Cutting edge: innate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimuli

Abstract

Innate memory phenotype (IMP) CD8(+) T cells are nonconventional αβ T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8(+) T cells in MHCI(-/-) recipients, resembling those in Itk(-/-) mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.

FIGURE 1. Development of IMP CD8⁺ T cells via hematopoietic MHCI selection independent of the thymus

(A) Representative flow cytometric analysis of IMP CD8⁺ T cells (upper panel, CD44^hiCD122⁺) and P/I induced IFN-γ production (lower panel). Donor TCRβ⁺CD8α⁺ cells from WT→WT (MHCI⁺CD45.2⁺), MHCI^−/−→WT (MHCI⁻CD45.2⁺) and WT→MHCI^−/− (MHCI⁺CD45.2⁻) chimeras are shown. (B&C) The thymus is not required for development of IMP CD8⁺ T cells. (B) Bone marrow chimeras were generated as indicated and donor TCRβ⁺CD8α⁺ cells analyzed by flow cytometry for CD44 and CD122, and IFN-γ producing capacity induced by P/I, compared to those from WT and Nude mice. (C) Percentages of IMP, naïve and P/I induced CD44^hi IFN-γ producing CD8⁺ T cells, along with numbers of total, IMP and naïve CD8⁺ T cells, in “WT→WT” and “WT→Nude” chimeras. n = 4 in each group. *p < 0.05 by Student’s t test.

FIGURE 2. IMP CD8⁺ T cells in WT→MHCI^−/− chimeras develop despite depletion of T cells from donor bone marrow

WT bone marrow was either left intact or depleted of T-cells, and used as donors to generate “WT→WT” and “WT→MHCI^−/−” chimeras. Donor TCRβ⁺CD8α⁺ cells were analyzed. (A) Flow cytometric analysis for CD44 and CD122, and percentage of CD44^hi IFN-γ producing CD8⁺ T cells in response to P/I. (B) Percentages and numbers of IMP and naïve CD8⁺ T cells, and percentage of P/I induced CD44^hi IFN-γ⁺ CD8⁺ T cells. n = 3 in each group. *p < 0.05, ns = not significant, by Student’s t test.

FIGURE 3. Hematopoietic MHCI dependent CD8⁺ T cells resemble innate memory CD8⁺ T cells in Itk^−/− mice, but are distinct from HP cells

Comparison of HP, Itk^−/− and WM (WT→MHCI^−/−) CD8⁺ T cells. (A) HP, Itk^−/− and WM CD8⁺ T cells share expression of CD44 and CD122. (B) Itk^−/− and WM CD8⁺ T cells show extremely high correlation in whole genome gene expression, which is distinct from HP CD8⁺ T cells. Samples were clustered based on the hierarchy of correlation. (C) HP but not Itk^−/− CD8⁺ T cells, exhibit significantly higher number of differentially expressed genes compared to WM CD8⁺ T cells. Genes with significant change (fold change > 2, corrected P < 0.05) are shown in red. Numbers in red indicate numbers of gene that significantly up- or down- regulated.

FIGURE 4. Hematopoietic MHCI dependent OTI IMP CD8⁺ T cells exhibit prompt and potent antigen specific response in vitro without primary antigen exposure

BMTs were done as indicated and donor TCR transgene positive CD8α⁺ cells from OTI→WT (CD45.2⁺MHCI⁺), MHCI^−/−OTI→WT (CD45.2⁺MHCI⁻) and OTI→MHCI^−/− (CD45.2⁺MHCI⁺) chimeras analyzed. (A) Flow cytometric analysis of CD8⁺ T cells for CD44 and CD122 and P/I induced IFN-γ production in chimeric spleens. (B) Percentages and numbers of CD44^hiCD122⁺ IMP CD8⁺ T cells, CD44^loCD122⁻ naïve CD8⁺ T cells, and proportion of CD44^hi IFN-γ producing cells in response to P/I. *p < 0.05, ns = not significant, by Student’s t test. Data represent results from three independent experiments. (C) Percentages of CD44^hi IFN-γ⁺, CD44^hi TNF-α⁺ and IFN-γ⁺/TNF-α⁺ double positive donor CD8⁺ T cells in response to OVA and OVA_257–264 peptide along the time course. All values were corrected for the response of non-stimulated controls. *p < 0.05, compared to “OTI→WT” chimeras, ^§p < 0.05, compared to “MHCI^−/−OTI→WT” chimeras, by two-way ANOVA. Data represent results from two independent experiments.