Quantitative protein mass-spectrometry requires a standardized pre-analytical phase.

OBJECTIVES: Quantitative protein mass-spectrometry (QPMS) in blood depends on tryptic digestion of proteins and subsequent measurement of representing peptides. Whether serum and plasma can be used interchangeably and whether in-vitro anticoagulants affect the recovery is unknown. In our laboratory serum samples are the preferred matrix for QPMS measurement of multiple apolipoproteins. In this study, we investigated the effect of different matrices on apolipoprotein quantification by mass spectrometry. METHODS: Blood samples were collected from 44 healthy donors in Beckton Dickinson blood tubes simultaneously for serum (with/without gel) and plasma (heparin, citrate or EDTA). Nine apolipoproteins were quantified according to standard operating procedure using value-assigned native serum calibrators for quantitation. Tryptic digestion kinetics were investigated in the different matrices by following formation of peptides for each apolipoprotein in time, up to 22 h. RESULTS: In citrate plasma recovery of apolipoproteins showed an overall reduction with a bias of -14.6%. For heparin plasma only -0.3% bias was found compared to serum, whereas for EDTA-plasma reduction was more pronounced (-5.3% bias) and variable with >14% reduction for peptides of apoA-I, A-II and C-III. Digestion kinetics revealed that especially slow forming peptides showed reduced formation in EDTA-plasma. CONCLUSIONS: Plasma anticoagulants affect QPMS test results. Heparin plasma showed comparable results to serum. Reduced concentrations in citrate plasma can be explained by dilution, whereas reduced recovery in EDTA-plasma is dependent on altered proteolytic digestion efficiency. The results highlight the importance of a standardized pre-analytical phase for accurate QPMS applications in clinical chemistry.

CD163+ cytokine-producing cDC2 stimulate intratumoral type 1 T cell responses in HPV16-induced oropharyngeal cancer.

BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative OPSCC, especially in patients with a concomitant intratumoral HPV-specific and type-1 cytokine-oriented T cell response. However, knowledge on the type of myeloid cells and their coordination with intratumoral T cells and influence on patient outcome in OPSCC is lacking. METHODS: We analyzed the presence of intratumoral myeloid cells and their relationship to tumor-infiltrating T cells and patient outcome in a well-described cohort of HPV16+ patients with OPSCC using multispectral immunofluorescence, flow cytometry and functional analyses. RESULTS: We show that the tumor microenvironment of HPV16+ OPSCC tumors with such an ongoing HPV16-specific T cell response is highly infiltrated with a newly defined CD163+ cytokine-producing subset of conventional dendritic cell type 2 (cDC2), called DC3. These CD163+ cDC2 predominantly stimulated type 1 T cell polarization and produced high levels of interleukin-12 (IL-12) and IL-18, required for IFNγ and IL-22 production by T cells after cognate antigen stimulation. Tumor-infiltration with these CD163+ cDC2 positively correlated with the infiltration by Tbet+ and tumor-specific T cells, and with prolonged survival. CONCLUSIONS: These data suggest an important role for intratumoral CD163+ cDC2 in stimulating tumor-infiltrating T cells to exert their antitumor effects.

High numbers of activated helper T cells are associated with better clinical outcome in early stage vulvar cancer, irrespective of HPV or p53 status.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed. METHODS: Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal expression of CD3, CD8, Foxp3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC (n = 14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls. RESULTS: Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3+PD-1+), specifically helper T cells (CD3+CD8-Foxp3-), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4+PD-1++CD161-CD38+HLA-DR+ and CD8+CD103+CD161-NKG2A+/-PD1++CD38++HLA-DR+) effector memory T cells. CONCLUSION: This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated.

The Anatomical Location Shapes the Immune Infiltrate in Tumors of Same Etiology and Affects Survival.

PURPOSE: The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood. EXPERIMENTAL DESIGN: We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)-induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC). RESULTS: Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8+CD103+CD161+ effector T cells and less CD4+CD161+ effector memory T cells than OPSCC. CD161+ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4+ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4+ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar. CONCLUSIONS: The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.

NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines.

Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

Intratumoral HPV16-Specific T Cells Constitute a Type I-Oriented Tumor Microenvironment to Improve Survival in HPV16-Driven Oropharyngeal Cancer.

Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) has a much better prognosis than HPV-negative OPSCC, and this is linked to dense tumor immune infiltration. As the viral antigens may trigger potent immunity, we studied the relationship between the presence of intratumoral HPV-specific T-cell responses, the immune contexture in the tumor microenvironment, and clinical outcome.Experimental Design: To this purpose, an in-depth analysis of tumor-infiltrating immune cells in a prospective cohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cell assays, mass cytometry (CyTOF), flow cytometry, and fluorescent immunostaining of tumor tissues. Key findings were validated in a cohort of 75 patients with HPV16-positive OPSCC present in the publicly available The Cancer Genome Atlas database.Results: In 64% of the HPV16-positive tumors, type I HPV16-specific T cells were present. Their presence was not only strongly related to a better overall survival, a smaller tumor size, and less lymph node metastases but also to a type I-oriented tumor microenvironment, including high numbers of activated CD161+ T cells, CD103+ tissue-resident T cells, dendritic cells (DC), and DC-like macrophages.Conclusions: The viral antigens trigger a tumor-specific T-cell response that shapes a favorable immune contexture for the response to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process. Clin Cancer Res; 24(3); 634-47. ©2017 AACRSee related commentary by Laban and Hoffmann, p. 505.

Potential use of lymph node-derived HPV-specific T cells for adoptive cell therapy of cervical cancer.

Adoptive transfer of tumor-specific T cells, expanded from tumor-infiltrating lymphocytes or from peripheral blood, is a promising immunotherapeutic approach for the treatment of cancer. Here, we studied whether the tumor-draining lymph nodes (TDLN) of patients with human papillomavirus (HPV)-induced cervical cancer can be used as a source for ACT. The objectives were to isolate lymph node mononuclear cells (LNMC) from TDLN and optimally expand HPV-specific CD4+ and CD8+ T cells under clinical grade conditions. TDLN were isolated from 11 patients with early-stage cervical cancer during radical surgery. Isolated lymphocytes were expanded in the presence of HPV16 E6 and E7 clinical grade synthetic long peptides and IL-2 for 22 days and then analyzed for HPV16 specificity by proliferation assay, multiparameter flow cytometry and cytokine analysis as well as for CD25 and FoxP3 expression. Stimulation of LNMC resulted in expansion of polyclonal HPV-specific T cells in all patients. On average a 36-fold expansion of a CD4+ and/or CD8+ HPV16-specific T cell population was observed, which maintained its capacity for secondary expansion. The T helper type 1 cytokine IFNγ was produced in all cell cultures and in some cases also the Th2 cytokines IL-10 and IL-5. The procedure was highly reproducible, as evidenced by complete repeats of the stimulation procedures under research and under full good manufacturing practice conditions. In conclusion, TDLN represent a rich source of polyclonal HPV16 E6- and E7-specific T cells, which can be expanded under clinical grade conditions for adoptive immunotherapy in patients with cervical cancer.

Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses.

Therapeutic vaccination with human papillomavirus type 16 synthetic long peptides (HPV16-SLPs) results in T cell-mediated regression of HPV16-induced premalignant lesions but fails to install clinically effective immunity in patients with HPV16-positive cervical cancer. We explored whether HPV16-SLP vaccination can be combined with standard carboplatin and paclitaxel chemotherapy to improve immunity and which time point would be optimal for vaccination. This was studied in the HPV16 E6/E7-positive TC-1 mouse tumor model and in patients with advanced cervical cancer. In mice and patients, the presence of a progressing tumor was associated with abnormal frequencies of circulating myeloid cells. Treatment of TC-1-bearing mice with chemotherapy and therapeutic vaccination resulted in superior survival and was directly related to a chemotherapy-mediated altered composition of the myeloid cell population in the blood and tumor. Chemotherapy had no effect on tumor-specific T cell responses. In advanced cervical cancer patients, carboplatin-paclitaxel also normalized the abnormal numbers of circulating myeloid cells, and this was associated with increased T cell reactivity to recall antigens. The effect was most pronounced starting 2 weeks after the second cycle of chemotherapy, providing an optimal immunological window for vaccination. This was validated with a single dose of HPV16-SLP vaccine given in this time window. The resulting proliferative HPV16-specific T cell responses were unusually strong and were retained after all cycles of chemotherapy. In conclusion, carboplatin-paclitaxel therapy fosters vigorous vaccine-induced T cell responses when vaccination is given after chemotherapy and has reset the tumor-induced abnormal myeloid cell composition to normal values.

Lethal giant larvae-1 deficiency enhances the CD8(+) effector T-cell response to antigen challenge in vivo.

Lethal giant larvae-1 (Lgl-1) is an evolutionary conserved protein that regulates cell polarity in diverse lineages; however, the role of Lgl-1 in the polarity and function of immune cells remains to be elucidated. To assess the role of Lgl-1 in T cells, we generated chimeric mice with a hematopoietic system deficient for Lgl-1. Lgl-1 deficiency did not impair the activation or function of peripheral CD8(+) T cells in response to antigen presentation in vitro, but did skew effector and memory T-cell differentiation. When challenged with antigen-expressing virus or tumor, Lgl-1-deficient mice displayed altered T-cell responses. This manifested in a stronger antiviral and antitumor effector CD8(+) T-cell response, the latter resulting in enhanced control of MC38-OVA tumors. These results reveal a novel role for Lgl-1 in the regulation of virus-specific T-cell responses and antitumor immunity.

Regulation of asymmetric cell division and polarity by Scribble is not required for humoral immunity.

The production of protective antibody requires effective signalling of naive B cells following encounter with antigen, and the divergence of responding B lymphocytes into distinct lineages. Polarity proteins have recently been proposed as important mediators of both the initial B cell response, and potentially of asymmetric cell division. Here we show that, although polarity proteins of the Scribble complex, Scribble, Dlg1 and Lgl1, are expressed and polarized during early B cell activation, their deficiency has no effect on the in vivo outcome of immunization or challenge with influenza infection. Furthermore, we find a striking correlation in the differentiation outcome of daughters of single founder B cells in vitro. Taken together, our results indicate that B cell differentiation does not require polarity proteins of the Scribble complex, and the findings do not support a role for asymmetric cell division in B cell activation and differentiation.

Asymmetric cell division of T cells upon antigen presentation uses multiple conserved mechanisms.

Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8(+) T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.

Immuno-histological analysis of dendritic cells in nasal biopsies of IgA nephropathy patients.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Intranasal vaccination of patients with IgAN has shown mucosal and systemic IgA hyporesponsiveness. Here, we investigated whether this IgA hyporesponse in IgAN patients can be explained by reduced numbers or altered subset distribution of dendritic cells (DCs) in nasal mucosa. METHODS: Eighteen IgAN patients and 18 healthy volunteers were recruited for this study. Nasal biopsies were taken, after local anaesthesia, from the lower edge of the inferior turbinate. Staining for different subsets of DCs was performed using specific monoclonal antibodies. To detect myeloid DCs, we used CD1a, DC-SIGN and blood dendritic cell antigen-1 (BDCA-1) as a marker and for plasmacytoid DCs we used BDCA-2. DC-cell numbers in the epithelium and in lamina propria were counted separately and expressed as positively stained cells per mm(2). RESULTS: Both myeloid and plasmacytoid DC could be demonstrated in nasal biopsies. Quantification showed that IgAN patients contained significantly more DC-SIGN-positive cells in the lamina propria compared to controls. In addition, in IgAN patients, we observed more CD1a-positive cells in the epithelium. No differences in BDCA-1 and BDCA-2-positive cells were found between patients and controls. The number of positively stained cells in the epithelial layer correlated strongly with the number of positively stained cells in the lamina propria. CONCLUSIONS: Patients with IgAN have higher numbers of CD1a-positive cells in the epithelial layer and more DC-SIGN-positive cells in the lamina propria. Therefore, the earlier observed IgA hyporesponsiveness in IgAN patients after mucosal vaccination cannot be explained by lower numbers of nasal DCs.

Homeostasis and function of goblet cells during rotavirus infection in mice.

Rotaviruses are the leading cause of severe viral gastroenteritis in young children. To gain insight in goblet cell homeostasis and intestinal mucin expression during rotavirus infection, 6-day-old mice were inoculated with murine rotavirus. To determine epithelial cell migration, mice were injected with BrdU just before inoculation. Small intestines were isolated at different days postinfection (dpi) and evaluated for rotavirus and goblet cell-specific gene expression. Small intestinal mucins of control and infected animals at 1, 2, and 4 dpi were isolated and tested for their capability to neutralize rotavirus infection in vitro. After inoculation, two peaks of viral replication were observed at 1 and 4 dpi. During infection, the number of goblet cells in infected mice was decreased in duodenum and jejunum, but was unaffected in the ileum. Goblet cells in infected animals accumulated at the tips of the villi. Muc2 mRNA levels were increased during the peak of viral replication at 1 dpi, whereas at other time points Muc2 and Tff3 mRNA levels were maintained at control levels. Muc2 protein levels in the tissue were also maintained, however Tff3 protein levels were strongly decreased. The number of goblet cells containing sulfated mucins was reduced during the two peaks of infection. Mucins isolated at 1 and 2 dpi from control and infected mice efficiently neutralized rotavirus infection in vitro. Moreover, mucins isolated from infected mice at 4 dpi were more potent in inhibiting rotavirus infection than mucins from control mice at 4 dpi. In conclusion, these data show that during rotavirus infection, goblet cells, in contrast to enterocytes, are relatively spared from apoptosis especially in the ileum. Goblet cell-specific Muc2 expression is increased and mucin structure is modified in the course of infection. This suggests that goblet cells and mucins play a role in the active defense against rotavirus infection and that age-dependent differences in mucin quantities, composition, and/or structure alter the anti-viral capabilities of small intestinal mucins.

Antibodies against mesangial cells in a rat model of chronic renal allograft rejection.

BACKGROUND: Chronic renal allograft rejection (CR) is the leading cause of late renal transplant failure. The histological lesions of CR may comprise glomerular basement membrane (GBM) duplications and mesangiolysis. Its pathogenesis is not yet completely understood, although lately humoral responses have been suggested to be important. Recently, we identified antibody responses directed against GBM antigens in the Fischer (F344) to Lewis (LEW) renal transplantation model. Immunofluorescent studies in this model also suggested deposition of antibodies on mesangial cells. Therefore, we hypothesized that antibodies were not only directed at GBM antigens but also to mesangial cell antigens. METHODS: F344 to LEW renal transplantations were performed and sera were collected. Pre- and post-transplantation sera were tested for antibody binding to donor rat mesangial cells (RMCs) cultured from F344 kidneys. Anti-mesangial cell antibodies were compared with anti-GBM antibodies measured in the same sera. RESULTS: Post-transplant sera of F344 to LEW renal transplantations, but not LEW to F344, bound to F344 RMC in a dose-dependent manner. Whereas antibodies reactive with RMCs were not present before transplantation, all rats with CR developed antibodies. The antibodies were predominantly of the IgG1 isotype. Antibody binding to RMCs correlated with binding to F344 GBM. Pre-incubation with RMCs partially inhibited GBM binding, and RMC binding was inhibited by GBM. Antibody binding to RMCs did not result in complement activation or cell lysis. CONCLUSION: LEW recipients of F344 grafts produce antibodies reactive with F344 RMCs. The antigens involved are similar to or at least share antigenic epitopes with antigens recognized in the GBM.

Antibody response against the glomerular basement membrane protein agrin in patients with transplant glomerulopathy.

Chronic allograft nephropathy (CAN) of renal allografts is still the most important cause of graft loss. A subset of these patients have transplant glomerulopathy (TGP), characterized by glomerular basement membrane (GBM) duplications, but of unknown etiology. Recently, a role for the immune system in the pathogenesis of TGP has been suggested. In 11 of 16 patients with TGP and in 3 of 16 controls with CAN in the absence of TGP we demonstrate circulating antibodies reactive with GBM isolates. The presence of anti-GBM antibodies was associated with the number of rejection episodes prior to diagnosis of TGP. Sera from the TGP patients also reacted with highly purified GBM heparan sulphate proteoglycans (HSPG). Indirect immunofluorescence with patient IgG showed a GBM-like staining pattern and colocalization with the HSPGs perlecan and especially agrin. Using patient IgG, we affinity purified the antigen and identified it as agrin. Reactivity with agrin was found in 7 of 16 (44%) of patients with TGP and in 7 of 11 (64%) patients with anti-GBM reactivity. In conclusion, we have identified a humoral response against the GBM-HSPG agrin in patients with TGP, which may play a role in the pathogenesis of TGP.

Anti-tubular basement membrane antibodies and giant cell formation in a model of chronic renal allograft rejection.

BACKGROUND: In the Fisher (F344)-to-Lewis (LEW) kidney transplantation (Tx) model of chronic rejection, antibodies reactive with tubular basement membranes have been found. We investigated whether giant cells, typical for a granulomatous inflammatory reaction, can be found during allograft rejection. METHODS: F344-to-LEW renal Txs were performed, and kidneys were removed at various time points. Kidneys were analyzed by histology and immunohistochemistry. Interferon-gamma levels were measured by reverse-transcriptase polymerase chain reaction. RESULTS: Multinucleated Langhans'-type giant cells were found in F344 allografts at days 21 and 30 and disappeared thereafter. Giant cells were not observed in LEW allografts. The giant cells were ED1, OX-43, and major histocompatibility complex class II positive. All grafts showed transient expression of interferon-gamma, peaking at days 7 to 14 post-Tx. CONCLUSION: Granulomatous inflammation with giant cell formation occurs in the F344-to-LEW model and may represent an alloimmune response against tissue-specific antigens.

Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice.

Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.

Telomere shortening and cellular senescence in a model of chronic renal allograft rejection.

Cellular senescence has been suggested to play a role in the deterioration of renal graft function and has been linked to telomere shortening. We have investigated markers of cellular senescence in the F344 to LEW rat model of chronic renal transplant rejection. Syngeneic and LEW to F344 transplants were used as controls. Substantial telomere shortening was observed in all transplants, including allogeneic and syngeneic grafts from day 7 post-transplant onwards. Ischemia of native F344 kidneys was already sufficient to induce telomere shortening. It is known that shortened telomeres can activate cell cycle regulators, such as p21 and p16. Accordingly, all cases showed a transient p21 increase, with a maximum at day 7 and a sustained expression of p16. Importantly, senescence-associated beta-galactosidase staining, a cytological marker for senescence, was only observed in tubular epithelial cells of chronically rejecting F344 allografts from day 30 post-transplantation onwards. Long-term surviving LEW allografts or syngeneic F344 grafts were negative for senescence-associated beta-galactosidase. In conclusion, ischemia during transplantation results in telomere shortening and subsequent activation of p21 and p16, whereas senescence-associated beta-galactosidase staining is only present in chronically rejecting kidney grafts.