The FAT10- and ubiquitin-dependent degradation machineries exhibit common and distinct requirements for MHC class I antigen presentation

Like ubiquitin (Ub), the ubiquitin-like protein FAT10 can serve as a signal for proteasome-dependent protein degradation. Here, we investigated the contribution of FAT10 substrate modification to MHC class I antigen presentation. We show that N-terminal modification of the human cytomegalovirus-derived pp65 antigen to FAT10 facilitates direct presentation and dendritic cell-mediated cross-presentation of the HLA-A2 restricted pp65495–503 epitope. Interestingly, our data indicate that the pp65 presentation initiated by either FAT10 or Ub partially relied on the 19S proteasome subunit Rpn10 (S5a). However, FAT10 distinguished itself from Ub in that it promoted a pp65 response which was not influenced by immunoproteasomes or PA28. Further divergence occurred at the level of Ub-binding proteins with NUB1 supporting the pp65 presentation arising from FAT10, while it exerted no effect on that initiated by Ub. Collectively, our data establish FAT10 modification as a distinct and alternative signal for facilitated MHC class I antigen presentation

High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations

T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent

A Broad Profile of Co-Dominant Epitopes Shapes the Peripheral Mycobacterium tuberculosis Specific CD8+ T-Cell Immune Response in South African Patients with Active Tuberculosis.

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition

Peripheral and systemic antigens elicit an expandable pool of resident memory CD8(+) T cells in the bone marrow

BM has been put forward as a major reservoir for memory CD8+  T cells. In order to fulfill that function, BM should "store" memory CD8+ T cells, which in biological terms would require these "stored" memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue-resident memory CD8+ T (TRM ) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8+ TRM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL-15, Blimp-1, and Hobit. CD8+ TRM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size-restricted and expands upon peripheral antigenic re-challenge. This works extends the role of the BM in the maintenance of CD8+ T cell memory to include the preservation of an expandable reservoir of functional, non-recirculating memory CD8+ T cells, which develop in response to a large variety of peripheral antigens

CD28-mediated costimulation impacts on the differentiation of DC vaccination-induced T cell responses

Costimulatory signals such as the ones elicited by CD28/B7 receptor ligation are essential for efficient T cell activation but their role in anti-tumour immune responses remains controversial. In the present study we compared the efficacy of DC vaccination-induced melanoma specific T cell responses to control the development of subcutaneous tumours and pulmonary metastases in CD28-deficient mice. Lack of CD28-mediated costimulatory signals accelerated tumour development in both model systems and also the load of pulmonary metastases was strongly increased by the end of the observation period. To scrutinize whether lack of CD28 signalling influences priming, homing or effector function of Trp-2(180−188)/K(b)-reactive T cells we investigated the characteristics of circulating and tumour infiltrating T cells. No difference in the frequency of Trp-2(180−188)/K(b)-reactive CD8(+) T cells could be demonstrated among the cellular infiltrate of subcutaneous tumours after DC vaccination between both genotypes. However, the number of IFN-γ-producing Trp-2-reactive cells was substantially lower in CD28-deficient mice and also their cytotoxicity was reduced. This suggests that CD28-mediated costimulatory signals are essential for differentiation of functional tumour-specific CD8(+) T-effector cells despite having no impact on the homing of primed CD8(+) T cells