T follicular helper cell responses to SARS‐CoV‐2 vaccination among healthy and immunocompromised adults

Abstract The worldwide rollout of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccinations in the last 2 years has produced a multitude of studies investigating T‐cell responses in the peripheral blood and a limited number in secondary lymphoid tissues. As a key component to an effective immune response, vaccine‐specific T follicular helper (Tfh) cells are localized in the draining lymph node (LN) and assist in the selection of highly specific B‐cell clones for the production of neutralizing antibodies. While these cells have been noted in the blood as circulating Tfh (cTfh) cells, they are not often taken into consideration when examining effective CD4+ T‐cell responses, particularly in immunocompromised groups. Furthermore, site‐specific analyses in locations such as the LN have recently become an attractive area of investigation. This is mainly a result of improved sampling methods via ultrasound‐guided fine‐needle biopsy (FNB)/fine‐needle aspiration (FNA), which are less invasive than LN excision and able to be performed longitudinally. While these studies have been undertaken in healthy individuals, data from immunocompromised groups are lacking. This review will focus on both Tfh and cTfh responses after SARS‐CoV‐2 vaccination in healthy and immunocompromised individuals. This area of investigation could identify key characteristics of a successful LN response required for the prevention of infection and viral clearance. This furthermore may highlight responses that could be fine‐tuned to improve vaccine efficacy within immunocompromised groups that are at a risk of more severe disease.


INTRODUCTION
The effort of vaccination and its global application is one of the best ways to reduce the burden of infectious disease. Implemented first to eliminate the spread of smallpox, regular vaccination for diseases such as polio and measles has led to their elimination in developed countries, significantly reducing morbidity and mortality (particularly in children). 1 More recently, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination has played a substantial role in significantly reducing the severe consequences of this infection. This is particularly important for vulnerable groups such as the immune compromised, who are at most risk of complications from disease. 2,3 While this group receives a substantial benefit, some individuals are unable to mount an appropriate response to vaccination. [4][5][6] Those undergoing immunosuppressive treatment (such as monoclonal antibody therapy, alkylating agents or organ transplant recipients) and patients with cancer often have issues with seroconversion. 4,7 In the case of the SARS-CoV-2 vaccinations, these communities often developed lower antibody levels after vaccination and, in some studies, lower levels of vaccine-specific T cells. 6,8 T follicular helper (Tfh) cells are a specialized CD4 + T-cell subset found in secondary lymphoid organs such as the lymph node (LN). They play a vital role in B-cell survival, clonal expansion, somatic hypermutation and affinity maturation, leading to highly specific and class-switched antibodies. 9 While responses to vaccination occur in the LN, access to this environment for analysis of the human immune response is difficult and can be invasive. Circulating Tfh cells (cTfh) have been used as a surrogate for LN Tfh cells and are easily measured in the peripheral blood. 10 This subset has been shown to correlate with neutralizing antibody (NAb) levels following various vaccinations, which are key for the prevention of infection and clearance of acute viral infections. [10][11][12] In this review, we focus on Tfh cells in response to SARS-CoV-2 vaccinations in healthy and immunocompromised individuals in both the site-specific LN and peripheral blood. This comparison could assist in understanding the differences between these groups, which may be fine-tuned to improve vaccine efficacy within immunocompromised individuals that are at a risk of more severe disease.

T FOLLICULAR HELPER CELLS
In the T-cell zone of the LN, na€ ıve CD4 + T cells are primed by antigen-presenting dendritic cells via peptide engagement of the cognate T-cell receptor (TCR), leading to the downregulation of C-C chemokine receptor type 7 (CCR7). 13 The presence of particular cytokines, such as interleukin-6 and interleukin-21 in the LN microenvironment at the time of priming, lead these cells toward a Tfh differentiation pathway, hallmarked by the upregulation of C-X-C chemokine receptor type 5 (CXCR5) on the cell surface. 14 Allowing for the migration of these CD4 + T cells from the T-cell zone to the T-B-cell border, where they express the transcription factor B-cell lymphoma 6 (BCL-6) and the key molecule inducible T-cell costimulator (ICOS), 15,16 it is in this location they are termed pre-Tfh cells. 17 Upon interaction with activated B cells presenting the cognate antigen and critical signaling through molecules such as ICOS/ICOS ligand, CD40 ligand/CD40 and CD28/CD86, the pre-Tfh cells undergo differentiation into Tfh cells and are able to migrate into the B-cell follicle to provide B-cell help and underpin the formation of germinal centres. [18][19][20][21] It is at this location that the activated Tfh cells engage with B cells to support their expansion and increase antibody specificity. 19,22 It is believed that some of these germinal center Tfh cells migrate out of the LN and can be found in the peripheral blood as cTfh cells. 10,23,24 The source of cTfh is, however, not entirely clear. An alternative proposal by He et al. demonstrated via murine model that cTfh cells were generated from pre-Tfh cells prior to their migration to the germinal center of the LN. 22 cTfh cells have been shown to express similar phenotypic markers as bone fide LN Tfh cells, such as CXCR5, programmed cell death protein 1 (PD-1) and often ICOS; however, they lack BCL-6 lymphoma 6 expression. 12,25 Defined by their expression of CXCR3 and CCR6, cTfh can be further subtyped into cTfh-1, cTfh-2 and cTfh-17 ( Figure 1). These cTfh subsets have been detected in response to several vaccinations and infections (summarized in Table 1). [26][27][28] Similar subtyping has been performed in macaque LNs with the separation of Tfh cells into Tfh-1, Tfh-2 and Tfh-17, as has been performed in human blood previously (Table 1). 29 CXCR3 + Tfh cells have also been observed in the human tonsil but were not defined as Tfh-1 in this study. 23 This subtyping could assist in understanding Tfh migration within and from the LN, and their relationship to peripheral blood cTfh cells. 29,30 RESPONSES TO SARS-COV-2 VACCINATION AMONG HEALTHY ADULTS SARS-CoV-2 emerged in late 2019 and quickly spread across the globe, causing approximately 674 million infections and over 6.8 million deaths at the time of writing. 31 Less than a year later, multiple SARS-CoV-2 vaccinations were developed and rolled out, resulting in substantially reduced morbidity and mortality of infection. [32][33][34][35][36][37]

Peripheral blood responses in healthy individuals
Analyzing the expansion of antigen-specific cTfh cells in the blood has proven valuable for understanding the immune response to yellow fever vaccination (YFV) and hepatitis B vaccination, with these responses positively correlated with NAb levels. 10,11 In a secondary response to influenza vaccination, cTfh cells peaked 7 days after vaccination compared with 14 days after vaccination in a primary response to YFV and SARS-CoV-2 vaccination. 10,12,25,38,39 Antigen-specific cTfh cells were found to correlate with spike immunoglobulin G antibody levels in infection-na€ ıve healthy individuals 10-12 days following the second SARS-CoV-2 messenger RNA (mRNA) vaccination. 40 Increased frequencies of ICOS + cTfh-1 and ICOS + CD38 + cTfh cells have also been correlated with higher immunoglobulin G antibody levels in separate studies of influenza and pneumococcal vaccination. [41][42][43] Interestingly, these cells have been shown to display increased TCR clonality, indicating their probable antigen specificity compared to their ICOS -CD38 -cTfh counterparts, emphasizing the importance of these markers in the immune response to vaccination. 44 To detect SARS-CoV-2 spike-specific CD4 + T cells in the blood, Wragg et al. 45 used an HLA-DRB1*15:01restricted tetramer (S 751-767 ). 45 Following initial SARS-CoV-2 vaccination [n = 7 BNT162b2 (Pfizer), n = 2 ChAdOx nCov-19 (AstraZeneca/Oxford), n = 1 NVX-Cov2372 (Novavax)], expansions of tetramer-specific cTfh cells were identified in SARS-CoV-2 infection-na€ ıve individuals but upon secondary vaccination the same level of expansion was not observed. 45 Interestingly, however, Painter et al. 46 identified an expansion of SARS-CoV-2-specific cTfh cells (following peptide pool stimulation) 1 week after secondary vaccination in 36 individuals that received the mRNA SARS-CoV-2 vaccination (94% received BNT162b2 and 6% mRNA-1273). 46 The differences in results could be a result of several factors such as the flow cytometry gating, both studies defined cTfh as CXCR5 + memory CD4 + T cells, with Painter et al. excluding the CD45RA + CD27 + CCR7 + -na€ ıve T-cell population and Wragg et al. excluding the CD45RA + CCR7 + population. Further differences could be a result of smaller cohort size in the latter study and the detection of antigenspecific cTfh cells via tetramer, compared with peptide pool stimulation, as the true magnitude of these cells could be underestimated because of their restriction to a particular SARS-CoV-2 TCR epitope.
An interesting observation was made by Zhang et al. 38 who studied cTfh cells in response to four different coronavirus disease 2019 (COVID-19) vaccines [mRNA-1273 (Moderna), BNT162b2 (Pfizer), Ad26.COV2.S (Janssen) and NVX-Cov2372 (Novavax)]. 38 A SARS-CoV-2 spike peptide pool was used to expand and detect antigen-specific cTfh cells and showed that the percentage of these cells was significantly lower following the Janssen vaccination compared with the other vaccines tested at all timepoints. A nonsignificant difference was also observed between the median percentage of cTfh cells between the two mRNA vaccines. Principal component analysis of cellular and neutralization data revealed that while the Moderna and Pfizer vaccines generate similar cellular responses, the latter tends to have more "heterogeneity" in both the CD4 + and CD8 + T cells over time, with regard to intracellular cytokine expression and activation. This flags potential sources of variability when investigating in-depth COVID-19 vaccine cellular immune responses, even between the two mRNA-based vaccines, and should be taken into consideration in future studies.
The aforesaid studies highlight the different approaches used to identify SARS-CoV-2 spike-specific cTfh following vaccination. Tetramer-based studies are performed ex vivo and hence allow further phenotypic characterization of the spike-specific cTfh; markers for activation and chemokine receptors such as CXCR3 and CCR6 may be included with tetramer staining. Peptide stimulation-based assays can be performed in vitro without HLA-typing and hence can include larger numbers of vaccinees. This methodology allows the assessment of a broader SARS-CoV-2 spike-specific cTfh response; however, further phenotypic characterization maybe limited because of the changes in certain markers during antigen stimulation. In healthy individuals, cTfh type 1 (PD-1 + CXCR5 + CXCR3 + ) cells increase at day 14 of the primary immune response and at day 7 in the secondary response (in yellow fever and influenza vaccination). This cell type has been noted to correlate with higher levels of neutralizing antibodies after vaccination. This figure was created with BioRender. CCR6, C-C chemokine receptor type 6; CXCR3, C-X-C chemokine receptor type 3; CXCR5, C-X-C chemokine receptor type 5; PD-1, programmed cell death protein 1.
By incorporating the data from both tetramer and peptide stimulation-based assays, a more comprehensive picture of how SARS-CoV-2 vaccines elicit cTfh responses can be gleaned. The subdivision of antigenspecific cTfh into cTfh-1, cTfh-2 and cTfh-17 (Table 1) provides further characterization of the cTfh vaccine response. Previous vaccination studies have found that antigen-specific cTfh-1 were positively correlated with NAbs following YFV. 10 In that same study, the antigenspecific cTfh-1 were detected increasingly until 14 days following YFV and in other studies, it has been seen 7 days following influenza and human papillomavirus vaccinations. 10,25,47 This cTfh-1 skewing of antigen-specific cTfh was also observed by Wragg et al. 45 following the second dose of SARS-CoV-2 vaccination. 45 This was similar in a study that measured cTfh numbers via cells/lL, in which the authors found a significant increase in the cTfh-1 counts per lL of blood, but was limited to the first dose of the mRNA-1273 vaccination. 48 There was a twofold increase also noted by Samanovic et al. after the first dose of BNT162b2 vaccination (and a slight increase upon second dose) among the cTfh cells (PD-1 + CXCR5 + ) that expressed ICOS + CD38 + , wherein these activated cells have been correlated with antibody responses and overlapping clonality after successive influenza vaccinations. 49,50 The polarization of the cTfh cells seems to occur after multiple different vaccinations, including the SARS-CoV-2 mRNA platform, and at times correlates with increased protection in the form of spike-immunoglobulin G and neutralizing antibodies. 50 cTfh-1 polarization, however, has not been found ubiquitously across all cTfh SARS-CoV-2 vaccination responses. Following the third dose of CoronaVac, antigen-specific cTfh-17 expansion dominated the total spike-specific cTfh cell response at 2-and 8-weeks after vaccination. 51 It is unclear whether this was observed following the first or second CoronaVac vaccinations, as this study only investigated these cTfh subtypes after the third dose. cTfh-1 and cTfh-17 cells have further been investigated after two doses of the NVX-CoV2373 (Novavax) vaccination, with higher spike-specific cTfh-17 cells seen at day 7 after the first dose that slightly correlated (r = 0.35, P = 0.044) with spike immunoglobulin G titers 14 days later (day 21). 52 A recent study concluded that cTfh-17 cells are able to maintain better immune memory than the cTfh-1 and cTfh-2 subsets, as they made up the majority of the Table 1. Summary of immunophenotyping markers to identify T follicular (Tfh) and circulating T follicular helper (cTfh) cells in the immune response to vaccination and infection.

Lymph node responses in healthy adults
Following deltoid intramuscular vaccination, establishment of the immune response occurs in the axillary draining LN. While there is an abundance of research on vaccine responses in peripheral blood, human LN studies are limited. Investigating this crucial location would allow for more in-depth characterization of the cellular response to vaccination in both healthy and immunocompromised groups.
It is only in recent years that ultrasound-guided fineneedle biopsies (FNBs;sampling via capillary action) or fine-needle aspirates (FNA; sampling via aspiration) have been used to collect human LN cells during infection (such as HIV) or following vaccination. [54][55][56] In 2020, Turner et al. 54 applied this technique to study LN responses following influenza vaccination, in which their main focus was to investigate the complexities and clonality of germinal center B cells and plasmablasts. 54 While there was no focus on Tfh or CD4 + T cells in general, this was one of the first papers to use this sample type after vaccination in humans.
A comprehensive analysis of CD4 + Tfh cells was recently reported by our group in which we performed FNBs of the draining and contralateral axillary LNs preand 5 days after seasonal influenza vaccination. 55 An increase in the absolute number of GC-Tfh cells was observed exclusively in the draining LN after vaccination compared with before vaccination. These cells were highly activated and had recently proliferated, as measured by the coexpression of CD38, ICOS and Ki-67, respectively. Prior to the COVID-19 pandemic and the subsequent delivery of multiple SARS-CoV-2 vaccinations, there were very few studies investigating the LN through FNB/FNA following vaccination.
There has only been one FNA study focused on Tfh cells in healthy adults following SARS-CoV-2 vaccination (BNT162b2 mRNA). 57 Another recently published study investigated the LN in healthy and SARS-CoV-2-infected individuals after SARS-CoV-2 vaccination, but used core biopsy samples to analyze the LN architecture. 58 Mudd et al. 57 sampled draining axillary LNs of SARS-CoV-2-na€ ıve volunteers (n = 15) with longitudinal LN FNA samples taken before and after primary and secondary vaccination, and at increasing intervals up to 200 days after the first vaccination (n = 6). 57 An HLA-DPB1*04restricted SARS-CoV-2 tetramer (S 167-180 ) was used to detect S 167-180 -specific Tfh cells present in the draining LN at day 40 after secondary vaccination in five individuals, and up to day 200 in two individuals ( Figure 2). In peripheral blood the S 167-180 -specific cTfh cells peaked 1 week after the second vaccination and then decreased thereafter (becoming undetectable at the later timepoints in some volunteers). TCR sequencing of the LN tetramer-specific Tfh cells was also performed in four participants at day 60, and in another three at day 110 (days 39 and 89 after secondary vaccination, respectively). Although specific dominant TCR clones remained consistent in the LN despite a 50-day gap between sampling, a comparative analysis was not performed in the blood, which would have provided valuable insight into the clonal dynamics of both the memory CD4 + and the cTfh cells over time.
These studies emphasize a clear need for investigating T-cell responses after vaccination at the site of induction across multiple different vaccines and cohorts. This could assist in settling the conflicting results in terms of the relationship between Tfh and cTfh cell lineages, but also further our understanding of the mechanisms of action by novel mRNA vaccines, which are now a highly attractive platform for immunization.

RESPONSES TO SARS-COV-2 VACCINATION AMONG IMMUNOCOMPROMISED ADULTS
With increased diversity in SARS-CoV-2 variants and waning vaccination rates, immunocompromised patients are still at risk for more severe COVID-19 symptoms and mortality. 59 While antiviral treatments such as nirmatrelvir/ritonavir (Paxlovid) are effective in these groups, this protease inhibitor needs to be commenced within 5 days of symptom onset to have an optimal effect. 60,61 Monoclonal antibody therapy is also an important prevention modality to reduce mortality and disease burden; however, the rapid growth of new SARS-CoV-2 variants has reduced their efficacy and they are often expensive to manufacture and problematic to administer en mass. 62 Vaccination therefore remains an important tool for reducing the effect of COVID-19 with issues such as efficacy and appropriate seroconversion being key in these groups.

Peripheral blood responses in immunocompromised individuals
There are limited studies focusing on the cTfh responses after vaccination in immunocompromised adults, therefore this section will include a number of studies describing vaccine-specific CD4 + T cells and not particularly antigenspecific cTfh in this population of interest.
In a recent study by Gao et al. 63 spike-specific CD4 + T-cell responses were investigated in a number of immunocompromised patients, including those with solid organ transplants (n = 41), hematopoietic stem cell transplants (n = 43), chronic lymphocytic leukemia (n = 53), primary immunodeficiency (n = 48) and HIV infection (n = 50). 63 The responses were compared across two BNT162b2 doses and followed out to 6 months. The patients with hematopoietic stem cell transplant, primary immunodeficiency and HIV had generally detectable and preserved spike-specific CD4 + T-cell responses, which have also been seen in both treatment-na€ ıve and immunochemotherapy-treated individuals with lymphoma. 64 Interestingly, only after the second vaccine dose was there a substantial and likely meaningful increase in spike-specific CD4 + T cells recorded in the solid organ transplant and leukemia patients. At the 6-month timepoint, the longevity of the CD4 + T-cell responses in these latter groups (solid organ transplant and chronic lymphocytic leukemia) was not sustained as well as responses seen in healthy individuals or the other immunocompromised groups. While this study did not investigate the cTfh cells specifically, it does highlight meaningful differences between these patient groups with respect to the antigen-specific CD4 + T-cell responses, and this may flag which groups require more focus on to improve vaccine responses.
Apostolidis et al. 40 recently analyzed cTfh cells from patients with multiple sclerosis treated with anti-CD20 therapy (n = 20). 40 In this patient group, the antigenspecific cTfh cells, determined by SARS-CoV-2 peptide vaccination in healthy individuals have revealed a large increase in Tfh cells that are proliferating and highly activated. Tfh cells that are specific to antigens presented by messenger RNA (mRNA) vaccination in particular have considerable longevity in this site. In immunocompromised individuals after vaccination, these cells are reduced in number but also lack the expression of key markers for activation, migration and B-cell interaction. The tetramer-specific Tfh graph is adapted from Mudd et al. 57 The image was created with BioRender. CXCR3, C-X-C chemokine receptor type 3; CXCR5, C-X-C chemokine receptor type 5; FNA, fine-needle aspirate; FNB, fine-needle biopsy; ICOS, inducible T-cell costimulator; LN, lymph node; PD-1, programmed cell death protein 1; T mem , T memory cell.
pool stimulation, had a nonsignificant reduction at all timepoints compared with healthy adults. Verstegen et al. 48 have reported similar results when comparing patients with multiple sclerosis treated with anti-CD20 (n = 7) and patients with rheumatoid arthritis on methotrexate (n = 14), both infected with SARS-CoV-2 prior to vaccination. 48 They found a negligible or minimal increase in the cTfh and cTfh-1 cell compartment (per lL of blood and as a percentage of memory CD4 + cells) following the first mRNA vaccination compared with a previously infected healthy control group. These findings could suggest that the recall of cTfh cells upon the reintroduction of SARS-CoV-2 could be impaired, although this finding could also be due to the lower cell numbers seen before vaccination compared with the healthy group in the latter paper.
All of these findings indicate potential deficiencies within the LN response, specifically as anti-CD20 monoclonal antibody treatment reduces the number of B cells in the blood over time. 65 The lack of interaction between LN Tfh and B cells could lead to reduced trafficking of the cTfh into the peripheral blood. Sitespecific analysis of secondary lymphoid organs would likely unveil possible issues with cell interactions within patients with multiple sclerosis treated with anti-CD20. This also emphasizes the importance of undertaking thorough analyses of all subsets of memory CD4 + T cells when studying T-cell responses and their possible functionality. As cTfh cells have been shown to positively correlate with NAb titers and are likely representative of Tfh cell action, this lack of investigation could potentially lead to an overestimation of effective T-cell activity.
Overall, these studies emphasize the complexities across multiple immunocompromised patient groups. Differences in their specific T-cell and antibody responses could potentially help explain the genesis of differences in vaccine efficacy. Despite the value of this work, it is clear that investigation needs to be performed in the sitespecific areas of the LN. As the cTfh cell compartment is reduced in those on anti-CD20 therapy, the logical next step would be to perform similar LN studies in these patient groups to those performed in healthy volunteers. This would provide invaluable data on the interaction between B and T cells in the LN and likely inform the deficiencies within this area that could be manipulated to possibly increase vaccine efficacy.

Lymph node responses in immunocompromised adults
As noted previously, there are limited studies that have investigated the LN immune response to vaccination, while these studies in immunocompromised adults are even rarer. Prior to the release of the SARS-CoV-2 vaccinations, there was only one such study investigating the LN responses in people living with HIV and on long-term therapy. Moysi et al. 66 performed core biopsies on the draining and nondraining inguinal LNs before and after influenza vaccination. 66 Their study highlighted that there were cell expansions and gene expression differences in Tfh cells, after vaccination in people living with HIV and uninfected adults, despite long-term therapy.
Furthermore, at time of writing, there was currently only one published study of LN FNBs on immunocompromised participants. Lederer et al. 67 compared LN FNB samples from healthy participants (n = 15) and those on immunosuppressant drugs following kidney transplant (n = 13). 67 Draining and nondraining (n = 4) LNs were sampled 14 days following primary and 8 days following secondary SARS-CoV-2 mRNA vaccination. In healthy individuals, similar to our previous LN study, 55 they found that Tfh cell expansion was exclusively localized in the draining LN, and there was an increased percentage of these cells (from the total CD4 + CD45RA À memory T-cell population) upon secondary vaccination. However, this was not the case for the kidney transplant group, which had a significant reduction in Tfh cells as the percentage of total CD4 + T cells in the LN. Clustering analysis of flow cytometry data using t-distributed stochastic neighbor embedding of LN memory CXCR5 + CD4 + T cells revealed a clear reduction in the expression of PD-1, Bcl-6, ICOS and CD38 in the memory T cells from kidney transplant participants compared with healthy individuals. This emphasizes the marked failure of Tfh cell induction after vaccination in these immunocompromised individuals.
This decrease in Tfh cells was also reflected in the peripheral blood of the kidney transplant group, where peptide stimulation identified a reduction in the CD4 + T cells responding to SARS-CoV-2 across all vaccine timepoints in the immunocompromised group (including the third dose). These findings were similar to what was observed in the peripheral blood by Apostolidis et al. 40 These and future studies could aid in the exploration of the effects of various adjuvants to supplement current vaccine formats aimed at improving efficacy or to enhance Tfh cell expansion.
LN follicle issues have also been observed in those undergoing antitumor necrosis factor-a treatment for conditions such as inflammatory bowel disease. 68 This type of immunosuppressive therapy decreases follicle numbers in the LN and leads to disrupted LN architecture as a whole. While this group mostly has an intact peripheral blood T-cell response after SARS-CoV-2 vaccination, there could be underlying issues within the LN itself, making it an interesting area for FNB analysis. 68,69 Further research into SARS-CoV-2-specific germinal center Tfh cells and their implications on NAb levels is warranted. While investigating highly immunosuppressed patients such as organ transplant recipients provides valuable insight into improving severely depleted SARS-CoV-2 responses, there is a great need to improve vaccine efficacy in those that have autoimmune diseases or are on treatments such as anti-CD20 monoclonal therapy. This would be assisted by investigating the immune responses at its source rather than repeated peripheral blood studies.

CONCLUSION
It is clear that despite the large number of studies investigating peripheral blood spike-specific CD4 + and cTfh cells from healthy and immunocompromised adults after SARS-CoV-2 vaccination, there are areas that need further investigation. Standardization of the phenotypic markers used to define cTfh cells would be highly beneficial at identifying this CD4 + T-cell subset, as several of the papers referenced in this review use CXCR5 exclusively, without incorporating PD-1. Future clonality studies involving the LN would give more clarity to this standardization, as well as possibly incorporating markers such as CXCR3, ICOS, CD38 or HLA-DR to identify acute specific cTfh cells after infection or vaccination. In addition, investigating approaches to increase vaccine efficacy in immunocompromised individuals would be highly beneficial. As these groups are often the most impacted by infections such as SARS-CoV-2, research to increase effective seroconversion would greatly reduce associated mortality and morbidity. Further research into vaccine efficacy would be greatly assisted by site-specific LN analyses, which then has the potential to be modified to improve the immune response.