-SSO oligotyping procedure that is able to discriminate ten HLA-A2 (2 PCR/11 probes), two HLA-A3 (1 PCR/1 probe), and two HLA-B44 subtypes (1 PCR/2 probes). The frequency of these subtypes has been determined in a large panel of local blood donors and leukemic patients in combination with their unrelated potential donors. A*0201 and A*0301 were the predominant subtypes (> 95%) for A2 and A3, respectively. B*4402 occurred twice as frequently as B*4403. A2 and B44 subtype mismatches were analyzed in a group of 30 patients and their 116 unrelated potential donors who were matched serologically (low-stringency matching: AB without splits, DR1-10). For seven patients (23%) at least one A2- or B44-subtype-mismatched donor was found. For two of these patients (7%), the subtype-mismatched donor would have been considered as compatible on the basis of high stringency matching (AB splits, DRB1 subtypes, DRB3/B5). For one patient of Mediterranean origin, all five donors recruited from a north European registry (matched with high stringency) appeared to be subtype incompatible (A*0201/A*0205). The rather low percentage of A2- and B4-subtype mismatches in DRB1/B3/B5 matched combinations confirms the significance of linkage disequilibria of HLA antigens. Because unrelated donor selection is done through international registries, however, class I subtyping might be necessary when individuals originate from different geographic areas.

High-resolution histocompatibility testing of a group of sixteen B44-positive, ABDR serologically matched unrelated donor-recipient pairs. Analysis of serologically undisclosed incompatibilities by cellular techniques, isoelectrofocusing, and HLA oligotyping.

We have characterized HLA incompatibilities in a group of 16 B44-positive patients who were serologically ABDR matched with their 23 (unrelated) potential bone marrow donors. After analysis with a combination of cellular techniques, IEF for HLA-A/B and oligotyping for class II and HLA-B44, 44% of the patients revealed one or more HLA incompatibility with at least one of their potential donors. CTL activity was detected in 12 of the 22 combinations tested. CTL incompatibility occurred more frequently in DR subtype-mismatched combinations, but CTL reactivity was always directed against class I. To characterize these incompatibilities between matched unrelated individuals, we analyzed the specificity of T-cell clones from seven primary CTL cultures. In three combinations, CTL reactivity was directed against a subtype of B44. In two combinations, the CTL reactivity was directed against a non-B44 class I subtype. In two of seven combinations, the CTLs recognized an antigen that, though unconditionally associated with B4403, was expressed by 60% of the B4403+ cells only. Because all 12 of these B4403+ targets recognized could be typed for one HLA-C allele only (Cwl-Cw8), we believe that this alloreactivity might be directed against a serologically undefined Cw antigen.