Johanna Olweus

Targeting of cancer neoantigens with donor-derived T cell receptor repertoires

Outsourcing cancer immunotherapy Successful cancer immunotherapy depends on a patients T cells recognizing tumor-specific mutations and then waging a lethal attack. Despite tumors harboring many mutations, most individuals have very few T cells that respond to these so-called “neo-antigens.” Strønen et al. isolated T cells from healthy donors that responded to predicted neo-antigens expressed by melanomas taken from three patients, sometimes including neo-antigens that the patients own T cells ignored (see the Perspective by Yadav and Delamarre). Testing whether such an outsourcing strategy could improve clinical outcomes will be an important next step. Science, this issue p. 1337; see also p. 1275 T cells from healthy human donors may be an important resource for outsourcing cancer immunotherapy. Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation–derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen-specific T cell responses are therefore attractive. We found that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11 of 57 predicted human leukocyte antigen (HLA)– A*02:01–binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells redirected with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such “outsourced” immune responses in cancer immunotherapy.

High‐throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV‐reactive CD8+ T cells

Epstein‐Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high‐throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV‐reactivity of the T‐cell receptor (TCR) repertoires in MS. TCR‐β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T‐cell clones, represented by TCR‐β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR‐β libraries generated from peripheral blood T cells reactive against autologous EBV‐transformed B cells were highly enriched for public EBV‐specific sequences and were used to quantify EBV‐reactive TCR‐β sequences in CSF. TCR‐β sequences of EBV‐reactive CD8+ T cells, including several public EBV‐specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV‐reactive CD4+ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T‐cell response in MS. The presented strategy links TCR sequence to intrathecal T‐cell specificity, demonstrating enrichment of EBV‐reactive CD8+ T cells in MS.

A Practical Approach to T-Cell Receptor Cloning and Expression

Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5′-RACE amplification. We here present an improved 5′-RACE protocol that represents a fast and reliable way to identify a TcR from 105 cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality.

Dendritic cells in cytomegalovirus infection: viral evasion and host countermeasures.

Human cytomegalovirus (HCMV) is a β‐herpesvirus that infects the majority of the population during early childhood and thereafter establishes life‐long latency. Primary infection as well as spontaneous reactivation usually remains asymptomatic in healthy hosts but can, in the context of systemic immunosuppression, result in substantial morbidity and mortality. HCMV counteracts the host immune response by interfering with the recognition of infected cells. A growing body of literature has also suggested that the virus evades the immune system by paralyzing the initiators of antiviral immune responses – the dendritic cells (DCs). In the current review, we discuss the effects of CMV (HCMV and murine CMV) on various DC subsets and the ensuing innate and adaptive immune responses. The impact of HCMV on DCs has mainly been investigated using monocyte‐derived DCs, which are rendered functionally impaired by infection. In mouse models, DCs are targets of viral evasion as well, but the complex cross‐talk between DCs and natural killer cells has, however, demonstrated an instrumental role for DCs in the control and clearance of viral infection. Fewer studies address the role of peripheral blood DC subsets, plasmacytoid DCs and CD11c+ myeloid DCs in the response against HCMV. These DCs, rather than being paralyzed by HCMV, are largely resistant to infection, mount a vigorous first‐line defense and induce T‐cell responses to the virus. This possibly provides a partial explanation for an intriguing conundrum: the highly efficient control of viral infection and reactivation in immunocompetent hosts in spite of multi‐layered viral evasion mechanisms.

Sequential intranodal immunotherapy induces antitumor immunity and correlated regression of disseminated follicular lymphoma

Advanced stage follicular lymphoma (FL) is incurable by conventional therapies. In the present pilot clinical trial, we explored the efficacy and immunogenicity of a novel in situ immunotherapeutic strategy. Fourteen patients with untreated or relapsed stage III/IV FL were included and received local radiotherapy to solitary lymphoma nodes and intranodal injections of low-dose rituximab (5 mg), immature autologous dendritic cells, and granulocyte-macrophage colony-stimulating factor at the same site. The treatment was repeated 3 times targeting different lymphoma nodes. Primary end points were clinical responses and induction of systemic immunity. Five out of 14 patients (36%) displayed objective clinical responses, including 1 patient with cutaneous FL who showed regression of skin lesions. Two of the patients had durable complete remissions. Notably, the magnitude of vaccination-induced systemic CD8 T-cell-mediated responses correlated closely with reduction in total tumor area (r = 0.71, P = .006), and immune responders showed prolonged time to next treatment. Clinical responders did not have a lower tumor burden than nonresponders pretreatment, suggesting that the T cells could eliminate large tumor masses once immune responses were induced. In conclusion, the combined use of 3 treatment modalities, and in situ administration in single lymphoma nodes, mediated systemic T-cell immunity accompanied by regression of disseminated FL. The trial was registered at www.clinicaltrials.gov as #NCT01926639.

High-throughput T-cell receptor sequencing across chronic liver diseases reveals distinct disease-associated repertoires.

Hepatic T‐cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune‐mediated liver diseases. Conceptually the presence of disease‐associated antigens is predicted to be reflected in T‐cell receptor (TCR) repertoires. Here, we aimed to determine if disease‐associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high‐throughput sequencing of the TCRβ chain complementarity‐determining region 3 of liver‐infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen‐driven selection. In PSC and PBC, disease‐associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles. Conclusion: We demonstrate liver‐infiltrating disease–associated clonotypes in all three diseases evaluated, and evidence for antigen‐driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high‐throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease‐relevant T cells in order to better understand and treat liver disease. (Hepatology 2016;63:1608‐1619)

Alloreactive cytotoxic T cells provide means to decipher the immunopeptidome and reveal a plethora of tumor-associated self-epitopes

Significance T cells recognize fragments of cellular peptides when presented at the cell surface by HLA molecules. Knowledge of which peptides derived from cellular proteins that are available at the cell surface for T-cell recognition is central to our understanding of T-cell tolerance and immunity. Here, we used alloreactive T cells as tools for detection of self-peptides bound to foreign HLA-A2. Our results indicate that the self-immunopeptidome is far more diverse than previously estimated. Furthermore, our data demonstrate that such self-peptides represent highly attractive targets for T-cell–based cancer immunotherapy. HLA molecules presenting peptides derived from tumor-associated self-antigens (self-TAA) are attractive targets for T-cell–based immunotherapy of cancer. However, detection of such epitopes is hampered by self-tolerance and limitations in the sensitivity of mass spectrometry. Here, we used T cells from HLA-A2–negative donors as tools to detect HLA-A2–bound peptides from two leukemia-associated differentiation antigens; CD20 and the previously undescribed cancer target myeloperoxidase. A high-throughput platform for epitope discovery was designed using dendritic cells cotransfected with full-length transcripts of self-TAA and HLA-A2 to allow presentation of all naturally processed peptides from a predefined self-protein on foreign HLA. Antigen-reactive T cells were directly detected using panels of color-coded peptide–HLA multimers containing epitopes predicted by a computer algorithm. Strikingly, cytotoxic T cells were generated against 37 out of 50 peptides predicted to bind HLA-A2. Among these, 36 epitopes were previously undescribed. The allorestricted T cells were exquisitely peptide- and HLA-specific and responded strongly to HLA-A2–positive leukemic cells with endogenous expression of CD20 or myeloperoxidase. These results indicate that the repertoire of self-peptides presented on HLA class I has been underestimated and that a wealth of self-TAA can be targeted by T cells when using nontolerized T-cell repertoires.

Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumour cells.

Most tumour‐associated antigens (TAA) are non‐mutated self‐antigens. The peripheral T cell repertoire is devoid of high‐avidity TAA‐specific cytotoxic T lymphocytes (CTL) due to self‐tolerance. As tolerance is major histocompatibility complex‐restricted, T cells may be immunized against TAA presented by a non‐self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo‐restricted CTL of high‐avidity and low cross‐reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA‐A*0201, efficiently present externally loaded peptides from the antigen, Melan‐A/MART‐1 to T cells from HLA‐A*0201‐negative donors. CD8+ T cells binding HLA‐A*0201/MART‐1 pentamers were detected already after 12 days of co‐culture in 11/11 donors. The majority of cells from pentamer+ cell lines were CTL and efficiently killed HLA‐A*0201+ melanoma cells, whilst sparing HLA‐A*0201+ B‐cells. Allo‐restricted CTL specific for peptides from the leukaemia‐associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo‐HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour‐associated antigens.

Gut and liver T-cells of common clonal origin in primary sclerosing cholangitis-inflammatory bowel disease

BACKGROUND & AIMS Recruitment of gut-derived memory T-cells to the liver is believed to drive hepatic inflammation in primary sclerosing cholangitis (PSC). However, whether gut-infiltrating and liver-infiltrating T-cells share T cell receptors (TCRs) and antigenic specificities is unknown. We used paired gut and liver samples from PSC patients with concurrent inflammatory bowel disease (PSC-IBD), and normal tissue samples from colon cancer controls, to assess potential T cell clonotype overlap between the two compartments. METHODS High-throughput sequencing of TCRβ repertoires was applied on matched colon, liver and blood samples from patients with PSC-IBD (n=10), and on paired tumor-adjacent normal gut and liver tissue samples from colon cancer patients (n=10). RESULTS An average of 9.7% (range: 4.7-19.9%) memory T cell clonotypes overlapped in paired PSC-IBD affected gut and liver samples, after excluding clonotypes present at similar frequencies in blood. Shared clonotypes constituted on average 16.0% (range: 8.7-32.6%) and 15.0% (range: 5.9-26.3%) of the liver and gut memory T-cells, respectively. A significantly higher overlap was observed between paired PSC-IBD affected samples (8.7%, p=0.0007) compared to paired normal gut and liver samples (3.6%), after downsampling to equal number of reads. CONCLUSION Memory T-cells of common clonal origin were detected in paired gut and liver samples of patients with PSC-IBD. Our data indicate that this is related to PSC-IBD pathogenesis, suggesting that memory T-cells driven by shared antigens are present in the gut and liver of PSC-IBD patients. Our findings support efforts to therapeutically target memory T cell recruitment in PSC-IBD. LAY SUMMARY Primary sclerosing cholangitis (PSC) is a devastating liver disease strongly associated with inflammatory bowel disease (IBD). The cause of PSC is unknown, but it has been suggested that the immune reactions in the gut and the liver are connected. Our data demonstrate for the first time that a proportion of the T-cells in the gut and the liver react to similar triggers, and that this proportion is particularly high in patients with PSC and IBD.

Targeting B cell leukemia with highly specific allogeneic T cells with a public recognition motif

The possibility that allogeneic T cells may be targeted to leukemia has important therapeutic implications. As most tumor antigens represent self-proteins, high-avidity tumor-specific T cells are largely deleted from the repertoire of the patient. In contrast, T cells from major histocompatibility complex (MHC)-mismatched donors provide naïve repertoires wherein such cells have not been systematically eliminated. Yet, evidence for peptide degeneracy or poly-specificity warrants caution in the use of foreign human leukocyte antigen (HLA) or peptide complexes as therapeutic targets. Here, we cocultured HLA-A*0201-negative T cells with autologous dendritic cells engineered to present HLA-A*0201 complexed with a peptide from the B cell antigen CD20 (CD20p). HLA-A*0201/CD20p pentamer-reactive CD8+ T cells were readily obtained from all donors. The polyclonal cells showed exquisite peptide and MHC specificity, and efficiently killed HLA-A*0201-positive B cells, including primary chronic lymphocytic leukemia cells. The T cell receptor (TCR) sequences displayed a novel type of conservation, with extensive homology in the TCR β chain complementarity-determining region 3 and in J, but not V, region. This is surprising, as the donors were HLA disparate and their TCR repertoires are expected to show little overlap. The results demonstrate the first public recognition motif for an allogeneic HLA/peptide complex. The allo-restricted T cells or TCRs could provide graft-versus-leukemia in the absence of graft-versus-host disease.