Early effects of different brain radiotherapy modalities on circulating leucocyte subpopulations in rodents.

PURPOSES: Lymphopenia is extensively studied, but not circulating leucocyte subpopulations, which however have distinct roles in tumor tolerance. Proton therapy has been shown to have a lesser impact on the immune system than conventional X-ray radiotherapy through lower dose exposure to healthy tissues. We explored the differential effects of brain X-ray and proton irradiation on circulating leucocyte subpopulations. MATERIALS AND METHODS: Leucocyte subpopulation counts from tumor-free mice were obtained 12 hours after 4 fractions of 2.5 Gy. The relationships between irradiation type (X-rays or protons), irradiated volume (whole-brain/hemi-brain) and dose rate (1 or 2 Gy/min) with circulating leucocyte subpopulations (T-CD4+, T-CD8+, B, and NK-cells, neutrophils, and monocytes) were investigated using linear regression and tree-based modeling approaches. Relationships between dose maps (brain, vessels, lymph nodes (LNs)) and leucocyte subpopulations were analyzed and applied to construct the blood dose model, assessing the hypothesis of a direct lymphocyte-killing effect in radiation-induced lymphopenia. RESULTS: Radiation-induced lymphopenia occurred after X-ray but not proton brain irradiation in lymphoid subpopulations (T-CD4+, T-CD8+, B, and NK-cells). There was an increase in neutrophil counts following protons but not X-rays. Monocytes remained unchanged under both X-rays and protons. Besides irradiation type, irradiated volume and dose rate had a significant impact on NK-cell, neutrophil and monocyte levels but not T-CD4+, T-CD8+, and B-cells. The dose to the blood had a heterogeneous impact on leucocyte subpopulations: neutrophil counts remained stable with increasing dose to the blood, while lymphocyte counts decreased with increasing dose (T-CD8+-cells > T-CD4+-cells > B-cells > NK-cells). Direct cell-killing effect of the dose to the blood mildly contributed to radiation-induced lymphopenia. LN exposure significantly contributed to lymphopenia and partially explained the distinct impact of irradiation type on circulating lymphocytes. CONCLUSIONS: Leucocyte subpopulations reacted differently to X-ray or proton brain irradiation. This difference could be partly explained by LN exposure to radiation dose. Further researches and analyses on other biological processes and interactions between leucocyte subpopulations are ongoing. The various mechanisms underlying leucocyte subpopulation changes under different irradiation modalities may have implications for the choice of radiotherapy modalities and their combination with immunotherapy in brain cancer treatment.

Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets.

Foxp3+ regulatory T cells (Tregs) in the colon are key to promoting peaceful coexistence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TFs; Helios, Rorγ, Gata3, and cMaf), but their interrelationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs (Rorγ, Helios, Gata3, and cMaf) play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR repertoires in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related and cannot be uniquely equated to tTreg and pTreg. Comparison of spleen and colon repertoires revealed that 2 to 5% of clonotypes are shared between the locations. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.

Sex- and age-specific aspects of human peripheral T-cell dynamics.

Background: The diversity of the antigenic T cell receptor (TCR) repertoire clonally expressed on T lymphocytes is a key element of the adaptive immune system protective functions. A decline in diversity in the older adults is associated with health deterioration. This diversity is generated by the rearrangement of TRB genes coding for TCR chains during lymphocyte differentiation in the thymus, but is essentially maintained by peripheral T lymphocytes proliferation for most of life. Deep sequencing of rearranged TRB genes from blood cells allows the monitoring of peripheral T cell repertoire dynamics. We analysed two aspects of rearranged TRB diversity, related to T lymphocyte proliferation and to the distribution of the T cell clone size, in a collection of repertoires obtained from 1 to 74 years-old donors. Results: Our results show that peripheral T lymphocytes expansion differs according to the recombination status of their TRB loci. Their proliferation rate changes with age, with different patterns in men and women. T cell clone size becomes more heterogeneous with time, and, in adults, is always more even in women. Importantly, a longitudinal analysis of TRB repertoires obtained at ten years intervals from individual men and women confirms the findings of this cross-sectional study. Conclusions: Peripheral T lymphocyte proliferation partially depends on their thymic developmental history. The rate of proliferation of T cells differing in their TRB rearrangement status is different in men and women before the age of 18 years old, but similar thereafter.

Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets.

Foxp3 + regulatory T cells (Tregs) in the colon are key to promoting peaceful co-existence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TF; Helios, Rorg, Gata3, cMaf), but their inter-relationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR clonotypes in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related, and cannot be uniquely equated to tTreg and pTreg. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.

Beyond lymphopenia, unraveling radiation-induced leucocyte subpopulation kinetics and mechanisms through modeling approaches.

Leucocyte subpopulations in both lymphoid and myeloid lineages have a significant impact on antitumor immune response. While radiation-induced lymphopenia is being studied extensively, radiation effects on lymphoid and myeloid subtypes have been relatively less addressed. Interactions between leucocyte subpopulations, their specific radiation sensitivity and the specific kinetics of each subpopulation can be modeled based on both experimental data and knowledge of physiological leucocyte depletion, production, proliferation, maturation and homeostasis. Modeling approaches of the leucocyte kinetics that may be used to unravel mechanisms underlying radiation induced-leucopenia and prediction of changes in cell counts and compositions after irradiation are presented in this review. The approaches described open up new possibilities for determining the influence of irradiation parameters both on a single-time point of acute effects and the subsequent recovery of leukocyte subpopulations. Utilization of these approaches to model kinetic data in post-radiotherapy states may be a useful tool for further development of new treatment strategies or for the combination of radiotherapy and immunotherapy.

Bystander signals from low- and high-dose irradiated human primary fibroblasts and keratinocytes modulate the inflammatory response of peripheral blood mononuclear cells.

Irradiated cells can propagate signals to neighboring cells. Manifestations of these so-called bystander effects (BEs) are thought to be relatively more important after exposure to low- vs high-dose radiation and can be mediated via the release of secreted molecules, including inflammatory cytokines, from irradiated cells. Thus, BEs can potentially modify the inflammatory environment of irradiated cells. To determine whether these modifications could affect the functionality of bystander immune cells and their inflammatory response, we analyzed and compared the in vitro response of primary human fibroblasts and keratinocytes to low and high doses of radiation and assessed their ability to modulate the inflammatory activation of peripheral blood mononuclear cells (PBMCs). Only high-dose exposure resulted in either up- or down-regulation of selected inflammatory genes. In conditioned culture media transfer experiments, radiation-induced bystander signals elicited from irradiated fibroblasts and keratinocytes were found to modulate the transcription of inflammatory mediator genes in resting PBMCs, and after activation of PBMCs stimulated with lipopolysaccharide (LPS), a strong inflammatory agent. Radiation-induced BEs induced from skin cells can therefore act as a modifier of the inflammatory response of bystander immune cells and affect their functionality.

Label-Free Direct Mass Spectrometry Analysis of the Bystander Effects Induced in Chondrocytes by Chondrosarcoma Cells Irradiated with X-rays and Carbon Ions.

BACKGROUND: Radiation-induced bystander effects are induced changes in cells that were not themselves directly irradiated but were in the vicinity of a radiation path. Such effects, which occur in the microenvironment of an irradiated tumor, remain poorly understood and depend on the cell type and irradiation quality. This study aimed to evaluate bystander effects in non-irradiated chondrocytes that received conditioned medium from irradiated chondrosarcoma cells. METHODS: SW1353 chondrosarcoma cells were irradiated with X-rays and carbon ions, each at 0.1 Gy and 2 Gy, and the conditioned media of the irradiated cells were transferred to T/C-28A2 chondrocytes and Human Umbilical Venous Endothelial Cells (HUVECs). The whole proteome of bystander chondrocytes was analyzed by label-free mass spectrometry, and a comparative study was performed by dose and irradiation quality. HUVECs were evaluated for inflammatory cytokine secretion. RESULTS: The bystander response of chondrocytes to X-ray irradiation primarily affected the protein translation pathway (DHX36, EIF3B, EIF3D, EIF3M, EIF5, RPL6, RPLP0, RPS24, SYNCRIP), IL-12 (AIP, BOLA2, MIF, GAS6, MIF, PDGFRB) and the oxidative stress pathway (MGST3, PRDX2, PXDN, SOD2, TXN, TXNL1). Following carbon-ion irradiation, the G1/S pathway (PCBP4, PSMD12, PSME, XIAP) and mitotic G2 DNA damage checkpoint pathway (MRE11, TAOK1, UIMC1) were engaged. Changes in the regulation of chromosome separation (BCL7C, BUB3, CENPF, DYNC1LI1, SMARCA4, SMC4) were associated with only low-dose X-ray and carbon-ion irradiation. Modification of the protein translation pathway represented at least 30% of bystander effects and could play a role, possibly along with stress granules, in reduction in cellular metabolism to protect proteins. Stress granules were significantly enriched according to an interaction map. CONCLUSIONS: All these accessions corresponded to a window of the proteins modulated in response to the bystander effect. Our chondrosarcoma model clarified the nature of the bystander response of chondrocytes and may suggest several interesting new mechanisms that are specific to particular irradiation doses and qualities.

Germline DNA Retention in Murine and Human Rearranged T Cell Receptor Gene Coding Joints: Alternative Recombination Signal Sequences and V(D)J Recombinase Errors.

The genes coding for the antigenic T cell receptor (TR) subunits are assembled in thymocytes from discrete V, D, and J genes by a site-specific recombination process. A tight control of this activity is required to prevent potentially detrimental recombination events. V, D, and J genes are flanked by semi-conserved nucleotide motives called recombination signal sequences (RSSs). V(D)J recombination is initiated by the precise introduction of a DNA double-strand break exactly at the border of the genes and their RSSs by the RAG recombinase. RSSs are therefore physically separated from the coding region of the genes before assembly of a rearranged TR gene. During a high throughput profiling of TRB genes in mice, we identified rearranged TRB genes in which part or all of a flanking RSS was retained in V-D or D-J coding joints. In some instances, this retention of germline DNA resulted from the use of an upstream alternative RSS. However, we also identified TRB sequences where retention of germline DNA occurred in the absence of alternative RSS, suggesting that RAG activity was mis-targeted during recombination. Similar events were also identified in human rearranged TRB and TRG genes. The use of alternative RSSs during V(D)J recombination illustrates the complexity of RAG-RSSs interactions during V(D)J recombination. While the frequency of errors resulting from mis-targeted RAG activity is very low, we believe that these RAG errors may be at the origin of oncogenic translocations and are a threat for genetic stability in developing lymphocytes.

Combining PARP inhibition, radiation, and immunotherapy: A possible strategy to improve the treatment of cancer?

Immunotherapy has revolutionized the practice of oncology, improving survival in certain groups of patients with cancer. Immunotherapy can synergize with radiation therapy, increase locoregional control, and have abscopal effects. Combining it with other treatments, such as targeted therapies, is a promising means of improving the efficacy of immunotherapy. Because the value of immunotherapy is amplified with the expression of tumor antigens, coupling poly(ADP-ribose) polymerase (PARP) inhibitors and immunotherapy might be a promising treatment for cancer. Further, PARP inhibitors (PARPis) are being combined with radiation therapy to inhibit DNA repair functions, thus enhancing the effects of radiation; this association might interact with the antitumor immune response. Cytotoxic T lymphocytes are central to the antitumor immune response. PARP inhibitors and ionizing radiation can enhance the infiltration of cytotoxic T lymphocytes into the tumor bed, but they can also enhance PD-1/PDL-1 expression. Thus, the addition of immune checkpoint inhibitors with PARP inhibitors and/or ionizing radiation could counterbalance such immunosuppressive effects. With the present review article, we proposed to evaluate some of these associated therapies, and we explored the biological mechanisms and medical benefits of the potential combination of radiation therapy, immunotherapy, and PARP inhibitors.

Influence of Confounding Factors on Radiation Dose Estimation Using In Vivo Validated Transcriptional Biomarkers.

For triage purposes following a nuclear accident, blood-based gene expression biomarkers can provide rapid dose estimates for a large number of individuals. Ionizing-radiation-responsive genes are regulated through the DNA damage-response pathway, which includes activation of multiple transcription factors. Modulators of this pathway could potentially affect the response of these biomarkers and consequently compromise accurate dose estimation calculations. In the present study, four potential confounding factors were selected: cancer condition, sex, simulated bacterial infection (lipopolysaccharide), and curcumin, an anti-inflammatory/antioxidant agent. Their potential influence on the transcriptional response to radiation of the genes CCNG1 and PHPT1, two biomarkers of radiation exposure ex vivo, was assessed. First, both CCNG1 and PHPT1 were detected in vivo in blood samples from radiotherapy patients and as such were validated as biomarkers of exposure. Importantly, their basal expression level was slightly but significantly affected in vivo by patients' cancer condition. Moreover, lipopolysaccharide stimulation of blood irradiated ex vivo led to a significant modification of CCNG1 and PHPT1 transcriptional response in a dose- and time-dependent manner with opposite regulatory effects. Curcumin also affected CCNG1 and PHPT1 transcriptional response counteracting some of the radiation induction. No differences were observed based on sex. Dose estimations calculated using linear regression were affected by lipopolysaccharide and curcumin. In conclusion, several confounding factors tested in this study can indeed modulate the transcriptional response of CCNG1 and PHPT1 and consequently can affect radiation exposure dose estimations but not to a level which should prevent the biomarkers' use for triage purposes.

Ionizing radiation does not impair the mechanisms controlling genetic stability during T cell receptor gene rearrangement in mice.

PURPOSE: To determine whether low dose/low dose rate radiation-induced genetic instability may result from radiation-induced inactivation of mechanisms induced by the ATM-dependent DNA damage response checkpoint. To this end, we analysed the faithfulness of T cell receptor (TR) gene rearrangement by V(D)J recombination in DNA from mice exposed to a single dose of X-ray or chronically exposed to low dose rate γ radiation. MATERIALS AND METHODS: Genomic DNA obtained from the blood or the thymus of wild type or Ogg1-deficient mice exposed to low (0.1) or intermediate/high (0.2-1 Gy) doses of radiation either by acute X-rays exposure or protracted exposure to low dose-rate γ-radiation was used to analyse by PCR the presence of illegitimate TR gene rearrangements. RESULTS: Radiation exposure does not increase the onset of TR gene trans-rearrangements in irradiated mice. In mice where it happens, trans-rearrangements remain sporadic events in developing T lymphocytes. CONCLUSION: We concluded that low dose/low dose rate ionizing radiation (IR) exposure does not lead to widespread inactivation of ATM-dependent mechanisms, and therefore that the mechanisms enforcing genetic stability are not impaired by IR in developing lymphocytes and lymphocyte progenitors, including BM-derived hematopoietic stem cells, in low dose/low dose rate exposed mice.

Low-dose radiation accelerates aging of the T-cell receptor repertoire in CBA/Ca mice.

While the biological effects of high-dose-ionizing radiation on human health are well characterized, the consequences of low-dose radiation exposure remain poorly defined, even though they are of major importance for radiological protection. Lymphocytes are very radiosensitive, and radiation-induced health effects may result from immune cell loss and/or immune system impairment. To decipher the mechanisms of effects of low doses, we analyzed the modulation of the T-cell receptor gene repertoire in mice exposed to a single low (0.1 Gy) or high (1 Gy) dose of radiation. High-throughput T-cell receptor gene profiling was used to visualize T-lymphocyte dynamics over time in control and irradiated mice. Radiation exposure induces "aging-like" effects on the T-cell receptor gene repertoire, detectable as early as 1 month post-exposure and for at least 6 months. Surprisingly, these effects are more pronounced in animals exposed to 0.1 Gy than to 1 Gy, where partial correction occurs over time. Importantly, we found that low-dose radiation effects are partially due to the hematopoietic stem cell impairment. Collectively, our findings show that acute low-dose radiation exposure specifically results in long-term alterations of the T-lymphocyte repertoire.

Key mechanisms involved in ionizing radiation-induced systemic effects. A current review.

Organisms respond to physical, chemical and biological threats by a potent inflammatory response, aimed at preserving tissue integrity and restoring tissue homeostasis and function. Systemic effects in an organism refer to an effect or phenomenon which originates at a specific point and can spread throughout the body affecting a group of organs or tissues. Ionizing radiation (IR)-induced systemic effects arise usually from a local exposure of an organ or part of the body. This stress induces a variety of responses in the irradiated cells/tissues, initiated by the DNA damage response and DNA repair (DDR/R), apoptosis or immune response, including inflammation. Activation of this IR-response (IRR) system, especially at the organism level, consists of several subsystems and exerts a variety of targeted and non-targeted effects. Based on the above, we believe that in order to understand this complex response system better one should follow a 'holistic' approach including all possible mechanisms and at all organization levels. In this review, we describe the current status of knowledge on the topic, as well as the key molecules and main mechanisms involved in the 'spreading' of the message throughout the body or cells. Last but not least, we discuss the danger-signal mediated systemic immune effects of radiotherapy for the clinical setup.

The Immune System in Cancer Prevention, Development and Therapy.

The immune system plays a pivotal role in the maintenance of the integrity of an organism. Besides the protection against pathogens, it is strongly involved in cancer prevention, development and defense. This review focuses on how the immune system protects against infections and trauma and on its role in cancer development and disease. Focus is set on the interactions of the innate and adaptive immune system and tumors. The role of IFN-γ as a pleiotropic cytokine that plays a very important role at the interface of innate and adaptive immune systems in tumor development and induction of anti-tumor immune responses is outlined. Further, immune cells as prognostic and predictive markers of cancer will be discussed. Data are provided that even the brain as immune privileged organ is subjected to immune surveillance and consequently also brain tumors. Immune therapeutic approaches for glioblastoma multiforme, the most frequent and malignant brain tumor, based on vaccination with dendritic cells are outlined and application of hyperthermia in form of magnetic nanoparticles is discussed. We conclude that the immune system and developing tumors are intimately intertwined. Anti-tumor immune responses can be prominently boosted by multimodal therapies aiming on the one hand to induce immunogenic tumor cell death forms and on the other hand to actively counteract the immune suppressive microenvironment based on the tumor itself.

The many interactions between the innate immune system and the response to radiation.

The role of the immune system in the protection of the organism against biological aggressions is long established and well-studied. A new role emerged more recently in the protection from - and the response to - physical trauma such as exposure to ionizing radiation. A pre-existing inflammation, induced by administration of an inflammatory cytokine or of a Toll-like receptor agonist, is indeed able to mitigate the toxic effects of acute radiation exposure. Conversely, it appears that the innate immune system can be activated during the course of the cellular response to radiation. Activation of different sensors and pattern recognition receptors by intra-cellular molecules such as HMGB1 or DNA released in the extra-cellular milieu or in the cytosol by irradiated cells induces the production of inflammatory and anti-viral cytokines. In addition, in human monocytes and macrophages, the expression of inflammatory cytokine genes can be directly induced by p53- and ATM-dependent mechanisms. This last finding establishes a direct link between radiation-induced DNA damage response and radiation-induced inflammation.

The toxicity redox mechanisms of cadmium alone or together with copper and zinc homeostasis alteration: its redox biomarkers.

Cadmium (Cd) is a toxic metal and can induce and/or promote diseases in humans (cancer, aging diseases, kidney and bone diseases, etc.). Its toxicity involves many mechanisms including the alteration of copper (Cu) and zinc (Zn) homeostasis leading to reactive oxygen species (ROS) production, either directly or through the inhibition of antioxidant activities. Importantly, ROS can induce oxidative damages in cells. Cadmium, Cu and Zn are also able to induce glutathione (GSH) and metallothioneins (MT) synthesis in a cell-type-dependent manner. As a consequence, the effects induced by these three metals result simultaneously from the inhibition of antioxidant activities and the induction of other factors such as GSH and MT synthesis. MT levels are regulated not only by the p53 protein in a cell-type-dependent manner, or by transcription factors such as metal-responsive transcription factor 1 (MTF-1) and cellular Zn levels but also by cellular GSH level. As described in the literature, DNA damage, GSH and MT levels are sensitive biomarkers used to identify Cd-induced toxicity alone or together with Cu and Zn homeostasis alteration.

Enhanced susceptibility of T lymphocytes to oxidative stress in the absence of the cellular prion protein.

The cellular prion glycoprotein (PrP(C)) is ubiquitously expressed but its physiologic functions remain enigmatic, particularly in the immune system. Here, we demonstrate in vitro and in vivo that PrP(C) is involved in T lymphocytes response to oxidative stress. By monitoring the intracellular level of reduced glutathione, we show that PrP(-/-) thymocytes display a higher susceptibility to H(2)O(2) exposure than PrP(+/+) cells. Furthermore, we find that in mice fed with a restricted diet, a regimen known to increase the intracellular level of ROS, PrP(-/-) thymocytes are more sensitive to oxidative stress. PrP(C) function appears to be specific for oxidative stress, since no significant differences are observed between PrP(-/-) and PrP(+/+) mice exposed to other kinds of stress. We also show a marked evolution of the redox status of T cells throughout differentiation in the thymus. Taken together, our results clearly ascribe to PrP(C) a protective function in thymocytes against oxidative stress.

Distinct effects of DNA-PKcs and Artemis inactivation on signal joint formation in vivo.

The assembly of functional immune receptor genes via V(D)J recombination in developing lymphocytes generates DNA double-stranded breaks intermediates that are repaired by non-homologous end joining (NHEJ). This repair pathway requires the sequential recruitment and activation onto coding and signal DNA ends of several proteins, including the DNA-dependent protein kinase and the nuclease Artemis. Artemis activity, triggered by the DNA-dependent protein kinase, is necessary to process the genes hairpin-sealed coding ends but appears dispensable for the ligation of the reciprocal phosphorylated, blunt-ended signal ends into a signal joint. The DNA-dependent protein kinase is however present on signal ends and could potentially recruit and activate Artemis during signal joint formation. To determine whether Artemis plays a role during the resolution of signal ends during V(D)J recombination, we analyzed the structure of signal joints generated in developing thymocytes during the rearrangement of T cell receptor genes in wild type mice and mice mutated for NHEJ factors. These joints exhibit junctional diversity resulting from N nucleotide polymerization by the terminal nucleotidyl transferase and nucleotide loss from one or both of the signal ends before they are ligated. Our results show that Artemis participates in the repair of signal ends in vivo. Furthermore, our results also show that while the DNA-dependent protein kinase complex protects signal ends from processing, including deletions, Artemis seems on the opposite to promote their accessibility to modifying enzymes. In addition, these data suggest that Artemis might be the nuclease responsible for nucleotide loss from signal ends during the repair process.

Gene-specific signal joint modifications during V(D)J recombination of TCRAD locus genes in murine and human thymocytes.

V(D)J recombination assembles functional T-cell receptor (TCR) genes from V, D and J components in developing thymocytes. Extensive processing of V, D and J extremities before they are ligated creates a high degree of junctional diversity which results in the generation of a large repertoire of different TCR chains. In contrast, the extremities of the intervening DNA segment, which bear the recombination signal sequences, are generally held to be monomorphic, so that signal joints (SJs) consist of the perfect head-to-head juxtaposition of recombination signal extremities. We analyzed the structure of SJs generated during the recombination of TCRAD locus genes in murine and human thymocytes. Junctional diversity resulting from N nucleotide additions or from N nucleotide additions and base loss was found for each type of SJ examined. Different patterns of processing/modification were found, suggesting that different enzymatic activities operate during recombination of TCRA and TCRD genes, although they are located within the same genetic locus. Recombination of the deltaRec-1 element generates a diverse repertoire of SJs exhibiting both combinatorial and junctional diversity in murine and human thymocytes. Therefore, SJ diversity appears to be an intrinsic feature of V(D)J recombination in unmanipulated thymocytes.