Outcome of TCR/pMHC interactions is presently unpredictable

This week journal Cell published a very important paper from KC Garcia's lab at Stanford. The focus of the study was to understand and determine, if possible, all the parameters that makes a given peptide/MHC and its similar looking variants stimulatory versus non-stimulatory to a given TCR molecule.

First, they compared two TCR molecules specific for class II HLA-DR4-HIV_{(Gag164–183)}. While both TCR expressing clones, TCR6 and TCR11, could bind pHLA tetramer, only TCR6 "proliferated, secreted cytokines, and downregulated TCR in response to stimulation with" HLA-DR4-HIV_{(Gag164–183)}. Both TCR clones could respond directly or after transducing them into TCR αβ-deficient cell line to non-specific stimulation such as anti-CD3 (OKT3).

Similar observation were made using TCRs specific for class I HLA-B35-HIV_{(Pol448–456)}. Here too, both TCR clones designated as TCR589 and TCR55 could bind specific pMHC tetramer but only TCR589 showed dose-dependent secretion of IL-2 in response to peptide-pulsed APCs.

Next, the authors determined TCR/pMHC affinity by surface plasmon resonance. For TCR55 (non-stimulatory) and TCR589 (stimulatory) clones it was 17_μM and 4_μM K_D, respectively. Surprisingly, for TCR6 (stimulatory) and TCR11 (non-stimulatory) clones affinity was 10_μM and 1_μM K_D, respectively.

Analysis of TCR55 (non-stimulatory) and TCR589 (stimulatory) TCR/pMHC interaction on lipid bilayers by total internal reflection fluorescence (TIRF) microscopy showed similar mean dwell times of 10.1 ± 2.0 s and 8.7 ± 2.1 s, respectively. Structural analysis of the crystal structures of TCR589 and TCR55 in complex with HLA-B35-HIV_{(Pol448–456)} also showed "no substantial differences in the conformations of the constant regions that would indicate differential allostery correlating with signaling."

Functional analysis of TCR proximal signaling molecules showed that stimulatory TCR589 but not TCR55 could up-regulate CD69, induce calcium release and recruit ZAP70 in response to HLA-B35-HIV_{(Pol448–456)}.

Next, they generated peptide library similar to HIV_{(Pol448–456)} with potencies "ranging from full agonism to partial agonism to no detectable signaling". Importantly, "the peptides that elicited these disparate signaling outcomes were remarkably similar in sequence."

One common feature of agonist peptides such as pep20 and SQL that could signal TCR55 was that such interaction excluded CD45 with varying degrees of efficiency compared to non-stimulatory peptide ligands in "a cell-free system using giant unilamellar vesicles (GUVs) to eliminate effects of signaling in live cells".

In addition, another common feature of stimulatory peptide ligands were so called catch bond formation between TCR and stimulatory pMHC "under conditions of force using a Biomembrane Force Probe (BFP) bond lifetime assay". In contrast, non-stimulatory ligands formed slip bond formations.

In summary, this study indicates that "catch bond formation and CD45 exclusion as principal emergent properties differentiating stimulatory from non-stimulatory TCR-pMHC interactions" but "the same pMHC can be an agonist or non-agonist for different TCRs, although it is difficult to assign causality to either the TCR or the peptide-MHC. The formation of a catch or slip bond is a collective property of the entire TCR-pMHC interface."

My opinion: one drawback of this study is that not many labs can verify its conclusions. I have not even heard of some of the techniques they used here. I could only trust that the authors tried to interpret their data as unbiased as it is rationally possible. Another weakness of the study is a failure to identify parameters that control CD45 exclusion or catch bond formation when TCR interacts with pMHC. Basically, with the exception of few very high affinity ligands (< 2_μM), we cannot predict in silico whether a given peptide will be stimulatory or non-stimulatory without actually doing an experiment on it.   

posted by David Usharauli

CD4 T cells silence innate over-activation to gut microbiota

A simple but very effective study was published in journal Nature from Ron Germain's lab. His group is known for publishing high quality in situ microscopy data combined with cellular analysis. New study continues this trend. 

In this study the authors analyzed pattern of phosphorylation of STAT3 transcription factor in small intestine derived from various immune deficient mouse strains. Compared to WT mice, pSTAT3 staining in RAG1 KO gut tissue (lacking adaptive immune system) was significantly up-regulated. 

Analysis of antibiotic-treated or germ-free mice indicated that pattern of pSTAT3 staining in RAG1-KO was correlated with the presence of gut microbiota.

Interestingly, longitudinal analysis showed that pSTAT3 staining inversely correlated with  maturation of adaptive immune system post weaning (between 4-20 weeks). 

Co-housing experiments showed that T cells, but not B cells, played a role in silencing innate pSTAT3 over-activation.

And out of T cells, it were CD4 T cells and class II antigen-presentation that played the role in pSTAT3 silencing.

Finally, both Tregs and SBF-specific Th17 cells (7B8 transgenic T cells) could mediate silencing of pSTAT3 over-activation. Both T cell type could down-regulate STAT3 phosphorylation in innate and epithelial cells but the mechanisms could be different. 

In summary, this study showed that persistent pSTAT3 over-activation observed in mice deficient for CD4 T cell function could explain some of chronic metabolic shifts observed in clinical settings. 

posted by David Usharauli

Foxp3+ Tregs control CD8 T cells, but not CD4 T cells, by IL-2 deprivation

This week Nature Immunology published another interesting study from Rudensky's lab done in collaboration with Jason Fontenot (who apparently moved from Biogen to Juno Therapeutics). So, if you are a fan of Tregs, below is my short scientific overview of that paper.

This study tries to answer what role IL-2 signaling plays in already established Foxp3+ Tregs. For such study mice with germline deficiency in IL-2 signaling cascade would have been impractical since IL-2's effect on thymocytes and etc. Instead, the authors went to already well established path of using mice lacking "molecule of interest" specifically in Tregs. In this case several Foxp3-cre mice were used, such as: l2rb^fl/flFoxp3^Cre, Il2ra^fl/flFoxp3^Cre, Stat5a^fl/flStat5b^fl/flFoxp3^Cre, Rosa26^Stat5bCAIl2rb^fl/flFoxp3^Cre and Rosa26^Stat5bCAIl2ra^fl/flFoxp3^Cre, Rosa26^Stat5bCAFoxp3^Cre–ERT2. All these gene-modified mice allows specific targeting of Foxp3+ Tregs.

Unsurprisingly, mice lacking IL-2Rβ or IL-2Rα or STAT5 signaling specifically in Tregs developed autoimmunity.

Surprisingly, however, while constitutive expression of STAT5 in Tregs lacking IL-2 signaling receptors (IL-2Rβ or IL-2Rα) could rescue mice from "CD4 effector phenotype" and early death, these mice still developed immunopathology later due to massive expansion of CD8 effector/memory cells, suggesting that sensing of IL-2 by Tregs, so called "IL-2 sink", was necessary to specifically control CD8 T cells, but not CD4 T cells.

In summary, this study suggests that Tregs are using distinct mechanisms to control CD4 and CD8 T cells. It kind of makes sense because MHC class-II restricted CD4+ Tregs cannot interact the same way with MHC class-I restricted CD8+ T cells as they could with conventional MHC class-II restricted conventional CD4+ T cells.

David Usharauli

Why anti-viral immune serum doesn't work in every infected patient?

Many viruses, such as Ebola or avian-origin Flu viruses, are extremely virulent and can cause death of infected individuals within days. However, some individuals are naturally resistant to such virulent infections and generate protective, neutralizing antibodies in response to them. Many organizations across world responsible for public health safety [and also military institutions] try to stockpile such anti-viral sera obtained from individuals who survive natural infection. It is believed that injection of anti-viral serum into infected individuals could help them to survive.

However, new study in journal Nature suggests that anti-viral serum effectiveness depends on viral tropism. It turns out that protection against viruses that infect so called immune privileged tissues, such as brain tissue, required presence of both anti-viral neutralizing antibodies and anti-viral CD4 T cells. Specifically, the authors, led by Akiko Iwasaki from Yale School of Medicine, showed that anti-viral CD4 T cell's role is to open the "gate" to privileged tissues for anti-viral antibodies that usually can not cross such barrier on their own.

For this study the authors used herpes simplex virus type 2 (HSV-2) virus challenge model that infects mouse immune privileged tissues such as the innervating neurons in the dorsal root ganglia (DRG). Initially the authors reported and re-confirmed that if mice were vaccinated with attenuated strain of HSV-2 before WT virus challenge, then these vaccinated mice were protected against WT virus challenge and this protection depended on antibody.

Strangely, however, immune serum could not protect naive, non-vaccinated mice against WT virus challenge. Moreover, immune serum protected vaccinated mice even if these mice did not make antibodies on their own. This suggested that something else  besides antibodies [generated during vaccination] needed alongside with anti-viral antibodies for virus protection. 

Indeed, vaccinated mice depleted of anti-viral memory CD4 T cells just prior to WT virus challenge were not protected.

Next, the authors found that presence of anti-viral CD4 T cells (but not of irrelevant CD4 T cells) were necessary for anti-viral antibodies to enter and accumulate within infected neuronal tissue.

Finally, the authors showed that neuronal tissue recruitment of anti-viral CD4 T cells depended on α4β1 (VLA4 integrin) interactions and subsequent CD4 T cell-derived IFN-γ secretion mediated local vascular permeability to enable antibody access to neuronal tissue.

In summary, this study explains why simple application of immune serum is not always sufficient for protection against neurotropic infections. Neutralizing immune serum on its own is not able to penetrate barrier, immune privileged tissues in infected recipients if they lack anti-viral CD4 T cells. This could explain why anti-Ebola serum was not effective in all Ebola-infected patients. This study also provides mechanistic explanation for current paradigm for rabies virus protection that requires application of both vaccine [to induce T cells] alongside anti-rabies immune serum.

David Usharauli

Anti-tumor effect of adoptive CD4 T cells positively correlates with high precursor frequency

It is now well accepted that similar to cytotoxic CD8 T cells, antigen-specific effector CD4 T cells can show a direct anti-tumor effect both in  mice and humans. This concept is still relatively new (actually first paper about it was published only in 2003). Incorporation of CD4 T cells and MHC II + peptides in tumor immunotherapy strategy increases chances of finding tumor-specific antigens (epitopes) relevant for personalized cancer medicine.

In this regard, new paper from journal Immunity is of interest. There, the authors showed that unlike CD8 T cells, intra-clonal competition of adoptive transfer CD4 T cells [at high precursor frequency] improved rather than diminished its anti-tumor effect and productive effector differentiation.  

In this study, the authors transferred different number (10³, 10⁴, 10⁵, 10⁶) of melanoma antigen, TRP-1 specific CD4 T cells into mice implanted with melanoma. Despite significant expansion at low precursor frequency, only high frequency transferred TRP-1 specific CD4 T cells managed to control and eradicate established tumors.

Moreover, the authors showed that in this settings, only TRP-1 specific CD4 T cells from high frequency adoptive transfer hosts underwent productive effector differentiation.

Even addition of anti-PD1 antibody to [low frequency] TRP-1 specific CD4 T cell adoptive transfer hosts failed to rescue their differentiation (increase in IL-21 is a marker of CD4 T cell exhaustion).

In summary, this study suggests that for CD4 T cells intra-clonal competition does not prevent efficient effector differentiation and establishment of productive anti-tumor immunity. It appears that CD4 T cells undergoing significant expansion at low precursor frequency develop "early and checkpoint inhibition-refractory exhaustion" preventing them to participate in effective anti-tumor response. This knowledge should be taken into account when considering adoptive T cell tumor therapy and checkpoint inhibition (according this study, anti-PD1 therapy could potentially accelerate tumor-specific CD4 T cell exhaustion at low precursor frequency).

David Usharauli

Thymic epitope expression determines pattern of CD4 T cell peripheral tolerance

A few months ago I posted my analysis of one of the important papers from journal of Immunity discussing mechanisms of CD4 T cell peripheral tolerance. In that paper, the authors led by J.J. Moon, showed that number and functionality of CRE recombinase-specific endogenous CD4 T cells were determined by antigen expression in the thymus. 

Today, journal Nature Immunology published very similar study from Marc Jenkins' lab (JJ Moon's former supervisor) where the authors showed that number and functionality of eGFP epitope-specific endogenous CD4 T cells were also determined by level of specific epitope expression in the thymus. These studies have important implications predicting the efficacy of vaccines and mechanisms of autoimmune diseases, so lets review it.

As some of you might know Marc Jenkins' lab pioneered technique for analysis of endogenous antigen[epitope]-specific T cells in WT mice. Here, the authors have analysed expansion of endogenous eGFP epitope:MHC II tetramer⁺ CD4 T cells in dozen of transgenic mice expressing eGFP protein under the guidance of different ubiquitous or tissue-specific promoters. 

This analysis revealed 3 patterns of T cell tolerance: ignorance, thymic Treg development and thymic deletion. Mechanistically, level of antigen expression in the thymus determined which out of these 3 tolerance patterns were operational.   

Tolerance by ignorance: Ins1^eGFP mice express eGFP exclusively in pancreas. Analysis showed that both Ins1^eGFP  and WT mice harbor similar number of eGFP-specific naive CD4 T cells, implying ignorance.

Tolerance by Treg induction: Ins2^eGFP mice express eGFP in pancreas and in the thymus in a AIRE-dependent manner.

Presence of eGFP in the thymus in Ins2^eGFP mice slightly reduced tet⁺ CD4 T cell numbers and correspondingly increased frequency of tet⁺ Foxp3⁺ CD4 T cells. 

Immunization with CFA-eGFP peptide showed that tolerance in Ins2^eGFP mice depended on AIRE-driven eGFP epitope expression in the thymus.

Tolerance by thymic deletion: UBC^eGFP mice displayed a profound thymic deletion of eGFP-specific tet⁺ CD4 T cells. 

The authors clearly showed that eGFP expression in the thymus inversely correlated with the number of tet⁺ CD4 T cells. 

Similar pattern of T cell "tolerance" were found for truly nonself- and self-epitope-specific T cells.

In summary, this study confirmed that level of antigen expression in the thymus (and not in periphery) determined overall T cell tolerance status. By analyzing the number of antigen-specific human T cells [in comparison to reference "self"-specific T cell numbers] the scientist could predict efficacy of vaccines, tumor vaccine for example, or predict the potential for development of autoimmune diseases.

David Usharauli

    

Reciprocal T subset-specific tumor protection by RNA vaccine encoding mutant MHC class I and II binding epitopes

Efficacy of solid cancer immunotherapy mostly depends on effector activity of T cells. Initially, CD8 T cells were thought to mediate primary anti-tumor activity. However, for the past 15 years, growing evidence pointed to a stand-alone CD4 T cell role in cancer protection.      

This new paper in journal Nature provided another example of CD4 T cell specific tumor protection. Strangely, the authors' data suggest that in silico generated MHC class I and II binding mutant epitopes reciprocally activated CD4 and CD8 T cell tumor responses, respectively.

Using three different mouse tumor models, the authors showed that surprisingly mice immunized either with MHC class I binding cancer-specific mutant 27-mer peptide + polyI:C or with MHC class I binding mutant epitope-encoding RNAs, generated predominantly CD4 T cell immunogenic response.   

One of the cancer epitope (B16-M30) encoding RNA even induced fully CD4 T cell-dependent 80% protection of cancer bearing mice.

Strangely, when the authors designed RNAs encoding several MHC class II binding mutant epitopes in combination with one class I binding epitope (synthetic RNA pentatope), anti-tumor protection was CD8 T cell-dependent.

Even more strangely, when the authors designed RNA pentatopes with only MHC class II binding epitopes (based on in silico algorithm and expression level), anti-tumor protection was also CD8 T cell-dependent.

In summary, the authors showed that tumors carry multiple (sometimes hundreds) of mutations that can specifically bind to MHC class II molecules. However, why were epitopes selected based on prediction to bind class I molecules induced CD4 T cell-dependent anti-tumor response (and vice versa for class II epitopes and CD8 T cells) are not clear.

David Usharauli    

Tumor immunotherapy: 1 year at a time

Tumor immunotherapy is both an old and new idea. Whenever there is a new discovery in immunology, people usually are asking 3 questions: can it cure HIV, cancer or allergy?

Recently, a promise for successful immunotherapeutic approach to cancer treatment was boosted by clinical introduction of anti-CTLA4 and anti-PDL1 humanized antibodies. These antibodies target and exploit the natural inhibitory checkpoints in T cell activation program relevant for natural tolerance process to self or food antigens. There is another approach as well, called CAR-T technology, that I will review later.

Still, the actual, long-term benefits of current immunotherapeutic approaches are debatable. Here is one example. This is a Science paper. The senior author on this paper is Steven Rosenberg. This is a guy who actually has started clinical immunotherapy with IL-2 in 1980's.

This paper describes a single patient's personalized tumor immunotherapy protocol. Initially, the authors sequenced a metastatic tumor obtained from resected lung tissue and found 26 mutations. Next, they have prepared 3 different tandem minigene constructs (8-9 mutations each), transfected them in autologous antigen-presenting cells and run ex vivo TIL (tumor-infiltrated lymphocytes) activation assay. Only one construct, TMG-1, showed TIL reactivity.

Next, from this tandem minegene construct (that contained 9 mutant minigens), the authors synthesized 9 individual minigene constructs with each containing reversion of one mutation back to wild-type sequence, so to permit detection TIL specificity by antigen elimination. Interestingly, TIL's reactivity was abolished when erbb2 interacting protein was reverted to wild-type sequence. 

In addition, TIL's tumor reactivity was localized in CD4 T cell subset and was made up mostly of Vbeta22+ CD4 T cells. 

Thus this patient's TIL (CD4 T cells) specificity was directed towards one mutation in erbb2ip.

Next, the authors has expanded patient's TIL in vitro and injected back 42.4 billion TIL (25% Vbeta22+) into patient. This adoptive transfer of tumor specific TIL produced >1 year tumor burden stabilization and regression of metastatic tumor in liver and lung.

After 1.5 year from the initial TIL transfer, tumor re-emerged, so the patient was re-injected with in vitro expanded 100 billion tumor specific Vbeta22+ TIL and again such transfer produced additional >6 months stabilization and regression of tumor metastasis.

It appears that the difference between this graph and Fig. 2 D has to do with the fact that the authors measured only those tumor lesions which were present during both treatments.

What we have learned from this study? First, tumor in this case did express neo-antigen detected by CD4 T cells. Second, in vitro expansion of tumor-specific TILs and their adoptive transfer back into patient yielded >1 year (after single transfer) and in total >2 years (after 2nd transfer) of tumor burden stabilization and metastatic regression. Third, this approach, however, did not provide a long-lasting tumor-protective effect, despite presence of transferred tumor-specific CD4 T cells.

Does it mean that TIL approach is not viable in a long run? Why were billions of tumor-specific T cells not able to eliminate tumor? Because of inhibitory milieu at tumor sites? Were they terminally-differentiated T cells lacking self-renewal properties? It seems that tumor was able to escape CD4 T cell control despite the fact it still continued to express neo-antigen. How? 

David Usharauli