Vaccination with post-translational modified, homocitrullinated peptides induces CD8 T-cell responses that mediate antitumor immunity.

BACKGROUND: Post-translational modification of proteins has the potential to alter the ability of T cells to recognize major histocompatibility complex (MHC) class -I and class-II restricted antigens, thereby resulting in altered immune responses. One such modification is carbamylation (homocitrullination) that results in the formation of homocitrulline (Hcit) residues in a non-enzymatic reaction of cyanate with the lysine residues in the polypeptide chain. Homocitrullination occurs in the tumor microenvironment and CD4-mediated immune responses to Hcit epitopes can target stressed tumor cells and provide a potent antitumor response in mouse models. METHODS: Homocitrullinated peptides were identified and assessed in vitro for HLA-A2 binding and in vivo in human leukocyte antigen (HLA) transgenic mouse models for immunogenicity. CD8 responses were assessed in vitro for cytotoxicity and in vivo tumor therapy. Human tumor samples were analyzed by targeted mass spectrometry for presence of homocitrullinated peptides. RESULTS: Homocitrullinated peptides from aldolase and cytokeratin were identified, that stimulated CD8-mediated responses in vivo. Modified peptides showed enhanced binding to HLA-A2 compared with the native sequences and immunization of HLA-A2 transgenic mice generated high avidity modification specific CD8 responses that killed peptide expressing target cells. Importantly, in vivo the homocitrullinated aldolase specific response was associated with efficient CD8 dependent antitumor therapy of the aggressive murine B16 tumor model indicating that this epitope is naturally presented in the tumor. In addition, the homocitrullinated aldolase epitope was also detected in human tumor samples. CONCLUSION: This is the first evidence that homocitrullinated peptides can be processed and presented via MHC-I and targeted for tumor therapy. Thus, Hcit-specific CD8 T-cell responses have potential in the development of future anticancer therapy.

Immune responses to citrullinated and homocitrullinated peptides in healthy donors are not restricted to the HLA SE shared allele and can be selected into the memory pool.

Citrullination and homocitrullination are stress induced post-translational modifications (siPTMs) which can be recognized by T cells. Peripheral blood mononuclear cells isolated from healthy donors and rheumatoid arthritis (RA) patients were stimulated with nine siPTM-peptides. CD45RA/CD45RO depletion was employed to determine if peptide-specific responses are naïve or memory. Human leucocyte antigen (HLA)-DP4 and HLA-DR4 transgenic mice were immunized with siPTM-peptides and immune responses were determined with ex vivo ELISpot assays. The majority (24 out of 25) of healthy donors showed CD4 T cell-specific proliferation to at least 1 siPTM-peptide, 19 to 2 siPTM-peptides, 14 to 3 siPTM-peptides, 9 to 4 siPTM-peptides, 6 to 5 siPTM-peptides and 4 to 6 siPTM-peptides. More donors responded to Vim28-49cit (68%) and Bip189-208cit (75%) compared with Vim415-433cit (33%). In RA patients, the presentation of citrullinated epitopes is associated with HLA-SE alleles; however, we witnessed responses in healthy donors who did not express the SE allele. The majority of responding T cells were effector memory cells with a Th1/cytotoxic phenotype. Responses to Vim28-49cit and Eno241-260cit originated in the memory pool, while the response to Vim415-433cit was naïve. In the HLA-DP4 and HLA-DR4 transgenic models, Vim28cit generated a memory response. Peptide-specific T cells were capable of Epstein-Barr virus transformed lymphoblastoid cell line recognition suggesting a link with stress due to infection. These results suggest siPTM-peptides are presented under conditions of cellular stress and inflammation and drive cytotoxic CD4 T cell responses that aid in the removal of stressed cells. The presentation of such siPTM-peptides is not restricted to HLA-SE in both humans and animal models.

What do cancer-specific T cells 'see'?

Complex cellular interactions between the immune system and cancer can impact tumour development, growth, and progression. T cells play a key role in these interactions; however, the challenge for T cells is to recognize tumour antigens whilst minimizing cross-reactivity with antigens associated with healthy tissue. Some tumour cells, including those associated with viral infections, have clear, tumour-specific antigens that can be targeted by T cells. A high mutational burden can lead to increased numbers of mutational neoantigens that allow very specific immune responses to be generated but also allow escape variants to develop. Other cancer indications and those with low mutational burden are less easily distinguished from normal tissue. Recent studies have suggested that cancer-associated alterations in tumour cell biology including changes in post-translational modification (PTM) patterns may also lead to novel antigens that can be directly recognized by T cells. The PTM-derived antigens provide tumour-specific T-cell responses that both escape central tolerance and avoid the necessity for individualized therapies. PTM-specific CD4 T-cell responses have shown tumour therapy in murine models and highlight the importance of CD4 T cells as well as CD8 T cells in reversing the immunosuppressive tumour microenvironment. Understanding which cancer-specific antigens can be recognized by T cells and the way that immune tolerance and the tumour microenvironment shape immune responses to cancer is vital for the future development of cancer therapies.

Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy.

Introduction: Post translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy. Methods: A citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model. Results: We show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide. Conclusion: We propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.

Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity.

Homocitrullination is the post translation modification (PTM) of the amino acid lysine to homocitrulline also referred to as carbamylation. This PTM has mainly been studied in relation to autoimmune diseases including rheumatoid arthritis. Homocitrullination of lysines alters their charge which can lead to generation of neoepitopes that are differentially presented by MHC-II and induce modification-specific immune responses. Homocitrullination is often considered a process which triggers autoimmune disease by bypassing self-tolerance however, we suggest that homocitrullination may also have an alternative role in immune responses including protection against cancer. Here we demonstrate that immune responses to homocitrullinated peptides from three different proteins can be induced via multiple HLA-types. Immunization of Balb/c or HLA-transgenic DR4 and DR1 mice can induce modification-specific CD4 mediated IFNγ responses. Healthy human donors show a clear repertoire for the homocitrullinated Vimentin peptide (Vim116-135Hcit), with modification-specific and oligoclonal responses. Importantly, in vivo homocitrulline specific Vim116-135Hcit,Cyk8 371-388Hcit and Aldo 140-157Hcit responses are able to confer an anti-tumor effect in the murine B16 melanoma model. The Vim116-135Hcit anti-tumor response was dependent upon tumor expression of MHC-II suggesting the direct recognition of PTMs on tumor is an important anti-tumor mechanism. Cancer patients also have a CD4 repertoire for Vim116-135Hcit. Together these results suggest that homocitrulline-specific immune responses can be generated in healthy mice and detected in human donors through a variety of HLA-restrictions. Immunization can induce responses to Vim116-135Hcit,Aldolase 140-157Hcit and Cyk8 371-388Hcit which provide anti-tumor therapy across several HLA-types. Our results advance our understanding of homocitrulline-specific immune responses, with implications for a number of fields beyond autoimmunity, including tumor immune surveillance.

Pulmonary Alveolar Microlithiasis: a Case Report in King Hussein Medical Center (KHMC), Amman, Jordan.

INTRODUCTION: Pulmonary Alveolar Microlithiasis (PAM) is a rare disorder that can affect patients at any age, although it is more common to present in the third and fourth decades of life. Most patients are asymptomatic at the time of diagnosis. However, some may present with symptoms of dyspnea or cough. PAM can be sporadic, or it can be hereditary. AIM: To focus on the importance of using chest CT scans along with bone scintigraphy to aid in the diagnosis of PAM. The importance of screening all family members is also addressed. CASE REPORT: In our case, the patient was a 21-year-old male, coming for routine check-up to be recruited in the army. He was referred to our clinic after the examining doctor noticed that his chest X-Ray was not normal. Upon revising his chest X-ray, he was found to have bilateral fine reticular infiltrates. His physical examination was unremarkable. His spirometry and DLCO were normal. A high-resolution chest CT scan was done, and showed diffuse bilateral microcalcifications with bilateral interstitial and septal thickening. To confirm the diagnosis of PAM, a Technetium-99m methylene diphosphonate (Tc-99m MDP) whole body bone scintigraphy was done, and it showed diffusely increased radiotracer uptake in both lungs. His family members were screened for PAM. His father and sister, who were completely asymptomatic and with normal pulmonary function tests, were found to have PAM as well. CONCLUSION: The use of bone scintigraphy plays an integral role in diagnosing patients with radiological findings consistent with PAM, and it can diagnose PAM without the need for invasive procedures. Once diagnosed, screening of all family members for PAM should be done, even when they are asymptomatic, as more than one-third of the cases have a familial pattern.

Left side approach for aortic valve replacement in patient with dextrocardia and situs inversus totalis.

Aortic valve replacement in patients with dextrocardia and situs inversus totalis is technically challenging due to anatomical considerations. Modifications of the cannulation strategy and operative tool sets are helpful. We report a 47-year-old man who had dextrocardia with situs inversus totalis with severe aortic regurgitation. Our approach was precisely planned depending on the clear anatomy outlined by preoperative contrast-enhanced computed tomography of the chest. We used a surgical approach in which the main surgeon was standing on the left side of the patient. Left sided approach provided excellent exposure for aortic valve replacement in this case scenario.

The Alvarado score versus computed tomography in the diagnosis of acute appendicitis: A prospective study.

BACKGROUND: To assess and compare performance of the Alvarado score and computed tomography scan in the diagnosis of acute appendicitis at King Hussein Medical Center. METHODS: A total of 320 patients with suspected acute appendicitis were included in this study over a period of 2 years. The Alvarado score was calculated for all of these patients and 112 CT scans were requested selectively by surgeons caring for the patients. The histopathology diagnosis was used as the gold standard against which diagnostic performance of Alvarado score and CT scan were compared. RESULTS: The complete data of 196 males and 124 females were analyzed at the end of the study period. The mean age was 26.1 ± 11.3 years. Appendectomy was performed in 263 patients with a negative appendectomy rate of 14.83% overall (12.28 in males and 19.56 in females). The remaining 57 patients were assumed to have no appendicitis. The diagnostic performance of CT scan was superior to that of Alvarado score with sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of 94.2 versus 85.4%, 90 versus 65%, 9.42 versus 2.44, and 0.065 versus 0.224, respectively (p-value < 0.05). The overall diagnostic accuracy of CT scan was 92.6% compared to 77.5% for Alvarado score. CONCLUSION: The Alvarado score was far from good and CT scan is more accurate in diagnosis of acute appendicitis in our patient population.