Antigen-specificity of human FOXP3+ Tregs

Foxp3+ regulatory T cells (Tregs) control immune response to prevent immunopathology. However, unlike conventional T cells, it is hard to determine antigen-specificity of Foxp3+ Tregs in a random T cell pool. Tregs do not secrete anything unique and they do not even proliferate when exposed to antigens in vitro, two functional readouts that are still used as a gold standard for determining antigen-specificity of conventional T cells. 

Hence, we have no clue as to antigen[epitope] specificity of vast majority of human Tregs. Specificity of T cells could be also determined by non-functional readout such as tetramer staining. This is what the authors in new PNAS paper have used to determine antigen-specificity of Tregs. 

They found that adult human peripheral blood contains FOXP3+ T cells that stain with tetramers specific for self as well as nonself peptides (Flu, melanoma protein, HIV epitopes).

Interestingly, frequency of antigen[epitope]-specific FOXP3+ Tregs varied among donors, but they were, on average, equally distributed among self and nonself [epitope]-specific Tregs, except Flu HA epitope.  

Finally, comparison of neonatal [cord blood] and adult blood revealed that actual number of [epitope]-specific Tregs / per ml of blood did not change much between newborn and adult indicating that most of Tregs tested in this study were generated already by the time of birth.

In summary, this study revealed that human peripheral blood contain Tregs specific for both self nonself antigens. Since tested donors were negative for some of the infection such as HSV, CMV or HIV, it begs the question what [cross-reactive?] antigens maintain CMV or HIV-specific Tregs in antigen-naive [antigen-unexposed] hosts?    

David Usharauli

Melanoma patients harbor tumor mutation-specific PD1+ CD8 T cells in the peripheral blood

Steven Rosenberg's research group at National Institutes of Health (NIH) continues to define and refine condition for T cell-based cancer-specific immunotherapy. (for example, introduction of high-throughput personalized screening strategy capable of evaluating T cell reactivity to neo-antigens presented on all of the HLA restriction elements of the individual).

This time, their group showed that peripheral blood of melanoma patients harbors small but distinct PD1+ CD8 T cell population enriched in specificities for cancer associated neo-antigens.

If one compares CD8 T cells from PBMC v Tumor sites (TIL), blood derived T cells contains few PD1⁺/PD1^high CD8 T cells.

However, when the authors has expanded in vitro those sorted PD1⁺ CD8 T cells and co-cultured them with autologous dendritic cells expressing tumor neo-antigens (as tandem minigenes, TMG), they could identify circulating neoantigen-reactive CD8 T cells in three of the four melanoma patients evaluated.

Then the authors re-constructed blood PD1⁺/PD1^high CD8 T cell TCR specificity by (a) pairing the sequences encoding the two most-dominant TCR-α and TCR-β sequences, (b) cloning them into retroviral vectors and (c) transducing autologous PBMC. This TCR construct could [for example] detect neo-antigens derived from mutations in the genes MAGE family member A6 (MAGEA6).

Importantly, both PD1⁺/PD1^high CD8 T cells enriched from peripheral blood or T lymphocytes transduced with retroviruses expressing neo-antigen-specific TCRs could detect autologous tumor cell lines.

Finally, the authors found that blood and tumor site derived PD1⁺ CD8 T cell showed high degree of overlap in their TCR specificity [to tumor neo-antigens], suggesting that analysis of peripheral PD1⁺ CD8 T cells from cancer patients could reveal TCR specificities of tumor infiltrated lymphocytes.

In summary, this study is another evidence that cancer immunotherapy holds great promise in providing cancer antigen-tailored treatments. Identification of cancer neo-antigen specific T cells (TCRs) as shown in this study, would accelerate development of tumor-specific TCR constructs and could contribute in overcoming precursor limitation inherent to endogenous T cell clones.

David Usharauli

Both non-self and self-specific CD8 T cell clones are found in equal frequency in human PBMCs

Clonal selection model of immune system ontogeny is the most widely accepted concept of immunology. Simply put, it postulates that T cell clones (B cells too) specific for self-antigens (epitopes) are purged from adaptive receptor repertoire during in utero development.

At the time of its formulation, there was no direct proof for this model. It was mainly supported by observations derived from organ transplantation studies. Later, research on TCR transgenic mice confirmed that self-specific T cells were indeed purged from T cell compartment. 

However, more recent studies questioned the validity of results obtain from transgenic mice, mainly due to non-physiological timing of TCR expression in transgenic T cells in the thymus. 

In this regard, this new, short study in journal Immunity is of interest. Using sensitive, tetramer-specific T cell enrichment method, the authors showed that human PBMC surprisingly contains equal numbers of both non-self and self-specific CD8 T cell clones.  

The authors showed that similar to the frequency of non-specific CD8 T cell clones, healthy human PBMCs contain self-specific CD8 T cell clones with frequency range between 1:10⁴ to 1:10⁶ (see below too).

Next, the authors showed that CD8 T cells derived from both female and male donors contained functionally active male antigen, HY-specific CD8 T cells (though, here CD8 T cells were expanded using non-physiological stimuli, such as anti-CD3/anti-CD28 antibodies or PHA).

However, as expected, further experiments with the pool of enriched self-specific CD8 T cell clones revealed that these self-specific CD8 T cells were anergic when stimulated with specific peptide and anti-CD28 antibody (resembling more physiological stimulation, hopefully).

In summary, these results suggest the following: immune system contains lots of self-specific CD8 T cells. These cells are anergic, i.e. non-responders in an ordinary sense. However, they are capable of becoming functionally active when stimulated by non-conventional ways, like PHA or therapeutic antibodies targeting its signaling molecules (CD28, for example) or during the process of autoimmunity?

Why immune system harbors so many self-specific T cells? It was really surprising that on average, PBMCs from male donors still had 1/3 of number of HY-specific CD8 T cells found in PBMCs from female donors. Even though almost every cell in male donors can express HY antigen, the authors still found so many high-affinity HY-specific CD8 T cells in PBMCs from male donors. Staining for mouse HY-specific CD8 T cells showed the same results.

Another interesting observation was the finding that PBMCs from healthy donors contained very high frequency of pre-proinsulin-specific CD8 T cells (almost 1:10⁴) that were significantly increased in donors with T1D. Is it possible that selection of high frequency of pre-proinsulin-specific CD8 T cells has to due with its cross-reactivity for certain evolutionary relevant pathogen-specific epitope?

David Usharauli