T-cell Clonotypes Research Articles

Rapid and Continued T-Cell Differentiation into Long-term Effector and Memory Stem Cells in Vaccinated Melanoma Patients.

Purpose: Patients with cancer benefit increasingly from T-cell-based therapies, such as adoptive T-cell transfer, checkpoint blockade, or vaccination. We have previously shown that serial vaccinations with Melan-AMART-126-35 peptide, CpG-B, and incomplete Freund adjuvant (IFA) generated robust tumor-specific CD8 T-cell responses in patients with melanoma. Here, we describe the detailed kinetics of early- and long-term establishment of T-cell frequency, differentiation (into memory and effector cells), polyfunctionality, and clonotype repertoire induced by vaccination.Experimental Design: Twenty-nine patients with melanoma were treated with multiple monthly subcutaneous vaccinations consisting of CpG-B, and either the native/EAA (n = 13) or the analogue/ELA (n = 16) Melan-AMART-126-35 peptide emulsified in IFA. Phenotypes and functionality of circulating Melan-A-specific CD8 T cells were assessed directly ex vivo by multiparameter flow cytometry, and TCR clonotypes were determined ex vivo by mRNA transcript analyses of individually sorted cells.Results: Our results highlight the determining impact of the initial vaccine injections on the rapid and strong induction of differentiated effector T cells in both patient cohorts. Moreover, long-term polyfunctional effector T-cell responses were associated with expansion of stem cell-like memory T cells over time along vaccination. Dominant TCR clonotypes emerged early and persisted throughout the entire period of observation. Interestingly, one highly dominant clonotype was found shared between memory and effector subsets.Conclusions: Peptide/CpG-B/IFA vaccination induced powerful long-term T-cell responses with robust effector cells and stem cell-like memory cells. These results support the further development of CpG-B-based cancer vaccines, either alone or as specific component of combination therapies. Clin Cancer Res; 23(13); 3285-96. ©2016 AACR.

Abstract 2988: Immunogenomic analyses of tumor cells and microenvironment in patients with advanced melanoma before and after treatment with nivolumab

Abstract Background: Response to checkpoint blockade may be dependent on tumor mutational load and the presence of antigen-specific effector T cells in the tumor microenvironment; however, how blockade modulates these features during therapy is unclear. We assessed genomic changes in tumors from patients (pts) with advanced melanoma receiving nivolumab (nivo) who progressed on ipilimumab (ipi-P) or were ipi-naive (ipi-N). Methods: Tumor biopsies were collected pretreatment and 4 weeks post first nivo dose from ipi-N or ipi-P pts treated with nivo 3 mg/kg Q2W in the phase 1 open-label CA209-038 study (NCT01621490). Biopsies from 68 pts were analyzed by whole exome, transcriptome, and/or TCR sequencing (paired biopsies from 41, 42, and 34 pts, respectively). Results: Objective response rate (ORR) in the overall cohort (n=85) was 27% with similar ORR in ipi-N and ipi-P cohorts. In the genomic cohort (n=68), ORR was 23% with a similar number of complete or partial responses (CR/PR) in ipi-N and ipi-P pts (n=7 and n=8, respectively). Prior to treatment, mutational and neoantigen load were comparable, regardless of previous treatment. Following nivo treatment, both mutational and neoantigen load were reduced 5-fold in pts who responded (CR/PR; n=9) and 1.2-fold in pts with stable disease (SD, n=13) compared with a 1.1-fold increase in pts with progressive disease (PD, n=19). Intratumoral heterogeneity analysis before and after nivo demonstrated that CR/PR pts generally lost tumor mutation clones/subclones. Novel tumor mutation clones were observed in on-treatment samples from 2 CR/PR pts and all pts who progressed on nivo. Transcriptome analyses revealed significant increases in distinct tumor immune cell subsets (CD8+ T cells and NK cells) and immune checkpoint gene expression (LAG3, CTLA4, PCDC1, and CD274 [PD-L1]) following nivo, which were more pronounced in pts with CR/PR vs PD (log2 fold-changes of 1.24, 1.07, 1.71, and 0.74, respectively). Consistent with the transcriptome analyses, tumor-infiltrating lymphocytes, as assessed by immunohistochemistry, generally increased following nivo in pts who responded: 2.8 vs 1.9-fold change in CR/PR/SD vs PD in the ipi-P cohort; 4.8 vs 1.8-fold change in CR/PR/SD vs PD in the ipi-N cohort. Differences in treatment-related TCR repertoire diversity changes were apparent between pts who responded within the ipi-N and ipi-P cohorts: a decrease in the evenness of T-cell clonotype distribution was observed among pts with CR/PR/SD relative to pts with PD in the ipi-N cohort (P=0.036), but not in the ipi-P cohort. Conclusion: Nivo and ipi modulate T-cell repertoire and tumor mutational heterogeneity in pts with advanced melanoma, presenting potential mechanisms of action underlying successful nivo therapy. These data also show that prior ipi treatment may influence biological response to nivo, but further investigation is warranted. Citation Format: Timothy A. Chan, Nadeem Riaz, Jonathan J. Havel, Vladimir Makarov, Alexis Desrichard, Jennifer S. Sims, F. Stephen Hodi, Salvador Martín-Algarra, William H. Sharfman, Shailender Bhatia, Wen-Jen Hwu, Thomas F. Gajewski, Craig L. Slingluff, Sviatoslav M. Kendall, Han Chang, John-William Sidhom, Jonathan P. Schneck, Nils Weinhold, Christine E. Horak, Walter J. Urba. Immunogenomic analyses of tumor cells and microenvironment in patients with advanced melanoma before and after treatment with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2017-2988

T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer

We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×1011 HLA-C*08:02–restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.

Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity—hence reactivity to self—and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 Ld-restricted epitope, derived from the Rop7 protein of Toxoplasma gondii. We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro. Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds.

Melanoma Lesions Independently Acquire T-cell Resistance during Metastatic Latency.

Melanoma often recurs after a latency period of several years, presenting a T cell-edited phenotype that reflects a role for CD8(+) T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen-specific CD8(+) T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8(+) T cell-resistant, HLA class I-negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8(+) T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Jun(high)/MITF(low) phenotype, possibly associated with immunosuppression, which contrasted with a c-Jun(low)/MITF(high) phenotype of T cell-edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347-58. ©2016 AACR.

Abstract LB-264: Landscape of tumor-infiltrating T-cell repertoire of human cancers

Abstract T cells play a critical role in antitumor immunity and are the primary targets in many immunotherapies. However, the study of tumor-infiltrating T cells has been complicated by the extremely diverse repertoire of T cell receptors, characterized by the complementarity determining region 3 (CDR3). Here using our newly developed algorithm, we studied the CDR3 sequences of tumor infiltrating T cells in 9,142 RNA-seq samples across 29 diseases. In total, we assembled over 600 thousands CDR3 sequences, covering the α, β, γ, δ chains. TRBV genes usage, CDR3 sequence length and conservation of infiltrating T cells resembled those in the peripheral blood of healthy donors in many tumors, except brain and kidney cancers. About 20% of the CDR3 calls were shared in multiple individuals. T cell clonotypes carrying these CDR3 sequences are known as the ‘public T cells’. In this work, we found that public T cell CDR3 amino acid (AA) sequences are shorter compared to the private ones and the middle 3 positions of the private CDR3 sequences were enriched for hydrophobic AAs. This finding potentially suggests that private T cell clonotypes in the tumors have greater potential to recognize neoantigens. We defined clonotype per kilo-reads (CPK) to measure the diversity of T cell repertoire and identified a strong positive association between CPK and tumor mutation load, implying that neoantigens may diversify the infiltrating T cell population. In addition, we predicted SPAG5 and TSSK6 as putative immunogenic cancer/testis antigens in multiple cancers based on their strong associations with CPK. Finally, we identified 3 potential immunogenic somatic mutations based on their co-occurrence with CDR3 sequences. One of them, PRAMEF4 F300V, was predicted to bind strongly to both MHC-I and MHC-II, with matched HLA types in its carriers. Our analyses have the potential to simultaneously identify immunogenic neoantigen and the tumor-reactive T-cell clonotype. Citation Format: Bo Li, Taiwen Liu, Binbin Wang, Jinzeng Wang, Sachet Shukla, Ruoxu Dou, Stephen Hodi, Catherine Wu, Nir Hacohen, Jun Liu, X. Shirley Liu. Landscape of tumor-infiltrating T-cell repertoire of human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-264.

Aire Enforces Immune Tolerance by Directing Autoreactive T Cells into the Regulatory T Cell Lineage

The promiscuous expression of tissue-restricted antigens in the thymus, driven in part by autoimmune regulator (Aire), is critical for the protection of peripheral tissues from autoimmune attack. Aire-dependent processes are thought to promote both clonal deletion and the development of Foxp3(+) regulatory T (Treg) cells, suggesting that autoimmunity associated with Aire deficiency results from two failed tolerance mechanisms. Here, examination of autoimmune lesions in Aire(-/-) mice revealed an unexpected third possibility. We found that the predominant conventional Tcell clonotypes infiltrating target lesions express antigen receptors that were preferentially expressed by Foxp3(+) Treg cells in Aire(+/+) mice. Thus, Aire enforces immune tolerance by ensuring that distinct autoreactive Tcell specificities differentiate into the Treg cell lineage; dysregulation of this process results in the diversion of Treg cell-biased clonotypes into pathogenic conventional Tcells.

Proinsulin Expression Shapes the TCR Repertoire but Fails to Control the Development of Low-Avidity Insulin-Reactive CD8+ T Cells

NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8+ T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8+ T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23–reactive TRBV19+CD8+ T cells and causes diabetes; 2) insulin B15-23–reactive TRBV19+CD8+ T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8+ T-cell compartment; 4) a biased repertoire of insulin-reactive CD8+ T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8+ T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes.

Functional evidence for TCR-intrinsic specificity for MHCII

How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR-pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4(+) or CD8(+) T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR-MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or "shaved" peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR-MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.

Stepwise B-cell-dependent expansion of T helper clonotypes diversifies the T-cell response.

Antigen receptor diversity underpins adaptive immunity by providing the ground for clonal selection of lymphocytes with the appropriate antigen reactivity. Current models attribute T cell clonal selection during the immune response to T-cell receptor (TCR) affinity for either foreign or self peptides. Here, we report that clonal selection of CD4+ T cells is also extrinsically regulated by B cells. In response to viral infection, the antigen-specific TCR repertoire is progressively diversified by staggered clonotypic expansion, according to functional avidity, which correlates with self-reactivity. Clonal expansion of lower-avidity T-cell clonotypes depends on availability of MHC II-expressing B cells, in turn influenced by B-cell activation. B cells clonotypically diversify the CD4+ T-cell response also to vaccination or tumour challenge, revealing a common effect.

Antigen Selection Shapes the T-cell Repertoire in Chronic Lymphocytic Leukemia.

The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia, although relevant for understanding malignant cell interactions with cognate T cells, is largely unexplored. Here we profiled the T-cell receptor β chain gene repertoire in 58 chronic lymphocytic leukemia patients, focusing on cases assigned to well-characterized subsets with stereotyped clonotypic B-cell receptor immunoglobulins, therefore those cases most evidently selected by antigen (subsets #1, #2, and #4). Remarkable repertoire skewing and oligoclonality were observed, and differences between subsets were noted regarding both T-cell receptor β chain gene usage and the extent of clonality, with subset #2 being the least oligoclonal. Longitudinal analysis of subset #4 cases revealed that although the repertoire may fluctuate over time, certain clonotypes persist, thus alluding to persistent antigenic stimulation. Shared ("stereotyped") clonotypes were found between different patients, reflecting selection by common antigenic elements. Cross-comparison of our dataset with public databases showed that some T-cell clonotypes may have expanded secondary to common viral infections; however, the majority of clonotypes proved to be disease-specific. Overall, the T-cell receptor β chain repertoire in chronic lymphocytic leukemia is likely shaped by antigen selection and the implicated antigenic elements may concern epitopes that also select the malignant B-cell progenitors or, more intriguingly, chronic lymphocytic leukemia-derived epitopes.

Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6.

The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells. T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells. We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines. These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers.

Deep sequencing of the TCRβ repertoire reveals T cell clonal expansion is associated with HTLV-1 proviral load in HAM/TSP patients

HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is associated with a chronic inflammatory central nervous system in which the host immune response against HTLV-1 infection is considered immunopathogenic. We have applied a new unbiased deep sequencing method of TCR repertoire to accurately measure the diversity and degree of clonal expansion of peripheral circulating T-cells in patients with HAM/TSP compared to age, gender, and ethnicity matched healthy controls. TCR libraries were generated using 5'rapid amplification of cDNA ends to amplify the V-D-J genes of unsorted T-cells obtained from peripheral blood. Over 100 million reads were analyzed using MiGec software (Shugay, et al., Nat Methods. 2014 Jun;11(6):653-5), that align and match the human TCR β-chain nucleotide sequences through IMGT database. T-cell clonotypes with high-quality CDR3 sequence were selected by a molecular identifier PCR strategy, which allowed for filtering PCR errors and correct for artificial sequences generated on a HiSeq 2000 Illumina system platform. By calculating the coefficient of variation of our dataset we considered clonal expansion as those unique TCR clonotypes showing reads >8. While we did not observe any significant statistical differences of the number of single clones (clonotypes with 1 read, P=0.62) between HAM/TSP patients and healthy controls groups, by contrast, a higher clonal expansion of the T-cell repertoire was observed in HAM/TSP patients when compared to healthy controls (unpaired parametric T-test with P=0.026). Interestingly, this clonal expansion of the HAM/TSP TCR repertoire correlated with the HTLV-I provirus load (as defined by digital PCR quantification of the HTLV-I tax gene) (r=0.64, P=0.008). Our findings strongly suggest that in this chronic neurological disorder, a strong dysregulated T-cell response may be driven by HTLV-I and that strategies to decrease virus may be of clinical benefit.

Changes in peanut-specific T-cell clonotype with oral immunotherapy

Changes in peanut-specific T-cell clonotype with oral immunotherapy

Use of High-Throughput Sequencing (HTS) of TCRß to Determine the Kinetics of Graft-Versus-Lymphoma (GVL) Effect and T-Cell Repertoire Profiles after Allogeneic Transplant

▪Introduction:While allogeneic transplant using non-myeloablative preparative regimen provides a viable alternative with low TRM, the kinetics of GVL effect and the T-cell reconstitution may differ from the myeloablative transplant. In general, the donor cell engraftment is slower using a non-myeloablative regimen and a delayed GVL effect is expected. In this study, we applied the TCRß HTS to determine (1) the kinetics of the GVL effect by quantifying the tumor cell burden prior to and after transplant and (2) the pace of the T-cell reconstitution after transplant.Method: This report included a cohort of 24 patients with advanced stage mycosis fungoides or Sézary Syndrome who underwent allogeneic transplant using a non-myeloablative regimen with total skin electron beam therapy (TSEBT, 24-36 Gy), total lymphoid irradiation (TLI, 8 Gy) and anti-thymocyte globulin (ATG). All patients received G-CSF-mobilized peripheral blood hematopoietic cells with a median donor CD34+ cell dose of 6.9 x 106/kg (range 2.0-12.4) and a median donor CD3+ cell dose of 278.1 x 106/kg (range 134.4-631.0). The unique malignant T-cell clonotype of each individual patient was identified from diagnostic blood/skin samples as the single dominant sequence by TCRß HTS. Blood samples were collected prior to and at different time points after transplant. DNA extracted from PBMC corresponding to approximately 200,000 genomes was used for HTS (Sci. Transl. Med.5:214ra171, 2013) (ImmunoSEQ, Adaptive Biotech). The tumor cell burden (including minimal residual disease, MRD) was expressed as percentage of the malignant clonotype found in the entire T-cell repertoire.Results: Prior to transplant, 21 patients (88%) had detectable disease by TCRß HTS in the blood (<1%: 10 patients, 1-5%: 3 patients, >5%: 8 patients), and 3 patients had no detectable disease in the blood. The percentage of malignant clone decreased in 19 of these 21 patients at day+30 post-transplant, and 2 patients showed stable minimal disease (0.03 and 0.30%, respectively). The reduction of tumor burden was most pronounced in patients with >5% involvement prior to transplant. In these 8 patients, the pre- and day+30 post-transplant disease burden decreased from 77.9 to 0.9%, 23.6 to 9.0%, 21.1 to 8.8%, 11.8 to 1.9%, 11.3 to 0.9%, 10.0 to 0.1%, 6.0 to 1.5% and 5.5 to 0.2%, respectively. This immediate post-transplant GVL effect was not associated with full donor T-cell engraftment (donor CD3+ >95%). The donor T-cell chimerism at day+30 was 94%, 79%, 1%, 90%, 93%, 93%, 23% and 91% for these 8 patients, respectively. Subsequently, 11 of the 24 patients achieved sustained molecular remission in the blood with a median time to achieve molecular remission of 60 days (range 30-540). Patients with full donor T-cell chimerism (n=16) had a higher chance of achieving molecular remission in the blood than those with mixed donor T-cell chimerism (69% vs 0%, p = 0.002). Of these 11 patients who achieved molecular remission in the blood, 8 also achieved molecular remission in the skin at the same time. Of the remaining 3 patients, 2 achieved molecular remission in the skin more than 4 months after achieving remission in the blood, while one patient has yet to achieve molecular remission in the skin.We then analyzed the “T-cell repertoire” at different time points post-transplant by assessing the number of unique T-cell clonotypes in each blood sample from 14 patients who had at least 1-year follow-up. While the size of the T-cell repertoire varied significantly between patients, we observed an overall upward trend within individual patients after transplant (Table). The size of T-cell repertoire did not correlate with the donor CD34+ or CD3+ cell dose in the allograft.Conclusion:By using an extremely sensitive and specific TCRß HTS, we have shown an immediate post-transplant GVL effect in which a full donor engraftment was not required, followed by a subsequent sustained GVL effect that may depend on full donor T-cell engraftment. Our results suggest a different kinetics of GVL effect in different compartments (blood vs skin). We also demonstrate continued expansion of T-cell repertoire profile after allogeneic transplant. Abstract 2473Table. The number of unique T-cell clonotypes at different time points post-transplantDay+30Day+60Day+90Day+180Day+270Day+360Median7,55010,4177,9428,40013,62820,057Range629 - 60,6441,916 - 63,6911,297 - 82,0591,572 - 66,5916,510 - 42,5651,914 - 57,358 DisclosuresNo relevant conflicts of interest to declare.

A Comparison of Human CMVpp65-Specific Central Memory and Effector Memory CD8 T-Cells When Stimulated in-Vivo with IL-2 or IL-15/IL-15Rα Complex in a Murine Xenograft Model of Adoptive Cell Therapy

Initial clinical trials of adoptive immunotherapy have shown that the efficacy of adoptively transferred T-cells in man is often limited by the failure of cultured T cells, particularly cloned CD8 T cells, to persist in vivo. These studies demonstrated that the transferred T cells induced only transient responses and that persistence of the transferred T-cell clonotypes correlated with disease regression. A previous study suggested that CMV virus-specific CD8 T cell clones derived from central memory T cells (TCM), but not effector memory T cells (TEM), persisted long-term in non-human primates. On the other hand, another study comparing TCM and TEM derived SIV virus specific CD8 T-cell clones that were adoptively transferred in non-human primates demonstrated limited persistence of both TCM and TEM derived transferred T cells, and failed to show any difference between the two cell types. Because of these conflicting data, we have reexamed the persistence of adoptively transferred viral antigen specific T-cells derived from TCM and TEM population. Accordingly, we developed a NOG mouse model for studying the ability of human CMVpp65-specific T cells derived from central memory and effector memory populations to migrate to and accumulate in human tumor xenografts expressing CMVpp65, to alter the growth of these tumors and to persist in the tumors. This model also allows us to test immunomodulating agents and their ability to enhance targeted T-cell accumulations, antitumor activity and persistence. We analyzed CMVpp65-specific CD8 T cells derived from TCM and TEM precursors in vitro and in vivo. To tract the T-cells in vivo, we transduced membrane-bound Gaussia luciferase into TCM and TEM populations and monitored T cell trafficking by in vivo bioluminescence. Contrary to expectation, our results initially showed no differences between TCM and TEM derived CMVpp65-specific T-cell in mice co-treated with IL-2 in the time to accumulation, ultimate level of accumulation, degree of CMVpp65+ tumor regression or T-cell persistence. However, in mice cotreated with IL-15/IL-15Rα complex, both TCM and TEM exhibited more sustained engraftment and more prolonged accumulation in both the targeted tumor and in the marrow. In mice treated with IL-15/IL-15Rα, TCM and TEM derived T cells showed a similar effector memory phenotype and a similar level of regression of tumor growth. Thus, adoptive transfer of CMVpp65 specific TCM or TEM when combined with IL-15/IL-15Rα complex may support better persistence of antigen-specific T-cells following adoptive immunotherapy. Studies comparing IL-15/IL-15Rα complex with IL-15 alone are in progress. DisclosuresNo relevant conflicts of interest to declare.

Complex T-cell receptor repertoire dynamics underlie the CD8+ T-cell response to HIV-1.

Although CD8(+) T cells are important for the control of HIV-1 in vivo, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naive individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8(+) T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, as evidenced by discordant clonotype-specific transitions directed against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8(+) T-cell clonotypes orchestrated at the TCR-antigen interface. We describe a relation between viral epitope mutation, antigen-specific T-cell expansion, and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provides insights into the process of coadaptation between the human immune system and a rapidly evolving lentivirus.

T-Cell Costimulatory Blockade in Organ Transplantation

Before it became possible to derive T-cell lines and clones, initial experimentation on the activation requirements of T lymphocytes was performed on transformed cell lines, such as Jurkat. These studies, although technically correct, proved misleading as most transformed T cells can be activated by stimulation of the clonotypic T-cell receptor (TCR) alone. In contrast, once it became possible to study nontransformed T cells, it quickly became clear that TCR stimulation by itself is insufficient for optimal activation of naïve T cells, but in fact, induces a state of anergy. It then became clear that functional activation of T cells requires not only recognition of major histocompatibility complex (MHC) and peptide by the TCR, but also requires ligation of costimulatory receptors expressed on the cell surface.

Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes

T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were in the range of 1–17 (median = 5), and in several patients, the number of clonotypes expanded within tumor lesions resembled that observed among autologous peripheral blood mononuclear cells. Moreover, several of these clonotypes were identical in TILs and PBMCs. Flow cytometry data demonstrated that the general differentiation status of CD8+ TILs differed from that of circulating CD8+ T cells. Furthermore, PD-1 and LAG-3 were expressed by a significantly higher percentage of CD8+ RCC-infiltrating lymphocytes as compared with PBMCs obtained from RCC patients or healthy individuals. Thus, CD8+ TILs display a differentiated phenotype and express activation markers as well as surface molecules associated with the inhibition of T-cell functions. However, TILs are characterized by a low amount of expanded T-cell clonotypes.

HLA-B27-mediated protection in HIV and hepatitis C virus infection and pathogenesis in spondyloarthritis

HLA-B27 is associated with low viral load and long-term nonprogression in HIV infection as well as spontaneous clearance of hepatitis C virus (HCV) infection. This review summarizes mechanisms that have been suggested to be involved in this protective effect of HLA-B27, and highlights possible lessons for the role of HLA-B27 in spondyloarthritis. Recent studies linked protection by HLA-B27 in HIV and HCV infection to virological mechanisms such as a complicated pathways of viral escape from immunodominant HLA-B27-restricted virus-specific CD8+ T-cell epitopes. In addition, several immunological mechanisms have been proposed, including CD8+ T-cell polyfunctionality and functional avidity, thymic selection of CD8+ T-cell precursors, specific T-cell receptor repertoires and clonotypes, efficient antigen processing, and evasion from regulatory T-cell-mediated suppression. Multiple virological and immunological mechanisms have been suggested to contribute to HLA-B27-mediated protection in HIV and HCV infection. Some of these mechanisms may also be involved in HLA-B27-associated pathogenesis in spondyloarthritis.