Broad cross-reactive TCR repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes

Abstract

Memory T cells cross-reactive with epitopes encoded by related or even unrelated viruses may alter the immune response and pathogenesis of infection by a process known as heterologous immunity. Because a challenge virus epitope may react with only a subset of the T cell repertoire in a cross-reactive epitope-specific memory pool, the vigorous cross-reactive response may be narrowly focused, or oligoclonal. We show in this article, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M1(58-66) epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1(280-288) epitope (GLCTLVAML), that, under some conditions, heterologous immunity can lead to a significant broadening, rather than a narrowing, of the TCR repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad, rather than a narrow, T cell repertoire if there is a lack of dominant high-affinity clones; this hypothesis is supported by computer simulation.

Figure 1. BMLF1-tetramer positive T cells can produce IFNγ following M1 stimulation

CD8 T cells isolated from patient D-002 were grown in the presence of M1 peptide for 3 weeks. After a 5 hour stimulation with M1 peptide, the M1-specific T cell line was stained extracellularly with either M1- or BMLF1-specific tetramer and intracellularly for IFNγ. The % of the M1-specific T cell line that stained with the indicated tetramer (top number) and the % of the tetramer-positive population that co-stained with anti-IFNγ (bottom number) are shown. Not shown are control peptide EBV-BRLF-1₁₉₀ stimulation, whereby <0.06% of the BMLF1 tetramer+ population produced IFNγ, and control tetramer staining, CMVpp65 <.001%.

Figure 2. The Vβ repertoire of a cultured T cell line accurately reflects the antigen-specific repertoire ex vivo

(a) CD8 T cells were isolated from the peripheral blood of donor D-002 and immediately co-stained with BMLF1-specific tetramer and Vβ-specific antibodies. The bar graph illustrates the % of BMLF1 tetramer+ cells using each respective Vβ family. We then sorted BMLF1+ T cells and sub-cloned the CDR3β region using primers specific for 5 different Vβ families, which are indicated by the shaded boxes. The ID of the predominant clonotype(s), its frequency among all sequences analyzed within that family, and the amino acid sequence of its CDR3β loop is provided. (b) CD8 T cells derived from D-002 were cultured for 4 weeks in the presence of BMLF1-pulsed T2 cells before being stained with Vβ-specific antibodies. The bar graph illustrates the % of the T cell line (CD8 cells) using each respective Vβ family. The line was >90% BMLF1 tetramer+ (data not shown). The predominant clonotype(s) found within each of the 5 shaded Vβ families is displayed as described above.

Figure 3. Each tetramer-defined sub-population of a T cell line has a distinct Vβ repertoire

(a) CD8 T cells isolated from patient E1101 were cultured for 3 weeks in the presence of M1-pulsed T2 cells before being co-stained with M1- and BMLF1-specific tetramers. (b) Following incubation with tetramers, cells (a) were stained with Vβ-specific antibodies. Each bar graph illustrates the % of cells within its respective tetramer-defined gate that use each Vβ family. (c) CD8 T cells isolated from patient E1101 were cultured for 3 weeks in the presence of BMLF1-pulsed T2 cells before being co-stained with M1- and BMLF1-specific tetramers. (d) Following incubation with tetramers, cells (c) were stained with Vβ-specific antibodies. The bar graph illustrates the % of BMLF1-tetramer+ cells that use each Vβ family. Control tetramer CMVpp65 in both M1 line and BMLF1 line was <.001%.

Figure 4. The Vβ repertoire of cross-reactive cells is often comprised of multiple Vβ families and is unique to each individual

CD8 T cells were isolated from 4 healthy donors and 5 patients with IM. (a-c) CD8 T cells were cultured for 3-4 weeks in the presence of M1-pulsed T2 cells before being co-stained with M1- and BMLF1-specific tetramers followed by Vβ-specific antibodies. Three tetramer-defined gates were analyzed separately: (a) M1+ BMLF1+ (CXR-1), (b) M1- BMLF1+ (CXR-2), and (c) M1+ BMLF1- (non-cross-reactive M1). (d) CD8 T cells were cultured for 3-4 weeks in the presence of BMLF1-pulsed T2 cells before being stained with tetramers and Vβ-specific antibodies. The Vβ usage of M1- BMLF1+ (non-cross-reactive BMLF1) cells is shown. The degree of shading within each box represents the % of tetramer-gated cells that express that Vβ family, and a red outline of a box indicates that the Vβ family was detected in the cross-reactive repertoire but not the non-cross-reactive repertoires of that individual. e) Increased breadth of Vβ usage in the cross-reactive population when compared to the non-cross-reactive IAV-M1 population (* p=.05, paired t test) and the non-cross-reactive EBV-BMLF1 population (** p=.06, paired t test).

Figure 5. The Vβ repertoire of cross-reactive cells is comprised of multiple unique clonotypes using a combination of M1- and BMLF1-specific TCR elements

(a) An M1-specific T cell line derived from donor D-002 was co-stained with M1- and BMLF1-specific tetramers. Two separate populations were sorted: M1- BMLF1+ (CXR-2) and M1+ BMLF1- (non-cross-reactive M1). (b) A BMLF1-specific T cell line derived from donor D-002 was co-stained with both tetramers and the BMLF1-tetramer+ (non-cross-reactive BMLF1) cells were collected (c) The CDR3β regions of cross-reactive Vβ14+ clonotypes were sequenced. Based on a unique nucleotide sequence, clonotypes were assigned an ID and their frequency is shown, i.e. the number of times that clonotype was detected among the total sequences analyzed. The length of the CDR3β region was determined according to Chothia et al., shown supported by two flanking framework regions (51). Bold indicates that the clonotype was previously detected within BMLF1-specific cell populations. (d) The CDR3β regions of cross-reactive Vβ17+ clonotypes were sequenced. Unique clonotypes are arranged based on their frequency among all sequences analyzed. Due to space limitations, the amino acid sequence of the CDR3β loop has been abbreviated where 4 residues represents a full length of 8. The bars are shaded to indicate simultaneous detection within an alternative cell population as follows: (x025A1) = unique to the cross-reactive population, = detection within the non-cross-reactive M1+ population, (x025A0) = detection within the non-cross-reactive BMLF1-specific cell populations. (e) The CDR3P regions of non-cross-reactive M1+ Vβ17+ clonotypes were sequenced and arranged as described above.

Figure 6. The Vα repertoire of cross-reactive cells is comprised of multiple Vα families and is distinct from that of non-cross-reactive M1+or BMLF1+populations

CDR3α spectratyping analysis was performed on (a) the cross-reactive population described in Fig. 5a where arrows indicate positive reactions, or on (b) the non-cross-reactive BMLF1-specific population described in Fig. 5b, or simultaneously on (c) all three tetramer-defined cross-reactive and non-cross-reactive populations described in Fig. 5a, b where B = non-cross-reactive BMLF1+ cells, X = cross-reactive cells, and M = non-cross-reactive M1+ cells. (d) The CDR3α regions of Vα15+ T cells found within each of the 3 separate T cell populations were sequenced. Clonotypes with unique nucleotide sequences were assigned an ID and their frequency among all sequences analyzed is shown. The length of the CDR3α region was determined according to Chothia et al., shown supported by two flanking framework regions (51). Bold indicates that the clonotype was simultaneously detected within the non-cross-reactive BMLF1+ population.

Figure 7. Computer simulation of cross-reactive response: Lack of high affinity cross-reactive clones leads to a broader repertoire distribution

a.) Frequency hierarchy of clones responding to the primary infection. The clonal affinity to the primary immunogen is represented on the left and the affinity to the cross-reactive challenging pathogen is represented on the right. The bars are in shades of black to white, with black being clones having the highest affinity and white being clones having no affinity to the particular antigen. The individual clones are identified by a 4 digit code. b.) Frequency hierarchy of clones responding to the primary infection that has been modified to represent the distribution of clonotypes after the removal of the three highest frequency clonotypes with high affinity to the cross-reacting pathogen, thus simulating responses to a cross-reactive infection that is structurally dissimilar from the original antigen. In Fig. 7b, no high frequency high affinity clones to the primary epitope were cross-reactive with the challenging infection; clones FFOF, FFAF and FF2F have been replaced. c) The resulting repertoire upon secondary challenge of an individual having a memory TCR repertoire as depicted in (a) with a “near” or similar cross-reactive epitope. d) The resulting repertoire upon secondary challenge of an individual having a modified memory TCR repertoire as depicted in (b) with a “far” or dissimilar cross-reactive epitope. These results are representative of 3 sets of 26 virtual individuals each.